PERCUSSION AS AN ALTERNATIVE SEED TREATMENT FOR
ROBINIA NEOMEXICANA (NEW MEXICO LOCUST)
BY
NABIL Y KHADDURI
A thesis submitted to the Graduate School
in partial fulfillment of the requirements
for the degree
Master of Science
Major subject Horticulture
New Mexico State University
Las Cruces New Mexico
May 2002
Percussion as an Alternative Seed Treatment for Robinia neomexicana (New Mexico
Locust) a thesis by Nabil Y Khadduri in partial fulfillment of the requirements for
the degree Master of Science has been approved and accepted by the following
Interim Dean of the Ora uate School
Chair ofExamining Committee
Date
Committee in charge
Dr John T Harrington Chair
Dr David R Dreesen
Dr Leigh Murray
Dr Rolston St Hilaire
ACKNOWLEDGMENTS
I would like to thank Dr John Harrington for his guidance sense ofhumor
and flexibility throughout my studies I would also like to thank the members ofmy
committee for their support especially Dr Murray who patiently taught me the
subtleties of categorical analysis of variance
I am also grateful to Dr Anne Wagner and Molycorp Mine who provided
funding for this research Thanks to Dr Bob Karrfali for his assistance with x-ray
radiography and to Mohamed Murshed and Dr Soumitra Ghoshroy for their
assistance with scanning electron microscopy Special thanks go to my colleague Lee
Rosner who assisted me in all phases of this project Finally I thank my family for
their enduring love Jill Walid Amin Rolla Khalid Piet Zach Charlie and above
all mywife Lucie
111
VITA
June 3 1972
June 1990
May 1994
July 1995
August 1994- March 1998
March 1998-August 2000
August 2000-May 2002
Born in Beirut Lebanon
Graduated from Georgetown Day High School Washington DC
Graduated from Pomona College Claremont CA with Bachelor ofArts Degree in Political Science
Graduated from University ofNew Mexico Albuquerque NM with Post-baccalaureate Teaching Degree
6th Grade TeacherBanta Fe Public Schools Santa Fe NM and Environmental Educator Open Space Division Albuquerque NM
Assistant Manager Santa Ana Native Plant Nursery Santa Ana Pueblo NM
Graduate Research Assistant at New Mexico State University
FIELD OF STUDY
Horticulture
iv
ABSTRACT
PERCUSSION AS AN ALTERNATIVE SEED TREATMENT FOR
ROBINIA NEOMEXICANA (NEW MEXICO LOCUST)
BY
NABIL Y KHADDURI
Master of Science in Horticulture
New Mexico State University 2002
Las Cruces New Mexico
Dr John T Harrington Chair
New Mexico locust (Robinia neomexicana A Gray) a small tree native to
montane regions of the southwestern United States fills a successional role in postshy
disturbance situations The ability to colonize rapidly and ameliorate harsh sites
contributes to the importance ofNew Mexico locust in reclamation efforts Seedling
production for outplanting on disturbed sites has been hampered by poor germination
This research sought to improve seed propagation techniques in New Mexico locust
Nine seed sources were collected across a range of latitudes within New
Mexico Seed treatments included a control three levels of hot water and four levels
of percussion scarification Hot water treatments included immersion at 100degC and
boiling for periods of 30 seconds and 60 seconds Percussion treatment levels were 1
2 4 and 8 minutes A total of four lOO-seed samples were used to test each source
by scarification treatment combination Categorical Analysis of Variance was used to
determine main effects and interactions Pairwise comparisons were conducted to
compare non-control treatment means using a conservative alpha value of 005
divided by the number of comparisons
Averaged over all sources and for each source percussion scarification
durations of4 or 8 minutes yielded greatest germination In addition to consistently
bigh germination 4- and 8-minute percussion-treated seeds germinated as fast or
faster than all other treatments Averaged over all sources germination increased
with increasing durations of percussion then leveled off for the 4- and 8-minute
treatments Immersion and 30-second boiling scarification maximized germination
for the hot water treatments with a decrease in germination when seeds were boiled
for 60 seconds
Percussion scarification specifically weakens the strophiole the natural
dormancy barrier in many papilionoid legumes without excessively damaging the
seed coat As with New Mexico locust papilionoid legumes often play an integral
role in the revegetation ofdisturbed lands The percussion scarification technique
developed in this research has the potential to facilitate the use of these valuable
species in reclamation projects
VI
CONTENTS
LIST OF TABLES ix
LIST OF FIGURES middot middotmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot x
INTRODUCTION 1
LITERATlTRE REVIEW 4
Seed Donnancy 4
Physical Donnancy 5
Evolutionary Role ofPhysical Donnancy 6
Methods for Breaking Donnancy 6
Acid Scarification 6
Hot Water Scarification 8
Mechanical Scarification 11
Alternative Method for Breaking Donnancy Percussion 12
Objectives 15
Hypothesis 15
MATERIALS AND METHODS 16
Seed Collection and Cleaining 16
Experimental Design and Treatment Structure 16
Treatments ~ bullbull bullbullbullbull - bullbull ~ ~ 20
G T ennlnatlon estmg 21
X-ray Radiography 22
Scanning Electron Microscope Photography 22
Analysis 22
RESULTS 24
DISCUSSION 36
Detennining the Optimal Treatment 36
Strophiole and Seed Coat Behavior 37
CONCLUSION 44
Appendices
A PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR NEW MEXICO LOCUST AND BLACK LOCUST SEEDS 47
B GERMINATION PERCENTS AND STANDARD ERRORS FOR SOURCE BLOCK AND TREATMENT FOR NEW MEXICO LOCUST 64
LITERATURE CITED 72
Vlll
LIST OF TABLES
Table Page
1 Latitude longitude location elevation and collection date ofNew Mexico locust seed sources 18
2 Mean seed weight and standard error by source 19
3 Categorical Analysis ofVariance table for germination response to seed treatment seed source block and the interaction of the factors 25
4 Effect of seed source and treatment on G50 (days to 50 germination) 32
B1 Germination percents and standard errors for source block and treatment for New Mexico locust 65
LIST OF FIGURES
Figures Page
1 Seedsourcecollectionmap 17
2 Effect ofNew Mexico locust seed source on germination for data averaged over scarification treatments 26
3 Effect of scarification on germination for New Mexico locust for data averaged over all seed sources 27
4 Effect of seed source by treatment interaction on New Mexico locust germination for percussion scarification pattern a and pattern b 28
5 Effect of source by treatment interaction on New Mexico locust germination for hot water scarification 29
6 Timecourse of New Mexico locust germination following scarification treatments 31
7 X-ray radiographs of Sacramento seed source perc ussed for 1 4 or 8 minutes 33
8 Seed damage close-ups 34
9 SEM images of untreated vs 4-minute percussion-treated strophiolar cells ofNew Mexico locust seeds 35
10 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (b c) 38
11 Damage to New Mexico locust seeds observed during germination counts following hot water scarification 40
12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources 42
New Mexico locust a primary invader following disturbance 50
Implementation of hot water and percussion scarification 52
Effect of scarification on germination response for New Mexico locust 55
x
A4 Effect of selected scarification treatments on germination speed for New Mexico locust 56
AS Effect of scarification on germination response for black locust 57
A6 Effect of selected scarification treatments on germination speed for black locust 59
A7 Damage to New Mexico locust seeds observed during germination counts following hot water scarification 60
AS New Mexico locust seeds following percussion treatment at time of germination counts 61
A9 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (bc) 63
INTRODUCTION
New Mexico locust (Robinia neomexicana A Gray) a small tree native to the
southwestern United States occurs at elevations from 1200 to 2800 meters This
species fills a successional role in post-disturbance situations As a primary invader
New Mexico locust quickly establishes on burned areas arid flood banks as well as
road cuts (Wagner et aI 1992) Rapid growth crown sprouting and prolific root
suckering favor the successful establishment ofNew Mexico locust on disturbed sites
(Simpson 1988 Gottfried 1980) These attributes also may make New Mexico locust
well suited for steep-slope revegetation where erosion is a problem
New Mexico locust is a nitrogen-fixing legume that tolerates and improves
nutrient-poor soils Stands ofNew Mexico locust increase Nitrogen (N) and Calcium
(Ca) in the forest floor Levels of Carbon (C) Phosphorous (P) Sulfur (8) and
Potassium (K) aIso have been shown to increase in soil beneath New Mexico locust
(Klemmedson 1994)
The ability to colonize rapidly and ameliorate harsh sites contributes to New
Mexico locusts potential as a nurse plant A nurse plant colonizes an inhospitable
site and creates an environment suitable for successional plant establishment New
Mexico locust demonstrates this role in the ponderosa pine (Pinus ponderosa Douglas
ex Lawson amp C Lawson)Gambel oak (Quercus gambelii Nutt) community After a
disturbance such as wildfire New Mexico locust and Gambel oak colonize and
dominate the site until shaded out by ponderosa pine Once shaded these species
become understory shrubs a process taking an average of 15-20 years (Dick-Peddie
1993 Hanks and Dick-Peddie 1974) New Mexico locust gradually declines with
small dense patches averaging about 005 ha beneath pine until the next disturbance
once again offers it a competitive advantage (Klemmedson 1994)
Gottfried (1980) expecting ponderosa pine regeneration to be greater where
New Mexico locust had been eradicated found that survival of planted pine seedlings
was greater where locust had not been removed In a follow-up study Gottfried
(1980) noted that soil moisture in the top 57 cm was highest in 5-year old locust sites
as compared to grass or 20-year old locust sites He concluded that managing an
appropriate cover ofNew Mexico locust could help regenerate pine the later
successional species
The ability ofNew Mexico locust to improve harsh sites also makes it a
candidate for reclaiming disturbances associated with mining Black locust (Robinia
pseudoacacia 1) has been used for decades to reclaim mine spoils and other
disturbed sites in the eastern US and around the world (Keresztesi 1988 Zimmerman
and Carpenter 1980) Ashby et al (1985) described similar positive attributes of
black locust also mentioned previously for New Mexico locust quick cover for
stabilization supply ofN and nutrient-rich litter to the soil and site improvement for
establishment of later successional trees Referring specifically to mine reclamation
the authors note the ability of black locust to grow on a wide range ofmine soil
conditions including extremely acid soils Black locust also shows some tolerance to
soils compacted by grading and topsoiling practices (Ashby et aL 1985) New
2
Mexico locust holds promise as a native southwe~tem counterpart to black locust in
mine reclamation but it has been used infrequently to date
Natural invasion and succession occur slowly on most mine sites (Monsen
1984) While New Mexico locust often colonizes sites naturally there is no
assurance a seed will reach a particular site and establish in a reasonable time frame
The challenge is to introduce New Mexico locust as mother plants to facilitate
colonization Seedling production for outplanting on disturbed sites has been
hampered by poor germination (Lin et al 1996) The goal then is to develop
techniques to overcome poor germination in New Mexico locust thereby facilitating
macropropagation
LITERATURE REVIEW
Seed Dormancy
Low gennination in New Mexico locust is primarily the result of seed
donnancy the failure of an intact viable seed to genninate under favorable
conditions (Bewley 1997) Seed donnancy also can be defined as the inability of the
embryo to genninate because of some inherent constraint (Bewley and Black 1994)
The constraint may originate within the embryo referred to as endogenous or embryo
donnancy or from the tissues surrounding the embryo known as exogenous or coatshy
imposed donnancy (Baskin and Baskin 1998 Leadem 1997 Bewley and Black
1994)
New Mexico locust seed is donnant when the impenneable seed coat does not
allow movement ofwater to the embryo This specific fonn of coat-imposed
donnancy where the restraining tissue involved is an impenneable seed coat is
known as physical donnancy (Baskin and Baskin 1998 Bewley and Black 1994)
Physical donnancy is relatively uncommon in seeds less than 10 of all woody
species exhibit this condition (USDA Forest Service 1948) However it is
characteristic of many genera of the Fabaceae (bean) family For example all
successional trees in the genera Gleditsia and Robinia (of which New Mexico locust
is a member) produce seeds that exhibit physical donnancy (Baskin and Baskin
4
Physical Dormancy
Seeds that exhibit physical dormancy are said to be hard seeded referring to
the thick impermeable seed coat (Leadem 1997) The first line of protection of the
testa in leguminous seeds is the outermost waxy cuticle followed by a subcuticle
consisting ofsuberin Next is a palisade layer consisting of sclereid cells that have
thick lignified secondary walls The most common type of sclereid cell in palisade
layers is the macrosclereid The macrosclereids contain water-repellent substances
including cutin lignin suberin and wax (Rolston 1978) This palisade layer is the
primary barrier to water uptake (Serrato-Valenti et al 1993 Tran and Cavanagh
1984)
All seed coat openings must be blocked to effectively prevent water uptake
The natural opening in a seed where it was attached to the mother plant is the weak
link where physical dormancy is usually broken (Hopkinson 1993 Kuo and Tam
1988) Anatomical structures associated with the seed coat opening vary from
family to family In the Papilionoideae the subfamily of the Fabaceae family
containing New Mexico locust the site ofwater entry is the strophiole The
strophiole is a swelling on the seed coat close to the stalk scar (Hopkinson 1997)
Kelly and van Staden (1987) refer to the strophiole site as the lens where
macrosclereid cells must separate and raise before water can pass through Since
middot6germination ofdormant seeds of the Papilionoideae takes place when the strophiole
permeable (Hopkinson 1993 Kuo and Tam 1988) a logical approach to
5
genninating New Mexico locust artificially would be to loosen the strophiolar cells
and allow water to pass through to the embryo
Evolutionary Role of Physical Dormancy
Why is physical dormancy a mechanism that blocks germination
ecologically advantageous When a plant produc~s seeds with different degrees of
dormancy seeds that are dispersed at one time will germinate over a range of time
(Bewley and Black 1994) Through variability of dormancy within a seed crop seeds
are exposed to changing environmental conditions improving the overall chances of
survival of the seed line (Bewley and Black 1994) Varying expression of dormancy
is under genetic control which indicates that this may be an evolved survival strategy
(Kigel and Galili 1995) A range of dormancy levels may be a particular advantage in
the southwestern US where extreme environmental variations exist
Methods for Breaking Dormancy
There are three common approaches to breaking dormancy in New Mexico
locust seed acid scarification hot water soaks and mechanical treatments In each
case the objective of the treatment is to scarify or abrade the seed coat to make it
permeable to water
Acid Scarification
Acid scarification consists of soaking seeds in concentrated sulfuric acid
rinsing thoroughly with water upon removal followed by subsequent drying before
PlaJlltmg Olson (1974) reports that this scarification method produces the highest
middot~~jllalUll results for all Robinia species Most literature refers to a seminal 1937
6
study conducted by HG Meginnis This stud~ t~ough conducted on black locust
seed is regularly cited in the suggested use of acid to treat New Mexico locust seed
as well (Olson 1974 Vines 1960)
Thee published studies document the specific use of sulfuric acid to treat
New Mexico locust seed Cox and Klett (1984) report that soaking seeds in
concentrated sulfuric acid for thirty minutes improved germination to 34 over an
untreated control of4 Lin et al (1996) found that for two seed lots acid
scarification for twenty-five to eighty minutes produced germination of 80 A third
lot however resulted in less than 20 germination at all durations of 10 to 80
minutes Hine et al (1997) showed that acid treatment levels of 1 2 5 and 10
minutes resulted in less than 20 germination across five sources However there
was an increasing trend in germination with acid soak duration and acid treatments
were not imposed for as long a time as the previous two studies (30 to 80 minutes)
The mixed results obtained by these investigators were probably due to the
widely varying germination response of seed lots to acid treatment For this reason
Olson (1974) recommends correlating duration of acid treatment to each seed lot If
treatment duration is too long the embryo will be permanently damaged If soak
duration is too short the seed will remain impermeable to water and fail to germinate
Compounding the problem of treatment duration is the negative effect acid
have on seedling growth Any type of scarification can injure seeds to some
by disrupting cells essential for growth leading to fungal invasion and
middot-uuu~w injury (Copeland and MacDonald 1995) Some legume seeds subjected
7
to 60 minutes of sulfuric acid scarification (to achieve maximum gennination results)
showed more than 50 seedling mortality after gennination (Kelly and van Staden
1987) Bewley and Black (1994) noted that fungal growth on seeds is an indication
of leakage of sugars organic acids amino acids and proteins
Perhaps the greatest drawback to acid scarification is the dangerous nature of
the treatment itself Difficulties associated with storage and trained use of
concentrated acid may discourage many propagators from using this option (Dreesen
and Harrington 1997) Consequently hot water and to a lesser extent mechanical
scarification are more commonly used and practical treatments for legume seeds
Most southwestern container growers use either of these two treatments to scarify
New Mexico locust seed in particular (Hine et al 1997)
Hot Water Scarification
The widespread use ofhot water scarification to treat locust seed is probably
due to its ease of application and efficacy in comparison to other scarification
treatments Although this practice had been in use for some time Wilson (1944) was
one of the first to report on its systematic application for black locust The treatment
consists of placing seeds in boiling water (4 parts water tomiddot 1 part seed by volume)
removing the vessel from the heat source and allowing the water to cool and seeds to
soak for 24 hours
Hine et al (1997) Lin et al (1996) and Cox and Klett (1984) have examined
the specific application ofhot water scarification on New Mexico locust seed Each
these authors compared hot water to acid scarification Lin et aL (1996) reported
8
60 gennination for two hot water-treated seed lots while a third lot germinated
around 50 (as reported above acid scarification germination ranged from 20 to
80 for these lots) It should be noted that the authors only soaked the seeds in water
from 10 to 80 minutes for this experiment as opposed to the standard 24 hours Cox
and Klett (1984) stated that using the standard 24-hour hot water treatment improved
germination over an acid pre-treatment (which produced less than 20 germination)
but they did not give specific gennination numbers for the hot water treatment Hine
et al (1997) found that across five seed lots germination was 66 for the standard
hot water treatment (as compared to less than 20 germination for acid scarification)
Variations in treatment intensity and duration and their subsequent effects on seed lot
appear to contribute to conflicting conclusions as to whether acid scarification or hot
water treatment is a more successful treatment
Given mixed results ease ofapplication is an important argument for use of
hot water application over acid soaks Safety concerns and material costs for hot
water treatments are low (Dreesen and Harrington 1997) An extension of this is the
practicality ofhot water for treating large batches of seed where a relative increase in
water volume is all that is needed However increases in water and seed even when
inthe same 4 1 proportion can result in dramatically different rates of cooling than in
atest tube experiment Different cooling rates can lead to contrasting results for the
treatment temperature duration and seed lot (USDA Forest Service 1948)
As with acid scarification parameters ofhot water treatment application may
to be optimized to maximize germination Hine et al (1997) found that as
9
temperature was increased from 70degC to 100degC overall germination in New Mexico
locust seed increased The 100degC treatment maximized germination for each of five
sources tested with germination usually above 60
Prolonged exposure of seeds ofmany woody legumes to boiling temperatures
also may improve germination (Briscoe 1996) This approach has not been tested on
New Mexico locust seed Exposing seed to prolonged boiling temperatures may
result in fungal infection and subsequent seedling mortality Marunda (1990) noted
severe fungal infections on both acid and hot water treated Acacia holoserica Cunn
ex G Don seeds attributing the infections to seed damage resulting from the
treatments Identifying an optimal hot water treatment level that does not result in a
high rate of seedling mortality is a logical goal
With hot water treatments as with acid there is also a danger of overtreating
Seeds and reducing germination (Gosling et al 1995) The legume Leucaena
leucocephala (Lam) de Wit germinated equally well after 7 seconds of exposure to
water at 100degC 7 seconds at 90degC 30 seconds at 80degC and 4 minutes at 70degC In
addition acceptably high germination was obtained from treatment at 70degC from 3
minutes to 1 hour whereas no seeds germinated following treatment at 100degC for 2
minutes Perhaps along with a greater margin of error for treatment duration treating
~ew Mexico locust seeds at a lower temperature may result in a comparatively lower
of seedling mortality In practice however nursery growers tend to use the
and quickest treatment available The short treatment length at near-boiling
whether recommended or not may be what is used most frequently
10
Fine-tuning a treatment in this range of treatment temperature and duration may be
the most logical approach
Mechanical Scarification
This treatment involves scratching the seed coat with an abrasive material
(USDA Forest Service 1948) One system employs a rotating drum lined with
sandpaper A paddle spinning at a given number ofrevolutions per minute throws the
seed against the sandpaper (Hine et al 1997) A second method dry tumbling
tumbles seed in a mixer with pea gravel or sharp sand for 5 to 7 days (Dreesen 2001)
These approaches have the advantage that control of temperature is not required as in
water treatments nor is safety an issue as with add scarification
Hine et al (1997) mechanically treated New Mexico locust seed using 100shy
grit sandpaper in a Forsberg (rotating drum) scarifier Gennination was less than
10 They concluded that the seed coats were too thin for their apparatus and
proposed that a finer grit sandpaper would produce more even abrasion Although
germination varied by seed lot Dreesen (2001) reported that using a slow-speed dry
tumbling technique for 5 to 7 days produced gennination rates as high as 90
As with acid and hot water treatments treatment duration must be specifically
shy
correlated to seed lot Treated seeds are also susceptible to fungal infection
iHopkinson 1997) In addition equipment is specialized and may not always be
ltU~CUJl~ to a nursery (USDA Forest Service 1948) Several studies have confinned
degrading ofstrophiolar cells due to acid and hot water scarification (Miklas et al
11
1987 Dell 1980 Hanna 1984) but the effect ofmechanical scarification on the
strophiole has not been studied
Alternative Method for Breaking Dormancy Percussion
Strophiole degradation has been examined in depth using an impaction
treatment more commonly known as percussion Hamly (1932) first proposed that
shaking or impacting seeds could split the macrosc1ereid cells at the strophiole
Hamly successfully released tension ofbent strophiolar cells of impermeable
Melilotus alba Medik (White sweet clover) seeds by delivering physical blows
resulting in cells that had split apart and were only slightly bent Ninety-two percent
oftreated seeds became permeable as compared to 05 of control seeds Barton
(1947) showed that percussion significantly increases permeability in black locust
seed Ballard (1976) reasoned that the integrated nature of the strophiole within the
seed coat means a blow on any part of the seed weakens the strophiole There are no
published studies on the use of percussion on New Mexico locust
Percussion methods vary and often seem SUbjective or ill-defined Hamly
(1932) placed seeds in a glass bottle and shook them three times a second for 20
minutes resulting in 180 oscillations per minute Ballard (1973) describes shaking
seeds in a closed metal container with at least fifty seeds per container at 850-900
JS(~l11alIOIlS per minute for 1 minute Kelly and van Staden (1987) placed seeds into
sealed glass flasks Flasks were shaken by a wrist-shaker at a rate of 250
~UUVJ~ per minute Subsequent germination rates were 45 after 2 hours of
55 after 3 hours 60 after 6 hours and 100 after 12 hours
12
There are several potential advantages of percussion over acid hot water and
mechanical scarification Kelly and van Staden (1987) note that a percussion
treatment only alters macrosclereid cells of the strophiole by loosening separating
and raising them They emphasize that minimal damage is done to the overall
anatomical structure of the seed In acid scarification of the same seed lot radicle
and cotyledon damage occurred Based on photographic evidence the authors
believe that this morphological damage was due to uneven pressure during
imbibition with water entering haphazardly wherever the acid had sufficiently
dissolved the coat This ultimately led to uneven hydration of the embryo In
percussed seeds water only entered through the s~ophiole when the entire seed coat
had been saturated resulting in controlled and even embryo hydration (Kelly and van
Staden 1987)
Although all successful scarification treatments may focus at least in part on
the strophiole for papilionoid legumes (Baskin and Baskin 1998) percussion may be
less damaging to seed coat structures particularly when compared with acid Kelly
and van Staden did not compare hot water or mechanical scarification methods
against percussion However the fungal infection associated with hot water and
mechanical scarification as with acid scarificatio~ have been directly associated with
seed damage (Rehman et al 1998 USDA Forest Service 1948) Embryo damage in
has been observed in hot-water scarified legume seeds (Rehman et al 1998
13
By limiting embryo damage percussion scarification reduces seedling
mortality compared to other treatments Kelly and van Staden (1987) noted that
seedlings from the percussion treatment were morphologically sound and vigorous
when compared to acid treatment seedlings The authors attribute this improved
seedling vigor to the controlled water uptake that limited anatomical damage
Despite a 12-hour percussion treatment morphological damage was not substantially
greater than untreated seeds In addition seedling mass measured 10 days following
germination was not significantly different between percussed and untreated seeds
A final advantage ofpercussion may be the ability to adequately treat the most
dormant seeds while avoiding harm to seeds that have already been made permeable
by the treatment or that were permeable to begin with Instead ofcorroding scalding
or scratching the seed coat percussion appears to be just a loosening of the
strophiole spring (Ballard 1973) Kelly and van Staden (1987) were able to
increase germination with a lengthy treatment (12 hours) Figuring out whether a
substantial cushion oftreatment durations exist for percussion ofNew Mexico locust
seed would be a significant improvement to current scarification methods where seed
damage and seedling health is an issue
The greatest challenge for applying percussion to New Mexico locust seeds is
standardizing a treatment that is both effective and readily reproducible by nurseries
with mechanical scarification this may require the development of specific
~UllDment to properly quantify and execute the treatment
14
f
Objectives
The first objective of the study is to detennine which scarification treatment
results in optimal gennination speed and percentage ofNew Mexico locust The
second objective is to describe treatment interactions with nine seed sources collected
in New Mexico
Hypothesis
A percussion treatment acts exclusively on the main area ofwater uptake in
New Mexico locust seed loosening the strophiole so that imbibition can occur This
direct mechanism ofdonnancy release will result in improved gennination rate and
overall gennination percentage for New Mexico locust seed compared to standard
treatments Standard treatments including acid hot water and mechanical
scarification while eventually weakening the strophiolar cleft cause excessive
morphological damage through generalized seed coat degradation These treatments
are therefore prone to inconsistency due to over and under-treatment
15
MATERIALS AND METHODS
Seed Collection and Cleaning
Seeds were collected from August 25 through September 15 2000 from nine
locations along a latitudinal gradient in New Mexico Seed source locations and
descriptive information are given in Figure 1 and Tables I and 2 Stands were
separated by at least 45 km
Pods were hand picked as they began to ripen and dehisce (open) Collections
were made from ground level to approximately 3 m from several trees at each site
Pods were dried in open-ended brown paper bags for two to four weeks at room
temperature (21-230C) to facilitate cleaning Dried pods were placed in a closed cloth
sack which was pounded against the ground to separate seeds from pods For pods
still containing seeds hand maceration (crunching pods with hands) completed seed
separation Debris was removed by threshing with the aid of a greenhouse fan A
South Dakota Seed Blower (Seedbureau Chicago Illinois) was used to remove
remaining chaff and light seeds by air density separation Seeds were placed in dry
storage in sealed quart-size zip-lock bags within a sealed plastic container Storage
temperatures ranged from 2-4oC Seeds remained in storage for approximately 7
months before the experiment
Experimental Design and Treatment Structure
The experiment compared the impact ofhot water bath and percussion
l11aLl11 on germination properties ofNew Mexico locust seed A single
16
-l
Figure 1 Seed source collection map All sources were collected within New Mexico USA separated by at least 4S lan
IIIRZltItW lIJPIirmiddot rllll1lbullbull_~~~_II
Table 1 Latitude longitude location elevation and collection date ofNew Mexico locust seed sources
Lot title Latitude (N) Longitude (W) Location Elevation (m) Collection Date
Raton 36deg5846 104deg2872 Raton Pass NM 2295 82500
Holman 36deg0306 105deg2429 HolmanNM 2476 911500
Jemez 35deg4500 106deg4100 Jemez Springs NM 1976 9900 00
Sandia 35deg1083 106deg2341 Cedar Crest NM 2432 82600
Manzano 34deg3689 106deg2494 ManzanoNM 2465 9200
Magdalena 33deg5953 107deg0903 Magdalena NM 2544 82900
Capitan 33deg2728 105deg4325 Alto NM 2189 911200
Sacramento 32deg5798 105deg4496 Cloudcroft NM 2630 91400 Gila 32deg5400 107deg4600 Kingston NM 2432 9700
f
Table 2 Mean seed weight and standard error by source
a
Source Mean seed weight (g) for Standard error 100 seeds n = 5
Raton Holman Jemez Sandia Manzano Magdalena Capitan Sacramento Gila
2872 b c 2522 d 1826 e 2753 c 2413 d 2559 d 2443 d 3130 a 3029 ab
00103 00403 00276 00301 00370 00374 00458 00279 00348
a Mean seed weights followed by the same letter are not significantly
different
19
experiment was conducted utilizing a randomized complete block design blocked by
time with a factorial treatment structure Experimental factors were seed source and
scarification treatment Nine seed sources from throughout New Mexico were used
(Figure 1 Table 1) Scarification treatments included a control three levels ofhot
water and four levels of percussion Hot water treatments included immersion at
100degC and boiling for periods of 30 seconds and 60 seconds Percussion treatment
levels were 1 2 4 and 8 minutes One hundred seeds per block were used to test
each treatment combination Four blocks of seed were tested one at a time (all seed
sources included in each run) in close chronological sequence (within 6 weeks) A
total of four 100-seed samples were used to test each source by scarification treatment
combination
Treatments
Hot water treatment of seeds utilized 50 ml test tubes filled with 30 ml
distilled water Each test tube was used to treat one 100-seed sample at a time
resulting in a ratio of4 parts water to 1 part seed by volume Test tubes were placed
in a beaker with the beaker filled with tap water to the same level as the water within
test tubes Nine test tubes (one per seed source) were placed in the beaker
simultaneously so that one replication of each seed sourcescarification treatment
cotnblnatlon was conducted simultaneously Hot water treatments include immersion
boiling For immersion treatments seeds were added to test tubes with the water
(boiling) and the test tubes were immediately removed from the beaker to
For the boiling treatments test tubes remained in the beaker with water at
20
boiling temperature for a set duration after the addition of seeds Following all
treatments test tubes with seeds cooled 24 hours in a test tube rack
Percussion treatment utilized a pneumatic paint shaker (Central Pneumatic
model 00422 Camarillo CA) Compressed air was maintained at 530 kPa plusmn30 kPa
(80 psi plusmn 5 psi) resulting in approximately 350 oscillations per minute Seeds were
placed in 118 ml (4 oz) soil tin cans (US Can Lobard IL) and the lids were secured
with duct tape Each can was used to treat one 100-seed sample To take advantage
ofgreatest lateral movement of the paint shaker a spacer (paint can) was used to
position the soil tin at the end of the shaking arm This allowed shaking to occur at
the greatest distance from the pivot point (fulcrum) Tins were placed perpendicular
to the direction of shaking motion allowing seeds to impact at greatest force by
hitting flat surfaces of the soil tin
Germination Testing
Before gennination testing all seeds were soaked 24 hours in reverse
osmosis-treated water (this was included in the process ofhot water treatment
jU~SCIlOea above) Following soaking seeds were placed in 100 cm Petri dishes lined
90 cm Whatman qualitative 1 filter paper circles (MG Scientific Pleasant
WI) One Petri dish was used for each treatment by source combination
papers were initially moistened with 5 mL reverse osmosis-treated water then
~VU1CUlJl moistened as needed to ensure adequate conditions for gennination
were monitored daily (at 300 PM) for gennination for 14 days in accordance
~UIUlnJlVUa Seed Testing Association (ISTA) standards for Robinia species
21
(1STA 1999) Tests were carried out at room temperature (22degC plusmnOSoC)
Germination was classified as protrusion of the radicle from the embryo by at least
1mm as viewed with the naked eye
X-ray Radiography
On August 132001 three lOO-seed samples from the Sacramento seed source
were treated for 14 or 8-minute percussion durations The seeds were sent
immediately by overnight delivery to the Faxitron X-Ray Corporation in Wheeling
Illinois where Dr Bob Karrfalt ofthe USDA Forest Service oversaw their imaging
Radiographs were made at SX with a Faxitron Specimen Radiography System Model
MX-20
Scanning Electron Microscope Photography
On February 192002 a Hitachi S-3200N Variable Pressure Scanning
Electron Microscope (Mountain View CA) was used to examine strophiolar cells of
untreated seeds and seeds percussed for 4 minutes Seeds were cut longitudinally
igt their entire length using a razor blade splitting the strophiolar region roughly in
Cross sections of seed samples magnifying the strophiolar region were viewed
600X under the microscope
Analysis
Categorical Analysis of Variance was used to determine treatment source and
(Le time) main effects and interactions The response variable was total
~vlI This procedure is a Chi-square test of homogeneity using the natural log
of germinated to non-germinated seed for each treatment Maximumshy
22
likelihood analysis was used to calculate Chi-square test statistics with their observed
significance levels A single contrast was conducted to cempare treatment means
against the control Observed significance levels less than or equal to 005 were
considered significant Pairwise comparisons were conducted between non-control
treatment mean percents using a conservative alp~a value of 005 divided by the
number of comparisons (Bonferroni approach) and approximate pairwise z statistics
Categorical analyses were performed using Proc Catmod (SAS Institute 1989)
RESULTS
Scarification seed source block and all interactions between factors
influenced germination (Table 3) The main effects are reported in the presence of
these interactions because the overall robustness of treatments and seed sources are of
interest Background levels ofnaturally occurring variation in seed sources are
seen in control germination percentages in Figure 2 In particular time variability
is unlikely to be under the control of the producer Appendix B details experimental
data for source by treatment gennination percents for each of the four blocks
Averaged over all treatments germination by source ranged from 40 to
79 (Figure 2) Averaged over all sources (Figure 3) and for each source (Figure 4
5) percussion scarification durations of4 or 8 minutes yielded greatest germination
Averaged over all sources germination increased with increasing durations of
percussion then leveled off for the 4- and 8-minute treatments (Figure 3) Immersion
and 30-second boiling scarification maximized germination for the hot water
treatments with a decrease in germination when seeds were boiled for 60 seconds
For all but two sources there is a similar ipcreasing trend in germination from
1to 4 minutes ofpercussion (Figure 4) The Holman source increased in germination
1 to 2 minutes then leveled off at 4 minutes The Jemez source changed little
1 to 4 minutes of treatment As treatment duration increased from 4 to 8
mutes five sources decreased in germination (Figure 4a) whereas three sources
24
Table 3 Categorical Analysis of Variance table for germination response to seed treatment seed source block and the interaction of the factors
Source of DF Chi-Square Observed Variability Significance Level
Source 8 98370 lt00001 Trt 7 233876 lt00001
contrast 1 16976 lt00001 SourceTrt 56 97077 lt00001 Block 3 5538 lt00001
SourceBlock 24 10162 lt00001 TrtBlock 21 7564 lt00001
SourceTrtBlock 168 34283 lt00001
S 4)
8 ~
0
sectg ~ 40 ~
N 0 0
20
o f0ltgt ~ ~4 jybshy ((iltgto ~~ K1ltgt 6~b-~ ~~ 0b-~~ ~~ ~b-~ ~b-~b
80
60 -I-- - ~- middotmiddotmiddotmiddotmiddot1
r -] Northern NM Sources
Central NM Sources
Southern NM Sources
sect-o ~
~b-v(i
SOURCE
Figure 2 Effect ofNew Mexico locust seed source on germination for data averaged over all scarification treatments Control germination is shown by solid line within bar
80
~ cd ~ ltl) () 60 ~ ~
N
t 0shyrti tshySltl)
40
0
20
o lmmersion Boiling 30 sec Boiling 60 sec Control 1 min 2min 4 min 8 min
Hot Water Treatments Percussion Treatments
Figure 3 Effect of scarification on germination ofNew Mexico locust for data averaged over all seed sources Means labelled with the same letter are not significantly different
100~----------------------~--~----------------~
80
60
ltU
~
40 IJ
sectgsI
l100
80
60
40
a I middotmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot middotImiddotmiddotmiddotmiddotmiddotmiddot E
_~iii-bull _ _ bull f --~--
t-- -~ ---- -shy ---- shyI ~ ---- - shy
L
fiII
~
b
- shy ---r
- Holman
Sacramento Sandia Gila Jemez
c ~
ltgt ~
Tmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot-I
j~=~=-~~-1
I middotmiddot ~ bull r
t tr
Y -------M-a-gd-a-Ie-n-a--
1 _ _
fCapitan
---- Manzano ~y -_ Raton
2 4 8
Time interval ofpercussion scarification (min)
Figure 4 Effect of seed source by treatment interaction on New Mexico locust germination for percussion scarification pattern a and pattern b
28
tv 0
~ d
~ A s o
1 c3
80
60
40
20
o
TIT
t------=middotfmiddotmiddot-middotmiddot-middotmiddot-middotmiddot 1- --- - - --=- - -shy--cE= - _--shy --shy ----shy ~ ~ ---shy ~ -~- _ --shy-------r 1
I
Raton Holman
-_ Jemez
--- Sandia Manzano Magdalena Capitan Sacramento Gila
Immersion Boiling 30 sec Boiling 60 sec
Hot Water Scarification
Figure 5 Effect of seed source by treatment interaction on New Mexico locust germination for hot water scarification
continued to increase in germination (Figure 4b) There was no difference between
the two treatment levels for the Manzano source (Figure 4b)
The effect ofhot water scarification also varied among sources with few
overall differences between hot water treatments (Figure 5) However boiling for 60
seconds yielded highest germination for the Jemez source whereas increasing
durations ofboiling decreased germination for the Raton source
In addition to consistently high germination 4- and 8-minute percussionshy
treated seeds germinated as fast or faster than all other treatments (Figure 6 Table 4)
050 measured as the number of days needed to complete 50 ofeventual
germination averaged 2 days for both ofthese treatments
X-ray radiographs were taken of seeds collected from the Sacramento source
percussed for 1 4 or 8 minutes (Figure 7) Seeds show signs of damage at 8 minutes
ofpercussion (Figure 8) Compared to an undamaged seed percussed for only 4
minutes (Figure 8a) damage at the 8-minute level includes fractured radicles pitting
of the seed coat and chips in seed coat structure (Figure 8b-d)
Scanning electron microscope (SEM) images at 600X reveal the direct effect
of 4 minutes of percussion on cells of the strophiolar region (Figure 9) Whereas
untreated seeds retain cellular organization in the strophiolar region strophiolar cells
seeds treated for 4 minutes ofpercussion separate and raise resulting in a
~-~e~U appearance
30
Umiddot-~middot--l-~---middotymiddotmiddotmiddot-----middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot--middot--middotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middotmiddotmiddotmiddotmiddotmiddotmiddot- -
050= gt1
-e-- Percussion 8 min -0- Percussion 4 min ---T- Percussion 2 min -----ltil- Percussion 1 min -- Immersion --0- Boiling 30 sec -+- Boiling 60 sec -ltgt- Control
2 4 6 8 10 12
Day Figure 6 Timecourse ofNew Mexico locust gennination following scarification
treatments G50 is the number of days to 50 of eventual germination
31
14
Table 4 Effect of seed source and treatment on G50 (days to 50 germination)
Seed Source Control Immersion B30sec B60sec Plmin P2min P4min P8min mean Raton 3 3 5 7 4 3 2 2 4 Holman 2 2 2 2 2 2 2 2 2
Jemez 4 6 6 5 3 2 2 2 4 Sandia 2 2 2 2 2 2 2 2 2
w tv Manzano 5 2 2 3 3 2 2 2 3
Magdalena 4 2 2 3 4 3 2 1 3
Capitan 5 4 2 2 3 2 2 2 3
Sacramento 3 2 2 2 4 3 2 2 3 Gila 3 2 2 2 2 2 2 2 2 mean 3 3 3 3 3 2 2 2 3 standard 11 14 16 18 09 05 0 03 08 deviation
Note Columns and rows may not agree due to rounding
Figure 7 X-ray radiographs ofSacramento seed source percussed for 1 4 or 8 minutes
33
Figure 8a-d Seed damage close-ups Figure a shows undamaged seed percussed for 4 minutes Figures b-d show seeds percussed for 8 minutes Damage includes fracturing ofradicle in b complete removal ofseed coat in c and pitting ofseed coat in d
34
Untreated Treated
Figure 9 SEM images ofuntreated vs 4-minute percussion-treated strophiolar cells ofNew Mexico locust seeds Treated cells are loosened separated and raised
DISCUSSION
Determining the Optimal Treatment
Averaging results of scarification treatmems across seed sources is a useful
tool to predict which treatment is most likely to be successful for an unknown seed
source Averaged over all sources four- and eight-minute percussion treatments
resulted in the highest germination 815 and 790 respectively and produced
germination that was as fast or faster than all other treatments Although there was no
difference between these two treatments on average an argument for 4 minutes of
percussion as a standard protocol for treating New Mexico locust seeds can be made
Descriptively 4 minutes of percussion improved germination over 8 minutes
ofpercussion for a majority of sources (five out of nine) 8 minutes of percussion
improved germination for three ofnine sources with no difference for a fmal source
For individual sources 4 minutes ofpercussion was always the best or secondmiddot best
treatment and was always equal to or better than all hot water treatments
Germination at 8 minutes ofpercussion was lower than all other percussion durations
for the Jemez and Gila sources and was lower than at least one hot water treatment for
these sources as well
The x-ray radiographs of the Sacramento seed source illustrate the excessive
internal and external seed damage that can occur with 8 minutes of percussion
germination counts 8-minute percussed seeds from Holman and Capitan
ClUll signs of damage that were visible to the naked eye (see below)
r~evertheless some of these seeds still fit the criteria for germination Neither x-ray
36
photography nor visible observations during seed counts revealed seed damage at the
4-minute percussion level In operational trials seeds percussed for 4 minutes
developed into healthy seedlings (Khadduri and Harrington unpublished data)
Strophiole and Seed Coat Behavior
Four minutes ofpercussion successfully breaks seed dormancy in a large
percentage ofNew Mexico locust seeds without excessively damaging the
surrounding seed coat or internal seed coat structures Most papilionoid legume
seeds including seeds ofNew Mexico locust have a specific region of the seed coat
known as the strophiole or lens (Hamly 1932) The strophiole is located on the
cotyledonary lobe of a locust seed (Figure 10 a-c) If seeds are not indiscriminately
damaged the strophiole is the first point of water entry to the embryo (Hopkinson
1997) When a papilionoid seed is percussed for the appropriate amount of time
repeated hits on the integrated seed coat loosen the constrained cells of the strophiolar
region without excessively damaging the rest of the seed coat (Ballard 1976) SEM
photos illuStrate that 4 minutes of percussion loosened and separated strophiolar cells
ofNew Mexico locust seeds when compared with constrained organized strophiolar
cells of untreated seeds (Figure 9) Research-has shown that water passes to the
embryo through the treated strophiole in a controlled manner and regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues
(Kelly and van Staden 1987)
37
10 Strophiole ofunimbibed New Mexico locust seed (a) and following uptake (bc)
38
In contrast hot-water treatments can excessively degrade the seed coat
(Rebman et al 1998 Marunda 1990) Random cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rebman et aI 1998 Marunda 1990) Along with a
proportionately large amount ofun-imbibed (thus under-treated) seeds (Figure l1a)
damage was observed in hot-water treated seeds from six sources during germination
counts Damage included seeds where the radicle failed to elongate (Figure 11 b)
seeds where the radicle elongated but was delayed in freeing itself from the seed coat
(Figure 11c) and seeds where the radicle completely broke off from the embryo
(Figure lId) In addition some seeds germinated in reverse (the cotyledon emerged
fIrst) If the radicle emerged completely from the seed coat they were counted as
germinated
While nearly all seeds treated for 8 minutes ofpercussion imbibed water
damage was evident in x-ray evaluations (Sacramento seed source) and in
germination counts (Holman and Capitan) at this treatment level Seed damage
during germination counts was similar to levels found in hot-water treated seeds
suggesting that over-treatment by both hot water and longer durations of percussion
may result in similar damage to seed structure
The Sacramento and Holman seed sources were two of the fIve sources that
opcrplltlpn in germination from the 4- to 8-minute durations of percussion
the benefIts ofloosening the strophiole as evidenced by imbibition of
all 8-minute treated seeds were outweighed by external and internal seed coat
39
a
b c d
11 Damage to New Mexico locust seeds observed during germination cotmts following hot water scarification 9a Hot water replicate including ~t seedlings and numerous unimbibed seeds 9b Close-up of seed where raallCle failed to elongate 9c Close-up ofseed where radicle elongated but was
-gtIn in freeing itself from the seed coat 9d Close-up ofseed where radicle broke off from embryo
40
damage (which also may be contributing to high imbibition) For the three sources
for which 8 minutes of percussion resulted in maximum germination extended
percussion of seeds necessary to loosen the strophiole may not have excessively
damaged the seed anatomy The Manzano source did not significantly change in
germination from 4 to 8 minutes of percussion suggesting that an increase in
permeable seeds at the 8-minute treatment duration was balanced by the number of
seeds that failed to germinate due to seed damage To summarize the 4-minute
percussion treatment improved germination over 8 minutes ofpercussion for a
majority of sources (five ofnine) the 8-minute percussion treatment improved
germination over 4-minutes ofpercussion for three sources and there was no
difference with the Manzano source Notably for the Raton source 8 minutes of
percussion increased germination by 235 percentage points over 4-minutes of
percussion (7875 vs 5525 respectively) the next best treatment Raton was the
only source to result in germination less than 75 for 4 minutes ofpercussion so a
grower may wish to apply longer percussion durations to lots that do not respond well
to this treatment level
Finally there was an interesting trend in the Jemez and Raton sources seed
that significantly contributed to the source by treatment interaction (Figure 12) A
~u test showed that the Jemez source had a significantly lighter seed weight than
other sources while the Raton source had an average seed weight For the Jemez
the longest duration of boiling increased germination Conversely only short
U4UVll) of percussion were needed to achieve high germination and the longest
41
80 _ Jemez a Raton
iD
~ C iD e iD
Q c 0 ~
c ~ iD 0
20
0------shy
Immersion Boiling 30 sec Boiling 60 sec
Hot Water Scarification
10degTr========~--------~------------------------------
80
60
40
20
_ Jemez Raton
0------shy
1 min 2 min 4 min 8 min
Percussion Scarification
Figure 12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources
42
b
--~----~~~~========~---- duration ofpercussion reduced germination For the Raton source longer durations
ofboiling consistently decreased germination whereas longer percussion treatments
consistently increased germination
A possible explanation is that a thick seed coat is associated with a loose
strophiole structure and that a thin seed coat is associated with a constrained
strophiole This contrast merits further study and may suggest an ecological
adaptation Further investigation ofthis phenomenon may contribute to successful
pre-screening ofunknown seed sources to determine optimal treatment leveL
43
CONCLUSION
Based on results obtained in pilot study (see Appendix) and thesis
experiments 4 minutes ofpercussion is recommended as a standard protocol for
treating an unknown source ofNew Mexico locust seed In addition to laboratory
results the 4-minute percussion treatment has resulted in germination greater than
80 and healthy seedling grow-outs for production purposes (unpublished data) In
laboratory experiments only one hundred seeds per 118 ml (40z) soil tin were
percussed at a time whereas for production we have treated up to five hundred seeds
per tin For large-scale production ofNew Mexico locust seedlings growers may
wish to experiment with larger quantities of seed and larger-size soil tins
Seed storage trials following percussion treatments were not conducted
However germination greater than 80 and healthy seedlings were obtained when
seeds were percussion-treated and subsequently stored at room-temperature (22degC)
for one week (unpublished data) Seed storage trials merit further study as the
standard hot water treatment leaves seeds in a softened condition that is not
conducive to storage
Hydroseeding is an effective method for direct seeding in reclamation
situations Percussion treatment may be conducive to hydro seeding as seeds remain
a dry relatively undamaged yet permeable condition following treatment Sulfuric
seeds also remain dry following treatment but extensive damage to seed coat
poor seedling vigor have been reported with this treatment (Kelly and van Staden
A grow-out study using a hydro seeder on a reclamation site testing percussion
44
against other treatments such as sulfuric acid and mechanical scarification also
merits further study
Papilionoids are the largest subfamily oflegumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust these legumes often play an integral role in the revegetation of
disturbed lands The pilot study (Appendix) indicates that percussion scarification
successfully treats the widely used black locust as well Developing superior
scarification methods to the standard hot water mechanical scarification and acid
treatments may facilitate the use ofpapilionoid legumes in reclamation projects
45
APPENDICES
APPENDIX A
PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR
NEW MEXICO LOCUST AND BLACK LOCUST SEEDS
Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4
Hot water and sulfuric acid soaks are traditional treatments for seeds of many
temperate woody legwnes including locusts However these scarification techniques
often produce inconsistent germination Percussion scarification where seeds are
repeatedly propelled against a hard surface was compared with hot water
scarification to evaluate treatment efficacy for New Mexico locust (Robinia
neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment
seeds were placed in a 98degC water bath which was immediately removed from the
heat source For percussion scarification seeds were placed in a soil sample tin and
agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the
control were followed by 24-hour water soaks Hot water baths resulted in 56 and
41 germination for New Mexico locust and black locust respectively For both
species nearly all durations ofpercussion increased germination over the hot water
treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3
4 and 5 minutes for black locust resulted in at least 90 germination Traditional
scarification treatments randomly degrade the entire seed coat which can lead to
tissue damage during water uptake Percussion scarification specifically weakens the
strophiole the natural source ofwater entry to the seed in papilionoid legwnes
INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom
2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031
48
Following percussion imbibition is controlled through the strophiole and underlying
tissue is protected
Keywords New Mexico locust black locust scarification percussion germination
Introduction
Locust trees (Robinia spp) are aggressive pioneer species that quickly
colonize disturbed land fix atmospheric nitrogen in the soil and stabilize slopes that
are prone to erosion (Klemmedson 1994 Ashby et al 1985) (Figure A1ab)
Recognition of these attributes has contributed to increased demand for locusts in
reclamation projects Locust seeds however exhibit physical dormancy where a
thick seed coat prevents the movement of water and gasses to the embryo (Leadem
1997)
Sulfuric acid or hot water soaks have traditionally been used to break seed
dormancy in macro-propagation of locusts Many growers are moving away from the
use of su1furic acid in treating locust seeds (Dreesen and Harrington 1997) In
addition to being dangerous acid soak durations must be specifically correlated to
seed lot (Olson 1974) Collections of native seed can vary tremendously in seed size
seed weight and hard seededness etc This variability limits the utility of sulfuric
acid soaks as even within a given lot some seeds may be damaged whereas others
are under-treated and fail to imbibe (take up water) A survey of Southwestern
container growers indicates that hot water baths are currently the most common
scarification method for locust seeds (Hine et al 1997) However hot water baths
also produce inconsistent germination (Lin et al 1996)
49
b
Figure AI New Mexico locust a primary invader following disturbance Ala Crown-sprouting at edge ofburn near Los Alamos NM--one year after Cerro Grande fire Alb Colonizing road cut Gila National Forest NM
50
Percussion scarification where seeds are repeatedly propelled against a hard
surface is an alternative dormancy-breaking method that has proven successful in
legumes related to locusts (Hamly 1932 Barton 1947) We compared this alternative
treatment to hot water scarification to evaluate treatment efficacy for New Mexico
locust and black locust seeds
Materials and Methods
Commercial New Mexico locust seeds (Western Native Seed Coaldale CO)
were collected fall 2000 in Huerfano County CO Black locust seeds were collected
September 2000 from Taos County NM by harvesting ripe pods from several trees at
a distance of up to 3 m from ground level Black locust seeds were allowed to air-dry
for 3 weeks and were threshed and separated from large chaffusing a greenhouse
fan A South Dakota Seed Blower (Chicago Illinltois) was used to remove fine chaff
from both seed lots Seeds were storedmiddotat 2-4C until the germination study was
conducted in March 2001
This study compared hot water scarification with percussion scarification
using treatment and species as experimental factors Along with a control
scarification treatments included immersion of seed in boiling water as well as
percussion durations of 1 2 3 45 or 10 minutes Four 100-seed samples were used
to test each species by scarification treatment combination
For the hot water treatment test tubes were filled with 30 mL water and
placed in a water-filled beaker (Figure A2a) The bath was raised to boiling (-98C at
51
b
c
Figure A2 Implementation ofhot water and percussion scarification A2a Hot water bath A2b Paint-shaker set-up A2c Placing soil tin containing seeds at end ofpaint shaker ann
52
Las Cruces NM elevation of 1300 meters) Each 100-seed replicate was placed in a
test tube and each test tube was immediately removed to cool at room temperature
Percussion scarification was implemented using a pneumatic paint shaker
(Central Pneumatic model 00422 Camarillo CA) (Figure A2b) This allowed
standardization of treatment intensity Compressed air pressure was maintained at
530kPa plusmn 30kPa (80psi plusmn 5psi) resulting in approximately 350 oscillations per
minute Each lOO-seed sample was placed in a 118 ml (4 oz) soil tin for shaking To
maximize lateral movement of paint shaker a paint can was used as a spacer to place
soil tins at the end of the shaking arm (Figure A2c) This allowed shaking to occur at
the greatest distance from the pivot point (fulcrum) Following all treatments
including the control seeds were soaked for twenty-four hours in distilled water prior
to plating out
Gennination was evaluated on lab benches at room temperature Each
replicate of seeds was placed in a 100 cm petri dish on moistened filter paper with
humidity maintained by enclosing the petri dishes in ziplock bags Gennination was
monitored daily for 14 days in accordance with International Seed Testing
Association standards for Robinia species (ISTA 1999) Gennination was defined as
protrusion of the radicle from the embryo by at least 1mm as viewed by the naked
eye
Categorical Analysis of Variance using Prpc Catmod (SAS Institute 1989)
was used to detennine treatment differences in germination percentages for each
source This procedure is an extension of a Chi-square test of homogeneity using the
53
natural log of the ratio of germinated to non-germinated seeds for each treatment Pshy
values less than 005 were considered significant A limited set of pairwiseshy
comparisons was conducted to compare treatment mean percents using a
conservative alpha value of 005 divided by the number of comparisons
Results
Percussion maximized germination for both New Mexico locust and black
locust There were three germination response levels for New Mexico locust (Figure
A3) First there was a low response in the control indicating a dormant seed lot
The hot water treatment and I-minute duration of percussion share a second
intermediate response Germination was highest at percussion levels from 2 to 10
minutes
A selected percussion treatment intermediate in time (5 minutes) produced the
steepest germination curve indicating the fastest germination rate (Figure A4) The
hot water treatment produced an intermediate curve or relatively slower germination
rate The control curve was very flat indicating the slowest germination rate for New
Mexico locust
Control seeds germinated poorly for black locust as well (Figure A5) Hot
water scarification improved germination over the control but all levels of percussion
improved germination over hot water The optimal durations of percussion treatment
were bracketed with a significant increase from the 1 to 2 minute percussion level a
high germination response from 2 to 5 minutes of percussion and finally a significant
54
~ d 25 CL)
p t1 0 ~ t1
g Vl ~ Vl
100~---------------------------------------------------------------
80
60
40
20
o Control Hot Water Percussion
I min Percussion
2 min Percussion
3 min
Scarification
Percussion 4 min
Figure A3 Effect of scarification on germination response for New Mexico locust
Percussion 5 min
=significant difference between adjacent treatment bars ns = no significant difference
Percussion 10 min
~ ~ () u agt
Il-t d o ~
~ ~ c3
100middotlr~~~------------------------------------e- Percussion 5min middot 0middot Hot Water -- Control
80-J=middot======
6b-middot-lmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middot--middotmiddot-middot~-middotmiddot -middotmiddot middot middotmiddot-middot 1
40
20
-----~-----------
0 0 0 middotmiddotmiddotmiddotmiddot0middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot0
0 --~-----y-----~------_---~--- T-----~
o shy -TDay 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A4 Effect of selected scarification treatments on germination speed for New Mexico locust First seven days of fourteen-day experiment are shown
Day 7
80
~ cd
5 60 ()
[) Cl d 0 1t1 d
40sect dUl
-J
20
o
ns
Scarification
Figure A5 Effect of scarification on germination response for black locust = significant difference between adjacent treatment bars ns = no significant difference
decrease at the 10-minute level This drop-off suggests that 10 minutes of percussion
over-treats seeds from this lot of-black locust
A selected percussion treatment intermediate in time (5 minutes) also had the
fastest germination rate for black locust (Figure A6)_ The hot water curve is
somewhat flatter indicating a slower germination rate The control curve is very flat
indicating a slow germination rate
A qualitative difference between hot water and percussion-treated seeds was
also observed Along with healthy germinants the hot water treatment produced
over-treated seeds (Figure A 7a) Examples of damaged germinants include seeds
where the radicle did not elongate (Figure A 7b) seeds where the radicle elongated
but was delayed in freeing itself from the seed coat (Figure A7c) and seeds where the
radicle broke off completely from the embryo (Figure A7d) In addition to these
examples of over-treatment there were numerous small dark seeds that failed to
imbibe water For percussion treatment nearly all seeds took up water (Figure A8a)
Germination was rapid and uniform with little damage evident (Figure A8b)
To summarize both New Mexico locust and black locust seed lots were
dormant The standard hot water scarification treatment improved germination but
only to 56 and 41 for New Mexico locust and black locust respectively Nearly
all percussion durations improved total germination as well as germination rate over
the hot water treatment Germination was greater than 90 for percussion durations
of 4 5 and 10 minutes for New Mexico locust and 3 4 and 5 minutes for black locust
58
CI)
sgt t CI) 0
igt 0
sect ttl t E
Vl 0
c3
100r==~==~~=r------------------------------------~ ___ Percussion 5 min
middotmiddotmiddot0middotmiddotmiddot Hot Water -lIf- Control
80r=========~--~~----------------------------~
60
40
20 -~-~---~---------- ---~------------I
omiddot
O
_~_----------lIf-----~bull -----_ shy---shy I
Day 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A6 Effect of selected scarification treatments on germination speed for black locust First seven days of fourteen-day experiment are shown
Day 7
a
b c d
Figure A7 Damage to New Mexico locust seeds observed during germination counts following hot water scarification A7a Hot water replicate including damaged seedlings and numerous unimbibed seeds A7b Close-up ofseed where radicle failed to elongate A7c Close-up ofseed where radicle elongated but was delayed in freeing itself from the seed coat A7d Close-up ofseed where radicle completely broke offfrom embryo
60
a
b
Figure A8 New Mexico locust seeds following percussion treatment at time of germination counts A8a Percussion replicate with uniformly healthy seedlings and near complete imbibition of seeds A8b Close-up ofpercussion-treated seedling
61
Discussion
Papilionoid seeds have a specific anatomical feature known as the strophiole
or lens-the natural site of water entry to the seed (Hamly 1932) The strophiole is
located on the cotyledonary lobe of a locust seed (Figure A9) When a papilionoid
legume seed is percussed for the appropriate amount of time repeated hits on the
integrated seed coat loosen the constrained cells of the strophiolar region without
excessively damaging the rest of the seed coat (Ballard 1976)
When a percussed seed is soaked water enters exclusively through the
strophiole in a controlled manner (Kelly and Staden 1987) This regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues This
contrasts with seeds that have been hot water or acid scarified treatments that can
randomly degrade the seed coat Unlocalized cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rehman et al 1998 Marunda 1990)
Papilionoids are the largest subfamily of legumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust and black locust these legumes often play an integral role in the
revegetation ofdisturbed lands Developing superior scarification methods to the
standard hot water and acid treatments should fadlitate the use ofthese legumes in
restoration projects
62
Figure A9 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (bc)
63
APPENDIXB
GERMINATION PERCENTS AND STANDARD ERRORS FOR SOURCE
BLOCK AND TREATMENT FOR NEW MEXICO LOCUST
Table B 1 Gennination percents and standard errors for source block and treatment for New Mexico locust
Source Block Treatment Germination Percentage Standard Error
Capitan 1 Immersion 74 437 Capitan 2 Immersion 69 462 Capitan 3 Immersion 71 454 Capitan 4 Immersion 67 470 Capitan Boiling 30 sec 79 407 Capitan 2 Boiling 30 sec 76 427 Capitan 3 Boiling 30 sec 72 449 Capitan 4 Boiling 30 sec 76 427 Capitan Boiling 60 sec 71 454 Capitan 2 Boiling 60 sec 54 498 Capitan 3 Boiling 60 sec 67 470 Capitan 4 Boiling 60 sec 76 427 Capitan I Control 5 218 Capitan 2 Control 7 255 Capitan 3 Control 4 196 Capitan 4 Control 5 218 Capitan 1 Percussion I min 47 499 Capitan 2 Percussion I min
470
Capitan Percussion I min 39 488
Capitan 4 Percussion I min 38 485 Capitan I Percussion 2 min 55 497 Capitan Capitan
2
Percussion 2 min Percussion 2 min
71 65
454 477
Capitan 4 Percussion 2 min 56 496 Capitan I Percussion 4 min 86 347 Capitan 2 Percussion 4 min 79 407 Capitan 3 Percussion 4 min 81 392 Capitan 4 Percussion 4 min 82 384 Capitan I Percussion 8 min 90 300 Capitan 2 Percussion 8 min 88 325 Capitan 3 Percussion 8 min 90 300 Capitan 4 Percussion 8 min 90 300 Gila I Immersion 71 454 Gila 2 Immersion 77 421 Gila
Immersion 75 433
Gila 4 Immersion 72 449
Gila I Boiling 30 sec 68 466
Gila 2 Boiling 30 sec 81 392
Gila 3 Boiling 30 sec 54 498
Gila 4 Boiling 30 sec 63 483
Gila I Boiling 60 sec 55 497
Gila 2 Boiling 60 sec 68 466
Gila 3 Boiling 60 sec 57 495
Gila 4 Boiling 60 sec 70 458
65
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Gila 2 Control 13 336 Gila 3 Control 13 336 Gila 4 Control 20 400 Gila 1 Percussion 1 min 66 474 Gila 2 Percussion 1 min 57 495 Gila 3 Percussion 1 min 73 444 Gila 4 Percussion 1 min 82 384 Gila 1 Percussion 2 min 81 392 Gila 2 Percussion 2 min 86 347 Gila 3 Percussion 2 min 85 357 Gila 4 Percussion 2 min 54 498 Gila 1 Percussion 4 min 79 407 Gila 2 Percussion 4 min 83 376 Gila 3 Percussion 4 min 84 367 Gila 4 Percussion 4 min 79 407 Gila 1 Percussion 8 min 51 500 Gila 2 Percussion 8 min 76 427 Gila 3 Percussion 8 min 52 500 Gila 4 Percussion 8 min 74 439 Holman 1 Immersion 83 376 Holman 2 Immersion 86 347 Holman 3 Immersion 81 392 Holman 4 Immersion 90 300 Holman 1 Boiling 30 sec 90 300 Holman 2 Boiling 30 sec 85 357 Holman 3 Boiling 30 sec 87 336 Holman 4 Boiling 30 sec 83 376 Holman 1 Boiling 60 sec 79 407 Holman 2 Boiling 60 sec 80 400 Holman 3 Boiling 60 sec 86 347 Holman 4 Boiling 60 sec 93 255 Holman Control 25 433 Holman 2 Control 19 392 Holman Control 21 407
Holman 4 Control 24 427
Holman Percussion 1 min 77 421
Holman 2 Percussion 1 min 85 357
Holman 3 Percussion 1 min 80 400
Holman 4 Percussion 1 min 89 313
Holman 1 Percussion 2 min 88 325
Holman 2 Percussion 2 min 96 196
Holman 3 Percussion 2 min 93 255
Holman 4 Percussion 2 min 96 196
Holman 1 Percussion 4 min 92 271
Holman 2 Percussion 4 min 97 171
66
I
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Holman 3 Percussion 4 min 91 286 Holman 4 Percussion 4 min 96 196 Holman 1 Percussion 8 min 88 325 Holman 2 Percussion 8 min 85 357 Holman 3 Percussion 8 min 83 376 Holman 4 Percussion 8 min 82 384 Jemez 1 Immersion 53 499 Jemez 2 Immersion 44 496 Jemez 3 Immersion 52 500 Jemez 4 Immersion 70 458 Jemez 1 Boiling 30 sec 58 494 Jemez 2 Boiling 30 sec 55 497 Jemez 3 Boiling 30 sec 48 500 Jemez 4 Boiling 30 sec 65 477 Jemez 1 Boiling 60 sec 68 466 Jemez 2 Boiling 60 sec 63 483 Jemez 3 Boiling 60 sec 73 444 Jemez 4 Boiling 60 sec 89 313 Jemez 1 Control 2 140 Jemez 2 Control 9 286 Jemez 3 Control 6 237 Jemez 4 Control 8 271 Jemez 1 Percussion 1 min 58 494 Jemez 2 Percussion 1 min 63 483 Jemez 3 Percussion 1 min 77 421 Jemez 4 Percussion 1 min 89 313 Jemez 1 Percussion 2 min 68 466 Jemez 2 Percussion 2 min 64 480 Jemez 3 Percussion 2 min 80 400 Jemez 4 Percussion 2 min 83 376 Jemez Percussion 4 min 68 466 Jemez 2 Percussion 4 min 79 407 Jemez 3 Percussion 4 min 70 458 Jemez 4 Percussion 4 min 83 376 Jemez 1 Percussion 8 min 52 500 Jemez 2 Percussion 8 min 59 492 Jemez 3 Percussion 8 min 61 488 Jemez 4 Percussion 8 min 70 458 Magdalena 1 Immersion 69 462 Magdalena 2 Immersion 69 462 Magdalena J Immersion 70 458 Magdalena 4 Immersion 85 357 Magdalena 1 Boiling 30 sec 65 477 Magdalena 2 Boiling 30 sec 71 454 Magdalena 3 Boiling 30 sec 68 466
67
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Magdalena 4 Boiling 30 sec 83 376 Magdalena 1 Boiling 60 sec 63 483 Magdalena 2 Boiling 60 sec 68 466 Magdalena 3 Boiling 60 sec 63 483 Magdalena 4 Boiling 60 sec 82 384 Magdalena 1 Control 11 313 Magdalena 2 Control 6 237 Magdalena 3 Control 5 218 Magdalena 4 Control 4 196 Magdalena Percussion 1 min 32 466 Magdalena 2 Percussion 1 min 45 497 Magdalena 3 Percussion 1 min 46 498 Magdalena 4 Percussion I min 36 480 Magdalena 1 Percussion 2 min 67 470 Magdalena 2 Percussion 2 min 68 466 Magdalena 3 Percussion 2 min 65 477 Magdalena 4 Percussion 2 min 81 392 Magdalena 1 Percussion 4 min 87 336 Magdalena 2 Percussion 4 min 97 171 Magdalena 3 Percussion 4 min 83 376 Magdalena 4 Percussion 4 min 97 171 Magdalena 1 Percussion 8 min 98 lAO Magdalena 2 Percussion 8 min 100 000 Magdalena 3 Percussion 8 min 96 196 Magdalena 4 Percussion 8 min 98 lAO Manzano 1 Immersion 67 470 Manzano 2 Immersion 57 495 Manzano 3 Immersion 72 4049 Manzano 4 Immersion 73 4044 Manzano 1 Boiling 30 sec 70 458 Manzano 2 Boiling 30 sec 64 480 Manzano 3 Boiling 30 sec 71 454 Manzano 4 Boiling 30 sec 80 400 Manzano I Boiling 60 sec 50 500 Manzano 2 Boiling 60 sec 53 499 Manzano 3 Boiling 60 sec 59 492 Manzano 4 Boiling 60 sec 74 439 Manzano I Control 4 196 Manzano 2 Control 8 271 Manzano 3 Control 1 099 Manzano 4 Control 6 237 Manzano 1 Percussion 1 min 52 500 Manzano 2 Percussion 1 min 39 488 Manzano 3 Percussion 1 min 36 480 Manzano 4 Percussion 1 min 51 500
68
Table B 1 Continued
Source Block Treatment Gerrni~ation Percentage Standard Error
Manzano Percussion 2 min 53 499 Manzano 2 Percussion 2 min 58 494 Manzano 3 Percussion 2 min 61 488 Manzano 4 Percussion 2 min 62 485 Manzano 1 Percussion 4 min 76 427 Manzano 2 Percussion 4 min 85 357 Manzano 3 Percussion 4 min 79 407 Manzano 4 Percussion 4 min 83 376 Manzano 1 Percussion 8 min 87 336 Manzano 2 Percussion 8 min 83 376 Manzano 3 Percussion 8 min 83 376 Manzano 4 Percussion 8 min 75 433 Raton 1 Immersion 54 498 Raton 2 Immersion 37 483 Raton 3 Immersion 39 488 Raton 4 Immersion 69 462 Raton 1 Boiling 30 sec 36 480 Raton 2 Boiling 30 sec 20 400 Raton 3 Boiling 30 sec 28 449 Raton 4 Boiling 30 sec 60 490 Raton 1 Boiling 60 sec 15 357 Raton 2 Boiling 60 sec 171 Raton 3 Boiling 60 sec 4 196 Raton 4 Boiling 60 sec 35 477 Raton 1 Control 4 196 Raton 2 Control 12 325 Raton 3 Control 2 140 Raton 4 Control 6 237 Raton 1 Percussion 1 min 52 500 Raton 2 Percussion I min 29 454 Raton 3 Percussion I min 35 477 Raton 4 Percussion 1 min 31 462 Raton 1 Percussion 2 min 55 497 Raton 2 Percussion 2 min 35 477 Raton 3 Percussion 2 min 33 470 Raton 4 Percussion 2 min 42 494 Raton 1 Percussion 4 min 65 477 Raton 2 Percussion 4 min 52 500 Raton Percussion 4 min 51 500 Raton 4 Percussion 4 min 53 499 Raton 1 Percussion 8 min 88 325 Raton 2 Percussion 8 min 80 400 Raton 3 Percussion 8 min 73 444 Raton 4 Percussion 8 min 74 439 Sacramento 1 Immersion 43 495
69
Table B 1 Continued
-Source Block Treatment Germination Percentage Standard Error
Sacramento 2 Immersion 30 458 Sacramento 3 Immersion 38 485 Sacramento 4 Immersion 56 496 Sacramento 1 Boiling 30 sec 47 499 Sacramento 2 Boiling 30 sec 48 500 Sacramento 3 Boiling 30 sec 48 500 Sacramento 4 Boiling 30 sec 56 496 Sacramento 1 Boiling 60 sec 52 500 Sacramento 2 Boiling 60 sec 67 470 Sacramento 3 Boiling 60 sec 43 495 Sacramento 4 Boiling 60 sec 48 500 Sacramento 1 Control 0 000 Sacramento 2 Control 3 171 Sacramento 3 Control 3 171 Sacramento 4 Control 099 Sacramento 1 Percussion 1 min 57 495 Sacramento 2 Percussion 1 min 61 488 Sacramento 3 Percussion 1 min 62 485 Sacramento 4 Percussion 1 min 55 497 Sacramento 1 Percussion 2 min 84 367 Sacramento 2 Percussion 2 min 70 458 Sacramento 3 Percussion 2 min 81 392 Sacramento 4 Percussion 2 min 69 462 Sacramento 1 Percussion 4 min 91 286 Sacramento 2 Percussion 4 min 84 367 Sacramento 3 Percussion 4 min 84 367 Sacramento 4 Percussion 4 min 94 237 Sacramento 1 Percussion 8 min 82 384 Sacramento 2 Percussion 8 min 81 392 Sacramento 3 Percussion 8 min 82 384 Sacramento 4 Percussion 8 min 81 392 Sandia 1 Immersion 76 427 Sandia 2 Immersion 70 458 Sandia 3 Immersion 70 458 Sandia 4 Immersion 81 392 Sandia 1 Boiling 30 sec 78 414 Sandia 2 Boiling 30 sec 71 454 Sandia 3 Boiling 30 sec 63 483 Sandia 4 Boiling 30 sec 78 414 Sandia 1 Boiling 60 sec 67 470 Sandia 2 Boiling 60 sec 65 477 Sandia 3 Boiling 60 sec 74 439 Sandia 4 Boiling 60 sec 66 474 Sandia 1 Control 16 367 Sandia 2 Control 7 255
70
Table B 1 Continued
Source Block Treatment Gennination Percentage Standard Error
Sandia 3 Control 8 271 Sandia 4 Control 16 367 Sandia 1 Percussion 1 min 67 470 Sandia 2 Percussion 1 min 61 488 Sandia 3 Percussion 1 min 66 474 Sandia 4 Percussion 1 min 59 492 Sandia I Percussion 2 min 82 384 Sandia 2 Percussion 2 min 83 376 Sandia 3 Percussion 2 min 81 392 Sandia 4 Percussion 2 min 84 367 Sandia 1 Percussion 4 min 89 313 Sandia 2 Percussion 4 min 86 347 Sandia 3 Percussion 4 min 89 313 Sandia 4 Percussion 4 min 85 357 Sandia 1 Percussion 8 min 80 400 Sandia 2 Percussion 8 min 77 421 Sandia 3 Percussion 8 min 69 462 Sandia 4 Percussion 8 min 66 474
LITERATURE CITED
Ashby WC Vogel WG Rogers NF 1985 Black locust in the reclamation equation General Technical Report Northeastern Forest Experiment Station USDA Forest Service NE-I05 12 p
Ballard LAT 1973 Physical barriers to gennination Seed Sci Technoll 285-303
___ 1976 Strophiolar water conduction in the seeds ofTrifolieae induced by action on the testa at nonstrophiolar sites Austr J Plant Physiol3 465-469
Barton LV 1947 Special studies on seed coat impenneability Contrib Boyce Thompson Inst 14 355-362
Baskin CC Baskin JM 1998 Seeds Ecology biogeography and evolution of donnancy and gennination Academic Press London UK 666 p
Bewley JD 1997 Seed germination and donnancy Plant Ce119 1055-1066
Bewley JD Black M 1994 Seeds physiology of development and germination 2nd ed Plenum Press New York NY 445 p
Briscoe CB 1996 Growing Nitrogen Fixing Trees in Acid Soils In MH Powell (ed) Nitrogen Fixing Trees for Acid Soils- A Field ManuaL Winrock International Morrilton AR 22 p
Copeland LO McDonald MB 1995 Principles of Seed Science and Technology 3rded Chapman amp Hall New York NY 409 p
Cox RE Klett JE 1984 Seed gennination requirements of native Colorado plants for use in the landscape Plant Propag 30(2) 6-10
Dell B 1980 Structure and function ofthe strophiolar plug in seeds ofAlbizia Lophantha Am J Bot 67 556-563
Dick-Peddie WA 1993 New Mexico Vegetation Past Present and Future University ofNew Mexico Press Albuquerque NM 244 p
Dreesen DR 2001 Propagation protocol for production ofcontainer Robinia neomexicana plants Los Lunas Plant Materials Center Los Lunas New Mexico In Native Plant Network URL httpwwwnativeplantnetworkorg (accessed 9 January 2002) Moscow (ID) University ofIdaho CoIl Natural Resources For Res Nursery
72
Dreesen DR Harrington JT 1997 Propagation of native plants for restoration projects in the Southwestern US- Preliminary Investigations In Landis TD Thompson JR tech coords National Proceedings Forest and Conservation Nursery Assocs Gen Tech Rep PNW-GTR-419 Portland OR USDA For Ser PNW Res Sta 77-88
Gottfried GJ 1980 Control ofNew Mexican locust and the effect on planted ponderosa pine in central Arizona US For Ser Res Note RM-386 Rocky Mountain For Range ExpStn Fort Collins CO
Gosling PG Samuel YK Jones SK 1995 A systematic examination of germination temperature chipping and water temperaturesoak duration pretreatments on the seeds ofLeucaena leucocephala Seed Sci amp Technol 23521-532
Hamly DH 1932 Softening of the seeds of Melilotus alba Bot Gazette 93 345shy375
Hanna PJ 1984 Anatomical features of the seed coat ofAcacia kempeana (Mueller) which relate to increased germination rate induced by heat treatment New Phytol 96 23-29
Hanks JP Dick-Peddie WA 1974 Vegetation patterns of the White Mountains New Mexico Southwest Naturalist 18(4) 371-382
Hine S Harrington JT Dreesen DR 1997 Seed scarification requirements for Robinia neomexicana In Harrington JTtech coord Proc Third SouthwestemContainer Growers Conf Feb 12-13 1997 Ft Collins CO
Hopkinson JM 1993 The strophiole of leguminous seeds Newsletter- IntI Herbage Seed Production Res Grp 18 7-8
---- 1997 Differences in hardseededness between Cunningham and Tarramba leucaena In Leucnet News The Newsletter of the International LeucaenaRes and Devlt Netwk No4
ISTA (International Seed Testing Association) 1999 International rules for seed testing rules Seed Sci amp Technol27 Supplement
Kelly KM van Staden J 1987 The lens as the site of permeability in the papilionoid seed Aspalathus linearis J Plant Physiol 128(45) 395-404
73
Keresztesi B 1988 Black locust the tree of agriculture Outlook on Agr 17(2) 77~85
Kigel 1 and Galili G 1995 Seed development and germination Marcel Dekker New York 853 p
Klemmedson 10 1994 New Mexico locust and parent material Influence on macronutrients of forest floor and soil Soil Sci Soc Am 1 58 974~980
Kuo WH Tam AW 1988 The pathway of water absorption of mung bean seeds Seed Sci amp Technol16(1) 139~144
Leadem CL 1997 Dormancy--unlocking seed secrets In Landis TD Thompson lR tech coords National Proceedings Forestand Conservation Nursery Assocs Gen Tech Rep PNW-G TR-419 Portland OR USDA For Ser PNW Res Sta 43-52
Lin Y Wagner MR Seweyestewa A Honahni MD DeGomez M McMillin JD 1996 Influence of cultural treatments on seed germination and seedling growth of Robinia neomexicana Forest Farm and Community Tree Res Reports Volume 1
Marunda CT 1990 Effects of seed pretreatment on the development ofAcacia auriculforinis and A holosericea seedlings ACIAR Proceedings Series No 28
Meginnis HG 1937 Sulphuric acid treatment to increase germination of black locust seed USDA Circ 453 34 p
Miklas PN Towensend CE Ladd SL 1987 Seedcoat anatomy and the scarification ofcicer milkvetch seed Crop Sci 27 766-772
Monsen SB 1984 Use of shrubs on mine spoils In The Challenge ofProducing Native Plants for the Intermountain Area Proceedings Intermountain Nurserymans Association 1983 conference Las Vegas NV compo PM Murphy 26-31 Ogden UT USDA For Ser Gen Tech Report INT-l68
Olson DF 1974 Robinia L Locust In Seeds of Woody Plants in the US tech coord CS Schopmeyer 728-731 USDA For Ser Agr Handbk No450
Rehman S Loescher RNl Harris P1C 1998 Dormancy breaking and germination ofAcacia salicina Lindl seeds Seed Sci amp Techno127 553-557
Rolston MP 1978 Water impermeable seed dormancy Bot Rev 44 365-396
74
SAS Institute Inc 1989 SASSTAT Users Guide Version 6 4th Edition Volume 1 Cary NC SAS Institute Inc 943 p
SerratoValenti G Conara L Ferrando M Modenesi P 1993 Structural and histochemical features ofStylosanthes scabra (Leguminosae Papilionoidae) seed coat as related to water entry Can J Bot 71 834-840
Simpson BJ 1988 A field guide to Texas trees Austin TX Texas Monthly Press 372p
Tran VN Cavanagh AK 1984 Structural aspects ofdormancy In Seed Physiology Vol 2 Murray DR (ed) p1-44 Academic Press Australia
USDA Forest Service 1948 Woody-plant seed manual USDA Misc Pub1654 416p
Vines RA 1960 Trees Shrubs and Woody Vines of the Southwest Austin Univ ofTexas Press 1104 p
Wagner MR Larson RE Craig TP 1992 Ecology and silvics ofNew Mexico locust (Robinia neomexicana) with an emphasis on the arboreal form p53-57 In JW Hanover K Miller and S Plesko (eds) Proc Intemat Confon Black Locust Biology Culture and Utilization Mich State Univ USA
Wilson JK 1944 Immersing seeds of species of Robinia in boiling water hastens germination J For 42 453-454
Zimmerman RW Carpenter SB 1980 First-year coppice production from a 5shyyear old black locust stand on surface mine spoil p309-314 Centr Hardwd For ConfProc University ofMissouri Columbia
Percussion as an Alternative Seed Treatment for Robinia neomexicana (New Mexico
Locust) a thesis by Nabil Y Khadduri in partial fulfillment of the requirements for
the degree Master of Science has been approved and accepted by the following
Interim Dean of the Ora uate School
Chair ofExamining Committee
Date
Committee in charge
Dr John T Harrington Chair
Dr David R Dreesen
Dr Leigh Murray
Dr Rolston St Hilaire
ACKNOWLEDGMENTS
I would like to thank Dr John Harrington for his guidance sense ofhumor
and flexibility throughout my studies I would also like to thank the members ofmy
committee for their support especially Dr Murray who patiently taught me the
subtleties of categorical analysis of variance
I am also grateful to Dr Anne Wagner and Molycorp Mine who provided
funding for this research Thanks to Dr Bob Karrfali for his assistance with x-ray
radiography and to Mohamed Murshed and Dr Soumitra Ghoshroy for their
assistance with scanning electron microscopy Special thanks go to my colleague Lee
Rosner who assisted me in all phases of this project Finally I thank my family for
their enduring love Jill Walid Amin Rolla Khalid Piet Zach Charlie and above
all mywife Lucie
111
VITA
June 3 1972
June 1990
May 1994
July 1995
August 1994- March 1998
March 1998-August 2000
August 2000-May 2002
Born in Beirut Lebanon
Graduated from Georgetown Day High School Washington DC
Graduated from Pomona College Claremont CA with Bachelor ofArts Degree in Political Science
Graduated from University ofNew Mexico Albuquerque NM with Post-baccalaureate Teaching Degree
6th Grade TeacherBanta Fe Public Schools Santa Fe NM and Environmental Educator Open Space Division Albuquerque NM
Assistant Manager Santa Ana Native Plant Nursery Santa Ana Pueblo NM
Graduate Research Assistant at New Mexico State University
FIELD OF STUDY
Horticulture
iv
ABSTRACT
PERCUSSION AS AN ALTERNATIVE SEED TREATMENT FOR
ROBINIA NEOMEXICANA (NEW MEXICO LOCUST)
BY
NABIL Y KHADDURI
Master of Science in Horticulture
New Mexico State University 2002
Las Cruces New Mexico
Dr John T Harrington Chair
New Mexico locust (Robinia neomexicana A Gray) a small tree native to
montane regions of the southwestern United States fills a successional role in postshy
disturbance situations The ability to colonize rapidly and ameliorate harsh sites
contributes to the importance ofNew Mexico locust in reclamation efforts Seedling
production for outplanting on disturbed sites has been hampered by poor germination
This research sought to improve seed propagation techniques in New Mexico locust
Nine seed sources were collected across a range of latitudes within New
Mexico Seed treatments included a control three levels of hot water and four levels
of percussion scarification Hot water treatments included immersion at 100degC and
boiling for periods of 30 seconds and 60 seconds Percussion treatment levels were 1
2 4 and 8 minutes A total of four lOO-seed samples were used to test each source
by scarification treatment combination Categorical Analysis of Variance was used to
determine main effects and interactions Pairwise comparisons were conducted to
compare non-control treatment means using a conservative alpha value of 005
divided by the number of comparisons
Averaged over all sources and for each source percussion scarification
durations of4 or 8 minutes yielded greatest germination In addition to consistently
bigh germination 4- and 8-minute percussion-treated seeds germinated as fast or
faster than all other treatments Averaged over all sources germination increased
with increasing durations of percussion then leveled off for the 4- and 8-minute
treatments Immersion and 30-second boiling scarification maximized germination
for the hot water treatments with a decrease in germination when seeds were boiled
for 60 seconds
Percussion scarification specifically weakens the strophiole the natural
dormancy barrier in many papilionoid legumes without excessively damaging the
seed coat As with New Mexico locust papilionoid legumes often play an integral
role in the revegetation ofdisturbed lands The percussion scarification technique
developed in this research has the potential to facilitate the use of these valuable
species in reclamation projects
VI
CONTENTS
LIST OF TABLES ix
LIST OF FIGURES middot middotmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot x
INTRODUCTION 1
LITERATlTRE REVIEW 4
Seed Donnancy 4
Physical Donnancy 5
Evolutionary Role ofPhysical Donnancy 6
Methods for Breaking Donnancy 6
Acid Scarification 6
Hot Water Scarification 8
Mechanical Scarification 11
Alternative Method for Breaking Donnancy Percussion 12
Objectives 15
Hypothesis 15
MATERIALS AND METHODS 16
Seed Collection and Cleaining 16
Experimental Design and Treatment Structure 16
Treatments ~ bullbull bullbullbullbull - bullbull ~ ~ 20
G T ennlnatlon estmg 21
X-ray Radiography 22
Scanning Electron Microscope Photography 22
Analysis 22
RESULTS 24
DISCUSSION 36
Detennining the Optimal Treatment 36
Strophiole and Seed Coat Behavior 37
CONCLUSION 44
Appendices
A PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR NEW MEXICO LOCUST AND BLACK LOCUST SEEDS 47
B GERMINATION PERCENTS AND STANDARD ERRORS FOR SOURCE BLOCK AND TREATMENT FOR NEW MEXICO LOCUST 64
LITERATURE CITED 72
Vlll
LIST OF TABLES
Table Page
1 Latitude longitude location elevation and collection date ofNew Mexico locust seed sources 18
2 Mean seed weight and standard error by source 19
3 Categorical Analysis ofVariance table for germination response to seed treatment seed source block and the interaction of the factors 25
4 Effect of seed source and treatment on G50 (days to 50 germination) 32
B1 Germination percents and standard errors for source block and treatment for New Mexico locust 65
LIST OF FIGURES
Figures Page
1 Seedsourcecollectionmap 17
2 Effect ofNew Mexico locust seed source on germination for data averaged over scarification treatments 26
3 Effect of scarification on germination for New Mexico locust for data averaged over all seed sources 27
4 Effect of seed source by treatment interaction on New Mexico locust germination for percussion scarification pattern a and pattern b 28
5 Effect of source by treatment interaction on New Mexico locust germination for hot water scarification 29
6 Timecourse of New Mexico locust germination following scarification treatments 31
7 X-ray radiographs of Sacramento seed source perc ussed for 1 4 or 8 minutes 33
8 Seed damage close-ups 34
9 SEM images of untreated vs 4-minute percussion-treated strophiolar cells ofNew Mexico locust seeds 35
10 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (b c) 38
11 Damage to New Mexico locust seeds observed during germination counts following hot water scarification 40
12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources 42
New Mexico locust a primary invader following disturbance 50
Implementation of hot water and percussion scarification 52
Effect of scarification on germination response for New Mexico locust 55
x
A4 Effect of selected scarification treatments on germination speed for New Mexico locust 56
AS Effect of scarification on germination response for black locust 57
A6 Effect of selected scarification treatments on germination speed for black locust 59
A7 Damage to New Mexico locust seeds observed during germination counts following hot water scarification 60
AS New Mexico locust seeds following percussion treatment at time of germination counts 61
A9 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (bc) 63
INTRODUCTION
New Mexico locust (Robinia neomexicana A Gray) a small tree native to the
southwestern United States occurs at elevations from 1200 to 2800 meters This
species fills a successional role in post-disturbance situations As a primary invader
New Mexico locust quickly establishes on burned areas arid flood banks as well as
road cuts (Wagner et aI 1992) Rapid growth crown sprouting and prolific root
suckering favor the successful establishment ofNew Mexico locust on disturbed sites
(Simpson 1988 Gottfried 1980) These attributes also may make New Mexico locust
well suited for steep-slope revegetation where erosion is a problem
New Mexico locust is a nitrogen-fixing legume that tolerates and improves
nutrient-poor soils Stands ofNew Mexico locust increase Nitrogen (N) and Calcium
(Ca) in the forest floor Levels of Carbon (C) Phosphorous (P) Sulfur (8) and
Potassium (K) aIso have been shown to increase in soil beneath New Mexico locust
(Klemmedson 1994)
The ability to colonize rapidly and ameliorate harsh sites contributes to New
Mexico locusts potential as a nurse plant A nurse plant colonizes an inhospitable
site and creates an environment suitable for successional plant establishment New
Mexico locust demonstrates this role in the ponderosa pine (Pinus ponderosa Douglas
ex Lawson amp C Lawson)Gambel oak (Quercus gambelii Nutt) community After a
disturbance such as wildfire New Mexico locust and Gambel oak colonize and
dominate the site until shaded out by ponderosa pine Once shaded these species
become understory shrubs a process taking an average of 15-20 years (Dick-Peddie
1993 Hanks and Dick-Peddie 1974) New Mexico locust gradually declines with
small dense patches averaging about 005 ha beneath pine until the next disturbance
once again offers it a competitive advantage (Klemmedson 1994)
Gottfried (1980) expecting ponderosa pine regeneration to be greater where
New Mexico locust had been eradicated found that survival of planted pine seedlings
was greater where locust had not been removed In a follow-up study Gottfried
(1980) noted that soil moisture in the top 57 cm was highest in 5-year old locust sites
as compared to grass or 20-year old locust sites He concluded that managing an
appropriate cover ofNew Mexico locust could help regenerate pine the later
successional species
The ability ofNew Mexico locust to improve harsh sites also makes it a
candidate for reclaiming disturbances associated with mining Black locust (Robinia
pseudoacacia 1) has been used for decades to reclaim mine spoils and other
disturbed sites in the eastern US and around the world (Keresztesi 1988 Zimmerman
and Carpenter 1980) Ashby et al (1985) described similar positive attributes of
black locust also mentioned previously for New Mexico locust quick cover for
stabilization supply ofN and nutrient-rich litter to the soil and site improvement for
establishment of later successional trees Referring specifically to mine reclamation
the authors note the ability of black locust to grow on a wide range ofmine soil
conditions including extremely acid soils Black locust also shows some tolerance to
soils compacted by grading and topsoiling practices (Ashby et aL 1985) New
2
Mexico locust holds promise as a native southwe~tem counterpart to black locust in
mine reclamation but it has been used infrequently to date
Natural invasion and succession occur slowly on most mine sites (Monsen
1984) While New Mexico locust often colonizes sites naturally there is no
assurance a seed will reach a particular site and establish in a reasonable time frame
The challenge is to introduce New Mexico locust as mother plants to facilitate
colonization Seedling production for outplanting on disturbed sites has been
hampered by poor germination (Lin et al 1996) The goal then is to develop
techniques to overcome poor germination in New Mexico locust thereby facilitating
macropropagation
LITERATURE REVIEW
Seed Dormancy
Low gennination in New Mexico locust is primarily the result of seed
donnancy the failure of an intact viable seed to genninate under favorable
conditions (Bewley 1997) Seed donnancy also can be defined as the inability of the
embryo to genninate because of some inherent constraint (Bewley and Black 1994)
The constraint may originate within the embryo referred to as endogenous or embryo
donnancy or from the tissues surrounding the embryo known as exogenous or coatshy
imposed donnancy (Baskin and Baskin 1998 Leadem 1997 Bewley and Black
1994)
New Mexico locust seed is donnant when the impenneable seed coat does not
allow movement ofwater to the embryo This specific fonn of coat-imposed
donnancy where the restraining tissue involved is an impenneable seed coat is
known as physical donnancy (Baskin and Baskin 1998 Bewley and Black 1994)
Physical donnancy is relatively uncommon in seeds less than 10 of all woody
species exhibit this condition (USDA Forest Service 1948) However it is
characteristic of many genera of the Fabaceae (bean) family For example all
successional trees in the genera Gleditsia and Robinia (of which New Mexico locust
is a member) produce seeds that exhibit physical donnancy (Baskin and Baskin
4
Physical Dormancy
Seeds that exhibit physical dormancy are said to be hard seeded referring to
the thick impermeable seed coat (Leadem 1997) The first line of protection of the
testa in leguminous seeds is the outermost waxy cuticle followed by a subcuticle
consisting ofsuberin Next is a palisade layer consisting of sclereid cells that have
thick lignified secondary walls The most common type of sclereid cell in palisade
layers is the macrosclereid The macrosclereids contain water-repellent substances
including cutin lignin suberin and wax (Rolston 1978) This palisade layer is the
primary barrier to water uptake (Serrato-Valenti et al 1993 Tran and Cavanagh
1984)
All seed coat openings must be blocked to effectively prevent water uptake
The natural opening in a seed where it was attached to the mother plant is the weak
link where physical dormancy is usually broken (Hopkinson 1993 Kuo and Tam
1988) Anatomical structures associated with the seed coat opening vary from
family to family In the Papilionoideae the subfamily of the Fabaceae family
containing New Mexico locust the site ofwater entry is the strophiole The
strophiole is a swelling on the seed coat close to the stalk scar (Hopkinson 1997)
Kelly and van Staden (1987) refer to the strophiole site as the lens where
macrosclereid cells must separate and raise before water can pass through Since
middot6germination ofdormant seeds of the Papilionoideae takes place when the strophiole
permeable (Hopkinson 1993 Kuo and Tam 1988) a logical approach to
5
genninating New Mexico locust artificially would be to loosen the strophiolar cells
and allow water to pass through to the embryo
Evolutionary Role of Physical Dormancy
Why is physical dormancy a mechanism that blocks germination
ecologically advantageous When a plant produc~s seeds with different degrees of
dormancy seeds that are dispersed at one time will germinate over a range of time
(Bewley and Black 1994) Through variability of dormancy within a seed crop seeds
are exposed to changing environmental conditions improving the overall chances of
survival of the seed line (Bewley and Black 1994) Varying expression of dormancy
is under genetic control which indicates that this may be an evolved survival strategy
(Kigel and Galili 1995) A range of dormancy levels may be a particular advantage in
the southwestern US where extreme environmental variations exist
Methods for Breaking Dormancy
There are three common approaches to breaking dormancy in New Mexico
locust seed acid scarification hot water soaks and mechanical treatments In each
case the objective of the treatment is to scarify or abrade the seed coat to make it
permeable to water
Acid Scarification
Acid scarification consists of soaking seeds in concentrated sulfuric acid
rinsing thoroughly with water upon removal followed by subsequent drying before
PlaJlltmg Olson (1974) reports that this scarification method produces the highest
middot~~jllalUll results for all Robinia species Most literature refers to a seminal 1937
6
study conducted by HG Meginnis This stud~ t~ough conducted on black locust
seed is regularly cited in the suggested use of acid to treat New Mexico locust seed
as well (Olson 1974 Vines 1960)
Thee published studies document the specific use of sulfuric acid to treat
New Mexico locust seed Cox and Klett (1984) report that soaking seeds in
concentrated sulfuric acid for thirty minutes improved germination to 34 over an
untreated control of4 Lin et al (1996) found that for two seed lots acid
scarification for twenty-five to eighty minutes produced germination of 80 A third
lot however resulted in less than 20 germination at all durations of 10 to 80
minutes Hine et al (1997) showed that acid treatment levels of 1 2 5 and 10
minutes resulted in less than 20 germination across five sources However there
was an increasing trend in germination with acid soak duration and acid treatments
were not imposed for as long a time as the previous two studies (30 to 80 minutes)
The mixed results obtained by these investigators were probably due to the
widely varying germination response of seed lots to acid treatment For this reason
Olson (1974) recommends correlating duration of acid treatment to each seed lot If
treatment duration is too long the embryo will be permanently damaged If soak
duration is too short the seed will remain impermeable to water and fail to germinate
Compounding the problem of treatment duration is the negative effect acid
have on seedling growth Any type of scarification can injure seeds to some
by disrupting cells essential for growth leading to fungal invasion and
middot-uuu~w injury (Copeland and MacDonald 1995) Some legume seeds subjected
7
to 60 minutes of sulfuric acid scarification (to achieve maximum gennination results)
showed more than 50 seedling mortality after gennination (Kelly and van Staden
1987) Bewley and Black (1994) noted that fungal growth on seeds is an indication
of leakage of sugars organic acids amino acids and proteins
Perhaps the greatest drawback to acid scarification is the dangerous nature of
the treatment itself Difficulties associated with storage and trained use of
concentrated acid may discourage many propagators from using this option (Dreesen
and Harrington 1997) Consequently hot water and to a lesser extent mechanical
scarification are more commonly used and practical treatments for legume seeds
Most southwestern container growers use either of these two treatments to scarify
New Mexico locust seed in particular (Hine et al 1997)
Hot Water Scarification
The widespread use ofhot water scarification to treat locust seed is probably
due to its ease of application and efficacy in comparison to other scarification
treatments Although this practice had been in use for some time Wilson (1944) was
one of the first to report on its systematic application for black locust The treatment
consists of placing seeds in boiling water (4 parts water tomiddot 1 part seed by volume)
removing the vessel from the heat source and allowing the water to cool and seeds to
soak for 24 hours
Hine et al (1997) Lin et al (1996) and Cox and Klett (1984) have examined
the specific application ofhot water scarification on New Mexico locust seed Each
these authors compared hot water to acid scarification Lin et aL (1996) reported
8
60 gennination for two hot water-treated seed lots while a third lot germinated
around 50 (as reported above acid scarification germination ranged from 20 to
80 for these lots) It should be noted that the authors only soaked the seeds in water
from 10 to 80 minutes for this experiment as opposed to the standard 24 hours Cox
and Klett (1984) stated that using the standard 24-hour hot water treatment improved
germination over an acid pre-treatment (which produced less than 20 germination)
but they did not give specific gennination numbers for the hot water treatment Hine
et al (1997) found that across five seed lots germination was 66 for the standard
hot water treatment (as compared to less than 20 germination for acid scarification)
Variations in treatment intensity and duration and their subsequent effects on seed lot
appear to contribute to conflicting conclusions as to whether acid scarification or hot
water treatment is a more successful treatment
Given mixed results ease ofapplication is an important argument for use of
hot water application over acid soaks Safety concerns and material costs for hot
water treatments are low (Dreesen and Harrington 1997) An extension of this is the
practicality ofhot water for treating large batches of seed where a relative increase in
water volume is all that is needed However increases in water and seed even when
inthe same 4 1 proportion can result in dramatically different rates of cooling than in
atest tube experiment Different cooling rates can lead to contrasting results for the
treatment temperature duration and seed lot (USDA Forest Service 1948)
As with acid scarification parameters ofhot water treatment application may
to be optimized to maximize germination Hine et al (1997) found that as
9
temperature was increased from 70degC to 100degC overall germination in New Mexico
locust seed increased The 100degC treatment maximized germination for each of five
sources tested with germination usually above 60
Prolonged exposure of seeds ofmany woody legumes to boiling temperatures
also may improve germination (Briscoe 1996) This approach has not been tested on
New Mexico locust seed Exposing seed to prolonged boiling temperatures may
result in fungal infection and subsequent seedling mortality Marunda (1990) noted
severe fungal infections on both acid and hot water treated Acacia holoserica Cunn
ex G Don seeds attributing the infections to seed damage resulting from the
treatments Identifying an optimal hot water treatment level that does not result in a
high rate of seedling mortality is a logical goal
With hot water treatments as with acid there is also a danger of overtreating
Seeds and reducing germination (Gosling et al 1995) The legume Leucaena
leucocephala (Lam) de Wit germinated equally well after 7 seconds of exposure to
water at 100degC 7 seconds at 90degC 30 seconds at 80degC and 4 minutes at 70degC In
addition acceptably high germination was obtained from treatment at 70degC from 3
minutes to 1 hour whereas no seeds germinated following treatment at 100degC for 2
minutes Perhaps along with a greater margin of error for treatment duration treating
~ew Mexico locust seeds at a lower temperature may result in a comparatively lower
of seedling mortality In practice however nursery growers tend to use the
and quickest treatment available The short treatment length at near-boiling
whether recommended or not may be what is used most frequently
10
Fine-tuning a treatment in this range of treatment temperature and duration may be
the most logical approach
Mechanical Scarification
This treatment involves scratching the seed coat with an abrasive material
(USDA Forest Service 1948) One system employs a rotating drum lined with
sandpaper A paddle spinning at a given number ofrevolutions per minute throws the
seed against the sandpaper (Hine et al 1997) A second method dry tumbling
tumbles seed in a mixer with pea gravel or sharp sand for 5 to 7 days (Dreesen 2001)
These approaches have the advantage that control of temperature is not required as in
water treatments nor is safety an issue as with add scarification
Hine et al (1997) mechanically treated New Mexico locust seed using 100shy
grit sandpaper in a Forsberg (rotating drum) scarifier Gennination was less than
10 They concluded that the seed coats were too thin for their apparatus and
proposed that a finer grit sandpaper would produce more even abrasion Although
germination varied by seed lot Dreesen (2001) reported that using a slow-speed dry
tumbling technique for 5 to 7 days produced gennination rates as high as 90
As with acid and hot water treatments treatment duration must be specifically
shy
correlated to seed lot Treated seeds are also susceptible to fungal infection
iHopkinson 1997) In addition equipment is specialized and may not always be
ltU~CUJl~ to a nursery (USDA Forest Service 1948) Several studies have confinned
degrading ofstrophiolar cells due to acid and hot water scarification (Miklas et al
11
1987 Dell 1980 Hanna 1984) but the effect ofmechanical scarification on the
strophiole has not been studied
Alternative Method for Breaking Dormancy Percussion
Strophiole degradation has been examined in depth using an impaction
treatment more commonly known as percussion Hamly (1932) first proposed that
shaking or impacting seeds could split the macrosc1ereid cells at the strophiole
Hamly successfully released tension ofbent strophiolar cells of impermeable
Melilotus alba Medik (White sweet clover) seeds by delivering physical blows
resulting in cells that had split apart and were only slightly bent Ninety-two percent
oftreated seeds became permeable as compared to 05 of control seeds Barton
(1947) showed that percussion significantly increases permeability in black locust
seed Ballard (1976) reasoned that the integrated nature of the strophiole within the
seed coat means a blow on any part of the seed weakens the strophiole There are no
published studies on the use of percussion on New Mexico locust
Percussion methods vary and often seem SUbjective or ill-defined Hamly
(1932) placed seeds in a glass bottle and shook them three times a second for 20
minutes resulting in 180 oscillations per minute Ballard (1973) describes shaking
seeds in a closed metal container with at least fifty seeds per container at 850-900
JS(~l11alIOIlS per minute for 1 minute Kelly and van Staden (1987) placed seeds into
sealed glass flasks Flasks were shaken by a wrist-shaker at a rate of 250
~UUVJ~ per minute Subsequent germination rates were 45 after 2 hours of
55 after 3 hours 60 after 6 hours and 100 after 12 hours
12
There are several potential advantages of percussion over acid hot water and
mechanical scarification Kelly and van Staden (1987) note that a percussion
treatment only alters macrosclereid cells of the strophiole by loosening separating
and raising them They emphasize that minimal damage is done to the overall
anatomical structure of the seed In acid scarification of the same seed lot radicle
and cotyledon damage occurred Based on photographic evidence the authors
believe that this morphological damage was due to uneven pressure during
imbibition with water entering haphazardly wherever the acid had sufficiently
dissolved the coat This ultimately led to uneven hydration of the embryo In
percussed seeds water only entered through the s~ophiole when the entire seed coat
had been saturated resulting in controlled and even embryo hydration (Kelly and van
Staden 1987)
Although all successful scarification treatments may focus at least in part on
the strophiole for papilionoid legumes (Baskin and Baskin 1998) percussion may be
less damaging to seed coat structures particularly when compared with acid Kelly
and van Staden did not compare hot water or mechanical scarification methods
against percussion However the fungal infection associated with hot water and
mechanical scarification as with acid scarificatio~ have been directly associated with
seed damage (Rehman et al 1998 USDA Forest Service 1948) Embryo damage in
has been observed in hot-water scarified legume seeds (Rehman et al 1998
13
By limiting embryo damage percussion scarification reduces seedling
mortality compared to other treatments Kelly and van Staden (1987) noted that
seedlings from the percussion treatment were morphologically sound and vigorous
when compared to acid treatment seedlings The authors attribute this improved
seedling vigor to the controlled water uptake that limited anatomical damage
Despite a 12-hour percussion treatment morphological damage was not substantially
greater than untreated seeds In addition seedling mass measured 10 days following
germination was not significantly different between percussed and untreated seeds
A final advantage ofpercussion may be the ability to adequately treat the most
dormant seeds while avoiding harm to seeds that have already been made permeable
by the treatment or that were permeable to begin with Instead ofcorroding scalding
or scratching the seed coat percussion appears to be just a loosening of the
strophiole spring (Ballard 1973) Kelly and van Staden (1987) were able to
increase germination with a lengthy treatment (12 hours) Figuring out whether a
substantial cushion oftreatment durations exist for percussion ofNew Mexico locust
seed would be a significant improvement to current scarification methods where seed
damage and seedling health is an issue
The greatest challenge for applying percussion to New Mexico locust seeds is
standardizing a treatment that is both effective and readily reproducible by nurseries
with mechanical scarification this may require the development of specific
~UllDment to properly quantify and execute the treatment
14
f
Objectives
The first objective of the study is to detennine which scarification treatment
results in optimal gennination speed and percentage ofNew Mexico locust The
second objective is to describe treatment interactions with nine seed sources collected
in New Mexico
Hypothesis
A percussion treatment acts exclusively on the main area ofwater uptake in
New Mexico locust seed loosening the strophiole so that imbibition can occur This
direct mechanism ofdonnancy release will result in improved gennination rate and
overall gennination percentage for New Mexico locust seed compared to standard
treatments Standard treatments including acid hot water and mechanical
scarification while eventually weakening the strophiolar cleft cause excessive
morphological damage through generalized seed coat degradation These treatments
are therefore prone to inconsistency due to over and under-treatment
15
MATERIALS AND METHODS
Seed Collection and Cleaning
Seeds were collected from August 25 through September 15 2000 from nine
locations along a latitudinal gradient in New Mexico Seed source locations and
descriptive information are given in Figure 1 and Tables I and 2 Stands were
separated by at least 45 km
Pods were hand picked as they began to ripen and dehisce (open) Collections
were made from ground level to approximately 3 m from several trees at each site
Pods were dried in open-ended brown paper bags for two to four weeks at room
temperature (21-230C) to facilitate cleaning Dried pods were placed in a closed cloth
sack which was pounded against the ground to separate seeds from pods For pods
still containing seeds hand maceration (crunching pods with hands) completed seed
separation Debris was removed by threshing with the aid of a greenhouse fan A
South Dakota Seed Blower (Seedbureau Chicago Illinois) was used to remove
remaining chaff and light seeds by air density separation Seeds were placed in dry
storage in sealed quart-size zip-lock bags within a sealed plastic container Storage
temperatures ranged from 2-4oC Seeds remained in storage for approximately 7
months before the experiment
Experimental Design and Treatment Structure
The experiment compared the impact ofhot water bath and percussion
l11aLl11 on germination properties ofNew Mexico locust seed A single
16
-l
Figure 1 Seed source collection map All sources were collected within New Mexico USA separated by at least 4S lan
IIIRZltItW lIJPIirmiddot rllll1lbullbull_~~~_II
Table 1 Latitude longitude location elevation and collection date ofNew Mexico locust seed sources
Lot title Latitude (N) Longitude (W) Location Elevation (m) Collection Date
Raton 36deg5846 104deg2872 Raton Pass NM 2295 82500
Holman 36deg0306 105deg2429 HolmanNM 2476 911500
Jemez 35deg4500 106deg4100 Jemez Springs NM 1976 9900 00
Sandia 35deg1083 106deg2341 Cedar Crest NM 2432 82600
Manzano 34deg3689 106deg2494 ManzanoNM 2465 9200
Magdalena 33deg5953 107deg0903 Magdalena NM 2544 82900
Capitan 33deg2728 105deg4325 Alto NM 2189 911200
Sacramento 32deg5798 105deg4496 Cloudcroft NM 2630 91400 Gila 32deg5400 107deg4600 Kingston NM 2432 9700
f
Table 2 Mean seed weight and standard error by source
a
Source Mean seed weight (g) for Standard error 100 seeds n = 5
Raton Holman Jemez Sandia Manzano Magdalena Capitan Sacramento Gila
2872 b c 2522 d 1826 e 2753 c 2413 d 2559 d 2443 d 3130 a 3029 ab
00103 00403 00276 00301 00370 00374 00458 00279 00348
a Mean seed weights followed by the same letter are not significantly
different
19
experiment was conducted utilizing a randomized complete block design blocked by
time with a factorial treatment structure Experimental factors were seed source and
scarification treatment Nine seed sources from throughout New Mexico were used
(Figure 1 Table 1) Scarification treatments included a control three levels ofhot
water and four levels of percussion Hot water treatments included immersion at
100degC and boiling for periods of 30 seconds and 60 seconds Percussion treatment
levels were 1 2 4 and 8 minutes One hundred seeds per block were used to test
each treatment combination Four blocks of seed were tested one at a time (all seed
sources included in each run) in close chronological sequence (within 6 weeks) A
total of four 100-seed samples were used to test each source by scarification treatment
combination
Treatments
Hot water treatment of seeds utilized 50 ml test tubes filled with 30 ml
distilled water Each test tube was used to treat one 100-seed sample at a time
resulting in a ratio of4 parts water to 1 part seed by volume Test tubes were placed
in a beaker with the beaker filled with tap water to the same level as the water within
test tubes Nine test tubes (one per seed source) were placed in the beaker
simultaneously so that one replication of each seed sourcescarification treatment
cotnblnatlon was conducted simultaneously Hot water treatments include immersion
boiling For immersion treatments seeds were added to test tubes with the water
(boiling) and the test tubes were immediately removed from the beaker to
For the boiling treatments test tubes remained in the beaker with water at
20
boiling temperature for a set duration after the addition of seeds Following all
treatments test tubes with seeds cooled 24 hours in a test tube rack
Percussion treatment utilized a pneumatic paint shaker (Central Pneumatic
model 00422 Camarillo CA) Compressed air was maintained at 530 kPa plusmn30 kPa
(80 psi plusmn 5 psi) resulting in approximately 350 oscillations per minute Seeds were
placed in 118 ml (4 oz) soil tin cans (US Can Lobard IL) and the lids were secured
with duct tape Each can was used to treat one 100-seed sample To take advantage
ofgreatest lateral movement of the paint shaker a spacer (paint can) was used to
position the soil tin at the end of the shaking arm This allowed shaking to occur at
the greatest distance from the pivot point (fulcrum) Tins were placed perpendicular
to the direction of shaking motion allowing seeds to impact at greatest force by
hitting flat surfaces of the soil tin
Germination Testing
Before gennination testing all seeds were soaked 24 hours in reverse
osmosis-treated water (this was included in the process ofhot water treatment
jU~SCIlOea above) Following soaking seeds were placed in 100 cm Petri dishes lined
90 cm Whatman qualitative 1 filter paper circles (MG Scientific Pleasant
WI) One Petri dish was used for each treatment by source combination
papers were initially moistened with 5 mL reverse osmosis-treated water then
~VU1CUlJl moistened as needed to ensure adequate conditions for gennination
were monitored daily (at 300 PM) for gennination for 14 days in accordance
~UIUlnJlVUa Seed Testing Association (ISTA) standards for Robinia species
21
(1STA 1999) Tests were carried out at room temperature (22degC plusmnOSoC)
Germination was classified as protrusion of the radicle from the embryo by at least
1mm as viewed with the naked eye
X-ray Radiography
On August 132001 three lOO-seed samples from the Sacramento seed source
were treated for 14 or 8-minute percussion durations The seeds were sent
immediately by overnight delivery to the Faxitron X-Ray Corporation in Wheeling
Illinois where Dr Bob Karrfalt ofthe USDA Forest Service oversaw their imaging
Radiographs were made at SX with a Faxitron Specimen Radiography System Model
MX-20
Scanning Electron Microscope Photography
On February 192002 a Hitachi S-3200N Variable Pressure Scanning
Electron Microscope (Mountain View CA) was used to examine strophiolar cells of
untreated seeds and seeds percussed for 4 minutes Seeds were cut longitudinally
igt their entire length using a razor blade splitting the strophiolar region roughly in
Cross sections of seed samples magnifying the strophiolar region were viewed
600X under the microscope
Analysis
Categorical Analysis of Variance was used to determine treatment source and
(Le time) main effects and interactions The response variable was total
~vlI This procedure is a Chi-square test of homogeneity using the natural log
of germinated to non-germinated seed for each treatment Maximumshy
22
likelihood analysis was used to calculate Chi-square test statistics with their observed
significance levels A single contrast was conducted to cempare treatment means
against the control Observed significance levels less than or equal to 005 were
considered significant Pairwise comparisons were conducted between non-control
treatment mean percents using a conservative alp~a value of 005 divided by the
number of comparisons (Bonferroni approach) and approximate pairwise z statistics
Categorical analyses were performed using Proc Catmod (SAS Institute 1989)
RESULTS
Scarification seed source block and all interactions between factors
influenced germination (Table 3) The main effects are reported in the presence of
these interactions because the overall robustness of treatments and seed sources are of
interest Background levels ofnaturally occurring variation in seed sources are
seen in control germination percentages in Figure 2 In particular time variability
is unlikely to be under the control of the producer Appendix B details experimental
data for source by treatment gennination percents for each of the four blocks
Averaged over all treatments germination by source ranged from 40 to
79 (Figure 2) Averaged over all sources (Figure 3) and for each source (Figure 4
5) percussion scarification durations of4 or 8 minutes yielded greatest germination
Averaged over all sources germination increased with increasing durations of
percussion then leveled off for the 4- and 8-minute treatments (Figure 3) Immersion
and 30-second boiling scarification maximized germination for the hot water
treatments with a decrease in germination when seeds were boiled for 60 seconds
For all but two sources there is a similar ipcreasing trend in germination from
1to 4 minutes ofpercussion (Figure 4) The Holman source increased in germination
1 to 2 minutes then leveled off at 4 minutes The Jemez source changed little
1 to 4 minutes of treatment As treatment duration increased from 4 to 8
mutes five sources decreased in germination (Figure 4a) whereas three sources
24
Table 3 Categorical Analysis of Variance table for germination response to seed treatment seed source block and the interaction of the factors
Source of DF Chi-Square Observed Variability Significance Level
Source 8 98370 lt00001 Trt 7 233876 lt00001
contrast 1 16976 lt00001 SourceTrt 56 97077 lt00001 Block 3 5538 lt00001
SourceBlock 24 10162 lt00001 TrtBlock 21 7564 lt00001
SourceTrtBlock 168 34283 lt00001
S 4)
8 ~
0
sectg ~ 40 ~
N 0 0
20
o f0ltgt ~ ~4 jybshy ((iltgto ~~ K1ltgt 6~b-~ ~~ 0b-~~ ~~ ~b-~ ~b-~b
80
60 -I-- - ~- middotmiddotmiddotmiddotmiddot1
r -] Northern NM Sources
Central NM Sources
Southern NM Sources
sect-o ~
~b-v(i
SOURCE
Figure 2 Effect ofNew Mexico locust seed source on germination for data averaged over all scarification treatments Control germination is shown by solid line within bar
80
~ cd ~ ltl) () 60 ~ ~
N
t 0shyrti tshySltl)
40
0
20
o lmmersion Boiling 30 sec Boiling 60 sec Control 1 min 2min 4 min 8 min
Hot Water Treatments Percussion Treatments
Figure 3 Effect of scarification on germination ofNew Mexico locust for data averaged over all seed sources Means labelled with the same letter are not significantly different
100~----------------------~--~----------------~
80
60
ltU
~
40 IJ
sectgsI
l100
80
60
40
a I middotmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot middotImiddotmiddotmiddotmiddotmiddotmiddot E
_~iii-bull _ _ bull f --~--
t-- -~ ---- -shy ---- shyI ~ ---- - shy
L
fiII
~
b
- shy ---r
- Holman
Sacramento Sandia Gila Jemez
c ~
ltgt ~
Tmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot-I
j~=~=-~~-1
I middotmiddot ~ bull r
t tr
Y -------M-a-gd-a-Ie-n-a--
1 _ _
fCapitan
---- Manzano ~y -_ Raton
2 4 8
Time interval ofpercussion scarification (min)
Figure 4 Effect of seed source by treatment interaction on New Mexico locust germination for percussion scarification pattern a and pattern b
28
tv 0
~ d
~ A s o
1 c3
80
60
40
20
o
TIT
t------=middotfmiddotmiddot-middotmiddot-middotmiddot-middotmiddot 1- --- - - --=- - -shy--cE= - _--shy --shy ----shy ~ ~ ---shy ~ -~- _ --shy-------r 1
I
Raton Holman
-_ Jemez
--- Sandia Manzano Magdalena Capitan Sacramento Gila
Immersion Boiling 30 sec Boiling 60 sec
Hot Water Scarification
Figure 5 Effect of seed source by treatment interaction on New Mexico locust germination for hot water scarification
continued to increase in germination (Figure 4b) There was no difference between
the two treatment levels for the Manzano source (Figure 4b)
The effect ofhot water scarification also varied among sources with few
overall differences between hot water treatments (Figure 5) However boiling for 60
seconds yielded highest germination for the Jemez source whereas increasing
durations ofboiling decreased germination for the Raton source
In addition to consistently high germination 4- and 8-minute percussionshy
treated seeds germinated as fast or faster than all other treatments (Figure 6 Table 4)
050 measured as the number of days needed to complete 50 ofeventual
germination averaged 2 days for both ofthese treatments
X-ray radiographs were taken of seeds collected from the Sacramento source
percussed for 1 4 or 8 minutes (Figure 7) Seeds show signs of damage at 8 minutes
ofpercussion (Figure 8) Compared to an undamaged seed percussed for only 4
minutes (Figure 8a) damage at the 8-minute level includes fractured radicles pitting
of the seed coat and chips in seed coat structure (Figure 8b-d)
Scanning electron microscope (SEM) images at 600X reveal the direct effect
of 4 minutes of percussion on cells of the strophiolar region (Figure 9) Whereas
untreated seeds retain cellular organization in the strophiolar region strophiolar cells
seeds treated for 4 minutes ofpercussion separate and raise resulting in a
~-~e~U appearance
30
Umiddot-~middot--l-~---middotymiddotmiddotmiddot-----middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot--middot--middotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middotmiddotmiddotmiddotmiddotmiddotmiddot- -
050= gt1
-e-- Percussion 8 min -0- Percussion 4 min ---T- Percussion 2 min -----ltil- Percussion 1 min -- Immersion --0- Boiling 30 sec -+- Boiling 60 sec -ltgt- Control
2 4 6 8 10 12
Day Figure 6 Timecourse ofNew Mexico locust gennination following scarification
treatments G50 is the number of days to 50 of eventual germination
31
14
Table 4 Effect of seed source and treatment on G50 (days to 50 germination)
Seed Source Control Immersion B30sec B60sec Plmin P2min P4min P8min mean Raton 3 3 5 7 4 3 2 2 4 Holman 2 2 2 2 2 2 2 2 2
Jemez 4 6 6 5 3 2 2 2 4 Sandia 2 2 2 2 2 2 2 2 2
w tv Manzano 5 2 2 3 3 2 2 2 3
Magdalena 4 2 2 3 4 3 2 1 3
Capitan 5 4 2 2 3 2 2 2 3
Sacramento 3 2 2 2 4 3 2 2 3 Gila 3 2 2 2 2 2 2 2 2 mean 3 3 3 3 3 2 2 2 3 standard 11 14 16 18 09 05 0 03 08 deviation
Note Columns and rows may not agree due to rounding
Figure 7 X-ray radiographs ofSacramento seed source percussed for 1 4 or 8 minutes
33
Figure 8a-d Seed damage close-ups Figure a shows undamaged seed percussed for 4 minutes Figures b-d show seeds percussed for 8 minutes Damage includes fracturing ofradicle in b complete removal ofseed coat in c and pitting ofseed coat in d
34
Untreated Treated
Figure 9 SEM images ofuntreated vs 4-minute percussion-treated strophiolar cells ofNew Mexico locust seeds Treated cells are loosened separated and raised
DISCUSSION
Determining the Optimal Treatment
Averaging results of scarification treatmems across seed sources is a useful
tool to predict which treatment is most likely to be successful for an unknown seed
source Averaged over all sources four- and eight-minute percussion treatments
resulted in the highest germination 815 and 790 respectively and produced
germination that was as fast or faster than all other treatments Although there was no
difference between these two treatments on average an argument for 4 minutes of
percussion as a standard protocol for treating New Mexico locust seeds can be made
Descriptively 4 minutes of percussion improved germination over 8 minutes
ofpercussion for a majority of sources (five out of nine) 8 minutes of percussion
improved germination for three ofnine sources with no difference for a fmal source
For individual sources 4 minutes ofpercussion was always the best or secondmiddot best
treatment and was always equal to or better than all hot water treatments
Germination at 8 minutes ofpercussion was lower than all other percussion durations
for the Jemez and Gila sources and was lower than at least one hot water treatment for
these sources as well
The x-ray radiographs of the Sacramento seed source illustrate the excessive
internal and external seed damage that can occur with 8 minutes of percussion
germination counts 8-minute percussed seeds from Holman and Capitan
ClUll signs of damage that were visible to the naked eye (see below)
r~evertheless some of these seeds still fit the criteria for germination Neither x-ray
36
photography nor visible observations during seed counts revealed seed damage at the
4-minute percussion level In operational trials seeds percussed for 4 minutes
developed into healthy seedlings (Khadduri and Harrington unpublished data)
Strophiole and Seed Coat Behavior
Four minutes ofpercussion successfully breaks seed dormancy in a large
percentage ofNew Mexico locust seeds without excessively damaging the
surrounding seed coat or internal seed coat structures Most papilionoid legume
seeds including seeds ofNew Mexico locust have a specific region of the seed coat
known as the strophiole or lens (Hamly 1932) The strophiole is located on the
cotyledonary lobe of a locust seed (Figure 10 a-c) If seeds are not indiscriminately
damaged the strophiole is the first point of water entry to the embryo (Hopkinson
1997) When a papilionoid seed is percussed for the appropriate amount of time
repeated hits on the integrated seed coat loosen the constrained cells of the strophiolar
region without excessively damaging the rest of the seed coat (Ballard 1976) SEM
photos illuStrate that 4 minutes of percussion loosened and separated strophiolar cells
ofNew Mexico locust seeds when compared with constrained organized strophiolar
cells of untreated seeds (Figure 9) Research-has shown that water passes to the
embryo through the treated strophiole in a controlled manner and regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues
(Kelly and van Staden 1987)
37
10 Strophiole ofunimbibed New Mexico locust seed (a) and following uptake (bc)
38
In contrast hot-water treatments can excessively degrade the seed coat
(Rebman et al 1998 Marunda 1990) Random cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rebman et aI 1998 Marunda 1990) Along with a
proportionately large amount ofun-imbibed (thus under-treated) seeds (Figure l1a)
damage was observed in hot-water treated seeds from six sources during germination
counts Damage included seeds where the radicle failed to elongate (Figure 11 b)
seeds where the radicle elongated but was delayed in freeing itself from the seed coat
(Figure 11c) and seeds where the radicle completely broke off from the embryo
(Figure lId) In addition some seeds germinated in reverse (the cotyledon emerged
fIrst) If the radicle emerged completely from the seed coat they were counted as
germinated
While nearly all seeds treated for 8 minutes ofpercussion imbibed water
damage was evident in x-ray evaluations (Sacramento seed source) and in
germination counts (Holman and Capitan) at this treatment level Seed damage
during germination counts was similar to levels found in hot-water treated seeds
suggesting that over-treatment by both hot water and longer durations of percussion
may result in similar damage to seed structure
The Sacramento and Holman seed sources were two of the fIve sources that
opcrplltlpn in germination from the 4- to 8-minute durations of percussion
the benefIts ofloosening the strophiole as evidenced by imbibition of
all 8-minute treated seeds were outweighed by external and internal seed coat
39
a
b c d
11 Damage to New Mexico locust seeds observed during germination cotmts following hot water scarification 9a Hot water replicate including ~t seedlings and numerous unimbibed seeds 9b Close-up of seed where raallCle failed to elongate 9c Close-up ofseed where radicle elongated but was
-gtIn in freeing itself from the seed coat 9d Close-up ofseed where radicle broke off from embryo
40
damage (which also may be contributing to high imbibition) For the three sources
for which 8 minutes of percussion resulted in maximum germination extended
percussion of seeds necessary to loosen the strophiole may not have excessively
damaged the seed anatomy The Manzano source did not significantly change in
germination from 4 to 8 minutes of percussion suggesting that an increase in
permeable seeds at the 8-minute treatment duration was balanced by the number of
seeds that failed to germinate due to seed damage To summarize the 4-minute
percussion treatment improved germination over 8 minutes ofpercussion for a
majority of sources (five ofnine) the 8-minute percussion treatment improved
germination over 4-minutes ofpercussion for three sources and there was no
difference with the Manzano source Notably for the Raton source 8 minutes of
percussion increased germination by 235 percentage points over 4-minutes of
percussion (7875 vs 5525 respectively) the next best treatment Raton was the
only source to result in germination less than 75 for 4 minutes ofpercussion so a
grower may wish to apply longer percussion durations to lots that do not respond well
to this treatment level
Finally there was an interesting trend in the Jemez and Raton sources seed
that significantly contributed to the source by treatment interaction (Figure 12) A
~u test showed that the Jemez source had a significantly lighter seed weight than
other sources while the Raton source had an average seed weight For the Jemez
the longest duration of boiling increased germination Conversely only short
U4UVll) of percussion were needed to achieve high germination and the longest
41
80 _ Jemez a Raton
iD
~ C iD e iD
Q c 0 ~
c ~ iD 0
20
0------shy
Immersion Boiling 30 sec Boiling 60 sec
Hot Water Scarification
10degTr========~--------~------------------------------
80
60
40
20
_ Jemez Raton
0------shy
1 min 2 min 4 min 8 min
Percussion Scarification
Figure 12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources
42
b
--~----~~~~========~---- duration ofpercussion reduced germination For the Raton source longer durations
ofboiling consistently decreased germination whereas longer percussion treatments
consistently increased germination
A possible explanation is that a thick seed coat is associated with a loose
strophiole structure and that a thin seed coat is associated with a constrained
strophiole This contrast merits further study and may suggest an ecological
adaptation Further investigation ofthis phenomenon may contribute to successful
pre-screening ofunknown seed sources to determine optimal treatment leveL
43
CONCLUSION
Based on results obtained in pilot study (see Appendix) and thesis
experiments 4 minutes ofpercussion is recommended as a standard protocol for
treating an unknown source ofNew Mexico locust seed In addition to laboratory
results the 4-minute percussion treatment has resulted in germination greater than
80 and healthy seedling grow-outs for production purposes (unpublished data) In
laboratory experiments only one hundred seeds per 118 ml (40z) soil tin were
percussed at a time whereas for production we have treated up to five hundred seeds
per tin For large-scale production ofNew Mexico locust seedlings growers may
wish to experiment with larger quantities of seed and larger-size soil tins
Seed storage trials following percussion treatments were not conducted
However germination greater than 80 and healthy seedlings were obtained when
seeds were percussion-treated and subsequently stored at room-temperature (22degC)
for one week (unpublished data) Seed storage trials merit further study as the
standard hot water treatment leaves seeds in a softened condition that is not
conducive to storage
Hydroseeding is an effective method for direct seeding in reclamation
situations Percussion treatment may be conducive to hydro seeding as seeds remain
a dry relatively undamaged yet permeable condition following treatment Sulfuric
seeds also remain dry following treatment but extensive damage to seed coat
poor seedling vigor have been reported with this treatment (Kelly and van Staden
A grow-out study using a hydro seeder on a reclamation site testing percussion
44
against other treatments such as sulfuric acid and mechanical scarification also
merits further study
Papilionoids are the largest subfamily oflegumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust these legumes often play an integral role in the revegetation of
disturbed lands The pilot study (Appendix) indicates that percussion scarification
successfully treats the widely used black locust as well Developing superior
scarification methods to the standard hot water mechanical scarification and acid
treatments may facilitate the use ofpapilionoid legumes in reclamation projects
45
APPENDICES
APPENDIX A
PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR
NEW MEXICO LOCUST AND BLACK LOCUST SEEDS
Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4
Hot water and sulfuric acid soaks are traditional treatments for seeds of many
temperate woody legwnes including locusts However these scarification techniques
often produce inconsistent germination Percussion scarification where seeds are
repeatedly propelled against a hard surface was compared with hot water
scarification to evaluate treatment efficacy for New Mexico locust (Robinia
neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment
seeds were placed in a 98degC water bath which was immediately removed from the
heat source For percussion scarification seeds were placed in a soil sample tin and
agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the
control were followed by 24-hour water soaks Hot water baths resulted in 56 and
41 germination for New Mexico locust and black locust respectively For both
species nearly all durations ofpercussion increased germination over the hot water
treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3
4 and 5 minutes for black locust resulted in at least 90 germination Traditional
scarification treatments randomly degrade the entire seed coat which can lead to
tissue damage during water uptake Percussion scarification specifically weakens the
strophiole the natural source ofwater entry to the seed in papilionoid legwnes
INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom
2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031
48
Following percussion imbibition is controlled through the strophiole and underlying
tissue is protected
Keywords New Mexico locust black locust scarification percussion germination
Introduction
Locust trees (Robinia spp) are aggressive pioneer species that quickly
colonize disturbed land fix atmospheric nitrogen in the soil and stabilize slopes that
are prone to erosion (Klemmedson 1994 Ashby et al 1985) (Figure A1ab)
Recognition of these attributes has contributed to increased demand for locusts in
reclamation projects Locust seeds however exhibit physical dormancy where a
thick seed coat prevents the movement of water and gasses to the embryo (Leadem
1997)
Sulfuric acid or hot water soaks have traditionally been used to break seed
dormancy in macro-propagation of locusts Many growers are moving away from the
use of su1furic acid in treating locust seeds (Dreesen and Harrington 1997) In
addition to being dangerous acid soak durations must be specifically correlated to
seed lot (Olson 1974) Collections of native seed can vary tremendously in seed size
seed weight and hard seededness etc This variability limits the utility of sulfuric
acid soaks as even within a given lot some seeds may be damaged whereas others
are under-treated and fail to imbibe (take up water) A survey of Southwestern
container growers indicates that hot water baths are currently the most common
scarification method for locust seeds (Hine et al 1997) However hot water baths
also produce inconsistent germination (Lin et al 1996)
49
b
Figure AI New Mexico locust a primary invader following disturbance Ala Crown-sprouting at edge ofburn near Los Alamos NM--one year after Cerro Grande fire Alb Colonizing road cut Gila National Forest NM
50
Percussion scarification where seeds are repeatedly propelled against a hard
surface is an alternative dormancy-breaking method that has proven successful in
legumes related to locusts (Hamly 1932 Barton 1947) We compared this alternative
treatment to hot water scarification to evaluate treatment efficacy for New Mexico
locust and black locust seeds
Materials and Methods
Commercial New Mexico locust seeds (Western Native Seed Coaldale CO)
were collected fall 2000 in Huerfano County CO Black locust seeds were collected
September 2000 from Taos County NM by harvesting ripe pods from several trees at
a distance of up to 3 m from ground level Black locust seeds were allowed to air-dry
for 3 weeks and were threshed and separated from large chaffusing a greenhouse
fan A South Dakota Seed Blower (Chicago Illinltois) was used to remove fine chaff
from both seed lots Seeds were storedmiddotat 2-4C until the germination study was
conducted in March 2001
This study compared hot water scarification with percussion scarification
using treatment and species as experimental factors Along with a control
scarification treatments included immersion of seed in boiling water as well as
percussion durations of 1 2 3 45 or 10 minutes Four 100-seed samples were used
to test each species by scarification treatment combination
For the hot water treatment test tubes were filled with 30 mL water and
placed in a water-filled beaker (Figure A2a) The bath was raised to boiling (-98C at
51
b
c
Figure A2 Implementation ofhot water and percussion scarification A2a Hot water bath A2b Paint-shaker set-up A2c Placing soil tin containing seeds at end ofpaint shaker ann
52
Las Cruces NM elevation of 1300 meters) Each 100-seed replicate was placed in a
test tube and each test tube was immediately removed to cool at room temperature
Percussion scarification was implemented using a pneumatic paint shaker
(Central Pneumatic model 00422 Camarillo CA) (Figure A2b) This allowed
standardization of treatment intensity Compressed air pressure was maintained at
530kPa plusmn 30kPa (80psi plusmn 5psi) resulting in approximately 350 oscillations per
minute Each lOO-seed sample was placed in a 118 ml (4 oz) soil tin for shaking To
maximize lateral movement of paint shaker a paint can was used as a spacer to place
soil tins at the end of the shaking arm (Figure A2c) This allowed shaking to occur at
the greatest distance from the pivot point (fulcrum) Following all treatments
including the control seeds were soaked for twenty-four hours in distilled water prior
to plating out
Gennination was evaluated on lab benches at room temperature Each
replicate of seeds was placed in a 100 cm petri dish on moistened filter paper with
humidity maintained by enclosing the petri dishes in ziplock bags Gennination was
monitored daily for 14 days in accordance with International Seed Testing
Association standards for Robinia species (ISTA 1999) Gennination was defined as
protrusion of the radicle from the embryo by at least 1mm as viewed by the naked
eye
Categorical Analysis of Variance using Prpc Catmod (SAS Institute 1989)
was used to detennine treatment differences in germination percentages for each
source This procedure is an extension of a Chi-square test of homogeneity using the
53
natural log of the ratio of germinated to non-germinated seeds for each treatment Pshy
values less than 005 were considered significant A limited set of pairwiseshy
comparisons was conducted to compare treatment mean percents using a
conservative alpha value of 005 divided by the number of comparisons
Results
Percussion maximized germination for both New Mexico locust and black
locust There were three germination response levels for New Mexico locust (Figure
A3) First there was a low response in the control indicating a dormant seed lot
The hot water treatment and I-minute duration of percussion share a second
intermediate response Germination was highest at percussion levels from 2 to 10
minutes
A selected percussion treatment intermediate in time (5 minutes) produced the
steepest germination curve indicating the fastest germination rate (Figure A4) The
hot water treatment produced an intermediate curve or relatively slower germination
rate The control curve was very flat indicating the slowest germination rate for New
Mexico locust
Control seeds germinated poorly for black locust as well (Figure A5) Hot
water scarification improved germination over the control but all levels of percussion
improved germination over hot water The optimal durations of percussion treatment
were bracketed with a significant increase from the 1 to 2 minute percussion level a
high germination response from 2 to 5 minutes of percussion and finally a significant
54
~ d 25 CL)
p t1 0 ~ t1
g Vl ~ Vl
100~---------------------------------------------------------------
80
60
40
20
o Control Hot Water Percussion
I min Percussion
2 min Percussion
3 min
Scarification
Percussion 4 min
Figure A3 Effect of scarification on germination response for New Mexico locust
Percussion 5 min
=significant difference between adjacent treatment bars ns = no significant difference
Percussion 10 min
~ ~ () u agt
Il-t d o ~
~ ~ c3
100middotlr~~~------------------------------------e- Percussion 5min middot 0middot Hot Water -- Control
80-J=middot======
6b-middot-lmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middot--middotmiddot-middot~-middotmiddot -middotmiddot middot middotmiddot-middot 1
40
20
-----~-----------
0 0 0 middotmiddotmiddotmiddotmiddot0middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot0
0 --~-----y-----~------_---~--- T-----~
o shy -TDay 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A4 Effect of selected scarification treatments on germination speed for New Mexico locust First seven days of fourteen-day experiment are shown
Day 7
80
~ cd
5 60 ()
[) Cl d 0 1t1 d
40sect dUl
-J
20
o
ns
Scarification
Figure A5 Effect of scarification on germination response for black locust = significant difference between adjacent treatment bars ns = no significant difference
decrease at the 10-minute level This drop-off suggests that 10 minutes of percussion
over-treats seeds from this lot of-black locust
A selected percussion treatment intermediate in time (5 minutes) also had the
fastest germination rate for black locust (Figure A6)_ The hot water curve is
somewhat flatter indicating a slower germination rate The control curve is very flat
indicating a slow germination rate
A qualitative difference between hot water and percussion-treated seeds was
also observed Along with healthy germinants the hot water treatment produced
over-treated seeds (Figure A 7a) Examples of damaged germinants include seeds
where the radicle did not elongate (Figure A 7b) seeds where the radicle elongated
but was delayed in freeing itself from the seed coat (Figure A7c) and seeds where the
radicle broke off completely from the embryo (Figure A7d) In addition to these
examples of over-treatment there were numerous small dark seeds that failed to
imbibe water For percussion treatment nearly all seeds took up water (Figure A8a)
Germination was rapid and uniform with little damage evident (Figure A8b)
To summarize both New Mexico locust and black locust seed lots were
dormant The standard hot water scarification treatment improved germination but
only to 56 and 41 for New Mexico locust and black locust respectively Nearly
all percussion durations improved total germination as well as germination rate over
the hot water treatment Germination was greater than 90 for percussion durations
of 4 5 and 10 minutes for New Mexico locust and 3 4 and 5 minutes for black locust
58
CI)
sgt t CI) 0
igt 0
sect ttl t E
Vl 0
c3
100r==~==~~=r------------------------------------~ ___ Percussion 5 min
middotmiddotmiddot0middotmiddotmiddot Hot Water -lIf- Control
80r=========~--~~----------------------------~
60
40
20 -~-~---~---------- ---~------------I
omiddot
O
_~_----------lIf-----~bull -----_ shy---shy I
Day 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A6 Effect of selected scarification treatments on germination speed for black locust First seven days of fourteen-day experiment are shown
Day 7
a
b c d
Figure A7 Damage to New Mexico locust seeds observed during germination counts following hot water scarification A7a Hot water replicate including damaged seedlings and numerous unimbibed seeds A7b Close-up ofseed where radicle failed to elongate A7c Close-up ofseed where radicle elongated but was delayed in freeing itself from the seed coat A7d Close-up ofseed where radicle completely broke offfrom embryo
60
a
b
Figure A8 New Mexico locust seeds following percussion treatment at time of germination counts A8a Percussion replicate with uniformly healthy seedlings and near complete imbibition of seeds A8b Close-up ofpercussion-treated seedling
61
Discussion
Papilionoid seeds have a specific anatomical feature known as the strophiole
or lens-the natural site of water entry to the seed (Hamly 1932) The strophiole is
located on the cotyledonary lobe of a locust seed (Figure A9) When a papilionoid
legume seed is percussed for the appropriate amount of time repeated hits on the
integrated seed coat loosen the constrained cells of the strophiolar region without
excessively damaging the rest of the seed coat (Ballard 1976)
When a percussed seed is soaked water enters exclusively through the
strophiole in a controlled manner (Kelly and Staden 1987) This regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues This
contrasts with seeds that have been hot water or acid scarified treatments that can
randomly degrade the seed coat Unlocalized cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rehman et al 1998 Marunda 1990)
Papilionoids are the largest subfamily of legumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust and black locust these legumes often play an integral role in the
revegetation ofdisturbed lands Developing superior scarification methods to the
standard hot water and acid treatments should fadlitate the use ofthese legumes in
restoration projects
62
Figure A9 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (bc)
63
APPENDIXB
GERMINATION PERCENTS AND STANDARD ERRORS FOR SOURCE
BLOCK AND TREATMENT FOR NEW MEXICO LOCUST
Table B 1 Gennination percents and standard errors for source block and treatment for New Mexico locust
Source Block Treatment Germination Percentage Standard Error
Capitan 1 Immersion 74 437 Capitan 2 Immersion 69 462 Capitan 3 Immersion 71 454 Capitan 4 Immersion 67 470 Capitan Boiling 30 sec 79 407 Capitan 2 Boiling 30 sec 76 427 Capitan 3 Boiling 30 sec 72 449 Capitan 4 Boiling 30 sec 76 427 Capitan Boiling 60 sec 71 454 Capitan 2 Boiling 60 sec 54 498 Capitan 3 Boiling 60 sec 67 470 Capitan 4 Boiling 60 sec 76 427 Capitan I Control 5 218 Capitan 2 Control 7 255 Capitan 3 Control 4 196 Capitan 4 Control 5 218 Capitan 1 Percussion I min 47 499 Capitan 2 Percussion I min
470
Capitan Percussion I min 39 488
Capitan 4 Percussion I min 38 485 Capitan I Percussion 2 min 55 497 Capitan Capitan
2
Percussion 2 min Percussion 2 min
71 65
454 477
Capitan 4 Percussion 2 min 56 496 Capitan I Percussion 4 min 86 347 Capitan 2 Percussion 4 min 79 407 Capitan 3 Percussion 4 min 81 392 Capitan 4 Percussion 4 min 82 384 Capitan I Percussion 8 min 90 300 Capitan 2 Percussion 8 min 88 325 Capitan 3 Percussion 8 min 90 300 Capitan 4 Percussion 8 min 90 300 Gila I Immersion 71 454 Gila 2 Immersion 77 421 Gila
Immersion 75 433
Gila 4 Immersion 72 449
Gila I Boiling 30 sec 68 466
Gila 2 Boiling 30 sec 81 392
Gila 3 Boiling 30 sec 54 498
Gila 4 Boiling 30 sec 63 483
Gila I Boiling 60 sec 55 497
Gila 2 Boiling 60 sec 68 466
Gila 3 Boiling 60 sec 57 495
Gila 4 Boiling 60 sec 70 458
65
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Gila 2 Control 13 336 Gila 3 Control 13 336 Gila 4 Control 20 400 Gila 1 Percussion 1 min 66 474 Gila 2 Percussion 1 min 57 495 Gila 3 Percussion 1 min 73 444 Gila 4 Percussion 1 min 82 384 Gila 1 Percussion 2 min 81 392 Gila 2 Percussion 2 min 86 347 Gila 3 Percussion 2 min 85 357 Gila 4 Percussion 2 min 54 498 Gila 1 Percussion 4 min 79 407 Gila 2 Percussion 4 min 83 376 Gila 3 Percussion 4 min 84 367 Gila 4 Percussion 4 min 79 407 Gila 1 Percussion 8 min 51 500 Gila 2 Percussion 8 min 76 427 Gila 3 Percussion 8 min 52 500 Gila 4 Percussion 8 min 74 439 Holman 1 Immersion 83 376 Holman 2 Immersion 86 347 Holman 3 Immersion 81 392 Holman 4 Immersion 90 300 Holman 1 Boiling 30 sec 90 300 Holman 2 Boiling 30 sec 85 357 Holman 3 Boiling 30 sec 87 336 Holman 4 Boiling 30 sec 83 376 Holman 1 Boiling 60 sec 79 407 Holman 2 Boiling 60 sec 80 400 Holman 3 Boiling 60 sec 86 347 Holman 4 Boiling 60 sec 93 255 Holman Control 25 433 Holman 2 Control 19 392 Holman Control 21 407
Holman 4 Control 24 427
Holman Percussion 1 min 77 421
Holman 2 Percussion 1 min 85 357
Holman 3 Percussion 1 min 80 400
Holman 4 Percussion 1 min 89 313
Holman 1 Percussion 2 min 88 325
Holman 2 Percussion 2 min 96 196
Holman 3 Percussion 2 min 93 255
Holman 4 Percussion 2 min 96 196
Holman 1 Percussion 4 min 92 271
Holman 2 Percussion 4 min 97 171
66
I
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Holman 3 Percussion 4 min 91 286 Holman 4 Percussion 4 min 96 196 Holman 1 Percussion 8 min 88 325 Holman 2 Percussion 8 min 85 357 Holman 3 Percussion 8 min 83 376 Holman 4 Percussion 8 min 82 384 Jemez 1 Immersion 53 499 Jemez 2 Immersion 44 496 Jemez 3 Immersion 52 500 Jemez 4 Immersion 70 458 Jemez 1 Boiling 30 sec 58 494 Jemez 2 Boiling 30 sec 55 497 Jemez 3 Boiling 30 sec 48 500 Jemez 4 Boiling 30 sec 65 477 Jemez 1 Boiling 60 sec 68 466 Jemez 2 Boiling 60 sec 63 483 Jemez 3 Boiling 60 sec 73 444 Jemez 4 Boiling 60 sec 89 313 Jemez 1 Control 2 140 Jemez 2 Control 9 286 Jemez 3 Control 6 237 Jemez 4 Control 8 271 Jemez 1 Percussion 1 min 58 494 Jemez 2 Percussion 1 min 63 483 Jemez 3 Percussion 1 min 77 421 Jemez 4 Percussion 1 min 89 313 Jemez 1 Percussion 2 min 68 466 Jemez 2 Percussion 2 min 64 480 Jemez 3 Percussion 2 min 80 400 Jemez 4 Percussion 2 min 83 376 Jemez Percussion 4 min 68 466 Jemez 2 Percussion 4 min 79 407 Jemez 3 Percussion 4 min 70 458 Jemez 4 Percussion 4 min 83 376 Jemez 1 Percussion 8 min 52 500 Jemez 2 Percussion 8 min 59 492 Jemez 3 Percussion 8 min 61 488 Jemez 4 Percussion 8 min 70 458 Magdalena 1 Immersion 69 462 Magdalena 2 Immersion 69 462 Magdalena J Immersion 70 458 Magdalena 4 Immersion 85 357 Magdalena 1 Boiling 30 sec 65 477 Magdalena 2 Boiling 30 sec 71 454 Magdalena 3 Boiling 30 sec 68 466
67
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Magdalena 4 Boiling 30 sec 83 376 Magdalena 1 Boiling 60 sec 63 483 Magdalena 2 Boiling 60 sec 68 466 Magdalena 3 Boiling 60 sec 63 483 Magdalena 4 Boiling 60 sec 82 384 Magdalena 1 Control 11 313 Magdalena 2 Control 6 237 Magdalena 3 Control 5 218 Magdalena 4 Control 4 196 Magdalena Percussion 1 min 32 466 Magdalena 2 Percussion 1 min 45 497 Magdalena 3 Percussion 1 min 46 498 Magdalena 4 Percussion I min 36 480 Magdalena 1 Percussion 2 min 67 470 Magdalena 2 Percussion 2 min 68 466 Magdalena 3 Percussion 2 min 65 477 Magdalena 4 Percussion 2 min 81 392 Magdalena 1 Percussion 4 min 87 336 Magdalena 2 Percussion 4 min 97 171 Magdalena 3 Percussion 4 min 83 376 Magdalena 4 Percussion 4 min 97 171 Magdalena 1 Percussion 8 min 98 lAO Magdalena 2 Percussion 8 min 100 000 Magdalena 3 Percussion 8 min 96 196 Magdalena 4 Percussion 8 min 98 lAO Manzano 1 Immersion 67 470 Manzano 2 Immersion 57 495 Manzano 3 Immersion 72 4049 Manzano 4 Immersion 73 4044 Manzano 1 Boiling 30 sec 70 458 Manzano 2 Boiling 30 sec 64 480 Manzano 3 Boiling 30 sec 71 454 Manzano 4 Boiling 30 sec 80 400 Manzano I Boiling 60 sec 50 500 Manzano 2 Boiling 60 sec 53 499 Manzano 3 Boiling 60 sec 59 492 Manzano 4 Boiling 60 sec 74 439 Manzano I Control 4 196 Manzano 2 Control 8 271 Manzano 3 Control 1 099 Manzano 4 Control 6 237 Manzano 1 Percussion 1 min 52 500 Manzano 2 Percussion 1 min 39 488 Manzano 3 Percussion 1 min 36 480 Manzano 4 Percussion 1 min 51 500
68
Table B 1 Continued
Source Block Treatment Gerrni~ation Percentage Standard Error
Manzano Percussion 2 min 53 499 Manzano 2 Percussion 2 min 58 494 Manzano 3 Percussion 2 min 61 488 Manzano 4 Percussion 2 min 62 485 Manzano 1 Percussion 4 min 76 427 Manzano 2 Percussion 4 min 85 357 Manzano 3 Percussion 4 min 79 407 Manzano 4 Percussion 4 min 83 376 Manzano 1 Percussion 8 min 87 336 Manzano 2 Percussion 8 min 83 376 Manzano 3 Percussion 8 min 83 376 Manzano 4 Percussion 8 min 75 433 Raton 1 Immersion 54 498 Raton 2 Immersion 37 483 Raton 3 Immersion 39 488 Raton 4 Immersion 69 462 Raton 1 Boiling 30 sec 36 480 Raton 2 Boiling 30 sec 20 400 Raton 3 Boiling 30 sec 28 449 Raton 4 Boiling 30 sec 60 490 Raton 1 Boiling 60 sec 15 357 Raton 2 Boiling 60 sec 171 Raton 3 Boiling 60 sec 4 196 Raton 4 Boiling 60 sec 35 477 Raton 1 Control 4 196 Raton 2 Control 12 325 Raton 3 Control 2 140 Raton 4 Control 6 237 Raton 1 Percussion 1 min 52 500 Raton 2 Percussion I min 29 454 Raton 3 Percussion I min 35 477 Raton 4 Percussion 1 min 31 462 Raton 1 Percussion 2 min 55 497 Raton 2 Percussion 2 min 35 477 Raton 3 Percussion 2 min 33 470 Raton 4 Percussion 2 min 42 494 Raton 1 Percussion 4 min 65 477 Raton 2 Percussion 4 min 52 500 Raton Percussion 4 min 51 500 Raton 4 Percussion 4 min 53 499 Raton 1 Percussion 8 min 88 325 Raton 2 Percussion 8 min 80 400 Raton 3 Percussion 8 min 73 444 Raton 4 Percussion 8 min 74 439 Sacramento 1 Immersion 43 495
69
Table B 1 Continued
-Source Block Treatment Germination Percentage Standard Error
Sacramento 2 Immersion 30 458 Sacramento 3 Immersion 38 485 Sacramento 4 Immersion 56 496 Sacramento 1 Boiling 30 sec 47 499 Sacramento 2 Boiling 30 sec 48 500 Sacramento 3 Boiling 30 sec 48 500 Sacramento 4 Boiling 30 sec 56 496 Sacramento 1 Boiling 60 sec 52 500 Sacramento 2 Boiling 60 sec 67 470 Sacramento 3 Boiling 60 sec 43 495 Sacramento 4 Boiling 60 sec 48 500 Sacramento 1 Control 0 000 Sacramento 2 Control 3 171 Sacramento 3 Control 3 171 Sacramento 4 Control 099 Sacramento 1 Percussion 1 min 57 495 Sacramento 2 Percussion 1 min 61 488 Sacramento 3 Percussion 1 min 62 485 Sacramento 4 Percussion 1 min 55 497 Sacramento 1 Percussion 2 min 84 367 Sacramento 2 Percussion 2 min 70 458 Sacramento 3 Percussion 2 min 81 392 Sacramento 4 Percussion 2 min 69 462 Sacramento 1 Percussion 4 min 91 286 Sacramento 2 Percussion 4 min 84 367 Sacramento 3 Percussion 4 min 84 367 Sacramento 4 Percussion 4 min 94 237 Sacramento 1 Percussion 8 min 82 384 Sacramento 2 Percussion 8 min 81 392 Sacramento 3 Percussion 8 min 82 384 Sacramento 4 Percussion 8 min 81 392 Sandia 1 Immersion 76 427 Sandia 2 Immersion 70 458 Sandia 3 Immersion 70 458 Sandia 4 Immersion 81 392 Sandia 1 Boiling 30 sec 78 414 Sandia 2 Boiling 30 sec 71 454 Sandia 3 Boiling 30 sec 63 483 Sandia 4 Boiling 30 sec 78 414 Sandia 1 Boiling 60 sec 67 470 Sandia 2 Boiling 60 sec 65 477 Sandia 3 Boiling 60 sec 74 439 Sandia 4 Boiling 60 sec 66 474 Sandia 1 Control 16 367 Sandia 2 Control 7 255
70
Table B 1 Continued
Source Block Treatment Gennination Percentage Standard Error
Sandia 3 Control 8 271 Sandia 4 Control 16 367 Sandia 1 Percussion 1 min 67 470 Sandia 2 Percussion 1 min 61 488 Sandia 3 Percussion 1 min 66 474 Sandia 4 Percussion 1 min 59 492 Sandia I Percussion 2 min 82 384 Sandia 2 Percussion 2 min 83 376 Sandia 3 Percussion 2 min 81 392 Sandia 4 Percussion 2 min 84 367 Sandia 1 Percussion 4 min 89 313 Sandia 2 Percussion 4 min 86 347 Sandia 3 Percussion 4 min 89 313 Sandia 4 Percussion 4 min 85 357 Sandia 1 Percussion 8 min 80 400 Sandia 2 Percussion 8 min 77 421 Sandia 3 Percussion 8 min 69 462 Sandia 4 Percussion 8 min 66 474
LITERATURE CITED
Ashby WC Vogel WG Rogers NF 1985 Black locust in the reclamation equation General Technical Report Northeastern Forest Experiment Station USDA Forest Service NE-I05 12 p
Ballard LAT 1973 Physical barriers to gennination Seed Sci Technoll 285-303
___ 1976 Strophiolar water conduction in the seeds ofTrifolieae induced by action on the testa at nonstrophiolar sites Austr J Plant Physiol3 465-469
Barton LV 1947 Special studies on seed coat impenneability Contrib Boyce Thompson Inst 14 355-362
Baskin CC Baskin JM 1998 Seeds Ecology biogeography and evolution of donnancy and gennination Academic Press London UK 666 p
Bewley JD 1997 Seed germination and donnancy Plant Ce119 1055-1066
Bewley JD Black M 1994 Seeds physiology of development and germination 2nd ed Plenum Press New York NY 445 p
Briscoe CB 1996 Growing Nitrogen Fixing Trees in Acid Soils In MH Powell (ed) Nitrogen Fixing Trees for Acid Soils- A Field ManuaL Winrock International Morrilton AR 22 p
Copeland LO McDonald MB 1995 Principles of Seed Science and Technology 3rded Chapman amp Hall New York NY 409 p
Cox RE Klett JE 1984 Seed gennination requirements of native Colorado plants for use in the landscape Plant Propag 30(2) 6-10
Dell B 1980 Structure and function ofthe strophiolar plug in seeds ofAlbizia Lophantha Am J Bot 67 556-563
Dick-Peddie WA 1993 New Mexico Vegetation Past Present and Future University ofNew Mexico Press Albuquerque NM 244 p
Dreesen DR 2001 Propagation protocol for production ofcontainer Robinia neomexicana plants Los Lunas Plant Materials Center Los Lunas New Mexico In Native Plant Network URL httpwwwnativeplantnetworkorg (accessed 9 January 2002) Moscow (ID) University ofIdaho CoIl Natural Resources For Res Nursery
72
Dreesen DR Harrington JT 1997 Propagation of native plants for restoration projects in the Southwestern US- Preliminary Investigations In Landis TD Thompson JR tech coords National Proceedings Forest and Conservation Nursery Assocs Gen Tech Rep PNW-GTR-419 Portland OR USDA For Ser PNW Res Sta 77-88
Gottfried GJ 1980 Control ofNew Mexican locust and the effect on planted ponderosa pine in central Arizona US For Ser Res Note RM-386 Rocky Mountain For Range ExpStn Fort Collins CO
Gosling PG Samuel YK Jones SK 1995 A systematic examination of germination temperature chipping and water temperaturesoak duration pretreatments on the seeds ofLeucaena leucocephala Seed Sci amp Technol 23521-532
Hamly DH 1932 Softening of the seeds of Melilotus alba Bot Gazette 93 345shy375
Hanna PJ 1984 Anatomical features of the seed coat ofAcacia kempeana (Mueller) which relate to increased germination rate induced by heat treatment New Phytol 96 23-29
Hanks JP Dick-Peddie WA 1974 Vegetation patterns of the White Mountains New Mexico Southwest Naturalist 18(4) 371-382
Hine S Harrington JT Dreesen DR 1997 Seed scarification requirements for Robinia neomexicana In Harrington JTtech coord Proc Third SouthwestemContainer Growers Conf Feb 12-13 1997 Ft Collins CO
Hopkinson JM 1993 The strophiole of leguminous seeds Newsletter- IntI Herbage Seed Production Res Grp 18 7-8
---- 1997 Differences in hardseededness between Cunningham and Tarramba leucaena In Leucnet News The Newsletter of the International LeucaenaRes and Devlt Netwk No4
ISTA (International Seed Testing Association) 1999 International rules for seed testing rules Seed Sci amp Technol27 Supplement
Kelly KM van Staden J 1987 The lens as the site of permeability in the papilionoid seed Aspalathus linearis J Plant Physiol 128(45) 395-404
73
Keresztesi B 1988 Black locust the tree of agriculture Outlook on Agr 17(2) 77~85
Kigel 1 and Galili G 1995 Seed development and germination Marcel Dekker New York 853 p
Klemmedson 10 1994 New Mexico locust and parent material Influence on macronutrients of forest floor and soil Soil Sci Soc Am 1 58 974~980
Kuo WH Tam AW 1988 The pathway of water absorption of mung bean seeds Seed Sci amp Technol16(1) 139~144
Leadem CL 1997 Dormancy--unlocking seed secrets In Landis TD Thompson lR tech coords National Proceedings Forestand Conservation Nursery Assocs Gen Tech Rep PNW-G TR-419 Portland OR USDA For Ser PNW Res Sta 43-52
Lin Y Wagner MR Seweyestewa A Honahni MD DeGomez M McMillin JD 1996 Influence of cultural treatments on seed germination and seedling growth of Robinia neomexicana Forest Farm and Community Tree Res Reports Volume 1
Marunda CT 1990 Effects of seed pretreatment on the development ofAcacia auriculforinis and A holosericea seedlings ACIAR Proceedings Series No 28
Meginnis HG 1937 Sulphuric acid treatment to increase germination of black locust seed USDA Circ 453 34 p
Miklas PN Towensend CE Ladd SL 1987 Seedcoat anatomy and the scarification ofcicer milkvetch seed Crop Sci 27 766-772
Monsen SB 1984 Use of shrubs on mine spoils In The Challenge ofProducing Native Plants for the Intermountain Area Proceedings Intermountain Nurserymans Association 1983 conference Las Vegas NV compo PM Murphy 26-31 Ogden UT USDA For Ser Gen Tech Report INT-l68
Olson DF 1974 Robinia L Locust In Seeds of Woody Plants in the US tech coord CS Schopmeyer 728-731 USDA For Ser Agr Handbk No450
Rehman S Loescher RNl Harris P1C 1998 Dormancy breaking and germination ofAcacia salicina Lindl seeds Seed Sci amp Techno127 553-557
Rolston MP 1978 Water impermeable seed dormancy Bot Rev 44 365-396
74
SAS Institute Inc 1989 SASSTAT Users Guide Version 6 4th Edition Volume 1 Cary NC SAS Institute Inc 943 p
SerratoValenti G Conara L Ferrando M Modenesi P 1993 Structural and histochemical features ofStylosanthes scabra (Leguminosae Papilionoidae) seed coat as related to water entry Can J Bot 71 834-840
Simpson BJ 1988 A field guide to Texas trees Austin TX Texas Monthly Press 372p
Tran VN Cavanagh AK 1984 Structural aspects ofdormancy In Seed Physiology Vol 2 Murray DR (ed) p1-44 Academic Press Australia
USDA Forest Service 1948 Woody-plant seed manual USDA Misc Pub1654 416p
Vines RA 1960 Trees Shrubs and Woody Vines of the Southwest Austin Univ ofTexas Press 1104 p
Wagner MR Larson RE Craig TP 1992 Ecology and silvics ofNew Mexico locust (Robinia neomexicana) with an emphasis on the arboreal form p53-57 In JW Hanover K Miller and S Plesko (eds) Proc Intemat Confon Black Locust Biology Culture and Utilization Mich State Univ USA
Wilson JK 1944 Immersing seeds of species of Robinia in boiling water hastens germination J For 42 453-454
Zimmerman RW Carpenter SB 1980 First-year coppice production from a 5shyyear old black locust stand on surface mine spoil p309-314 Centr Hardwd For ConfProc University ofMissouri Columbia
ACKNOWLEDGMENTS
I would like to thank Dr John Harrington for his guidance sense ofhumor
and flexibility throughout my studies I would also like to thank the members ofmy
committee for their support especially Dr Murray who patiently taught me the
subtleties of categorical analysis of variance
I am also grateful to Dr Anne Wagner and Molycorp Mine who provided
funding for this research Thanks to Dr Bob Karrfali for his assistance with x-ray
radiography and to Mohamed Murshed and Dr Soumitra Ghoshroy for their
assistance with scanning electron microscopy Special thanks go to my colleague Lee
Rosner who assisted me in all phases of this project Finally I thank my family for
their enduring love Jill Walid Amin Rolla Khalid Piet Zach Charlie and above
all mywife Lucie
111
VITA
June 3 1972
June 1990
May 1994
July 1995
August 1994- March 1998
March 1998-August 2000
August 2000-May 2002
Born in Beirut Lebanon
Graduated from Georgetown Day High School Washington DC
Graduated from Pomona College Claremont CA with Bachelor ofArts Degree in Political Science
Graduated from University ofNew Mexico Albuquerque NM with Post-baccalaureate Teaching Degree
6th Grade TeacherBanta Fe Public Schools Santa Fe NM and Environmental Educator Open Space Division Albuquerque NM
Assistant Manager Santa Ana Native Plant Nursery Santa Ana Pueblo NM
Graduate Research Assistant at New Mexico State University
FIELD OF STUDY
Horticulture
iv
ABSTRACT
PERCUSSION AS AN ALTERNATIVE SEED TREATMENT FOR
ROBINIA NEOMEXICANA (NEW MEXICO LOCUST)
BY
NABIL Y KHADDURI
Master of Science in Horticulture
New Mexico State University 2002
Las Cruces New Mexico
Dr John T Harrington Chair
New Mexico locust (Robinia neomexicana A Gray) a small tree native to
montane regions of the southwestern United States fills a successional role in postshy
disturbance situations The ability to colonize rapidly and ameliorate harsh sites
contributes to the importance ofNew Mexico locust in reclamation efforts Seedling
production for outplanting on disturbed sites has been hampered by poor germination
This research sought to improve seed propagation techniques in New Mexico locust
Nine seed sources were collected across a range of latitudes within New
Mexico Seed treatments included a control three levels of hot water and four levels
of percussion scarification Hot water treatments included immersion at 100degC and
boiling for periods of 30 seconds and 60 seconds Percussion treatment levels were 1
2 4 and 8 minutes A total of four lOO-seed samples were used to test each source
by scarification treatment combination Categorical Analysis of Variance was used to
determine main effects and interactions Pairwise comparisons were conducted to
compare non-control treatment means using a conservative alpha value of 005
divided by the number of comparisons
Averaged over all sources and for each source percussion scarification
durations of4 or 8 minutes yielded greatest germination In addition to consistently
bigh germination 4- and 8-minute percussion-treated seeds germinated as fast or
faster than all other treatments Averaged over all sources germination increased
with increasing durations of percussion then leveled off for the 4- and 8-minute
treatments Immersion and 30-second boiling scarification maximized germination
for the hot water treatments with a decrease in germination when seeds were boiled
for 60 seconds
Percussion scarification specifically weakens the strophiole the natural
dormancy barrier in many papilionoid legumes without excessively damaging the
seed coat As with New Mexico locust papilionoid legumes often play an integral
role in the revegetation ofdisturbed lands The percussion scarification technique
developed in this research has the potential to facilitate the use of these valuable
species in reclamation projects
VI
CONTENTS
LIST OF TABLES ix
LIST OF FIGURES middot middotmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot x
INTRODUCTION 1
LITERATlTRE REVIEW 4
Seed Donnancy 4
Physical Donnancy 5
Evolutionary Role ofPhysical Donnancy 6
Methods for Breaking Donnancy 6
Acid Scarification 6
Hot Water Scarification 8
Mechanical Scarification 11
Alternative Method for Breaking Donnancy Percussion 12
Objectives 15
Hypothesis 15
MATERIALS AND METHODS 16
Seed Collection and Cleaining 16
Experimental Design and Treatment Structure 16
Treatments ~ bullbull bullbullbullbull - bullbull ~ ~ 20
G T ennlnatlon estmg 21
X-ray Radiography 22
Scanning Electron Microscope Photography 22
Analysis 22
RESULTS 24
DISCUSSION 36
Detennining the Optimal Treatment 36
Strophiole and Seed Coat Behavior 37
CONCLUSION 44
Appendices
A PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR NEW MEXICO LOCUST AND BLACK LOCUST SEEDS 47
B GERMINATION PERCENTS AND STANDARD ERRORS FOR SOURCE BLOCK AND TREATMENT FOR NEW MEXICO LOCUST 64
LITERATURE CITED 72
Vlll
LIST OF TABLES
Table Page
1 Latitude longitude location elevation and collection date ofNew Mexico locust seed sources 18
2 Mean seed weight and standard error by source 19
3 Categorical Analysis ofVariance table for germination response to seed treatment seed source block and the interaction of the factors 25
4 Effect of seed source and treatment on G50 (days to 50 germination) 32
B1 Germination percents and standard errors for source block and treatment for New Mexico locust 65
LIST OF FIGURES
Figures Page
1 Seedsourcecollectionmap 17
2 Effect ofNew Mexico locust seed source on germination for data averaged over scarification treatments 26
3 Effect of scarification on germination for New Mexico locust for data averaged over all seed sources 27
4 Effect of seed source by treatment interaction on New Mexico locust germination for percussion scarification pattern a and pattern b 28
5 Effect of source by treatment interaction on New Mexico locust germination for hot water scarification 29
6 Timecourse of New Mexico locust germination following scarification treatments 31
7 X-ray radiographs of Sacramento seed source perc ussed for 1 4 or 8 minutes 33
8 Seed damage close-ups 34
9 SEM images of untreated vs 4-minute percussion-treated strophiolar cells ofNew Mexico locust seeds 35
10 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (b c) 38
11 Damage to New Mexico locust seeds observed during germination counts following hot water scarification 40
12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources 42
New Mexico locust a primary invader following disturbance 50
Implementation of hot water and percussion scarification 52
Effect of scarification on germination response for New Mexico locust 55
x
A4 Effect of selected scarification treatments on germination speed for New Mexico locust 56
AS Effect of scarification on germination response for black locust 57
A6 Effect of selected scarification treatments on germination speed for black locust 59
A7 Damage to New Mexico locust seeds observed during germination counts following hot water scarification 60
AS New Mexico locust seeds following percussion treatment at time of germination counts 61
A9 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (bc) 63
INTRODUCTION
New Mexico locust (Robinia neomexicana A Gray) a small tree native to the
southwestern United States occurs at elevations from 1200 to 2800 meters This
species fills a successional role in post-disturbance situations As a primary invader
New Mexico locust quickly establishes on burned areas arid flood banks as well as
road cuts (Wagner et aI 1992) Rapid growth crown sprouting and prolific root
suckering favor the successful establishment ofNew Mexico locust on disturbed sites
(Simpson 1988 Gottfried 1980) These attributes also may make New Mexico locust
well suited for steep-slope revegetation where erosion is a problem
New Mexico locust is a nitrogen-fixing legume that tolerates and improves
nutrient-poor soils Stands ofNew Mexico locust increase Nitrogen (N) and Calcium
(Ca) in the forest floor Levels of Carbon (C) Phosphorous (P) Sulfur (8) and
Potassium (K) aIso have been shown to increase in soil beneath New Mexico locust
(Klemmedson 1994)
The ability to colonize rapidly and ameliorate harsh sites contributes to New
Mexico locusts potential as a nurse plant A nurse plant colonizes an inhospitable
site and creates an environment suitable for successional plant establishment New
Mexico locust demonstrates this role in the ponderosa pine (Pinus ponderosa Douglas
ex Lawson amp C Lawson)Gambel oak (Quercus gambelii Nutt) community After a
disturbance such as wildfire New Mexico locust and Gambel oak colonize and
dominate the site until shaded out by ponderosa pine Once shaded these species
become understory shrubs a process taking an average of 15-20 years (Dick-Peddie
1993 Hanks and Dick-Peddie 1974) New Mexico locust gradually declines with
small dense patches averaging about 005 ha beneath pine until the next disturbance
once again offers it a competitive advantage (Klemmedson 1994)
Gottfried (1980) expecting ponderosa pine regeneration to be greater where
New Mexico locust had been eradicated found that survival of planted pine seedlings
was greater where locust had not been removed In a follow-up study Gottfried
(1980) noted that soil moisture in the top 57 cm was highest in 5-year old locust sites
as compared to grass or 20-year old locust sites He concluded that managing an
appropriate cover ofNew Mexico locust could help regenerate pine the later
successional species
The ability ofNew Mexico locust to improve harsh sites also makes it a
candidate for reclaiming disturbances associated with mining Black locust (Robinia
pseudoacacia 1) has been used for decades to reclaim mine spoils and other
disturbed sites in the eastern US and around the world (Keresztesi 1988 Zimmerman
and Carpenter 1980) Ashby et al (1985) described similar positive attributes of
black locust also mentioned previously for New Mexico locust quick cover for
stabilization supply ofN and nutrient-rich litter to the soil and site improvement for
establishment of later successional trees Referring specifically to mine reclamation
the authors note the ability of black locust to grow on a wide range ofmine soil
conditions including extremely acid soils Black locust also shows some tolerance to
soils compacted by grading and topsoiling practices (Ashby et aL 1985) New
2
Mexico locust holds promise as a native southwe~tem counterpart to black locust in
mine reclamation but it has been used infrequently to date
Natural invasion and succession occur slowly on most mine sites (Monsen
1984) While New Mexico locust often colonizes sites naturally there is no
assurance a seed will reach a particular site and establish in a reasonable time frame
The challenge is to introduce New Mexico locust as mother plants to facilitate
colonization Seedling production for outplanting on disturbed sites has been
hampered by poor germination (Lin et al 1996) The goal then is to develop
techniques to overcome poor germination in New Mexico locust thereby facilitating
macropropagation
LITERATURE REVIEW
Seed Dormancy
Low gennination in New Mexico locust is primarily the result of seed
donnancy the failure of an intact viable seed to genninate under favorable
conditions (Bewley 1997) Seed donnancy also can be defined as the inability of the
embryo to genninate because of some inherent constraint (Bewley and Black 1994)
The constraint may originate within the embryo referred to as endogenous or embryo
donnancy or from the tissues surrounding the embryo known as exogenous or coatshy
imposed donnancy (Baskin and Baskin 1998 Leadem 1997 Bewley and Black
1994)
New Mexico locust seed is donnant when the impenneable seed coat does not
allow movement ofwater to the embryo This specific fonn of coat-imposed
donnancy where the restraining tissue involved is an impenneable seed coat is
known as physical donnancy (Baskin and Baskin 1998 Bewley and Black 1994)
Physical donnancy is relatively uncommon in seeds less than 10 of all woody
species exhibit this condition (USDA Forest Service 1948) However it is
characteristic of many genera of the Fabaceae (bean) family For example all
successional trees in the genera Gleditsia and Robinia (of which New Mexico locust
is a member) produce seeds that exhibit physical donnancy (Baskin and Baskin
4
Physical Dormancy
Seeds that exhibit physical dormancy are said to be hard seeded referring to
the thick impermeable seed coat (Leadem 1997) The first line of protection of the
testa in leguminous seeds is the outermost waxy cuticle followed by a subcuticle
consisting ofsuberin Next is a palisade layer consisting of sclereid cells that have
thick lignified secondary walls The most common type of sclereid cell in palisade
layers is the macrosclereid The macrosclereids contain water-repellent substances
including cutin lignin suberin and wax (Rolston 1978) This palisade layer is the
primary barrier to water uptake (Serrato-Valenti et al 1993 Tran and Cavanagh
1984)
All seed coat openings must be blocked to effectively prevent water uptake
The natural opening in a seed where it was attached to the mother plant is the weak
link where physical dormancy is usually broken (Hopkinson 1993 Kuo and Tam
1988) Anatomical structures associated with the seed coat opening vary from
family to family In the Papilionoideae the subfamily of the Fabaceae family
containing New Mexico locust the site ofwater entry is the strophiole The
strophiole is a swelling on the seed coat close to the stalk scar (Hopkinson 1997)
Kelly and van Staden (1987) refer to the strophiole site as the lens where
macrosclereid cells must separate and raise before water can pass through Since
middot6germination ofdormant seeds of the Papilionoideae takes place when the strophiole
permeable (Hopkinson 1993 Kuo and Tam 1988) a logical approach to
5
genninating New Mexico locust artificially would be to loosen the strophiolar cells
and allow water to pass through to the embryo
Evolutionary Role of Physical Dormancy
Why is physical dormancy a mechanism that blocks germination
ecologically advantageous When a plant produc~s seeds with different degrees of
dormancy seeds that are dispersed at one time will germinate over a range of time
(Bewley and Black 1994) Through variability of dormancy within a seed crop seeds
are exposed to changing environmental conditions improving the overall chances of
survival of the seed line (Bewley and Black 1994) Varying expression of dormancy
is under genetic control which indicates that this may be an evolved survival strategy
(Kigel and Galili 1995) A range of dormancy levels may be a particular advantage in
the southwestern US where extreme environmental variations exist
Methods for Breaking Dormancy
There are three common approaches to breaking dormancy in New Mexico
locust seed acid scarification hot water soaks and mechanical treatments In each
case the objective of the treatment is to scarify or abrade the seed coat to make it
permeable to water
Acid Scarification
Acid scarification consists of soaking seeds in concentrated sulfuric acid
rinsing thoroughly with water upon removal followed by subsequent drying before
PlaJlltmg Olson (1974) reports that this scarification method produces the highest
middot~~jllalUll results for all Robinia species Most literature refers to a seminal 1937
6
study conducted by HG Meginnis This stud~ t~ough conducted on black locust
seed is regularly cited in the suggested use of acid to treat New Mexico locust seed
as well (Olson 1974 Vines 1960)
Thee published studies document the specific use of sulfuric acid to treat
New Mexico locust seed Cox and Klett (1984) report that soaking seeds in
concentrated sulfuric acid for thirty minutes improved germination to 34 over an
untreated control of4 Lin et al (1996) found that for two seed lots acid
scarification for twenty-five to eighty minutes produced germination of 80 A third
lot however resulted in less than 20 germination at all durations of 10 to 80
minutes Hine et al (1997) showed that acid treatment levels of 1 2 5 and 10
minutes resulted in less than 20 germination across five sources However there
was an increasing trend in germination with acid soak duration and acid treatments
were not imposed for as long a time as the previous two studies (30 to 80 minutes)
The mixed results obtained by these investigators were probably due to the
widely varying germination response of seed lots to acid treatment For this reason
Olson (1974) recommends correlating duration of acid treatment to each seed lot If
treatment duration is too long the embryo will be permanently damaged If soak
duration is too short the seed will remain impermeable to water and fail to germinate
Compounding the problem of treatment duration is the negative effect acid
have on seedling growth Any type of scarification can injure seeds to some
by disrupting cells essential for growth leading to fungal invasion and
middot-uuu~w injury (Copeland and MacDonald 1995) Some legume seeds subjected
7
to 60 minutes of sulfuric acid scarification (to achieve maximum gennination results)
showed more than 50 seedling mortality after gennination (Kelly and van Staden
1987) Bewley and Black (1994) noted that fungal growth on seeds is an indication
of leakage of sugars organic acids amino acids and proteins
Perhaps the greatest drawback to acid scarification is the dangerous nature of
the treatment itself Difficulties associated with storage and trained use of
concentrated acid may discourage many propagators from using this option (Dreesen
and Harrington 1997) Consequently hot water and to a lesser extent mechanical
scarification are more commonly used and practical treatments for legume seeds
Most southwestern container growers use either of these two treatments to scarify
New Mexico locust seed in particular (Hine et al 1997)
Hot Water Scarification
The widespread use ofhot water scarification to treat locust seed is probably
due to its ease of application and efficacy in comparison to other scarification
treatments Although this practice had been in use for some time Wilson (1944) was
one of the first to report on its systematic application for black locust The treatment
consists of placing seeds in boiling water (4 parts water tomiddot 1 part seed by volume)
removing the vessel from the heat source and allowing the water to cool and seeds to
soak for 24 hours
Hine et al (1997) Lin et al (1996) and Cox and Klett (1984) have examined
the specific application ofhot water scarification on New Mexico locust seed Each
these authors compared hot water to acid scarification Lin et aL (1996) reported
8
60 gennination for two hot water-treated seed lots while a third lot germinated
around 50 (as reported above acid scarification germination ranged from 20 to
80 for these lots) It should be noted that the authors only soaked the seeds in water
from 10 to 80 minutes for this experiment as opposed to the standard 24 hours Cox
and Klett (1984) stated that using the standard 24-hour hot water treatment improved
germination over an acid pre-treatment (which produced less than 20 germination)
but they did not give specific gennination numbers for the hot water treatment Hine
et al (1997) found that across five seed lots germination was 66 for the standard
hot water treatment (as compared to less than 20 germination for acid scarification)
Variations in treatment intensity and duration and their subsequent effects on seed lot
appear to contribute to conflicting conclusions as to whether acid scarification or hot
water treatment is a more successful treatment
Given mixed results ease ofapplication is an important argument for use of
hot water application over acid soaks Safety concerns and material costs for hot
water treatments are low (Dreesen and Harrington 1997) An extension of this is the
practicality ofhot water for treating large batches of seed where a relative increase in
water volume is all that is needed However increases in water and seed even when
inthe same 4 1 proportion can result in dramatically different rates of cooling than in
atest tube experiment Different cooling rates can lead to contrasting results for the
treatment temperature duration and seed lot (USDA Forest Service 1948)
As with acid scarification parameters ofhot water treatment application may
to be optimized to maximize germination Hine et al (1997) found that as
9
temperature was increased from 70degC to 100degC overall germination in New Mexico
locust seed increased The 100degC treatment maximized germination for each of five
sources tested with germination usually above 60
Prolonged exposure of seeds ofmany woody legumes to boiling temperatures
also may improve germination (Briscoe 1996) This approach has not been tested on
New Mexico locust seed Exposing seed to prolonged boiling temperatures may
result in fungal infection and subsequent seedling mortality Marunda (1990) noted
severe fungal infections on both acid and hot water treated Acacia holoserica Cunn
ex G Don seeds attributing the infections to seed damage resulting from the
treatments Identifying an optimal hot water treatment level that does not result in a
high rate of seedling mortality is a logical goal
With hot water treatments as with acid there is also a danger of overtreating
Seeds and reducing germination (Gosling et al 1995) The legume Leucaena
leucocephala (Lam) de Wit germinated equally well after 7 seconds of exposure to
water at 100degC 7 seconds at 90degC 30 seconds at 80degC and 4 minutes at 70degC In
addition acceptably high germination was obtained from treatment at 70degC from 3
minutes to 1 hour whereas no seeds germinated following treatment at 100degC for 2
minutes Perhaps along with a greater margin of error for treatment duration treating
~ew Mexico locust seeds at a lower temperature may result in a comparatively lower
of seedling mortality In practice however nursery growers tend to use the
and quickest treatment available The short treatment length at near-boiling
whether recommended or not may be what is used most frequently
10
Fine-tuning a treatment in this range of treatment temperature and duration may be
the most logical approach
Mechanical Scarification
This treatment involves scratching the seed coat with an abrasive material
(USDA Forest Service 1948) One system employs a rotating drum lined with
sandpaper A paddle spinning at a given number ofrevolutions per minute throws the
seed against the sandpaper (Hine et al 1997) A second method dry tumbling
tumbles seed in a mixer with pea gravel or sharp sand for 5 to 7 days (Dreesen 2001)
These approaches have the advantage that control of temperature is not required as in
water treatments nor is safety an issue as with add scarification
Hine et al (1997) mechanically treated New Mexico locust seed using 100shy
grit sandpaper in a Forsberg (rotating drum) scarifier Gennination was less than
10 They concluded that the seed coats were too thin for their apparatus and
proposed that a finer grit sandpaper would produce more even abrasion Although
germination varied by seed lot Dreesen (2001) reported that using a slow-speed dry
tumbling technique for 5 to 7 days produced gennination rates as high as 90
As with acid and hot water treatments treatment duration must be specifically
shy
correlated to seed lot Treated seeds are also susceptible to fungal infection
iHopkinson 1997) In addition equipment is specialized and may not always be
ltU~CUJl~ to a nursery (USDA Forest Service 1948) Several studies have confinned
degrading ofstrophiolar cells due to acid and hot water scarification (Miklas et al
11
1987 Dell 1980 Hanna 1984) but the effect ofmechanical scarification on the
strophiole has not been studied
Alternative Method for Breaking Dormancy Percussion
Strophiole degradation has been examined in depth using an impaction
treatment more commonly known as percussion Hamly (1932) first proposed that
shaking or impacting seeds could split the macrosc1ereid cells at the strophiole
Hamly successfully released tension ofbent strophiolar cells of impermeable
Melilotus alba Medik (White sweet clover) seeds by delivering physical blows
resulting in cells that had split apart and were only slightly bent Ninety-two percent
oftreated seeds became permeable as compared to 05 of control seeds Barton
(1947) showed that percussion significantly increases permeability in black locust
seed Ballard (1976) reasoned that the integrated nature of the strophiole within the
seed coat means a blow on any part of the seed weakens the strophiole There are no
published studies on the use of percussion on New Mexico locust
Percussion methods vary and often seem SUbjective or ill-defined Hamly
(1932) placed seeds in a glass bottle and shook them three times a second for 20
minutes resulting in 180 oscillations per minute Ballard (1973) describes shaking
seeds in a closed metal container with at least fifty seeds per container at 850-900
JS(~l11alIOIlS per minute for 1 minute Kelly and van Staden (1987) placed seeds into
sealed glass flasks Flasks were shaken by a wrist-shaker at a rate of 250
~UUVJ~ per minute Subsequent germination rates were 45 after 2 hours of
55 after 3 hours 60 after 6 hours and 100 after 12 hours
12
There are several potential advantages of percussion over acid hot water and
mechanical scarification Kelly and van Staden (1987) note that a percussion
treatment only alters macrosclereid cells of the strophiole by loosening separating
and raising them They emphasize that minimal damage is done to the overall
anatomical structure of the seed In acid scarification of the same seed lot radicle
and cotyledon damage occurred Based on photographic evidence the authors
believe that this morphological damage was due to uneven pressure during
imbibition with water entering haphazardly wherever the acid had sufficiently
dissolved the coat This ultimately led to uneven hydration of the embryo In
percussed seeds water only entered through the s~ophiole when the entire seed coat
had been saturated resulting in controlled and even embryo hydration (Kelly and van
Staden 1987)
Although all successful scarification treatments may focus at least in part on
the strophiole for papilionoid legumes (Baskin and Baskin 1998) percussion may be
less damaging to seed coat structures particularly when compared with acid Kelly
and van Staden did not compare hot water or mechanical scarification methods
against percussion However the fungal infection associated with hot water and
mechanical scarification as with acid scarificatio~ have been directly associated with
seed damage (Rehman et al 1998 USDA Forest Service 1948) Embryo damage in
has been observed in hot-water scarified legume seeds (Rehman et al 1998
13
By limiting embryo damage percussion scarification reduces seedling
mortality compared to other treatments Kelly and van Staden (1987) noted that
seedlings from the percussion treatment were morphologically sound and vigorous
when compared to acid treatment seedlings The authors attribute this improved
seedling vigor to the controlled water uptake that limited anatomical damage
Despite a 12-hour percussion treatment morphological damage was not substantially
greater than untreated seeds In addition seedling mass measured 10 days following
germination was not significantly different between percussed and untreated seeds
A final advantage ofpercussion may be the ability to adequately treat the most
dormant seeds while avoiding harm to seeds that have already been made permeable
by the treatment or that were permeable to begin with Instead ofcorroding scalding
or scratching the seed coat percussion appears to be just a loosening of the
strophiole spring (Ballard 1973) Kelly and van Staden (1987) were able to
increase germination with a lengthy treatment (12 hours) Figuring out whether a
substantial cushion oftreatment durations exist for percussion ofNew Mexico locust
seed would be a significant improvement to current scarification methods where seed
damage and seedling health is an issue
The greatest challenge for applying percussion to New Mexico locust seeds is
standardizing a treatment that is both effective and readily reproducible by nurseries
with mechanical scarification this may require the development of specific
~UllDment to properly quantify and execute the treatment
14
f
Objectives
The first objective of the study is to detennine which scarification treatment
results in optimal gennination speed and percentage ofNew Mexico locust The
second objective is to describe treatment interactions with nine seed sources collected
in New Mexico
Hypothesis
A percussion treatment acts exclusively on the main area ofwater uptake in
New Mexico locust seed loosening the strophiole so that imbibition can occur This
direct mechanism ofdonnancy release will result in improved gennination rate and
overall gennination percentage for New Mexico locust seed compared to standard
treatments Standard treatments including acid hot water and mechanical
scarification while eventually weakening the strophiolar cleft cause excessive
morphological damage through generalized seed coat degradation These treatments
are therefore prone to inconsistency due to over and under-treatment
15
MATERIALS AND METHODS
Seed Collection and Cleaning
Seeds were collected from August 25 through September 15 2000 from nine
locations along a latitudinal gradient in New Mexico Seed source locations and
descriptive information are given in Figure 1 and Tables I and 2 Stands were
separated by at least 45 km
Pods were hand picked as they began to ripen and dehisce (open) Collections
were made from ground level to approximately 3 m from several trees at each site
Pods were dried in open-ended brown paper bags for two to four weeks at room
temperature (21-230C) to facilitate cleaning Dried pods were placed in a closed cloth
sack which was pounded against the ground to separate seeds from pods For pods
still containing seeds hand maceration (crunching pods with hands) completed seed
separation Debris was removed by threshing with the aid of a greenhouse fan A
South Dakota Seed Blower (Seedbureau Chicago Illinois) was used to remove
remaining chaff and light seeds by air density separation Seeds were placed in dry
storage in sealed quart-size zip-lock bags within a sealed plastic container Storage
temperatures ranged from 2-4oC Seeds remained in storage for approximately 7
months before the experiment
Experimental Design and Treatment Structure
The experiment compared the impact ofhot water bath and percussion
l11aLl11 on germination properties ofNew Mexico locust seed A single
16
-l
Figure 1 Seed source collection map All sources were collected within New Mexico USA separated by at least 4S lan
IIIRZltItW lIJPIirmiddot rllll1lbullbull_~~~_II
Table 1 Latitude longitude location elevation and collection date ofNew Mexico locust seed sources
Lot title Latitude (N) Longitude (W) Location Elevation (m) Collection Date
Raton 36deg5846 104deg2872 Raton Pass NM 2295 82500
Holman 36deg0306 105deg2429 HolmanNM 2476 911500
Jemez 35deg4500 106deg4100 Jemez Springs NM 1976 9900 00
Sandia 35deg1083 106deg2341 Cedar Crest NM 2432 82600
Manzano 34deg3689 106deg2494 ManzanoNM 2465 9200
Magdalena 33deg5953 107deg0903 Magdalena NM 2544 82900
Capitan 33deg2728 105deg4325 Alto NM 2189 911200
Sacramento 32deg5798 105deg4496 Cloudcroft NM 2630 91400 Gila 32deg5400 107deg4600 Kingston NM 2432 9700
f
Table 2 Mean seed weight and standard error by source
a
Source Mean seed weight (g) for Standard error 100 seeds n = 5
Raton Holman Jemez Sandia Manzano Magdalena Capitan Sacramento Gila
2872 b c 2522 d 1826 e 2753 c 2413 d 2559 d 2443 d 3130 a 3029 ab
00103 00403 00276 00301 00370 00374 00458 00279 00348
a Mean seed weights followed by the same letter are not significantly
different
19
experiment was conducted utilizing a randomized complete block design blocked by
time with a factorial treatment structure Experimental factors were seed source and
scarification treatment Nine seed sources from throughout New Mexico were used
(Figure 1 Table 1) Scarification treatments included a control three levels ofhot
water and four levels of percussion Hot water treatments included immersion at
100degC and boiling for periods of 30 seconds and 60 seconds Percussion treatment
levels were 1 2 4 and 8 minutes One hundred seeds per block were used to test
each treatment combination Four blocks of seed were tested one at a time (all seed
sources included in each run) in close chronological sequence (within 6 weeks) A
total of four 100-seed samples were used to test each source by scarification treatment
combination
Treatments
Hot water treatment of seeds utilized 50 ml test tubes filled with 30 ml
distilled water Each test tube was used to treat one 100-seed sample at a time
resulting in a ratio of4 parts water to 1 part seed by volume Test tubes were placed
in a beaker with the beaker filled with tap water to the same level as the water within
test tubes Nine test tubes (one per seed source) were placed in the beaker
simultaneously so that one replication of each seed sourcescarification treatment
cotnblnatlon was conducted simultaneously Hot water treatments include immersion
boiling For immersion treatments seeds were added to test tubes with the water
(boiling) and the test tubes were immediately removed from the beaker to
For the boiling treatments test tubes remained in the beaker with water at
20
boiling temperature for a set duration after the addition of seeds Following all
treatments test tubes with seeds cooled 24 hours in a test tube rack
Percussion treatment utilized a pneumatic paint shaker (Central Pneumatic
model 00422 Camarillo CA) Compressed air was maintained at 530 kPa plusmn30 kPa
(80 psi plusmn 5 psi) resulting in approximately 350 oscillations per minute Seeds were
placed in 118 ml (4 oz) soil tin cans (US Can Lobard IL) and the lids were secured
with duct tape Each can was used to treat one 100-seed sample To take advantage
ofgreatest lateral movement of the paint shaker a spacer (paint can) was used to
position the soil tin at the end of the shaking arm This allowed shaking to occur at
the greatest distance from the pivot point (fulcrum) Tins were placed perpendicular
to the direction of shaking motion allowing seeds to impact at greatest force by
hitting flat surfaces of the soil tin
Germination Testing
Before gennination testing all seeds were soaked 24 hours in reverse
osmosis-treated water (this was included in the process ofhot water treatment
jU~SCIlOea above) Following soaking seeds were placed in 100 cm Petri dishes lined
90 cm Whatman qualitative 1 filter paper circles (MG Scientific Pleasant
WI) One Petri dish was used for each treatment by source combination
papers were initially moistened with 5 mL reverse osmosis-treated water then
~VU1CUlJl moistened as needed to ensure adequate conditions for gennination
were monitored daily (at 300 PM) for gennination for 14 days in accordance
~UIUlnJlVUa Seed Testing Association (ISTA) standards for Robinia species
21
(1STA 1999) Tests were carried out at room temperature (22degC plusmnOSoC)
Germination was classified as protrusion of the radicle from the embryo by at least
1mm as viewed with the naked eye
X-ray Radiography
On August 132001 three lOO-seed samples from the Sacramento seed source
were treated for 14 or 8-minute percussion durations The seeds were sent
immediately by overnight delivery to the Faxitron X-Ray Corporation in Wheeling
Illinois where Dr Bob Karrfalt ofthe USDA Forest Service oversaw their imaging
Radiographs were made at SX with a Faxitron Specimen Radiography System Model
MX-20
Scanning Electron Microscope Photography
On February 192002 a Hitachi S-3200N Variable Pressure Scanning
Electron Microscope (Mountain View CA) was used to examine strophiolar cells of
untreated seeds and seeds percussed for 4 minutes Seeds were cut longitudinally
igt their entire length using a razor blade splitting the strophiolar region roughly in
Cross sections of seed samples magnifying the strophiolar region were viewed
600X under the microscope
Analysis
Categorical Analysis of Variance was used to determine treatment source and
(Le time) main effects and interactions The response variable was total
~vlI This procedure is a Chi-square test of homogeneity using the natural log
of germinated to non-germinated seed for each treatment Maximumshy
22
likelihood analysis was used to calculate Chi-square test statistics with their observed
significance levels A single contrast was conducted to cempare treatment means
against the control Observed significance levels less than or equal to 005 were
considered significant Pairwise comparisons were conducted between non-control
treatment mean percents using a conservative alp~a value of 005 divided by the
number of comparisons (Bonferroni approach) and approximate pairwise z statistics
Categorical analyses were performed using Proc Catmod (SAS Institute 1989)
RESULTS
Scarification seed source block and all interactions between factors
influenced germination (Table 3) The main effects are reported in the presence of
these interactions because the overall robustness of treatments and seed sources are of
interest Background levels ofnaturally occurring variation in seed sources are
seen in control germination percentages in Figure 2 In particular time variability
is unlikely to be under the control of the producer Appendix B details experimental
data for source by treatment gennination percents for each of the four blocks
Averaged over all treatments germination by source ranged from 40 to
79 (Figure 2) Averaged over all sources (Figure 3) and for each source (Figure 4
5) percussion scarification durations of4 or 8 minutes yielded greatest germination
Averaged over all sources germination increased with increasing durations of
percussion then leveled off for the 4- and 8-minute treatments (Figure 3) Immersion
and 30-second boiling scarification maximized germination for the hot water
treatments with a decrease in germination when seeds were boiled for 60 seconds
For all but two sources there is a similar ipcreasing trend in germination from
1to 4 minutes ofpercussion (Figure 4) The Holman source increased in germination
1 to 2 minutes then leveled off at 4 minutes The Jemez source changed little
1 to 4 minutes of treatment As treatment duration increased from 4 to 8
mutes five sources decreased in germination (Figure 4a) whereas three sources
24
Table 3 Categorical Analysis of Variance table for germination response to seed treatment seed source block and the interaction of the factors
Source of DF Chi-Square Observed Variability Significance Level
Source 8 98370 lt00001 Trt 7 233876 lt00001
contrast 1 16976 lt00001 SourceTrt 56 97077 lt00001 Block 3 5538 lt00001
SourceBlock 24 10162 lt00001 TrtBlock 21 7564 lt00001
SourceTrtBlock 168 34283 lt00001
S 4)
8 ~
0
sectg ~ 40 ~
N 0 0
20
o f0ltgt ~ ~4 jybshy ((iltgto ~~ K1ltgt 6~b-~ ~~ 0b-~~ ~~ ~b-~ ~b-~b
80
60 -I-- - ~- middotmiddotmiddotmiddotmiddot1
r -] Northern NM Sources
Central NM Sources
Southern NM Sources
sect-o ~
~b-v(i
SOURCE
Figure 2 Effect ofNew Mexico locust seed source on germination for data averaged over all scarification treatments Control germination is shown by solid line within bar
80
~ cd ~ ltl) () 60 ~ ~
N
t 0shyrti tshySltl)
40
0
20
o lmmersion Boiling 30 sec Boiling 60 sec Control 1 min 2min 4 min 8 min
Hot Water Treatments Percussion Treatments
Figure 3 Effect of scarification on germination ofNew Mexico locust for data averaged over all seed sources Means labelled with the same letter are not significantly different
100~----------------------~--~----------------~
80
60
ltU
~
40 IJ
sectgsI
l100
80
60
40
a I middotmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot middotImiddotmiddotmiddotmiddotmiddotmiddot E
_~iii-bull _ _ bull f --~--
t-- -~ ---- -shy ---- shyI ~ ---- - shy
L
fiII
~
b
- shy ---r
- Holman
Sacramento Sandia Gila Jemez
c ~
ltgt ~
Tmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot-I
j~=~=-~~-1
I middotmiddot ~ bull r
t tr
Y -------M-a-gd-a-Ie-n-a--
1 _ _
fCapitan
---- Manzano ~y -_ Raton
2 4 8
Time interval ofpercussion scarification (min)
Figure 4 Effect of seed source by treatment interaction on New Mexico locust germination for percussion scarification pattern a and pattern b
28
tv 0
~ d
~ A s o
1 c3
80
60
40
20
o
TIT
t------=middotfmiddotmiddot-middotmiddot-middotmiddot-middotmiddot 1- --- - - --=- - -shy--cE= - _--shy --shy ----shy ~ ~ ---shy ~ -~- _ --shy-------r 1
I
Raton Holman
-_ Jemez
--- Sandia Manzano Magdalena Capitan Sacramento Gila
Immersion Boiling 30 sec Boiling 60 sec
Hot Water Scarification
Figure 5 Effect of seed source by treatment interaction on New Mexico locust germination for hot water scarification
continued to increase in germination (Figure 4b) There was no difference between
the two treatment levels for the Manzano source (Figure 4b)
The effect ofhot water scarification also varied among sources with few
overall differences between hot water treatments (Figure 5) However boiling for 60
seconds yielded highest germination for the Jemez source whereas increasing
durations ofboiling decreased germination for the Raton source
In addition to consistently high germination 4- and 8-minute percussionshy
treated seeds germinated as fast or faster than all other treatments (Figure 6 Table 4)
050 measured as the number of days needed to complete 50 ofeventual
germination averaged 2 days for both ofthese treatments
X-ray radiographs were taken of seeds collected from the Sacramento source
percussed for 1 4 or 8 minutes (Figure 7) Seeds show signs of damage at 8 minutes
ofpercussion (Figure 8) Compared to an undamaged seed percussed for only 4
minutes (Figure 8a) damage at the 8-minute level includes fractured radicles pitting
of the seed coat and chips in seed coat structure (Figure 8b-d)
Scanning electron microscope (SEM) images at 600X reveal the direct effect
of 4 minutes of percussion on cells of the strophiolar region (Figure 9) Whereas
untreated seeds retain cellular organization in the strophiolar region strophiolar cells
seeds treated for 4 minutes ofpercussion separate and raise resulting in a
~-~e~U appearance
30
Umiddot-~middot--l-~---middotymiddotmiddotmiddot-----middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot--middot--middotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middotmiddotmiddotmiddotmiddotmiddotmiddot- -
050= gt1
-e-- Percussion 8 min -0- Percussion 4 min ---T- Percussion 2 min -----ltil- Percussion 1 min -- Immersion --0- Boiling 30 sec -+- Boiling 60 sec -ltgt- Control
2 4 6 8 10 12
Day Figure 6 Timecourse ofNew Mexico locust gennination following scarification
treatments G50 is the number of days to 50 of eventual germination
31
14
Table 4 Effect of seed source and treatment on G50 (days to 50 germination)
Seed Source Control Immersion B30sec B60sec Plmin P2min P4min P8min mean Raton 3 3 5 7 4 3 2 2 4 Holman 2 2 2 2 2 2 2 2 2
Jemez 4 6 6 5 3 2 2 2 4 Sandia 2 2 2 2 2 2 2 2 2
w tv Manzano 5 2 2 3 3 2 2 2 3
Magdalena 4 2 2 3 4 3 2 1 3
Capitan 5 4 2 2 3 2 2 2 3
Sacramento 3 2 2 2 4 3 2 2 3 Gila 3 2 2 2 2 2 2 2 2 mean 3 3 3 3 3 2 2 2 3 standard 11 14 16 18 09 05 0 03 08 deviation
Note Columns and rows may not agree due to rounding
Figure 7 X-ray radiographs ofSacramento seed source percussed for 1 4 or 8 minutes
33
Figure 8a-d Seed damage close-ups Figure a shows undamaged seed percussed for 4 minutes Figures b-d show seeds percussed for 8 minutes Damage includes fracturing ofradicle in b complete removal ofseed coat in c and pitting ofseed coat in d
34
Untreated Treated
Figure 9 SEM images ofuntreated vs 4-minute percussion-treated strophiolar cells ofNew Mexico locust seeds Treated cells are loosened separated and raised
DISCUSSION
Determining the Optimal Treatment
Averaging results of scarification treatmems across seed sources is a useful
tool to predict which treatment is most likely to be successful for an unknown seed
source Averaged over all sources four- and eight-minute percussion treatments
resulted in the highest germination 815 and 790 respectively and produced
germination that was as fast or faster than all other treatments Although there was no
difference between these two treatments on average an argument for 4 minutes of
percussion as a standard protocol for treating New Mexico locust seeds can be made
Descriptively 4 minutes of percussion improved germination over 8 minutes
ofpercussion for a majority of sources (five out of nine) 8 minutes of percussion
improved germination for three ofnine sources with no difference for a fmal source
For individual sources 4 minutes ofpercussion was always the best or secondmiddot best
treatment and was always equal to or better than all hot water treatments
Germination at 8 minutes ofpercussion was lower than all other percussion durations
for the Jemez and Gila sources and was lower than at least one hot water treatment for
these sources as well
The x-ray radiographs of the Sacramento seed source illustrate the excessive
internal and external seed damage that can occur with 8 minutes of percussion
germination counts 8-minute percussed seeds from Holman and Capitan
ClUll signs of damage that were visible to the naked eye (see below)
r~evertheless some of these seeds still fit the criteria for germination Neither x-ray
36
photography nor visible observations during seed counts revealed seed damage at the
4-minute percussion level In operational trials seeds percussed for 4 minutes
developed into healthy seedlings (Khadduri and Harrington unpublished data)
Strophiole and Seed Coat Behavior
Four minutes ofpercussion successfully breaks seed dormancy in a large
percentage ofNew Mexico locust seeds without excessively damaging the
surrounding seed coat or internal seed coat structures Most papilionoid legume
seeds including seeds ofNew Mexico locust have a specific region of the seed coat
known as the strophiole or lens (Hamly 1932) The strophiole is located on the
cotyledonary lobe of a locust seed (Figure 10 a-c) If seeds are not indiscriminately
damaged the strophiole is the first point of water entry to the embryo (Hopkinson
1997) When a papilionoid seed is percussed for the appropriate amount of time
repeated hits on the integrated seed coat loosen the constrained cells of the strophiolar
region without excessively damaging the rest of the seed coat (Ballard 1976) SEM
photos illuStrate that 4 minutes of percussion loosened and separated strophiolar cells
ofNew Mexico locust seeds when compared with constrained organized strophiolar
cells of untreated seeds (Figure 9) Research-has shown that water passes to the
embryo through the treated strophiole in a controlled manner and regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues
(Kelly and van Staden 1987)
37
10 Strophiole ofunimbibed New Mexico locust seed (a) and following uptake (bc)
38
In contrast hot-water treatments can excessively degrade the seed coat
(Rebman et al 1998 Marunda 1990) Random cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rebman et aI 1998 Marunda 1990) Along with a
proportionately large amount ofun-imbibed (thus under-treated) seeds (Figure l1a)
damage was observed in hot-water treated seeds from six sources during germination
counts Damage included seeds where the radicle failed to elongate (Figure 11 b)
seeds where the radicle elongated but was delayed in freeing itself from the seed coat
(Figure 11c) and seeds where the radicle completely broke off from the embryo
(Figure lId) In addition some seeds germinated in reverse (the cotyledon emerged
fIrst) If the radicle emerged completely from the seed coat they were counted as
germinated
While nearly all seeds treated for 8 minutes ofpercussion imbibed water
damage was evident in x-ray evaluations (Sacramento seed source) and in
germination counts (Holman and Capitan) at this treatment level Seed damage
during germination counts was similar to levels found in hot-water treated seeds
suggesting that over-treatment by both hot water and longer durations of percussion
may result in similar damage to seed structure
The Sacramento and Holman seed sources were two of the fIve sources that
opcrplltlpn in germination from the 4- to 8-minute durations of percussion
the benefIts ofloosening the strophiole as evidenced by imbibition of
all 8-minute treated seeds were outweighed by external and internal seed coat
39
a
b c d
11 Damage to New Mexico locust seeds observed during germination cotmts following hot water scarification 9a Hot water replicate including ~t seedlings and numerous unimbibed seeds 9b Close-up of seed where raallCle failed to elongate 9c Close-up ofseed where radicle elongated but was
-gtIn in freeing itself from the seed coat 9d Close-up ofseed where radicle broke off from embryo
40
damage (which also may be contributing to high imbibition) For the three sources
for which 8 minutes of percussion resulted in maximum germination extended
percussion of seeds necessary to loosen the strophiole may not have excessively
damaged the seed anatomy The Manzano source did not significantly change in
germination from 4 to 8 minutes of percussion suggesting that an increase in
permeable seeds at the 8-minute treatment duration was balanced by the number of
seeds that failed to germinate due to seed damage To summarize the 4-minute
percussion treatment improved germination over 8 minutes ofpercussion for a
majority of sources (five ofnine) the 8-minute percussion treatment improved
germination over 4-minutes ofpercussion for three sources and there was no
difference with the Manzano source Notably for the Raton source 8 minutes of
percussion increased germination by 235 percentage points over 4-minutes of
percussion (7875 vs 5525 respectively) the next best treatment Raton was the
only source to result in germination less than 75 for 4 minutes ofpercussion so a
grower may wish to apply longer percussion durations to lots that do not respond well
to this treatment level
Finally there was an interesting trend in the Jemez and Raton sources seed
that significantly contributed to the source by treatment interaction (Figure 12) A
~u test showed that the Jemez source had a significantly lighter seed weight than
other sources while the Raton source had an average seed weight For the Jemez
the longest duration of boiling increased germination Conversely only short
U4UVll) of percussion were needed to achieve high germination and the longest
41
80 _ Jemez a Raton
iD
~ C iD e iD
Q c 0 ~
c ~ iD 0
20
0------shy
Immersion Boiling 30 sec Boiling 60 sec
Hot Water Scarification
10degTr========~--------~------------------------------
80
60
40
20
_ Jemez Raton
0------shy
1 min 2 min 4 min 8 min
Percussion Scarification
Figure 12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources
42
b
--~----~~~~========~---- duration ofpercussion reduced germination For the Raton source longer durations
ofboiling consistently decreased germination whereas longer percussion treatments
consistently increased germination
A possible explanation is that a thick seed coat is associated with a loose
strophiole structure and that a thin seed coat is associated with a constrained
strophiole This contrast merits further study and may suggest an ecological
adaptation Further investigation ofthis phenomenon may contribute to successful
pre-screening ofunknown seed sources to determine optimal treatment leveL
43
CONCLUSION
Based on results obtained in pilot study (see Appendix) and thesis
experiments 4 minutes ofpercussion is recommended as a standard protocol for
treating an unknown source ofNew Mexico locust seed In addition to laboratory
results the 4-minute percussion treatment has resulted in germination greater than
80 and healthy seedling grow-outs for production purposes (unpublished data) In
laboratory experiments only one hundred seeds per 118 ml (40z) soil tin were
percussed at a time whereas for production we have treated up to five hundred seeds
per tin For large-scale production ofNew Mexico locust seedlings growers may
wish to experiment with larger quantities of seed and larger-size soil tins
Seed storage trials following percussion treatments were not conducted
However germination greater than 80 and healthy seedlings were obtained when
seeds were percussion-treated and subsequently stored at room-temperature (22degC)
for one week (unpublished data) Seed storage trials merit further study as the
standard hot water treatment leaves seeds in a softened condition that is not
conducive to storage
Hydroseeding is an effective method for direct seeding in reclamation
situations Percussion treatment may be conducive to hydro seeding as seeds remain
a dry relatively undamaged yet permeable condition following treatment Sulfuric
seeds also remain dry following treatment but extensive damage to seed coat
poor seedling vigor have been reported with this treatment (Kelly and van Staden
A grow-out study using a hydro seeder on a reclamation site testing percussion
44
against other treatments such as sulfuric acid and mechanical scarification also
merits further study
Papilionoids are the largest subfamily oflegumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust these legumes often play an integral role in the revegetation of
disturbed lands The pilot study (Appendix) indicates that percussion scarification
successfully treats the widely used black locust as well Developing superior
scarification methods to the standard hot water mechanical scarification and acid
treatments may facilitate the use ofpapilionoid legumes in reclamation projects
45
APPENDICES
APPENDIX A
PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR
NEW MEXICO LOCUST AND BLACK LOCUST SEEDS
Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4
Hot water and sulfuric acid soaks are traditional treatments for seeds of many
temperate woody legwnes including locusts However these scarification techniques
often produce inconsistent germination Percussion scarification where seeds are
repeatedly propelled against a hard surface was compared with hot water
scarification to evaluate treatment efficacy for New Mexico locust (Robinia
neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment
seeds were placed in a 98degC water bath which was immediately removed from the
heat source For percussion scarification seeds were placed in a soil sample tin and
agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the
control were followed by 24-hour water soaks Hot water baths resulted in 56 and
41 germination for New Mexico locust and black locust respectively For both
species nearly all durations ofpercussion increased germination over the hot water
treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3
4 and 5 minutes for black locust resulted in at least 90 germination Traditional
scarification treatments randomly degrade the entire seed coat which can lead to
tissue damage during water uptake Percussion scarification specifically weakens the
strophiole the natural source ofwater entry to the seed in papilionoid legwnes
INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom
2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031
48
Following percussion imbibition is controlled through the strophiole and underlying
tissue is protected
Keywords New Mexico locust black locust scarification percussion germination
Introduction
Locust trees (Robinia spp) are aggressive pioneer species that quickly
colonize disturbed land fix atmospheric nitrogen in the soil and stabilize slopes that
are prone to erosion (Klemmedson 1994 Ashby et al 1985) (Figure A1ab)
Recognition of these attributes has contributed to increased demand for locusts in
reclamation projects Locust seeds however exhibit physical dormancy where a
thick seed coat prevents the movement of water and gasses to the embryo (Leadem
1997)
Sulfuric acid or hot water soaks have traditionally been used to break seed
dormancy in macro-propagation of locusts Many growers are moving away from the
use of su1furic acid in treating locust seeds (Dreesen and Harrington 1997) In
addition to being dangerous acid soak durations must be specifically correlated to
seed lot (Olson 1974) Collections of native seed can vary tremendously in seed size
seed weight and hard seededness etc This variability limits the utility of sulfuric
acid soaks as even within a given lot some seeds may be damaged whereas others
are under-treated and fail to imbibe (take up water) A survey of Southwestern
container growers indicates that hot water baths are currently the most common
scarification method for locust seeds (Hine et al 1997) However hot water baths
also produce inconsistent germination (Lin et al 1996)
49
b
Figure AI New Mexico locust a primary invader following disturbance Ala Crown-sprouting at edge ofburn near Los Alamos NM--one year after Cerro Grande fire Alb Colonizing road cut Gila National Forest NM
50
Percussion scarification where seeds are repeatedly propelled against a hard
surface is an alternative dormancy-breaking method that has proven successful in
legumes related to locusts (Hamly 1932 Barton 1947) We compared this alternative
treatment to hot water scarification to evaluate treatment efficacy for New Mexico
locust and black locust seeds
Materials and Methods
Commercial New Mexico locust seeds (Western Native Seed Coaldale CO)
were collected fall 2000 in Huerfano County CO Black locust seeds were collected
September 2000 from Taos County NM by harvesting ripe pods from several trees at
a distance of up to 3 m from ground level Black locust seeds were allowed to air-dry
for 3 weeks and were threshed and separated from large chaffusing a greenhouse
fan A South Dakota Seed Blower (Chicago Illinltois) was used to remove fine chaff
from both seed lots Seeds were storedmiddotat 2-4C until the germination study was
conducted in March 2001
This study compared hot water scarification with percussion scarification
using treatment and species as experimental factors Along with a control
scarification treatments included immersion of seed in boiling water as well as
percussion durations of 1 2 3 45 or 10 minutes Four 100-seed samples were used
to test each species by scarification treatment combination
For the hot water treatment test tubes were filled with 30 mL water and
placed in a water-filled beaker (Figure A2a) The bath was raised to boiling (-98C at
51
b
c
Figure A2 Implementation ofhot water and percussion scarification A2a Hot water bath A2b Paint-shaker set-up A2c Placing soil tin containing seeds at end ofpaint shaker ann
52
Las Cruces NM elevation of 1300 meters) Each 100-seed replicate was placed in a
test tube and each test tube was immediately removed to cool at room temperature
Percussion scarification was implemented using a pneumatic paint shaker
(Central Pneumatic model 00422 Camarillo CA) (Figure A2b) This allowed
standardization of treatment intensity Compressed air pressure was maintained at
530kPa plusmn 30kPa (80psi plusmn 5psi) resulting in approximately 350 oscillations per
minute Each lOO-seed sample was placed in a 118 ml (4 oz) soil tin for shaking To
maximize lateral movement of paint shaker a paint can was used as a spacer to place
soil tins at the end of the shaking arm (Figure A2c) This allowed shaking to occur at
the greatest distance from the pivot point (fulcrum) Following all treatments
including the control seeds were soaked for twenty-four hours in distilled water prior
to plating out
Gennination was evaluated on lab benches at room temperature Each
replicate of seeds was placed in a 100 cm petri dish on moistened filter paper with
humidity maintained by enclosing the petri dishes in ziplock bags Gennination was
monitored daily for 14 days in accordance with International Seed Testing
Association standards for Robinia species (ISTA 1999) Gennination was defined as
protrusion of the radicle from the embryo by at least 1mm as viewed by the naked
eye
Categorical Analysis of Variance using Prpc Catmod (SAS Institute 1989)
was used to detennine treatment differences in germination percentages for each
source This procedure is an extension of a Chi-square test of homogeneity using the
53
natural log of the ratio of germinated to non-germinated seeds for each treatment Pshy
values less than 005 were considered significant A limited set of pairwiseshy
comparisons was conducted to compare treatment mean percents using a
conservative alpha value of 005 divided by the number of comparisons
Results
Percussion maximized germination for both New Mexico locust and black
locust There were three germination response levels for New Mexico locust (Figure
A3) First there was a low response in the control indicating a dormant seed lot
The hot water treatment and I-minute duration of percussion share a second
intermediate response Germination was highest at percussion levels from 2 to 10
minutes
A selected percussion treatment intermediate in time (5 minutes) produced the
steepest germination curve indicating the fastest germination rate (Figure A4) The
hot water treatment produced an intermediate curve or relatively slower germination
rate The control curve was very flat indicating the slowest germination rate for New
Mexico locust
Control seeds germinated poorly for black locust as well (Figure A5) Hot
water scarification improved germination over the control but all levels of percussion
improved germination over hot water The optimal durations of percussion treatment
were bracketed with a significant increase from the 1 to 2 minute percussion level a
high germination response from 2 to 5 minutes of percussion and finally a significant
54
~ d 25 CL)
p t1 0 ~ t1
g Vl ~ Vl
100~---------------------------------------------------------------
80
60
40
20
o Control Hot Water Percussion
I min Percussion
2 min Percussion
3 min
Scarification
Percussion 4 min
Figure A3 Effect of scarification on germination response for New Mexico locust
Percussion 5 min
=significant difference between adjacent treatment bars ns = no significant difference
Percussion 10 min
~ ~ () u agt
Il-t d o ~
~ ~ c3
100middotlr~~~------------------------------------e- Percussion 5min middot 0middot Hot Water -- Control
80-J=middot======
6b-middot-lmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middot--middotmiddot-middot~-middotmiddot -middotmiddot middot middotmiddot-middot 1
40
20
-----~-----------
0 0 0 middotmiddotmiddotmiddotmiddot0middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot0
0 --~-----y-----~------_---~--- T-----~
o shy -TDay 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A4 Effect of selected scarification treatments on germination speed for New Mexico locust First seven days of fourteen-day experiment are shown
Day 7
80
~ cd
5 60 ()
[) Cl d 0 1t1 d
40sect dUl
-J
20
o
ns
Scarification
Figure A5 Effect of scarification on germination response for black locust = significant difference between adjacent treatment bars ns = no significant difference
decrease at the 10-minute level This drop-off suggests that 10 minutes of percussion
over-treats seeds from this lot of-black locust
A selected percussion treatment intermediate in time (5 minutes) also had the
fastest germination rate for black locust (Figure A6)_ The hot water curve is
somewhat flatter indicating a slower germination rate The control curve is very flat
indicating a slow germination rate
A qualitative difference between hot water and percussion-treated seeds was
also observed Along with healthy germinants the hot water treatment produced
over-treated seeds (Figure A 7a) Examples of damaged germinants include seeds
where the radicle did not elongate (Figure A 7b) seeds where the radicle elongated
but was delayed in freeing itself from the seed coat (Figure A7c) and seeds where the
radicle broke off completely from the embryo (Figure A7d) In addition to these
examples of over-treatment there were numerous small dark seeds that failed to
imbibe water For percussion treatment nearly all seeds took up water (Figure A8a)
Germination was rapid and uniform with little damage evident (Figure A8b)
To summarize both New Mexico locust and black locust seed lots were
dormant The standard hot water scarification treatment improved germination but
only to 56 and 41 for New Mexico locust and black locust respectively Nearly
all percussion durations improved total germination as well as germination rate over
the hot water treatment Germination was greater than 90 for percussion durations
of 4 5 and 10 minutes for New Mexico locust and 3 4 and 5 minutes for black locust
58
CI)
sgt t CI) 0
igt 0
sect ttl t E
Vl 0
c3
100r==~==~~=r------------------------------------~ ___ Percussion 5 min
middotmiddotmiddot0middotmiddotmiddot Hot Water -lIf- Control
80r=========~--~~----------------------------~
60
40
20 -~-~---~---------- ---~------------I
omiddot
O
_~_----------lIf-----~bull -----_ shy---shy I
Day 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A6 Effect of selected scarification treatments on germination speed for black locust First seven days of fourteen-day experiment are shown
Day 7
a
b c d
Figure A7 Damage to New Mexico locust seeds observed during germination counts following hot water scarification A7a Hot water replicate including damaged seedlings and numerous unimbibed seeds A7b Close-up ofseed where radicle failed to elongate A7c Close-up ofseed where radicle elongated but was delayed in freeing itself from the seed coat A7d Close-up ofseed where radicle completely broke offfrom embryo
60
a
b
Figure A8 New Mexico locust seeds following percussion treatment at time of germination counts A8a Percussion replicate with uniformly healthy seedlings and near complete imbibition of seeds A8b Close-up ofpercussion-treated seedling
61
Discussion
Papilionoid seeds have a specific anatomical feature known as the strophiole
or lens-the natural site of water entry to the seed (Hamly 1932) The strophiole is
located on the cotyledonary lobe of a locust seed (Figure A9) When a papilionoid
legume seed is percussed for the appropriate amount of time repeated hits on the
integrated seed coat loosen the constrained cells of the strophiolar region without
excessively damaging the rest of the seed coat (Ballard 1976)
When a percussed seed is soaked water enters exclusively through the
strophiole in a controlled manner (Kelly and Staden 1987) This regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues This
contrasts with seeds that have been hot water or acid scarified treatments that can
randomly degrade the seed coat Unlocalized cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rehman et al 1998 Marunda 1990)
Papilionoids are the largest subfamily of legumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust and black locust these legumes often play an integral role in the
revegetation ofdisturbed lands Developing superior scarification methods to the
standard hot water and acid treatments should fadlitate the use ofthese legumes in
restoration projects
62
Figure A9 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (bc)
63
APPENDIXB
GERMINATION PERCENTS AND STANDARD ERRORS FOR SOURCE
BLOCK AND TREATMENT FOR NEW MEXICO LOCUST
Table B 1 Gennination percents and standard errors for source block and treatment for New Mexico locust
Source Block Treatment Germination Percentage Standard Error
Capitan 1 Immersion 74 437 Capitan 2 Immersion 69 462 Capitan 3 Immersion 71 454 Capitan 4 Immersion 67 470 Capitan Boiling 30 sec 79 407 Capitan 2 Boiling 30 sec 76 427 Capitan 3 Boiling 30 sec 72 449 Capitan 4 Boiling 30 sec 76 427 Capitan Boiling 60 sec 71 454 Capitan 2 Boiling 60 sec 54 498 Capitan 3 Boiling 60 sec 67 470 Capitan 4 Boiling 60 sec 76 427 Capitan I Control 5 218 Capitan 2 Control 7 255 Capitan 3 Control 4 196 Capitan 4 Control 5 218 Capitan 1 Percussion I min 47 499 Capitan 2 Percussion I min
470
Capitan Percussion I min 39 488
Capitan 4 Percussion I min 38 485 Capitan I Percussion 2 min 55 497 Capitan Capitan
2
Percussion 2 min Percussion 2 min
71 65
454 477
Capitan 4 Percussion 2 min 56 496 Capitan I Percussion 4 min 86 347 Capitan 2 Percussion 4 min 79 407 Capitan 3 Percussion 4 min 81 392 Capitan 4 Percussion 4 min 82 384 Capitan I Percussion 8 min 90 300 Capitan 2 Percussion 8 min 88 325 Capitan 3 Percussion 8 min 90 300 Capitan 4 Percussion 8 min 90 300 Gila I Immersion 71 454 Gila 2 Immersion 77 421 Gila
Immersion 75 433
Gila 4 Immersion 72 449
Gila I Boiling 30 sec 68 466
Gila 2 Boiling 30 sec 81 392
Gila 3 Boiling 30 sec 54 498
Gila 4 Boiling 30 sec 63 483
Gila I Boiling 60 sec 55 497
Gila 2 Boiling 60 sec 68 466
Gila 3 Boiling 60 sec 57 495
Gila 4 Boiling 60 sec 70 458
65
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Gila 2 Control 13 336 Gila 3 Control 13 336 Gila 4 Control 20 400 Gila 1 Percussion 1 min 66 474 Gila 2 Percussion 1 min 57 495 Gila 3 Percussion 1 min 73 444 Gila 4 Percussion 1 min 82 384 Gila 1 Percussion 2 min 81 392 Gila 2 Percussion 2 min 86 347 Gila 3 Percussion 2 min 85 357 Gila 4 Percussion 2 min 54 498 Gila 1 Percussion 4 min 79 407 Gila 2 Percussion 4 min 83 376 Gila 3 Percussion 4 min 84 367 Gila 4 Percussion 4 min 79 407 Gila 1 Percussion 8 min 51 500 Gila 2 Percussion 8 min 76 427 Gila 3 Percussion 8 min 52 500 Gila 4 Percussion 8 min 74 439 Holman 1 Immersion 83 376 Holman 2 Immersion 86 347 Holman 3 Immersion 81 392 Holman 4 Immersion 90 300 Holman 1 Boiling 30 sec 90 300 Holman 2 Boiling 30 sec 85 357 Holman 3 Boiling 30 sec 87 336 Holman 4 Boiling 30 sec 83 376 Holman 1 Boiling 60 sec 79 407 Holman 2 Boiling 60 sec 80 400 Holman 3 Boiling 60 sec 86 347 Holman 4 Boiling 60 sec 93 255 Holman Control 25 433 Holman 2 Control 19 392 Holman Control 21 407
Holman 4 Control 24 427
Holman Percussion 1 min 77 421
Holman 2 Percussion 1 min 85 357
Holman 3 Percussion 1 min 80 400
Holman 4 Percussion 1 min 89 313
Holman 1 Percussion 2 min 88 325
Holman 2 Percussion 2 min 96 196
Holman 3 Percussion 2 min 93 255
Holman 4 Percussion 2 min 96 196
Holman 1 Percussion 4 min 92 271
Holman 2 Percussion 4 min 97 171
66
I
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Holman 3 Percussion 4 min 91 286 Holman 4 Percussion 4 min 96 196 Holman 1 Percussion 8 min 88 325 Holman 2 Percussion 8 min 85 357 Holman 3 Percussion 8 min 83 376 Holman 4 Percussion 8 min 82 384 Jemez 1 Immersion 53 499 Jemez 2 Immersion 44 496 Jemez 3 Immersion 52 500 Jemez 4 Immersion 70 458 Jemez 1 Boiling 30 sec 58 494 Jemez 2 Boiling 30 sec 55 497 Jemez 3 Boiling 30 sec 48 500 Jemez 4 Boiling 30 sec 65 477 Jemez 1 Boiling 60 sec 68 466 Jemez 2 Boiling 60 sec 63 483 Jemez 3 Boiling 60 sec 73 444 Jemez 4 Boiling 60 sec 89 313 Jemez 1 Control 2 140 Jemez 2 Control 9 286 Jemez 3 Control 6 237 Jemez 4 Control 8 271 Jemez 1 Percussion 1 min 58 494 Jemez 2 Percussion 1 min 63 483 Jemez 3 Percussion 1 min 77 421 Jemez 4 Percussion 1 min 89 313 Jemez 1 Percussion 2 min 68 466 Jemez 2 Percussion 2 min 64 480 Jemez 3 Percussion 2 min 80 400 Jemez 4 Percussion 2 min 83 376 Jemez Percussion 4 min 68 466 Jemez 2 Percussion 4 min 79 407 Jemez 3 Percussion 4 min 70 458 Jemez 4 Percussion 4 min 83 376 Jemez 1 Percussion 8 min 52 500 Jemez 2 Percussion 8 min 59 492 Jemez 3 Percussion 8 min 61 488 Jemez 4 Percussion 8 min 70 458 Magdalena 1 Immersion 69 462 Magdalena 2 Immersion 69 462 Magdalena J Immersion 70 458 Magdalena 4 Immersion 85 357 Magdalena 1 Boiling 30 sec 65 477 Magdalena 2 Boiling 30 sec 71 454 Magdalena 3 Boiling 30 sec 68 466
67
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Magdalena 4 Boiling 30 sec 83 376 Magdalena 1 Boiling 60 sec 63 483 Magdalena 2 Boiling 60 sec 68 466 Magdalena 3 Boiling 60 sec 63 483 Magdalena 4 Boiling 60 sec 82 384 Magdalena 1 Control 11 313 Magdalena 2 Control 6 237 Magdalena 3 Control 5 218 Magdalena 4 Control 4 196 Magdalena Percussion 1 min 32 466 Magdalena 2 Percussion 1 min 45 497 Magdalena 3 Percussion 1 min 46 498 Magdalena 4 Percussion I min 36 480 Magdalena 1 Percussion 2 min 67 470 Magdalena 2 Percussion 2 min 68 466 Magdalena 3 Percussion 2 min 65 477 Magdalena 4 Percussion 2 min 81 392 Magdalena 1 Percussion 4 min 87 336 Magdalena 2 Percussion 4 min 97 171 Magdalena 3 Percussion 4 min 83 376 Magdalena 4 Percussion 4 min 97 171 Magdalena 1 Percussion 8 min 98 lAO Magdalena 2 Percussion 8 min 100 000 Magdalena 3 Percussion 8 min 96 196 Magdalena 4 Percussion 8 min 98 lAO Manzano 1 Immersion 67 470 Manzano 2 Immersion 57 495 Manzano 3 Immersion 72 4049 Manzano 4 Immersion 73 4044 Manzano 1 Boiling 30 sec 70 458 Manzano 2 Boiling 30 sec 64 480 Manzano 3 Boiling 30 sec 71 454 Manzano 4 Boiling 30 sec 80 400 Manzano I Boiling 60 sec 50 500 Manzano 2 Boiling 60 sec 53 499 Manzano 3 Boiling 60 sec 59 492 Manzano 4 Boiling 60 sec 74 439 Manzano I Control 4 196 Manzano 2 Control 8 271 Manzano 3 Control 1 099 Manzano 4 Control 6 237 Manzano 1 Percussion 1 min 52 500 Manzano 2 Percussion 1 min 39 488 Manzano 3 Percussion 1 min 36 480 Manzano 4 Percussion 1 min 51 500
68
Table B 1 Continued
Source Block Treatment Gerrni~ation Percentage Standard Error
Manzano Percussion 2 min 53 499 Manzano 2 Percussion 2 min 58 494 Manzano 3 Percussion 2 min 61 488 Manzano 4 Percussion 2 min 62 485 Manzano 1 Percussion 4 min 76 427 Manzano 2 Percussion 4 min 85 357 Manzano 3 Percussion 4 min 79 407 Manzano 4 Percussion 4 min 83 376 Manzano 1 Percussion 8 min 87 336 Manzano 2 Percussion 8 min 83 376 Manzano 3 Percussion 8 min 83 376 Manzano 4 Percussion 8 min 75 433 Raton 1 Immersion 54 498 Raton 2 Immersion 37 483 Raton 3 Immersion 39 488 Raton 4 Immersion 69 462 Raton 1 Boiling 30 sec 36 480 Raton 2 Boiling 30 sec 20 400 Raton 3 Boiling 30 sec 28 449 Raton 4 Boiling 30 sec 60 490 Raton 1 Boiling 60 sec 15 357 Raton 2 Boiling 60 sec 171 Raton 3 Boiling 60 sec 4 196 Raton 4 Boiling 60 sec 35 477 Raton 1 Control 4 196 Raton 2 Control 12 325 Raton 3 Control 2 140 Raton 4 Control 6 237 Raton 1 Percussion 1 min 52 500 Raton 2 Percussion I min 29 454 Raton 3 Percussion I min 35 477 Raton 4 Percussion 1 min 31 462 Raton 1 Percussion 2 min 55 497 Raton 2 Percussion 2 min 35 477 Raton 3 Percussion 2 min 33 470 Raton 4 Percussion 2 min 42 494 Raton 1 Percussion 4 min 65 477 Raton 2 Percussion 4 min 52 500 Raton Percussion 4 min 51 500 Raton 4 Percussion 4 min 53 499 Raton 1 Percussion 8 min 88 325 Raton 2 Percussion 8 min 80 400 Raton 3 Percussion 8 min 73 444 Raton 4 Percussion 8 min 74 439 Sacramento 1 Immersion 43 495
69
Table B 1 Continued
-Source Block Treatment Germination Percentage Standard Error
Sacramento 2 Immersion 30 458 Sacramento 3 Immersion 38 485 Sacramento 4 Immersion 56 496 Sacramento 1 Boiling 30 sec 47 499 Sacramento 2 Boiling 30 sec 48 500 Sacramento 3 Boiling 30 sec 48 500 Sacramento 4 Boiling 30 sec 56 496 Sacramento 1 Boiling 60 sec 52 500 Sacramento 2 Boiling 60 sec 67 470 Sacramento 3 Boiling 60 sec 43 495 Sacramento 4 Boiling 60 sec 48 500 Sacramento 1 Control 0 000 Sacramento 2 Control 3 171 Sacramento 3 Control 3 171 Sacramento 4 Control 099 Sacramento 1 Percussion 1 min 57 495 Sacramento 2 Percussion 1 min 61 488 Sacramento 3 Percussion 1 min 62 485 Sacramento 4 Percussion 1 min 55 497 Sacramento 1 Percussion 2 min 84 367 Sacramento 2 Percussion 2 min 70 458 Sacramento 3 Percussion 2 min 81 392 Sacramento 4 Percussion 2 min 69 462 Sacramento 1 Percussion 4 min 91 286 Sacramento 2 Percussion 4 min 84 367 Sacramento 3 Percussion 4 min 84 367 Sacramento 4 Percussion 4 min 94 237 Sacramento 1 Percussion 8 min 82 384 Sacramento 2 Percussion 8 min 81 392 Sacramento 3 Percussion 8 min 82 384 Sacramento 4 Percussion 8 min 81 392 Sandia 1 Immersion 76 427 Sandia 2 Immersion 70 458 Sandia 3 Immersion 70 458 Sandia 4 Immersion 81 392 Sandia 1 Boiling 30 sec 78 414 Sandia 2 Boiling 30 sec 71 454 Sandia 3 Boiling 30 sec 63 483 Sandia 4 Boiling 30 sec 78 414 Sandia 1 Boiling 60 sec 67 470 Sandia 2 Boiling 60 sec 65 477 Sandia 3 Boiling 60 sec 74 439 Sandia 4 Boiling 60 sec 66 474 Sandia 1 Control 16 367 Sandia 2 Control 7 255
70
Table B 1 Continued
Source Block Treatment Gennination Percentage Standard Error
Sandia 3 Control 8 271 Sandia 4 Control 16 367 Sandia 1 Percussion 1 min 67 470 Sandia 2 Percussion 1 min 61 488 Sandia 3 Percussion 1 min 66 474 Sandia 4 Percussion 1 min 59 492 Sandia I Percussion 2 min 82 384 Sandia 2 Percussion 2 min 83 376 Sandia 3 Percussion 2 min 81 392 Sandia 4 Percussion 2 min 84 367 Sandia 1 Percussion 4 min 89 313 Sandia 2 Percussion 4 min 86 347 Sandia 3 Percussion 4 min 89 313 Sandia 4 Percussion 4 min 85 357 Sandia 1 Percussion 8 min 80 400 Sandia 2 Percussion 8 min 77 421 Sandia 3 Percussion 8 min 69 462 Sandia 4 Percussion 8 min 66 474
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Meginnis HG 1937 Sulphuric acid treatment to increase germination of black locust seed USDA Circ 453 34 p
Miklas PN Towensend CE Ladd SL 1987 Seedcoat anatomy and the scarification ofcicer milkvetch seed Crop Sci 27 766-772
Monsen SB 1984 Use of shrubs on mine spoils In The Challenge ofProducing Native Plants for the Intermountain Area Proceedings Intermountain Nurserymans Association 1983 conference Las Vegas NV compo PM Murphy 26-31 Ogden UT USDA For Ser Gen Tech Report INT-l68
Olson DF 1974 Robinia L Locust In Seeds of Woody Plants in the US tech coord CS Schopmeyer 728-731 USDA For Ser Agr Handbk No450
Rehman S Loescher RNl Harris P1C 1998 Dormancy breaking and germination ofAcacia salicina Lindl seeds Seed Sci amp Techno127 553-557
Rolston MP 1978 Water impermeable seed dormancy Bot Rev 44 365-396
74
SAS Institute Inc 1989 SASSTAT Users Guide Version 6 4th Edition Volume 1 Cary NC SAS Institute Inc 943 p
SerratoValenti G Conara L Ferrando M Modenesi P 1993 Structural and histochemical features ofStylosanthes scabra (Leguminosae Papilionoidae) seed coat as related to water entry Can J Bot 71 834-840
Simpson BJ 1988 A field guide to Texas trees Austin TX Texas Monthly Press 372p
Tran VN Cavanagh AK 1984 Structural aspects ofdormancy In Seed Physiology Vol 2 Murray DR (ed) p1-44 Academic Press Australia
USDA Forest Service 1948 Woody-plant seed manual USDA Misc Pub1654 416p
Vines RA 1960 Trees Shrubs and Woody Vines of the Southwest Austin Univ ofTexas Press 1104 p
Wagner MR Larson RE Craig TP 1992 Ecology and silvics ofNew Mexico locust (Robinia neomexicana) with an emphasis on the arboreal form p53-57 In JW Hanover K Miller and S Plesko (eds) Proc Intemat Confon Black Locust Biology Culture and Utilization Mich State Univ USA
Wilson JK 1944 Immersing seeds of species of Robinia in boiling water hastens germination J For 42 453-454
Zimmerman RW Carpenter SB 1980 First-year coppice production from a 5shyyear old black locust stand on surface mine spoil p309-314 Centr Hardwd For ConfProc University ofMissouri Columbia
VITA
June 3 1972
June 1990
May 1994
July 1995
August 1994- March 1998
March 1998-August 2000
August 2000-May 2002
Born in Beirut Lebanon
Graduated from Georgetown Day High School Washington DC
Graduated from Pomona College Claremont CA with Bachelor ofArts Degree in Political Science
Graduated from University ofNew Mexico Albuquerque NM with Post-baccalaureate Teaching Degree
6th Grade TeacherBanta Fe Public Schools Santa Fe NM and Environmental Educator Open Space Division Albuquerque NM
Assistant Manager Santa Ana Native Plant Nursery Santa Ana Pueblo NM
Graduate Research Assistant at New Mexico State University
FIELD OF STUDY
Horticulture
iv
ABSTRACT
PERCUSSION AS AN ALTERNATIVE SEED TREATMENT FOR
ROBINIA NEOMEXICANA (NEW MEXICO LOCUST)
BY
NABIL Y KHADDURI
Master of Science in Horticulture
New Mexico State University 2002
Las Cruces New Mexico
Dr John T Harrington Chair
New Mexico locust (Robinia neomexicana A Gray) a small tree native to
montane regions of the southwestern United States fills a successional role in postshy
disturbance situations The ability to colonize rapidly and ameliorate harsh sites
contributes to the importance ofNew Mexico locust in reclamation efforts Seedling
production for outplanting on disturbed sites has been hampered by poor germination
This research sought to improve seed propagation techniques in New Mexico locust
Nine seed sources were collected across a range of latitudes within New
Mexico Seed treatments included a control three levels of hot water and four levels
of percussion scarification Hot water treatments included immersion at 100degC and
boiling for periods of 30 seconds and 60 seconds Percussion treatment levels were 1
2 4 and 8 minutes A total of four lOO-seed samples were used to test each source
by scarification treatment combination Categorical Analysis of Variance was used to
determine main effects and interactions Pairwise comparisons were conducted to
compare non-control treatment means using a conservative alpha value of 005
divided by the number of comparisons
Averaged over all sources and for each source percussion scarification
durations of4 or 8 minutes yielded greatest germination In addition to consistently
bigh germination 4- and 8-minute percussion-treated seeds germinated as fast or
faster than all other treatments Averaged over all sources germination increased
with increasing durations of percussion then leveled off for the 4- and 8-minute
treatments Immersion and 30-second boiling scarification maximized germination
for the hot water treatments with a decrease in germination when seeds were boiled
for 60 seconds
Percussion scarification specifically weakens the strophiole the natural
dormancy barrier in many papilionoid legumes without excessively damaging the
seed coat As with New Mexico locust papilionoid legumes often play an integral
role in the revegetation ofdisturbed lands The percussion scarification technique
developed in this research has the potential to facilitate the use of these valuable
species in reclamation projects
VI
CONTENTS
LIST OF TABLES ix
LIST OF FIGURES middot middotmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot x
INTRODUCTION 1
LITERATlTRE REVIEW 4
Seed Donnancy 4
Physical Donnancy 5
Evolutionary Role ofPhysical Donnancy 6
Methods for Breaking Donnancy 6
Acid Scarification 6
Hot Water Scarification 8
Mechanical Scarification 11
Alternative Method for Breaking Donnancy Percussion 12
Objectives 15
Hypothesis 15
MATERIALS AND METHODS 16
Seed Collection and Cleaining 16
Experimental Design and Treatment Structure 16
Treatments ~ bullbull bullbullbullbull - bullbull ~ ~ 20
G T ennlnatlon estmg 21
X-ray Radiography 22
Scanning Electron Microscope Photography 22
Analysis 22
RESULTS 24
DISCUSSION 36
Detennining the Optimal Treatment 36
Strophiole and Seed Coat Behavior 37
CONCLUSION 44
Appendices
A PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR NEW MEXICO LOCUST AND BLACK LOCUST SEEDS 47
B GERMINATION PERCENTS AND STANDARD ERRORS FOR SOURCE BLOCK AND TREATMENT FOR NEW MEXICO LOCUST 64
LITERATURE CITED 72
Vlll
LIST OF TABLES
Table Page
1 Latitude longitude location elevation and collection date ofNew Mexico locust seed sources 18
2 Mean seed weight and standard error by source 19
3 Categorical Analysis ofVariance table for germination response to seed treatment seed source block and the interaction of the factors 25
4 Effect of seed source and treatment on G50 (days to 50 germination) 32
B1 Germination percents and standard errors for source block and treatment for New Mexico locust 65
LIST OF FIGURES
Figures Page
1 Seedsourcecollectionmap 17
2 Effect ofNew Mexico locust seed source on germination for data averaged over scarification treatments 26
3 Effect of scarification on germination for New Mexico locust for data averaged over all seed sources 27
4 Effect of seed source by treatment interaction on New Mexico locust germination for percussion scarification pattern a and pattern b 28
5 Effect of source by treatment interaction on New Mexico locust germination for hot water scarification 29
6 Timecourse of New Mexico locust germination following scarification treatments 31
7 X-ray radiographs of Sacramento seed source perc ussed for 1 4 or 8 minutes 33
8 Seed damage close-ups 34
9 SEM images of untreated vs 4-minute percussion-treated strophiolar cells ofNew Mexico locust seeds 35
10 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (b c) 38
11 Damage to New Mexico locust seeds observed during germination counts following hot water scarification 40
12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources 42
New Mexico locust a primary invader following disturbance 50
Implementation of hot water and percussion scarification 52
Effect of scarification on germination response for New Mexico locust 55
x
A4 Effect of selected scarification treatments on germination speed for New Mexico locust 56
AS Effect of scarification on germination response for black locust 57
A6 Effect of selected scarification treatments on germination speed for black locust 59
A7 Damage to New Mexico locust seeds observed during germination counts following hot water scarification 60
AS New Mexico locust seeds following percussion treatment at time of germination counts 61
A9 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (bc) 63
INTRODUCTION
New Mexico locust (Robinia neomexicana A Gray) a small tree native to the
southwestern United States occurs at elevations from 1200 to 2800 meters This
species fills a successional role in post-disturbance situations As a primary invader
New Mexico locust quickly establishes on burned areas arid flood banks as well as
road cuts (Wagner et aI 1992) Rapid growth crown sprouting and prolific root
suckering favor the successful establishment ofNew Mexico locust on disturbed sites
(Simpson 1988 Gottfried 1980) These attributes also may make New Mexico locust
well suited for steep-slope revegetation where erosion is a problem
New Mexico locust is a nitrogen-fixing legume that tolerates and improves
nutrient-poor soils Stands ofNew Mexico locust increase Nitrogen (N) and Calcium
(Ca) in the forest floor Levels of Carbon (C) Phosphorous (P) Sulfur (8) and
Potassium (K) aIso have been shown to increase in soil beneath New Mexico locust
(Klemmedson 1994)
The ability to colonize rapidly and ameliorate harsh sites contributes to New
Mexico locusts potential as a nurse plant A nurse plant colonizes an inhospitable
site and creates an environment suitable for successional plant establishment New
Mexico locust demonstrates this role in the ponderosa pine (Pinus ponderosa Douglas
ex Lawson amp C Lawson)Gambel oak (Quercus gambelii Nutt) community After a
disturbance such as wildfire New Mexico locust and Gambel oak colonize and
dominate the site until shaded out by ponderosa pine Once shaded these species
become understory shrubs a process taking an average of 15-20 years (Dick-Peddie
1993 Hanks and Dick-Peddie 1974) New Mexico locust gradually declines with
small dense patches averaging about 005 ha beneath pine until the next disturbance
once again offers it a competitive advantage (Klemmedson 1994)
Gottfried (1980) expecting ponderosa pine regeneration to be greater where
New Mexico locust had been eradicated found that survival of planted pine seedlings
was greater where locust had not been removed In a follow-up study Gottfried
(1980) noted that soil moisture in the top 57 cm was highest in 5-year old locust sites
as compared to grass or 20-year old locust sites He concluded that managing an
appropriate cover ofNew Mexico locust could help regenerate pine the later
successional species
The ability ofNew Mexico locust to improve harsh sites also makes it a
candidate for reclaiming disturbances associated with mining Black locust (Robinia
pseudoacacia 1) has been used for decades to reclaim mine spoils and other
disturbed sites in the eastern US and around the world (Keresztesi 1988 Zimmerman
and Carpenter 1980) Ashby et al (1985) described similar positive attributes of
black locust also mentioned previously for New Mexico locust quick cover for
stabilization supply ofN and nutrient-rich litter to the soil and site improvement for
establishment of later successional trees Referring specifically to mine reclamation
the authors note the ability of black locust to grow on a wide range ofmine soil
conditions including extremely acid soils Black locust also shows some tolerance to
soils compacted by grading and topsoiling practices (Ashby et aL 1985) New
2
Mexico locust holds promise as a native southwe~tem counterpart to black locust in
mine reclamation but it has been used infrequently to date
Natural invasion and succession occur slowly on most mine sites (Monsen
1984) While New Mexico locust often colonizes sites naturally there is no
assurance a seed will reach a particular site and establish in a reasonable time frame
The challenge is to introduce New Mexico locust as mother plants to facilitate
colonization Seedling production for outplanting on disturbed sites has been
hampered by poor germination (Lin et al 1996) The goal then is to develop
techniques to overcome poor germination in New Mexico locust thereby facilitating
macropropagation
LITERATURE REVIEW
Seed Dormancy
Low gennination in New Mexico locust is primarily the result of seed
donnancy the failure of an intact viable seed to genninate under favorable
conditions (Bewley 1997) Seed donnancy also can be defined as the inability of the
embryo to genninate because of some inherent constraint (Bewley and Black 1994)
The constraint may originate within the embryo referred to as endogenous or embryo
donnancy or from the tissues surrounding the embryo known as exogenous or coatshy
imposed donnancy (Baskin and Baskin 1998 Leadem 1997 Bewley and Black
1994)
New Mexico locust seed is donnant when the impenneable seed coat does not
allow movement ofwater to the embryo This specific fonn of coat-imposed
donnancy where the restraining tissue involved is an impenneable seed coat is
known as physical donnancy (Baskin and Baskin 1998 Bewley and Black 1994)
Physical donnancy is relatively uncommon in seeds less than 10 of all woody
species exhibit this condition (USDA Forest Service 1948) However it is
characteristic of many genera of the Fabaceae (bean) family For example all
successional trees in the genera Gleditsia and Robinia (of which New Mexico locust
is a member) produce seeds that exhibit physical donnancy (Baskin and Baskin
4
Physical Dormancy
Seeds that exhibit physical dormancy are said to be hard seeded referring to
the thick impermeable seed coat (Leadem 1997) The first line of protection of the
testa in leguminous seeds is the outermost waxy cuticle followed by a subcuticle
consisting ofsuberin Next is a palisade layer consisting of sclereid cells that have
thick lignified secondary walls The most common type of sclereid cell in palisade
layers is the macrosclereid The macrosclereids contain water-repellent substances
including cutin lignin suberin and wax (Rolston 1978) This palisade layer is the
primary barrier to water uptake (Serrato-Valenti et al 1993 Tran and Cavanagh
1984)
All seed coat openings must be blocked to effectively prevent water uptake
The natural opening in a seed where it was attached to the mother plant is the weak
link where physical dormancy is usually broken (Hopkinson 1993 Kuo and Tam
1988) Anatomical structures associated with the seed coat opening vary from
family to family In the Papilionoideae the subfamily of the Fabaceae family
containing New Mexico locust the site ofwater entry is the strophiole The
strophiole is a swelling on the seed coat close to the stalk scar (Hopkinson 1997)
Kelly and van Staden (1987) refer to the strophiole site as the lens where
macrosclereid cells must separate and raise before water can pass through Since
middot6germination ofdormant seeds of the Papilionoideae takes place when the strophiole
permeable (Hopkinson 1993 Kuo and Tam 1988) a logical approach to
5
genninating New Mexico locust artificially would be to loosen the strophiolar cells
and allow water to pass through to the embryo
Evolutionary Role of Physical Dormancy
Why is physical dormancy a mechanism that blocks germination
ecologically advantageous When a plant produc~s seeds with different degrees of
dormancy seeds that are dispersed at one time will germinate over a range of time
(Bewley and Black 1994) Through variability of dormancy within a seed crop seeds
are exposed to changing environmental conditions improving the overall chances of
survival of the seed line (Bewley and Black 1994) Varying expression of dormancy
is under genetic control which indicates that this may be an evolved survival strategy
(Kigel and Galili 1995) A range of dormancy levels may be a particular advantage in
the southwestern US where extreme environmental variations exist
Methods for Breaking Dormancy
There are three common approaches to breaking dormancy in New Mexico
locust seed acid scarification hot water soaks and mechanical treatments In each
case the objective of the treatment is to scarify or abrade the seed coat to make it
permeable to water
Acid Scarification
Acid scarification consists of soaking seeds in concentrated sulfuric acid
rinsing thoroughly with water upon removal followed by subsequent drying before
PlaJlltmg Olson (1974) reports that this scarification method produces the highest
middot~~jllalUll results for all Robinia species Most literature refers to a seminal 1937
6
study conducted by HG Meginnis This stud~ t~ough conducted on black locust
seed is regularly cited in the suggested use of acid to treat New Mexico locust seed
as well (Olson 1974 Vines 1960)
Thee published studies document the specific use of sulfuric acid to treat
New Mexico locust seed Cox and Klett (1984) report that soaking seeds in
concentrated sulfuric acid for thirty minutes improved germination to 34 over an
untreated control of4 Lin et al (1996) found that for two seed lots acid
scarification for twenty-five to eighty minutes produced germination of 80 A third
lot however resulted in less than 20 germination at all durations of 10 to 80
minutes Hine et al (1997) showed that acid treatment levels of 1 2 5 and 10
minutes resulted in less than 20 germination across five sources However there
was an increasing trend in germination with acid soak duration and acid treatments
were not imposed for as long a time as the previous two studies (30 to 80 minutes)
The mixed results obtained by these investigators were probably due to the
widely varying germination response of seed lots to acid treatment For this reason
Olson (1974) recommends correlating duration of acid treatment to each seed lot If
treatment duration is too long the embryo will be permanently damaged If soak
duration is too short the seed will remain impermeable to water and fail to germinate
Compounding the problem of treatment duration is the negative effect acid
have on seedling growth Any type of scarification can injure seeds to some
by disrupting cells essential for growth leading to fungal invasion and
middot-uuu~w injury (Copeland and MacDonald 1995) Some legume seeds subjected
7
to 60 minutes of sulfuric acid scarification (to achieve maximum gennination results)
showed more than 50 seedling mortality after gennination (Kelly and van Staden
1987) Bewley and Black (1994) noted that fungal growth on seeds is an indication
of leakage of sugars organic acids amino acids and proteins
Perhaps the greatest drawback to acid scarification is the dangerous nature of
the treatment itself Difficulties associated with storage and trained use of
concentrated acid may discourage many propagators from using this option (Dreesen
and Harrington 1997) Consequently hot water and to a lesser extent mechanical
scarification are more commonly used and practical treatments for legume seeds
Most southwestern container growers use either of these two treatments to scarify
New Mexico locust seed in particular (Hine et al 1997)
Hot Water Scarification
The widespread use ofhot water scarification to treat locust seed is probably
due to its ease of application and efficacy in comparison to other scarification
treatments Although this practice had been in use for some time Wilson (1944) was
one of the first to report on its systematic application for black locust The treatment
consists of placing seeds in boiling water (4 parts water tomiddot 1 part seed by volume)
removing the vessel from the heat source and allowing the water to cool and seeds to
soak for 24 hours
Hine et al (1997) Lin et al (1996) and Cox and Klett (1984) have examined
the specific application ofhot water scarification on New Mexico locust seed Each
these authors compared hot water to acid scarification Lin et aL (1996) reported
8
60 gennination for two hot water-treated seed lots while a third lot germinated
around 50 (as reported above acid scarification germination ranged from 20 to
80 for these lots) It should be noted that the authors only soaked the seeds in water
from 10 to 80 minutes for this experiment as opposed to the standard 24 hours Cox
and Klett (1984) stated that using the standard 24-hour hot water treatment improved
germination over an acid pre-treatment (which produced less than 20 germination)
but they did not give specific gennination numbers for the hot water treatment Hine
et al (1997) found that across five seed lots germination was 66 for the standard
hot water treatment (as compared to less than 20 germination for acid scarification)
Variations in treatment intensity and duration and their subsequent effects on seed lot
appear to contribute to conflicting conclusions as to whether acid scarification or hot
water treatment is a more successful treatment
Given mixed results ease ofapplication is an important argument for use of
hot water application over acid soaks Safety concerns and material costs for hot
water treatments are low (Dreesen and Harrington 1997) An extension of this is the
practicality ofhot water for treating large batches of seed where a relative increase in
water volume is all that is needed However increases in water and seed even when
inthe same 4 1 proportion can result in dramatically different rates of cooling than in
atest tube experiment Different cooling rates can lead to contrasting results for the
treatment temperature duration and seed lot (USDA Forest Service 1948)
As with acid scarification parameters ofhot water treatment application may
to be optimized to maximize germination Hine et al (1997) found that as
9
temperature was increased from 70degC to 100degC overall germination in New Mexico
locust seed increased The 100degC treatment maximized germination for each of five
sources tested with germination usually above 60
Prolonged exposure of seeds ofmany woody legumes to boiling temperatures
also may improve germination (Briscoe 1996) This approach has not been tested on
New Mexico locust seed Exposing seed to prolonged boiling temperatures may
result in fungal infection and subsequent seedling mortality Marunda (1990) noted
severe fungal infections on both acid and hot water treated Acacia holoserica Cunn
ex G Don seeds attributing the infections to seed damage resulting from the
treatments Identifying an optimal hot water treatment level that does not result in a
high rate of seedling mortality is a logical goal
With hot water treatments as with acid there is also a danger of overtreating
Seeds and reducing germination (Gosling et al 1995) The legume Leucaena
leucocephala (Lam) de Wit germinated equally well after 7 seconds of exposure to
water at 100degC 7 seconds at 90degC 30 seconds at 80degC and 4 minutes at 70degC In
addition acceptably high germination was obtained from treatment at 70degC from 3
minutes to 1 hour whereas no seeds germinated following treatment at 100degC for 2
minutes Perhaps along with a greater margin of error for treatment duration treating
~ew Mexico locust seeds at a lower temperature may result in a comparatively lower
of seedling mortality In practice however nursery growers tend to use the
and quickest treatment available The short treatment length at near-boiling
whether recommended or not may be what is used most frequently
10
Fine-tuning a treatment in this range of treatment temperature and duration may be
the most logical approach
Mechanical Scarification
This treatment involves scratching the seed coat with an abrasive material
(USDA Forest Service 1948) One system employs a rotating drum lined with
sandpaper A paddle spinning at a given number ofrevolutions per minute throws the
seed against the sandpaper (Hine et al 1997) A second method dry tumbling
tumbles seed in a mixer with pea gravel or sharp sand for 5 to 7 days (Dreesen 2001)
These approaches have the advantage that control of temperature is not required as in
water treatments nor is safety an issue as with add scarification
Hine et al (1997) mechanically treated New Mexico locust seed using 100shy
grit sandpaper in a Forsberg (rotating drum) scarifier Gennination was less than
10 They concluded that the seed coats were too thin for their apparatus and
proposed that a finer grit sandpaper would produce more even abrasion Although
germination varied by seed lot Dreesen (2001) reported that using a slow-speed dry
tumbling technique for 5 to 7 days produced gennination rates as high as 90
As with acid and hot water treatments treatment duration must be specifically
shy
correlated to seed lot Treated seeds are also susceptible to fungal infection
iHopkinson 1997) In addition equipment is specialized and may not always be
ltU~CUJl~ to a nursery (USDA Forest Service 1948) Several studies have confinned
degrading ofstrophiolar cells due to acid and hot water scarification (Miklas et al
11
1987 Dell 1980 Hanna 1984) but the effect ofmechanical scarification on the
strophiole has not been studied
Alternative Method for Breaking Dormancy Percussion
Strophiole degradation has been examined in depth using an impaction
treatment more commonly known as percussion Hamly (1932) first proposed that
shaking or impacting seeds could split the macrosc1ereid cells at the strophiole
Hamly successfully released tension ofbent strophiolar cells of impermeable
Melilotus alba Medik (White sweet clover) seeds by delivering physical blows
resulting in cells that had split apart and were only slightly bent Ninety-two percent
oftreated seeds became permeable as compared to 05 of control seeds Barton
(1947) showed that percussion significantly increases permeability in black locust
seed Ballard (1976) reasoned that the integrated nature of the strophiole within the
seed coat means a blow on any part of the seed weakens the strophiole There are no
published studies on the use of percussion on New Mexico locust
Percussion methods vary and often seem SUbjective or ill-defined Hamly
(1932) placed seeds in a glass bottle and shook them three times a second for 20
minutes resulting in 180 oscillations per minute Ballard (1973) describes shaking
seeds in a closed metal container with at least fifty seeds per container at 850-900
JS(~l11alIOIlS per minute for 1 minute Kelly and van Staden (1987) placed seeds into
sealed glass flasks Flasks were shaken by a wrist-shaker at a rate of 250
~UUVJ~ per minute Subsequent germination rates were 45 after 2 hours of
55 after 3 hours 60 after 6 hours and 100 after 12 hours
12
There are several potential advantages of percussion over acid hot water and
mechanical scarification Kelly and van Staden (1987) note that a percussion
treatment only alters macrosclereid cells of the strophiole by loosening separating
and raising them They emphasize that minimal damage is done to the overall
anatomical structure of the seed In acid scarification of the same seed lot radicle
and cotyledon damage occurred Based on photographic evidence the authors
believe that this morphological damage was due to uneven pressure during
imbibition with water entering haphazardly wherever the acid had sufficiently
dissolved the coat This ultimately led to uneven hydration of the embryo In
percussed seeds water only entered through the s~ophiole when the entire seed coat
had been saturated resulting in controlled and even embryo hydration (Kelly and van
Staden 1987)
Although all successful scarification treatments may focus at least in part on
the strophiole for papilionoid legumes (Baskin and Baskin 1998) percussion may be
less damaging to seed coat structures particularly when compared with acid Kelly
and van Staden did not compare hot water or mechanical scarification methods
against percussion However the fungal infection associated with hot water and
mechanical scarification as with acid scarificatio~ have been directly associated with
seed damage (Rehman et al 1998 USDA Forest Service 1948) Embryo damage in
has been observed in hot-water scarified legume seeds (Rehman et al 1998
13
By limiting embryo damage percussion scarification reduces seedling
mortality compared to other treatments Kelly and van Staden (1987) noted that
seedlings from the percussion treatment were morphologically sound and vigorous
when compared to acid treatment seedlings The authors attribute this improved
seedling vigor to the controlled water uptake that limited anatomical damage
Despite a 12-hour percussion treatment morphological damage was not substantially
greater than untreated seeds In addition seedling mass measured 10 days following
germination was not significantly different between percussed and untreated seeds
A final advantage ofpercussion may be the ability to adequately treat the most
dormant seeds while avoiding harm to seeds that have already been made permeable
by the treatment or that were permeable to begin with Instead ofcorroding scalding
or scratching the seed coat percussion appears to be just a loosening of the
strophiole spring (Ballard 1973) Kelly and van Staden (1987) were able to
increase germination with a lengthy treatment (12 hours) Figuring out whether a
substantial cushion oftreatment durations exist for percussion ofNew Mexico locust
seed would be a significant improvement to current scarification methods where seed
damage and seedling health is an issue
The greatest challenge for applying percussion to New Mexico locust seeds is
standardizing a treatment that is both effective and readily reproducible by nurseries
with mechanical scarification this may require the development of specific
~UllDment to properly quantify and execute the treatment
14
f
Objectives
The first objective of the study is to detennine which scarification treatment
results in optimal gennination speed and percentage ofNew Mexico locust The
second objective is to describe treatment interactions with nine seed sources collected
in New Mexico
Hypothesis
A percussion treatment acts exclusively on the main area ofwater uptake in
New Mexico locust seed loosening the strophiole so that imbibition can occur This
direct mechanism ofdonnancy release will result in improved gennination rate and
overall gennination percentage for New Mexico locust seed compared to standard
treatments Standard treatments including acid hot water and mechanical
scarification while eventually weakening the strophiolar cleft cause excessive
morphological damage through generalized seed coat degradation These treatments
are therefore prone to inconsistency due to over and under-treatment
15
MATERIALS AND METHODS
Seed Collection and Cleaning
Seeds were collected from August 25 through September 15 2000 from nine
locations along a latitudinal gradient in New Mexico Seed source locations and
descriptive information are given in Figure 1 and Tables I and 2 Stands were
separated by at least 45 km
Pods were hand picked as they began to ripen and dehisce (open) Collections
were made from ground level to approximately 3 m from several trees at each site
Pods were dried in open-ended brown paper bags for two to four weeks at room
temperature (21-230C) to facilitate cleaning Dried pods were placed in a closed cloth
sack which was pounded against the ground to separate seeds from pods For pods
still containing seeds hand maceration (crunching pods with hands) completed seed
separation Debris was removed by threshing with the aid of a greenhouse fan A
South Dakota Seed Blower (Seedbureau Chicago Illinois) was used to remove
remaining chaff and light seeds by air density separation Seeds were placed in dry
storage in sealed quart-size zip-lock bags within a sealed plastic container Storage
temperatures ranged from 2-4oC Seeds remained in storage for approximately 7
months before the experiment
Experimental Design and Treatment Structure
The experiment compared the impact ofhot water bath and percussion
l11aLl11 on germination properties ofNew Mexico locust seed A single
16
-l
Figure 1 Seed source collection map All sources were collected within New Mexico USA separated by at least 4S lan
IIIRZltItW lIJPIirmiddot rllll1lbullbull_~~~_II
Table 1 Latitude longitude location elevation and collection date ofNew Mexico locust seed sources
Lot title Latitude (N) Longitude (W) Location Elevation (m) Collection Date
Raton 36deg5846 104deg2872 Raton Pass NM 2295 82500
Holman 36deg0306 105deg2429 HolmanNM 2476 911500
Jemez 35deg4500 106deg4100 Jemez Springs NM 1976 9900 00
Sandia 35deg1083 106deg2341 Cedar Crest NM 2432 82600
Manzano 34deg3689 106deg2494 ManzanoNM 2465 9200
Magdalena 33deg5953 107deg0903 Magdalena NM 2544 82900
Capitan 33deg2728 105deg4325 Alto NM 2189 911200
Sacramento 32deg5798 105deg4496 Cloudcroft NM 2630 91400 Gila 32deg5400 107deg4600 Kingston NM 2432 9700
f
Table 2 Mean seed weight and standard error by source
a
Source Mean seed weight (g) for Standard error 100 seeds n = 5
Raton Holman Jemez Sandia Manzano Magdalena Capitan Sacramento Gila
2872 b c 2522 d 1826 e 2753 c 2413 d 2559 d 2443 d 3130 a 3029 ab
00103 00403 00276 00301 00370 00374 00458 00279 00348
a Mean seed weights followed by the same letter are not significantly
different
19
experiment was conducted utilizing a randomized complete block design blocked by
time with a factorial treatment structure Experimental factors were seed source and
scarification treatment Nine seed sources from throughout New Mexico were used
(Figure 1 Table 1) Scarification treatments included a control three levels ofhot
water and four levels of percussion Hot water treatments included immersion at
100degC and boiling for periods of 30 seconds and 60 seconds Percussion treatment
levels were 1 2 4 and 8 minutes One hundred seeds per block were used to test
each treatment combination Four blocks of seed were tested one at a time (all seed
sources included in each run) in close chronological sequence (within 6 weeks) A
total of four 100-seed samples were used to test each source by scarification treatment
combination
Treatments
Hot water treatment of seeds utilized 50 ml test tubes filled with 30 ml
distilled water Each test tube was used to treat one 100-seed sample at a time
resulting in a ratio of4 parts water to 1 part seed by volume Test tubes were placed
in a beaker with the beaker filled with tap water to the same level as the water within
test tubes Nine test tubes (one per seed source) were placed in the beaker
simultaneously so that one replication of each seed sourcescarification treatment
cotnblnatlon was conducted simultaneously Hot water treatments include immersion
boiling For immersion treatments seeds were added to test tubes with the water
(boiling) and the test tubes were immediately removed from the beaker to
For the boiling treatments test tubes remained in the beaker with water at
20
boiling temperature for a set duration after the addition of seeds Following all
treatments test tubes with seeds cooled 24 hours in a test tube rack
Percussion treatment utilized a pneumatic paint shaker (Central Pneumatic
model 00422 Camarillo CA) Compressed air was maintained at 530 kPa plusmn30 kPa
(80 psi plusmn 5 psi) resulting in approximately 350 oscillations per minute Seeds were
placed in 118 ml (4 oz) soil tin cans (US Can Lobard IL) and the lids were secured
with duct tape Each can was used to treat one 100-seed sample To take advantage
ofgreatest lateral movement of the paint shaker a spacer (paint can) was used to
position the soil tin at the end of the shaking arm This allowed shaking to occur at
the greatest distance from the pivot point (fulcrum) Tins were placed perpendicular
to the direction of shaking motion allowing seeds to impact at greatest force by
hitting flat surfaces of the soil tin
Germination Testing
Before gennination testing all seeds were soaked 24 hours in reverse
osmosis-treated water (this was included in the process ofhot water treatment
jU~SCIlOea above) Following soaking seeds were placed in 100 cm Petri dishes lined
90 cm Whatman qualitative 1 filter paper circles (MG Scientific Pleasant
WI) One Petri dish was used for each treatment by source combination
papers were initially moistened with 5 mL reverse osmosis-treated water then
~VU1CUlJl moistened as needed to ensure adequate conditions for gennination
were monitored daily (at 300 PM) for gennination for 14 days in accordance
~UIUlnJlVUa Seed Testing Association (ISTA) standards for Robinia species
21
(1STA 1999) Tests were carried out at room temperature (22degC plusmnOSoC)
Germination was classified as protrusion of the radicle from the embryo by at least
1mm as viewed with the naked eye
X-ray Radiography
On August 132001 three lOO-seed samples from the Sacramento seed source
were treated for 14 or 8-minute percussion durations The seeds were sent
immediately by overnight delivery to the Faxitron X-Ray Corporation in Wheeling
Illinois where Dr Bob Karrfalt ofthe USDA Forest Service oversaw their imaging
Radiographs were made at SX with a Faxitron Specimen Radiography System Model
MX-20
Scanning Electron Microscope Photography
On February 192002 a Hitachi S-3200N Variable Pressure Scanning
Electron Microscope (Mountain View CA) was used to examine strophiolar cells of
untreated seeds and seeds percussed for 4 minutes Seeds were cut longitudinally
igt their entire length using a razor blade splitting the strophiolar region roughly in
Cross sections of seed samples magnifying the strophiolar region were viewed
600X under the microscope
Analysis
Categorical Analysis of Variance was used to determine treatment source and
(Le time) main effects and interactions The response variable was total
~vlI This procedure is a Chi-square test of homogeneity using the natural log
of germinated to non-germinated seed for each treatment Maximumshy
22
likelihood analysis was used to calculate Chi-square test statistics with their observed
significance levels A single contrast was conducted to cempare treatment means
against the control Observed significance levels less than or equal to 005 were
considered significant Pairwise comparisons were conducted between non-control
treatment mean percents using a conservative alp~a value of 005 divided by the
number of comparisons (Bonferroni approach) and approximate pairwise z statistics
Categorical analyses were performed using Proc Catmod (SAS Institute 1989)
RESULTS
Scarification seed source block and all interactions between factors
influenced germination (Table 3) The main effects are reported in the presence of
these interactions because the overall robustness of treatments and seed sources are of
interest Background levels ofnaturally occurring variation in seed sources are
seen in control germination percentages in Figure 2 In particular time variability
is unlikely to be under the control of the producer Appendix B details experimental
data for source by treatment gennination percents for each of the four blocks
Averaged over all treatments germination by source ranged from 40 to
79 (Figure 2) Averaged over all sources (Figure 3) and for each source (Figure 4
5) percussion scarification durations of4 or 8 minutes yielded greatest germination
Averaged over all sources germination increased with increasing durations of
percussion then leveled off for the 4- and 8-minute treatments (Figure 3) Immersion
and 30-second boiling scarification maximized germination for the hot water
treatments with a decrease in germination when seeds were boiled for 60 seconds
For all but two sources there is a similar ipcreasing trend in germination from
1to 4 minutes ofpercussion (Figure 4) The Holman source increased in germination
1 to 2 minutes then leveled off at 4 minutes The Jemez source changed little
1 to 4 minutes of treatment As treatment duration increased from 4 to 8
mutes five sources decreased in germination (Figure 4a) whereas three sources
24
Table 3 Categorical Analysis of Variance table for germination response to seed treatment seed source block and the interaction of the factors
Source of DF Chi-Square Observed Variability Significance Level
Source 8 98370 lt00001 Trt 7 233876 lt00001
contrast 1 16976 lt00001 SourceTrt 56 97077 lt00001 Block 3 5538 lt00001
SourceBlock 24 10162 lt00001 TrtBlock 21 7564 lt00001
SourceTrtBlock 168 34283 lt00001
S 4)
8 ~
0
sectg ~ 40 ~
N 0 0
20
o f0ltgt ~ ~4 jybshy ((iltgto ~~ K1ltgt 6~b-~ ~~ 0b-~~ ~~ ~b-~ ~b-~b
80
60 -I-- - ~- middotmiddotmiddotmiddotmiddot1
r -] Northern NM Sources
Central NM Sources
Southern NM Sources
sect-o ~
~b-v(i
SOURCE
Figure 2 Effect ofNew Mexico locust seed source on germination for data averaged over all scarification treatments Control germination is shown by solid line within bar
80
~ cd ~ ltl) () 60 ~ ~
N
t 0shyrti tshySltl)
40
0
20
o lmmersion Boiling 30 sec Boiling 60 sec Control 1 min 2min 4 min 8 min
Hot Water Treatments Percussion Treatments
Figure 3 Effect of scarification on germination ofNew Mexico locust for data averaged over all seed sources Means labelled with the same letter are not significantly different
100~----------------------~--~----------------~
80
60
ltU
~
40 IJ
sectgsI
l100
80
60
40
a I middotmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot middotImiddotmiddotmiddotmiddotmiddotmiddot E
_~iii-bull _ _ bull f --~--
t-- -~ ---- -shy ---- shyI ~ ---- - shy
L
fiII
~
b
- shy ---r
- Holman
Sacramento Sandia Gila Jemez
c ~
ltgt ~
Tmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot-I
j~=~=-~~-1
I middotmiddot ~ bull r
t tr
Y -------M-a-gd-a-Ie-n-a--
1 _ _
fCapitan
---- Manzano ~y -_ Raton
2 4 8
Time interval ofpercussion scarification (min)
Figure 4 Effect of seed source by treatment interaction on New Mexico locust germination for percussion scarification pattern a and pattern b
28
tv 0
~ d
~ A s o
1 c3
80
60
40
20
o
TIT
t------=middotfmiddotmiddot-middotmiddot-middotmiddot-middotmiddot 1- --- - - --=- - -shy--cE= - _--shy --shy ----shy ~ ~ ---shy ~ -~- _ --shy-------r 1
I
Raton Holman
-_ Jemez
--- Sandia Manzano Magdalena Capitan Sacramento Gila
Immersion Boiling 30 sec Boiling 60 sec
Hot Water Scarification
Figure 5 Effect of seed source by treatment interaction on New Mexico locust germination for hot water scarification
continued to increase in germination (Figure 4b) There was no difference between
the two treatment levels for the Manzano source (Figure 4b)
The effect ofhot water scarification also varied among sources with few
overall differences between hot water treatments (Figure 5) However boiling for 60
seconds yielded highest germination for the Jemez source whereas increasing
durations ofboiling decreased germination for the Raton source
In addition to consistently high germination 4- and 8-minute percussionshy
treated seeds germinated as fast or faster than all other treatments (Figure 6 Table 4)
050 measured as the number of days needed to complete 50 ofeventual
germination averaged 2 days for both ofthese treatments
X-ray radiographs were taken of seeds collected from the Sacramento source
percussed for 1 4 or 8 minutes (Figure 7) Seeds show signs of damage at 8 minutes
ofpercussion (Figure 8) Compared to an undamaged seed percussed for only 4
minutes (Figure 8a) damage at the 8-minute level includes fractured radicles pitting
of the seed coat and chips in seed coat structure (Figure 8b-d)
Scanning electron microscope (SEM) images at 600X reveal the direct effect
of 4 minutes of percussion on cells of the strophiolar region (Figure 9) Whereas
untreated seeds retain cellular organization in the strophiolar region strophiolar cells
seeds treated for 4 minutes ofpercussion separate and raise resulting in a
~-~e~U appearance
30
Umiddot-~middot--l-~---middotymiddotmiddotmiddot-----middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot--middot--middotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middotmiddotmiddotmiddotmiddotmiddotmiddot- -
050= gt1
-e-- Percussion 8 min -0- Percussion 4 min ---T- Percussion 2 min -----ltil- Percussion 1 min -- Immersion --0- Boiling 30 sec -+- Boiling 60 sec -ltgt- Control
2 4 6 8 10 12
Day Figure 6 Timecourse ofNew Mexico locust gennination following scarification
treatments G50 is the number of days to 50 of eventual germination
31
14
Table 4 Effect of seed source and treatment on G50 (days to 50 germination)
Seed Source Control Immersion B30sec B60sec Plmin P2min P4min P8min mean Raton 3 3 5 7 4 3 2 2 4 Holman 2 2 2 2 2 2 2 2 2
Jemez 4 6 6 5 3 2 2 2 4 Sandia 2 2 2 2 2 2 2 2 2
w tv Manzano 5 2 2 3 3 2 2 2 3
Magdalena 4 2 2 3 4 3 2 1 3
Capitan 5 4 2 2 3 2 2 2 3
Sacramento 3 2 2 2 4 3 2 2 3 Gila 3 2 2 2 2 2 2 2 2 mean 3 3 3 3 3 2 2 2 3 standard 11 14 16 18 09 05 0 03 08 deviation
Note Columns and rows may not agree due to rounding
Figure 7 X-ray radiographs ofSacramento seed source percussed for 1 4 or 8 minutes
33
Figure 8a-d Seed damage close-ups Figure a shows undamaged seed percussed for 4 minutes Figures b-d show seeds percussed for 8 minutes Damage includes fracturing ofradicle in b complete removal ofseed coat in c and pitting ofseed coat in d
34
Untreated Treated
Figure 9 SEM images ofuntreated vs 4-minute percussion-treated strophiolar cells ofNew Mexico locust seeds Treated cells are loosened separated and raised
DISCUSSION
Determining the Optimal Treatment
Averaging results of scarification treatmems across seed sources is a useful
tool to predict which treatment is most likely to be successful for an unknown seed
source Averaged over all sources four- and eight-minute percussion treatments
resulted in the highest germination 815 and 790 respectively and produced
germination that was as fast or faster than all other treatments Although there was no
difference between these two treatments on average an argument for 4 minutes of
percussion as a standard protocol for treating New Mexico locust seeds can be made
Descriptively 4 minutes of percussion improved germination over 8 minutes
ofpercussion for a majority of sources (five out of nine) 8 minutes of percussion
improved germination for three ofnine sources with no difference for a fmal source
For individual sources 4 minutes ofpercussion was always the best or secondmiddot best
treatment and was always equal to or better than all hot water treatments
Germination at 8 minutes ofpercussion was lower than all other percussion durations
for the Jemez and Gila sources and was lower than at least one hot water treatment for
these sources as well
The x-ray radiographs of the Sacramento seed source illustrate the excessive
internal and external seed damage that can occur with 8 minutes of percussion
germination counts 8-minute percussed seeds from Holman and Capitan
ClUll signs of damage that were visible to the naked eye (see below)
r~evertheless some of these seeds still fit the criteria for germination Neither x-ray
36
photography nor visible observations during seed counts revealed seed damage at the
4-minute percussion level In operational trials seeds percussed for 4 minutes
developed into healthy seedlings (Khadduri and Harrington unpublished data)
Strophiole and Seed Coat Behavior
Four minutes ofpercussion successfully breaks seed dormancy in a large
percentage ofNew Mexico locust seeds without excessively damaging the
surrounding seed coat or internal seed coat structures Most papilionoid legume
seeds including seeds ofNew Mexico locust have a specific region of the seed coat
known as the strophiole or lens (Hamly 1932) The strophiole is located on the
cotyledonary lobe of a locust seed (Figure 10 a-c) If seeds are not indiscriminately
damaged the strophiole is the first point of water entry to the embryo (Hopkinson
1997) When a papilionoid seed is percussed for the appropriate amount of time
repeated hits on the integrated seed coat loosen the constrained cells of the strophiolar
region without excessively damaging the rest of the seed coat (Ballard 1976) SEM
photos illuStrate that 4 minutes of percussion loosened and separated strophiolar cells
ofNew Mexico locust seeds when compared with constrained organized strophiolar
cells of untreated seeds (Figure 9) Research-has shown that water passes to the
embryo through the treated strophiole in a controlled manner and regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues
(Kelly and van Staden 1987)
37
10 Strophiole ofunimbibed New Mexico locust seed (a) and following uptake (bc)
38
In contrast hot-water treatments can excessively degrade the seed coat
(Rebman et al 1998 Marunda 1990) Random cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rebman et aI 1998 Marunda 1990) Along with a
proportionately large amount ofun-imbibed (thus under-treated) seeds (Figure l1a)
damage was observed in hot-water treated seeds from six sources during germination
counts Damage included seeds where the radicle failed to elongate (Figure 11 b)
seeds where the radicle elongated but was delayed in freeing itself from the seed coat
(Figure 11c) and seeds where the radicle completely broke off from the embryo
(Figure lId) In addition some seeds germinated in reverse (the cotyledon emerged
fIrst) If the radicle emerged completely from the seed coat they were counted as
germinated
While nearly all seeds treated for 8 minutes ofpercussion imbibed water
damage was evident in x-ray evaluations (Sacramento seed source) and in
germination counts (Holman and Capitan) at this treatment level Seed damage
during germination counts was similar to levels found in hot-water treated seeds
suggesting that over-treatment by both hot water and longer durations of percussion
may result in similar damage to seed structure
The Sacramento and Holman seed sources were two of the fIve sources that
opcrplltlpn in germination from the 4- to 8-minute durations of percussion
the benefIts ofloosening the strophiole as evidenced by imbibition of
all 8-minute treated seeds were outweighed by external and internal seed coat
39
a
b c d
11 Damage to New Mexico locust seeds observed during germination cotmts following hot water scarification 9a Hot water replicate including ~t seedlings and numerous unimbibed seeds 9b Close-up of seed where raallCle failed to elongate 9c Close-up ofseed where radicle elongated but was
-gtIn in freeing itself from the seed coat 9d Close-up ofseed where radicle broke off from embryo
40
damage (which also may be contributing to high imbibition) For the three sources
for which 8 minutes of percussion resulted in maximum germination extended
percussion of seeds necessary to loosen the strophiole may not have excessively
damaged the seed anatomy The Manzano source did not significantly change in
germination from 4 to 8 minutes of percussion suggesting that an increase in
permeable seeds at the 8-minute treatment duration was balanced by the number of
seeds that failed to germinate due to seed damage To summarize the 4-minute
percussion treatment improved germination over 8 minutes ofpercussion for a
majority of sources (five ofnine) the 8-minute percussion treatment improved
germination over 4-minutes ofpercussion for three sources and there was no
difference with the Manzano source Notably for the Raton source 8 minutes of
percussion increased germination by 235 percentage points over 4-minutes of
percussion (7875 vs 5525 respectively) the next best treatment Raton was the
only source to result in germination less than 75 for 4 minutes ofpercussion so a
grower may wish to apply longer percussion durations to lots that do not respond well
to this treatment level
Finally there was an interesting trend in the Jemez and Raton sources seed
that significantly contributed to the source by treatment interaction (Figure 12) A
~u test showed that the Jemez source had a significantly lighter seed weight than
other sources while the Raton source had an average seed weight For the Jemez
the longest duration of boiling increased germination Conversely only short
U4UVll) of percussion were needed to achieve high germination and the longest
41
80 _ Jemez a Raton
iD
~ C iD e iD
Q c 0 ~
c ~ iD 0
20
0------shy
Immersion Boiling 30 sec Boiling 60 sec
Hot Water Scarification
10degTr========~--------~------------------------------
80
60
40
20
_ Jemez Raton
0------shy
1 min 2 min 4 min 8 min
Percussion Scarification
Figure 12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources
42
b
--~----~~~~========~---- duration ofpercussion reduced germination For the Raton source longer durations
ofboiling consistently decreased germination whereas longer percussion treatments
consistently increased germination
A possible explanation is that a thick seed coat is associated with a loose
strophiole structure and that a thin seed coat is associated with a constrained
strophiole This contrast merits further study and may suggest an ecological
adaptation Further investigation ofthis phenomenon may contribute to successful
pre-screening ofunknown seed sources to determine optimal treatment leveL
43
CONCLUSION
Based on results obtained in pilot study (see Appendix) and thesis
experiments 4 minutes ofpercussion is recommended as a standard protocol for
treating an unknown source ofNew Mexico locust seed In addition to laboratory
results the 4-minute percussion treatment has resulted in germination greater than
80 and healthy seedling grow-outs for production purposes (unpublished data) In
laboratory experiments only one hundred seeds per 118 ml (40z) soil tin were
percussed at a time whereas for production we have treated up to five hundred seeds
per tin For large-scale production ofNew Mexico locust seedlings growers may
wish to experiment with larger quantities of seed and larger-size soil tins
Seed storage trials following percussion treatments were not conducted
However germination greater than 80 and healthy seedlings were obtained when
seeds were percussion-treated and subsequently stored at room-temperature (22degC)
for one week (unpublished data) Seed storage trials merit further study as the
standard hot water treatment leaves seeds in a softened condition that is not
conducive to storage
Hydroseeding is an effective method for direct seeding in reclamation
situations Percussion treatment may be conducive to hydro seeding as seeds remain
a dry relatively undamaged yet permeable condition following treatment Sulfuric
seeds also remain dry following treatment but extensive damage to seed coat
poor seedling vigor have been reported with this treatment (Kelly and van Staden
A grow-out study using a hydro seeder on a reclamation site testing percussion
44
against other treatments such as sulfuric acid and mechanical scarification also
merits further study
Papilionoids are the largest subfamily oflegumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust these legumes often play an integral role in the revegetation of
disturbed lands The pilot study (Appendix) indicates that percussion scarification
successfully treats the widely used black locust as well Developing superior
scarification methods to the standard hot water mechanical scarification and acid
treatments may facilitate the use ofpapilionoid legumes in reclamation projects
45
APPENDICES
APPENDIX A
PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR
NEW MEXICO LOCUST AND BLACK LOCUST SEEDS
Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4
Hot water and sulfuric acid soaks are traditional treatments for seeds of many
temperate woody legwnes including locusts However these scarification techniques
often produce inconsistent germination Percussion scarification where seeds are
repeatedly propelled against a hard surface was compared with hot water
scarification to evaluate treatment efficacy for New Mexico locust (Robinia
neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment
seeds were placed in a 98degC water bath which was immediately removed from the
heat source For percussion scarification seeds were placed in a soil sample tin and
agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the
control were followed by 24-hour water soaks Hot water baths resulted in 56 and
41 germination for New Mexico locust and black locust respectively For both
species nearly all durations ofpercussion increased germination over the hot water
treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3
4 and 5 minutes for black locust resulted in at least 90 germination Traditional
scarification treatments randomly degrade the entire seed coat which can lead to
tissue damage during water uptake Percussion scarification specifically weakens the
strophiole the natural source ofwater entry to the seed in papilionoid legwnes
INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom
2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031
48
Following percussion imbibition is controlled through the strophiole and underlying
tissue is protected
Keywords New Mexico locust black locust scarification percussion germination
Introduction
Locust trees (Robinia spp) are aggressive pioneer species that quickly
colonize disturbed land fix atmospheric nitrogen in the soil and stabilize slopes that
are prone to erosion (Klemmedson 1994 Ashby et al 1985) (Figure A1ab)
Recognition of these attributes has contributed to increased demand for locusts in
reclamation projects Locust seeds however exhibit physical dormancy where a
thick seed coat prevents the movement of water and gasses to the embryo (Leadem
1997)
Sulfuric acid or hot water soaks have traditionally been used to break seed
dormancy in macro-propagation of locusts Many growers are moving away from the
use of su1furic acid in treating locust seeds (Dreesen and Harrington 1997) In
addition to being dangerous acid soak durations must be specifically correlated to
seed lot (Olson 1974) Collections of native seed can vary tremendously in seed size
seed weight and hard seededness etc This variability limits the utility of sulfuric
acid soaks as even within a given lot some seeds may be damaged whereas others
are under-treated and fail to imbibe (take up water) A survey of Southwestern
container growers indicates that hot water baths are currently the most common
scarification method for locust seeds (Hine et al 1997) However hot water baths
also produce inconsistent germination (Lin et al 1996)
49
b
Figure AI New Mexico locust a primary invader following disturbance Ala Crown-sprouting at edge ofburn near Los Alamos NM--one year after Cerro Grande fire Alb Colonizing road cut Gila National Forest NM
50
Percussion scarification where seeds are repeatedly propelled against a hard
surface is an alternative dormancy-breaking method that has proven successful in
legumes related to locusts (Hamly 1932 Barton 1947) We compared this alternative
treatment to hot water scarification to evaluate treatment efficacy for New Mexico
locust and black locust seeds
Materials and Methods
Commercial New Mexico locust seeds (Western Native Seed Coaldale CO)
were collected fall 2000 in Huerfano County CO Black locust seeds were collected
September 2000 from Taos County NM by harvesting ripe pods from several trees at
a distance of up to 3 m from ground level Black locust seeds were allowed to air-dry
for 3 weeks and were threshed and separated from large chaffusing a greenhouse
fan A South Dakota Seed Blower (Chicago Illinltois) was used to remove fine chaff
from both seed lots Seeds were storedmiddotat 2-4C until the germination study was
conducted in March 2001
This study compared hot water scarification with percussion scarification
using treatment and species as experimental factors Along with a control
scarification treatments included immersion of seed in boiling water as well as
percussion durations of 1 2 3 45 or 10 minutes Four 100-seed samples were used
to test each species by scarification treatment combination
For the hot water treatment test tubes were filled with 30 mL water and
placed in a water-filled beaker (Figure A2a) The bath was raised to boiling (-98C at
51
b
c
Figure A2 Implementation ofhot water and percussion scarification A2a Hot water bath A2b Paint-shaker set-up A2c Placing soil tin containing seeds at end ofpaint shaker ann
52
Las Cruces NM elevation of 1300 meters) Each 100-seed replicate was placed in a
test tube and each test tube was immediately removed to cool at room temperature
Percussion scarification was implemented using a pneumatic paint shaker
(Central Pneumatic model 00422 Camarillo CA) (Figure A2b) This allowed
standardization of treatment intensity Compressed air pressure was maintained at
530kPa plusmn 30kPa (80psi plusmn 5psi) resulting in approximately 350 oscillations per
minute Each lOO-seed sample was placed in a 118 ml (4 oz) soil tin for shaking To
maximize lateral movement of paint shaker a paint can was used as a spacer to place
soil tins at the end of the shaking arm (Figure A2c) This allowed shaking to occur at
the greatest distance from the pivot point (fulcrum) Following all treatments
including the control seeds were soaked for twenty-four hours in distilled water prior
to plating out
Gennination was evaluated on lab benches at room temperature Each
replicate of seeds was placed in a 100 cm petri dish on moistened filter paper with
humidity maintained by enclosing the petri dishes in ziplock bags Gennination was
monitored daily for 14 days in accordance with International Seed Testing
Association standards for Robinia species (ISTA 1999) Gennination was defined as
protrusion of the radicle from the embryo by at least 1mm as viewed by the naked
eye
Categorical Analysis of Variance using Prpc Catmod (SAS Institute 1989)
was used to detennine treatment differences in germination percentages for each
source This procedure is an extension of a Chi-square test of homogeneity using the
53
natural log of the ratio of germinated to non-germinated seeds for each treatment Pshy
values less than 005 were considered significant A limited set of pairwiseshy
comparisons was conducted to compare treatment mean percents using a
conservative alpha value of 005 divided by the number of comparisons
Results
Percussion maximized germination for both New Mexico locust and black
locust There were three germination response levels for New Mexico locust (Figure
A3) First there was a low response in the control indicating a dormant seed lot
The hot water treatment and I-minute duration of percussion share a second
intermediate response Germination was highest at percussion levels from 2 to 10
minutes
A selected percussion treatment intermediate in time (5 minutes) produced the
steepest germination curve indicating the fastest germination rate (Figure A4) The
hot water treatment produced an intermediate curve or relatively slower germination
rate The control curve was very flat indicating the slowest germination rate for New
Mexico locust
Control seeds germinated poorly for black locust as well (Figure A5) Hot
water scarification improved germination over the control but all levels of percussion
improved germination over hot water The optimal durations of percussion treatment
were bracketed with a significant increase from the 1 to 2 minute percussion level a
high germination response from 2 to 5 minutes of percussion and finally a significant
54
~ d 25 CL)
p t1 0 ~ t1
g Vl ~ Vl
100~---------------------------------------------------------------
80
60
40
20
o Control Hot Water Percussion
I min Percussion
2 min Percussion
3 min
Scarification
Percussion 4 min
Figure A3 Effect of scarification on germination response for New Mexico locust
Percussion 5 min
=significant difference between adjacent treatment bars ns = no significant difference
Percussion 10 min
~ ~ () u agt
Il-t d o ~
~ ~ c3
100middotlr~~~------------------------------------e- Percussion 5min middot 0middot Hot Water -- Control
80-J=middot======
6b-middot-lmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middot--middotmiddot-middot~-middotmiddot -middotmiddot middot middotmiddot-middot 1
40
20
-----~-----------
0 0 0 middotmiddotmiddotmiddotmiddot0middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot0
0 --~-----y-----~------_---~--- T-----~
o shy -TDay 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A4 Effect of selected scarification treatments on germination speed for New Mexico locust First seven days of fourteen-day experiment are shown
Day 7
80
~ cd
5 60 ()
[) Cl d 0 1t1 d
40sect dUl
-J
20
o
ns
Scarification
Figure A5 Effect of scarification on germination response for black locust = significant difference between adjacent treatment bars ns = no significant difference
decrease at the 10-minute level This drop-off suggests that 10 minutes of percussion
over-treats seeds from this lot of-black locust
A selected percussion treatment intermediate in time (5 minutes) also had the
fastest germination rate for black locust (Figure A6)_ The hot water curve is
somewhat flatter indicating a slower germination rate The control curve is very flat
indicating a slow germination rate
A qualitative difference between hot water and percussion-treated seeds was
also observed Along with healthy germinants the hot water treatment produced
over-treated seeds (Figure A 7a) Examples of damaged germinants include seeds
where the radicle did not elongate (Figure A 7b) seeds where the radicle elongated
but was delayed in freeing itself from the seed coat (Figure A7c) and seeds where the
radicle broke off completely from the embryo (Figure A7d) In addition to these
examples of over-treatment there were numerous small dark seeds that failed to
imbibe water For percussion treatment nearly all seeds took up water (Figure A8a)
Germination was rapid and uniform with little damage evident (Figure A8b)
To summarize both New Mexico locust and black locust seed lots were
dormant The standard hot water scarification treatment improved germination but
only to 56 and 41 for New Mexico locust and black locust respectively Nearly
all percussion durations improved total germination as well as germination rate over
the hot water treatment Germination was greater than 90 for percussion durations
of 4 5 and 10 minutes for New Mexico locust and 3 4 and 5 minutes for black locust
58
CI)
sgt t CI) 0
igt 0
sect ttl t E
Vl 0
c3
100r==~==~~=r------------------------------------~ ___ Percussion 5 min
middotmiddotmiddot0middotmiddotmiddot Hot Water -lIf- Control
80r=========~--~~----------------------------~
60
40
20 -~-~---~---------- ---~------------I
omiddot
O
_~_----------lIf-----~bull -----_ shy---shy I
Day 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A6 Effect of selected scarification treatments on germination speed for black locust First seven days of fourteen-day experiment are shown
Day 7
a
b c d
Figure A7 Damage to New Mexico locust seeds observed during germination counts following hot water scarification A7a Hot water replicate including damaged seedlings and numerous unimbibed seeds A7b Close-up ofseed where radicle failed to elongate A7c Close-up ofseed where radicle elongated but was delayed in freeing itself from the seed coat A7d Close-up ofseed where radicle completely broke offfrom embryo
60
a
b
Figure A8 New Mexico locust seeds following percussion treatment at time of germination counts A8a Percussion replicate with uniformly healthy seedlings and near complete imbibition of seeds A8b Close-up ofpercussion-treated seedling
61
Discussion
Papilionoid seeds have a specific anatomical feature known as the strophiole
or lens-the natural site of water entry to the seed (Hamly 1932) The strophiole is
located on the cotyledonary lobe of a locust seed (Figure A9) When a papilionoid
legume seed is percussed for the appropriate amount of time repeated hits on the
integrated seed coat loosen the constrained cells of the strophiolar region without
excessively damaging the rest of the seed coat (Ballard 1976)
When a percussed seed is soaked water enters exclusively through the
strophiole in a controlled manner (Kelly and Staden 1987) This regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues This
contrasts with seeds that have been hot water or acid scarified treatments that can
randomly degrade the seed coat Unlocalized cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rehman et al 1998 Marunda 1990)
Papilionoids are the largest subfamily of legumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust and black locust these legumes often play an integral role in the
revegetation ofdisturbed lands Developing superior scarification methods to the
standard hot water and acid treatments should fadlitate the use ofthese legumes in
restoration projects
62
Figure A9 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (bc)
63
APPENDIXB
GERMINATION PERCENTS AND STANDARD ERRORS FOR SOURCE
BLOCK AND TREATMENT FOR NEW MEXICO LOCUST
Table B 1 Gennination percents and standard errors for source block and treatment for New Mexico locust
Source Block Treatment Germination Percentage Standard Error
Capitan 1 Immersion 74 437 Capitan 2 Immersion 69 462 Capitan 3 Immersion 71 454 Capitan 4 Immersion 67 470 Capitan Boiling 30 sec 79 407 Capitan 2 Boiling 30 sec 76 427 Capitan 3 Boiling 30 sec 72 449 Capitan 4 Boiling 30 sec 76 427 Capitan Boiling 60 sec 71 454 Capitan 2 Boiling 60 sec 54 498 Capitan 3 Boiling 60 sec 67 470 Capitan 4 Boiling 60 sec 76 427 Capitan I Control 5 218 Capitan 2 Control 7 255 Capitan 3 Control 4 196 Capitan 4 Control 5 218 Capitan 1 Percussion I min 47 499 Capitan 2 Percussion I min
470
Capitan Percussion I min 39 488
Capitan 4 Percussion I min 38 485 Capitan I Percussion 2 min 55 497 Capitan Capitan
2
Percussion 2 min Percussion 2 min
71 65
454 477
Capitan 4 Percussion 2 min 56 496 Capitan I Percussion 4 min 86 347 Capitan 2 Percussion 4 min 79 407 Capitan 3 Percussion 4 min 81 392 Capitan 4 Percussion 4 min 82 384 Capitan I Percussion 8 min 90 300 Capitan 2 Percussion 8 min 88 325 Capitan 3 Percussion 8 min 90 300 Capitan 4 Percussion 8 min 90 300 Gila I Immersion 71 454 Gila 2 Immersion 77 421 Gila
Immersion 75 433
Gila 4 Immersion 72 449
Gila I Boiling 30 sec 68 466
Gila 2 Boiling 30 sec 81 392
Gila 3 Boiling 30 sec 54 498
Gila 4 Boiling 30 sec 63 483
Gila I Boiling 60 sec 55 497
Gila 2 Boiling 60 sec 68 466
Gila 3 Boiling 60 sec 57 495
Gila 4 Boiling 60 sec 70 458
65
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Gila 2 Control 13 336 Gila 3 Control 13 336 Gila 4 Control 20 400 Gila 1 Percussion 1 min 66 474 Gila 2 Percussion 1 min 57 495 Gila 3 Percussion 1 min 73 444 Gila 4 Percussion 1 min 82 384 Gila 1 Percussion 2 min 81 392 Gila 2 Percussion 2 min 86 347 Gila 3 Percussion 2 min 85 357 Gila 4 Percussion 2 min 54 498 Gila 1 Percussion 4 min 79 407 Gila 2 Percussion 4 min 83 376 Gila 3 Percussion 4 min 84 367 Gila 4 Percussion 4 min 79 407 Gila 1 Percussion 8 min 51 500 Gila 2 Percussion 8 min 76 427 Gila 3 Percussion 8 min 52 500 Gila 4 Percussion 8 min 74 439 Holman 1 Immersion 83 376 Holman 2 Immersion 86 347 Holman 3 Immersion 81 392 Holman 4 Immersion 90 300 Holman 1 Boiling 30 sec 90 300 Holman 2 Boiling 30 sec 85 357 Holman 3 Boiling 30 sec 87 336 Holman 4 Boiling 30 sec 83 376 Holman 1 Boiling 60 sec 79 407 Holman 2 Boiling 60 sec 80 400 Holman 3 Boiling 60 sec 86 347 Holman 4 Boiling 60 sec 93 255 Holman Control 25 433 Holman 2 Control 19 392 Holman Control 21 407
Holman 4 Control 24 427
Holman Percussion 1 min 77 421
Holman 2 Percussion 1 min 85 357
Holman 3 Percussion 1 min 80 400
Holman 4 Percussion 1 min 89 313
Holman 1 Percussion 2 min 88 325
Holman 2 Percussion 2 min 96 196
Holman 3 Percussion 2 min 93 255
Holman 4 Percussion 2 min 96 196
Holman 1 Percussion 4 min 92 271
Holman 2 Percussion 4 min 97 171
66
I
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Holman 3 Percussion 4 min 91 286 Holman 4 Percussion 4 min 96 196 Holman 1 Percussion 8 min 88 325 Holman 2 Percussion 8 min 85 357 Holman 3 Percussion 8 min 83 376 Holman 4 Percussion 8 min 82 384 Jemez 1 Immersion 53 499 Jemez 2 Immersion 44 496 Jemez 3 Immersion 52 500 Jemez 4 Immersion 70 458 Jemez 1 Boiling 30 sec 58 494 Jemez 2 Boiling 30 sec 55 497 Jemez 3 Boiling 30 sec 48 500 Jemez 4 Boiling 30 sec 65 477 Jemez 1 Boiling 60 sec 68 466 Jemez 2 Boiling 60 sec 63 483 Jemez 3 Boiling 60 sec 73 444 Jemez 4 Boiling 60 sec 89 313 Jemez 1 Control 2 140 Jemez 2 Control 9 286 Jemez 3 Control 6 237 Jemez 4 Control 8 271 Jemez 1 Percussion 1 min 58 494 Jemez 2 Percussion 1 min 63 483 Jemez 3 Percussion 1 min 77 421 Jemez 4 Percussion 1 min 89 313 Jemez 1 Percussion 2 min 68 466 Jemez 2 Percussion 2 min 64 480 Jemez 3 Percussion 2 min 80 400 Jemez 4 Percussion 2 min 83 376 Jemez Percussion 4 min 68 466 Jemez 2 Percussion 4 min 79 407 Jemez 3 Percussion 4 min 70 458 Jemez 4 Percussion 4 min 83 376 Jemez 1 Percussion 8 min 52 500 Jemez 2 Percussion 8 min 59 492 Jemez 3 Percussion 8 min 61 488 Jemez 4 Percussion 8 min 70 458 Magdalena 1 Immersion 69 462 Magdalena 2 Immersion 69 462 Magdalena J Immersion 70 458 Magdalena 4 Immersion 85 357 Magdalena 1 Boiling 30 sec 65 477 Magdalena 2 Boiling 30 sec 71 454 Magdalena 3 Boiling 30 sec 68 466
67
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Magdalena 4 Boiling 30 sec 83 376 Magdalena 1 Boiling 60 sec 63 483 Magdalena 2 Boiling 60 sec 68 466 Magdalena 3 Boiling 60 sec 63 483 Magdalena 4 Boiling 60 sec 82 384 Magdalena 1 Control 11 313 Magdalena 2 Control 6 237 Magdalena 3 Control 5 218 Magdalena 4 Control 4 196 Magdalena Percussion 1 min 32 466 Magdalena 2 Percussion 1 min 45 497 Magdalena 3 Percussion 1 min 46 498 Magdalena 4 Percussion I min 36 480 Magdalena 1 Percussion 2 min 67 470 Magdalena 2 Percussion 2 min 68 466 Magdalena 3 Percussion 2 min 65 477 Magdalena 4 Percussion 2 min 81 392 Magdalena 1 Percussion 4 min 87 336 Magdalena 2 Percussion 4 min 97 171 Magdalena 3 Percussion 4 min 83 376 Magdalena 4 Percussion 4 min 97 171 Magdalena 1 Percussion 8 min 98 lAO Magdalena 2 Percussion 8 min 100 000 Magdalena 3 Percussion 8 min 96 196 Magdalena 4 Percussion 8 min 98 lAO Manzano 1 Immersion 67 470 Manzano 2 Immersion 57 495 Manzano 3 Immersion 72 4049 Manzano 4 Immersion 73 4044 Manzano 1 Boiling 30 sec 70 458 Manzano 2 Boiling 30 sec 64 480 Manzano 3 Boiling 30 sec 71 454 Manzano 4 Boiling 30 sec 80 400 Manzano I Boiling 60 sec 50 500 Manzano 2 Boiling 60 sec 53 499 Manzano 3 Boiling 60 sec 59 492 Manzano 4 Boiling 60 sec 74 439 Manzano I Control 4 196 Manzano 2 Control 8 271 Manzano 3 Control 1 099 Manzano 4 Control 6 237 Manzano 1 Percussion 1 min 52 500 Manzano 2 Percussion 1 min 39 488 Manzano 3 Percussion 1 min 36 480 Manzano 4 Percussion 1 min 51 500
68
Table B 1 Continued
Source Block Treatment Gerrni~ation Percentage Standard Error
Manzano Percussion 2 min 53 499 Manzano 2 Percussion 2 min 58 494 Manzano 3 Percussion 2 min 61 488 Manzano 4 Percussion 2 min 62 485 Manzano 1 Percussion 4 min 76 427 Manzano 2 Percussion 4 min 85 357 Manzano 3 Percussion 4 min 79 407 Manzano 4 Percussion 4 min 83 376 Manzano 1 Percussion 8 min 87 336 Manzano 2 Percussion 8 min 83 376 Manzano 3 Percussion 8 min 83 376 Manzano 4 Percussion 8 min 75 433 Raton 1 Immersion 54 498 Raton 2 Immersion 37 483 Raton 3 Immersion 39 488 Raton 4 Immersion 69 462 Raton 1 Boiling 30 sec 36 480 Raton 2 Boiling 30 sec 20 400 Raton 3 Boiling 30 sec 28 449 Raton 4 Boiling 30 sec 60 490 Raton 1 Boiling 60 sec 15 357 Raton 2 Boiling 60 sec 171 Raton 3 Boiling 60 sec 4 196 Raton 4 Boiling 60 sec 35 477 Raton 1 Control 4 196 Raton 2 Control 12 325 Raton 3 Control 2 140 Raton 4 Control 6 237 Raton 1 Percussion 1 min 52 500 Raton 2 Percussion I min 29 454 Raton 3 Percussion I min 35 477 Raton 4 Percussion 1 min 31 462 Raton 1 Percussion 2 min 55 497 Raton 2 Percussion 2 min 35 477 Raton 3 Percussion 2 min 33 470 Raton 4 Percussion 2 min 42 494 Raton 1 Percussion 4 min 65 477 Raton 2 Percussion 4 min 52 500 Raton Percussion 4 min 51 500 Raton 4 Percussion 4 min 53 499 Raton 1 Percussion 8 min 88 325 Raton 2 Percussion 8 min 80 400 Raton 3 Percussion 8 min 73 444 Raton 4 Percussion 8 min 74 439 Sacramento 1 Immersion 43 495
69
Table B 1 Continued
-Source Block Treatment Germination Percentage Standard Error
Sacramento 2 Immersion 30 458 Sacramento 3 Immersion 38 485 Sacramento 4 Immersion 56 496 Sacramento 1 Boiling 30 sec 47 499 Sacramento 2 Boiling 30 sec 48 500 Sacramento 3 Boiling 30 sec 48 500 Sacramento 4 Boiling 30 sec 56 496 Sacramento 1 Boiling 60 sec 52 500 Sacramento 2 Boiling 60 sec 67 470 Sacramento 3 Boiling 60 sec 43 495 Sacramento 4 Boiling 60 sec 48 500 Sacramento 1 Control 0 000 Sacramento 2 Control 3 171 Sacramento 3 Control 3 171 Sacramento 4 Control 099 Sacramento 1 Percussion 1 min 57 495 Sacramento 2 Percussion 1 min 61 488 Sacramento 3 Percussion 1 min 62 485 Sacramento 4 Percussion 1 min 55 497 Sacramento 1 Percussion 2 min 84 367 Sacramento 2 Percussion 2 min 70 458 Sacramento 3 Percussion 2 min 81 392 Sacramento 4 Percussion 2 min 69 462 Sacramento 1 Percussion 4 min 91 286 Sacramento 2 Percussion 4 min 84 367 Sacramento 3 Percussion 4 min 84 367 Sacramento 4 Percussion 4 min 94 237 Sacramento 1 Percussion 8 min 82 384 Sacramento 2 Percussion 8 min 81 392 Sacramento 3 Percussion 8 min 82 384 Sacramento 4 Percussion 8 min 81 392 Sandia 1 Immersion 76 427 Sandia 2 Immersion 70 458 Sandia 3 Immersion 70 458 Sandia 4 Immersion 81 392 Sandia 1 Boiling 30 sec 78 414 Sandia 2 Boiling 30 sec 71 454 Sandia 3 Boiling 30 sec 63 483 Sandia 4 Boiling 30 sec 78 414 Sandia 1 Boiling 60 sec 67 470 Sandia 2 Boiling 60 sec 65 477 Sandia 3 Boiling 60 sec 74 439 Sandia 4 Boiling 60 sec 66 474 Sandia 1 Control 16 367 Sandia 2 Control 7 255
70
Table B 1 Continued
Source Block Treatment Gennination Percentage Standard Error
Sandia 3 Control 8 271 Sandia 4 Control 16 367 Sandia 1 Percussion 1 min 67 470 Sandia 2 Percussion 1 min 61 488 Sandia 3 Percussion 1 min 66 474 Sandia 4 Percussion 1 min 59 492 Sandia I Percussion 2 min 82 384 Sandia 2 Percussion 2 min 83 376 Sandia 3 Percussion 2 min 81 392 Sandia 4 Percussion 2 min 84 367 Sandia 1 Percussion 4 min 89 313 Sandia 2 Percussion 4 min 86 347 Sandia 3 Percussion 4 min 89 313 Sandia 4 Percussion 4 min 85 357 Sandia 1 Percussion 8 min 80 400 Sandia 2 Percussion 8 min 77 421 Sandia 3 Percussion 8 min 69 462 Sandia 4 Percussion 8 min 66 474
LITERATURE CITED
Ashby WC Vogel WG Rogers NF 1985 Black locust in the reclamation equation General Technical Report Northeastern Forest Experiment Station USDA Forest Service NE-I05 12 p
Ballard LAT 1973 Physical barriers to gennination Seed Sci Technoll 285-303
___ 1976 Strophiolar water conduction in the seeds ofTrifolieae induced by action on the testa at nonstrophiolar sites Austr J Plant Physiol3 465-469
Barton LV 1947 Special studies on seed coat impenneability Contrib Boyce Thompson Inst 14 355-362
Baskin CC Baskin JM 1998 Seeds Ecology biogeography and evolution of donnancy and gennination Academic Press London UK 666 p
Bewley JD 1997 Seed germination and donnancy Plant Ce119 1055-1066
Bewley JD Black M 1994 Seeds physiology of development and germination 2nd ed Plenum Press New York NY 445 p
Briscoe CB 1996 Growing Nitrogen Fixing Trees in Acid Soils In MH Powell (ed) Nitrogen Fixing Trees for Acid Soils- A Field ManuaL Winrock International Morrilton AR 22 p
Copeland LO McDonald MB 1995 Principles of Seed Science and Technology 3rded Chapman amp Hall New York NY 409 p
Cox RE Klett JE 1984 Seed gennination requirements of native Colorado plants for use in the landscape Plant Propag 30(2) 6-10
Dell B 1980 Structure and function ofthe strophiolar plug in seeds ofAlbizia Lophantha Am J Bot 67 556-563
Dick-Peddie WA 1993 New Mexico Vegetation Past Present and Future University ofNew Mexico Press Albuquerque NM 244 p
Dreesen DR 2001 Propagation protocol for production ofcontainer Robinia neomexicana plants Los Lunas Plant Materials Center Los Lunas New Mexico In Native Plant Network URL httpwwwnativeplantnetworkorg (accessed 9 January 2002) Moscow (ID) University ofIdaho CoIl Natural Resources For Res Nursery
72
Dreesen DR Harrington JT 1997 Propagation of native plants for restoration projects in the Southwestern US- Preliminary Investigations In Landis TD Thompson JR tech coords National Proceedings Forest and Conservation Nursery Assocs Gen Tech Rep PNW-GTR-419 Portland OR USDA For Ser PNW Res Sta 77-88
Gottfried GJ 1980 Control ofNew Mexican locust and the effect on planted ponderosa pine in central Arizona US For Ser Res Note RM-386 Rocky Mountain For Range ExpStn Fort Collins CO
Gosling PG Samuel YK Jones SK 1995 A systematic examination of germination temperature chipping and water temperaturesoak duration pretreatments on the seeds ofLeucaena leucocephala Seed Sci amp Technol 23521-532
Hamly DH 1932 Softening of the seeds of Melilotus alba Bot Gazette 93 345shy375
Hanna PJ 1984 Anatomical features of the seed coat ofAcacia kempeana (Mueller) which relate to increased germination rate induced by heat treatment New Phytol 96 23-29
Hanks JP Dick-Peddie WA 1974 Vegetation patterns of the White Mountains New Mexico Southwest Naturalist 18(4) 371-382
Hine S Harrington JT Dreesen DR 1997 Seed scarification requirements for Robinia neomexicana In Harrington JTtech coord Proc Third SouthwestemContainer Growers Conf Feb 12-13 1997 Ft Collins CO
Hopkinson JM 1993 The strophiole of leguminous seeds Newsletter- IntI Herbage Seed Production Res Grp 18 7-8
---- 1997 Differences in hardseededness between Cunningham and Tarramba leucaena In Leucnet News The Newsletter of the International LeucaenaRes and Devlt Netwk No4
ISTA (International Seed Testing Association) 1999 International rules for seed testing rules Seed Sci amp Technol27 Supplement
Kelly KM van Staden J 1987 The lens as the site of permeability in the papilionoid seed Aspalathus linearis J Plant Physiol 128(45) 395-404
73
Keresztesi B 1988 Black locust the tree of agriculture Outlook on Agr 17(2) 77~85
Kigel 1 and Galili G 1995 Seed development and germination Marcel Dekker New York 853 p
Klemmedson 10 1994 New Mexico locust and parent material Influence on macronutrients of forest floor and soil Soil Sci Soc Am 1 58 974~980
Kuo WH Tam AW 1988 The pathway of water absorption of mung bean seeds Seed Sci amp Technol16(1) 139~144
Leadem CL 1997 Dormancy--unlocking seed secrets In Landis TD Thompson lR tech coords National Proceedings Forestand Conservation Nursery Assocs Gen Tech Rep PNW-G TR-419 Portland OR USDA For Ser PNW Res Sta 43-52
Lin Y Wagner MR Seweyestewa A Honahni MD DeGomez M McMillin JD 1996 Influence of cultural treatments on seed germination and seedling growth of Robinia neomexicana Forest Farm and Community Tree Res Reports Volume 1
Marunda CT 1990 Effects of seed pretreatment on the development ofAcacia auriculforinis and A holosericea seedlings ACIAR Proceedings Series No 28
Meginnis HG 1937 Sulphuric acid treatment to increase germination of black locust seed USDA Circ 453 34 p
Miklas PN Towensend CE Ladd SL 1987 Seedcoat anatomy and the scarification ofcicer milkvetch seed Crop Sci 27 766-772
Monsen SB 1984 Use of shrubs on mine spoils In The Challenge ofProducing Native Plants for the Intermountain Area Proceedings Intermountain Nurserymans Association 1983 conference Las Vegas NV compo PM Murphy 26-31 Ogden UT USDA For Ser Gen Tech Report INT-l68
Olson DF 1974 Robinia L Locust In Seeds of Woody Plants in the US tech coord CS Schopmeyer 728-731 USDA For Ser Agr Handbk No450
Rehman S Loescher RNl Harris P1C 1998 Dormancy breaking and germination ofAcacia salicina Lindl seeds Seed Sci amp Techno127 553-557
Rolston MP 1978 Water impermeable seed dormancy Bot Rev 44 365-396
74
SAS Institute Inc 1989 SASSTAT Users Guide Version 6 4th Edition Volume 1 Cary NC SAS Institute Inc 943 p
SerratoValenti G Conara L Ferrando M Modenesi P 1993 Structural and histochemical features ofStylosanthes scabra (Leguminosae Papilionoidae) seed coat as related to water entry Can J Bot 71 834-840
Simpson BJ 1988 A field guide to Texas trees Austin TX Texas Monthly Press 372p
Tran VN Cavanagh AK 1984 Structural aspects ofdormancy In Seed Physiology Vol 2 Murray DR (ed) p1-44 Academic Press Australia
USDA Forest Service 1948 Woody-plant seed manual USDA Misc Pub1654 416p
Vines RA 1960 Trees Shrubs and Woody Vines of the Southwest Austin Univ ofTexas Press 1104 p
Wagner MR Larson RE Craig TP 1992 Ecology and silvics ofNew Mexico locust (Robinia neomexicana) with an emphasis on the arboreal form p53-57 In JW Hanover K Miller and S Plesko (eds) Proc Intemat Confon Black Locust Biology Culture and Utilization Mich State Univ USA
Wilson JK 1944 Immersing seeds of species of Robinia in boiling water hastens germination J For 42 453-454
Zimmerman RW Carpenter SB 1980 First-year coppice production from a 5shyyear old black locust stand on surface mine spoil p309-314 Centr Hardwd For ConfProc University ofMissouri Columbia
ABSTRACT
PERCUSSION AS AN ALTERNATIVE SEED TREATMENT FOR
ROBINIA NEOMEXICANA (NEW MEXICO LOCUST)
BY
NABIL Y KHADDURI
Master of Science in Horticulture
New Mexico State University 2002
Las Cruces New Mexico
Dr John T Harrington Chair
New Mexico locust (Robinia neomexicana A Gray) a small tree native to
montane regions of the southwestern United States fills a successional role in postshy
disturbance situations The ability to colonize rapidly and ameliorate harsh sites
contributes to the importance ofNew Mexico locust in reclamation efforts Seedling
production for outplanting on disturbed sites has been hampered by poor germination
This research sought to improve seed propagation techniques in New Mexico locust
Nine seed sources were collected across a range of latitudes within New
Mexico Seed treatments included a control three levels of hot water and four levels
of percussion scarification Hot water treatments included immersion at 100degC and
boiling for periods of 30 seconds and 60 seconds Percussion treatment levels were 1
2 4 and 8 minutes A total of four lOO-seed samples were used to test each source
by scarification treatment combination Categorical Analysis of Variance was used to
determine main effects and interactions Pairwise comparisons were conducted to
compare non-control treatment means using a conservative alpha value of 005
divided by the number of comparisons
Averaged over all sources and for each source percussion scarification
durations of4 or 8 minutes yielded greatest germination In addition to consistently
bigh germination 4- and 8-minute percussion-treated seeds germinated as fast or
faster than all other treatments Averaged over all sources germination increased
with increasing durations of percussion then leveled off for the 4- and 8-minute
treatments Immersion and 30-second boiling scarification maximized germination
for the hot water treatments with a decrease in germination when seeds were boiled
for 60 seconds
Percussion scarification specifically weakens the strophiole the natural
dormancy barrier in many papilionoid legumes without excessively damaging the
seed coat As with New Mexico locust papilionoid legumes often play an integral
role in the revegetation ofdisturbed lands The percussion scarification technique
developed in this research has the potential to facilitate the use of these valuable
species in reclamation projects
VI
CONTENTS
LIST OF TABLES ix
LIST OF FIGURES middot middotmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot x
INTRODUCTION 1
LITERATlTRE REVIEW 4
Seed Donnancy 4
Physical Donnancy 5
Evolutionary Role ofPhysical Donnancy 6
Methods for Breaking Donnancy 6
Acid Scarification 6
Hot Water Scarification 8
Mechanical Scarification 11
Alternative Method for Breaking Donnancy Percussion 12
Objectives 15
Hypothesis 15
MATERIALS AND METHODS 16
Seed Collection and Cleaining 16
Experimental Design and Treatment Structure 16
Treatments ~ bullbull bullbullbullbull - bullbull ~ ~ 20
G T ennlnatlon estmg 21
X-ray Radiography 22
Scanning Electron Microscope Photography 22
Analysis 22
RESULTS 24
DISCUSSION 36
Detennining the Optimal Treatment 36
Strophiole and Seed Coat Behavior 37
CONCLUSION 44
Appendices
A PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR NEW MEXICO LOCUST AND BLACK LOCUST SEEDS 47
B GERMINATION PERCENTS AND STANDARD ERRORS FOR SOURCE BLOCK AND TREATMENT FOR NEW MEXICO LOCUST 64
LITERATURE CITED 72
Vlll
LIST OF TABLES
Table Page
1 Latitude longitude location elevation and collection date ofNew Mexico locust seed sources 18
2 Mean seed weight and standard error by source 19
3 Categorical Analysis ofVariance table for germination response to seed treatment seed source block and the interaction of the factors 25
4 Effect of seed source and treatment on G50 (days to 50 germination) 32
B1 Germination percents and standard errors for source block and treatment for New Mexico locust 65
LIST OF FIGURES
Figures Page
1 Seedsourcecollectionmap 17
2 Effect ofNew Mexico locust seed source on germination for data averaged over scarification treatments 26
3 Effect of scarification on germination for New Mexico locust for data averaged over all seed sources 27
4 Effect of seed source by treatment interaction on New Mexico locust germination for percussion scarification pattern a and pattern b 28
5 Effect of source by treatment interaction on New Mexico locust germination for hot water scarification 29
6 Timecourse of New Mexico locust germination following scarification treatments 31
7 X-ray radiographs of Sacramento seed source perc ussed for 1 4 or 8 minutes 33
8 Seed damage close-ups 34
9 SEM images of untreated vs 4-minute percussion-treated strophiolar cells ofNew Mexico locust seeds 35
10 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (b c) 38
11 Damage to New Mexico locust seeds observed during germination counts following hot water scarification 40
12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources 42
New Mexico locust a primary invader following disturbance 50
Implementation of hot water and percussion scarification 52
Effect of scarification on germination response for New Mexico locust 55
x
A4 Effect of selected scarification treatments on germination speed for New Mexico locust 56
AS Effect of scarification on germination response for black locust 57
A6 Effect of selected scarification treatments on germination speed for black locust 59
A7 Damage to New Mexico locust seeds observed during germination counts following hot water scarification 60
AS New Mexico locust seeds following percussion treatment at time of germination counts 61
A9 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (bc) 63
INTRODUCTION
New Mexico locust (Robinia neomexicana A Gray) a small tree native to the
southwestern United States occurs at elevations from 1200 to 2800 meters This
species fills a successional role in post-disturbance situations As a primary invader
New Mexico locust quickly establishes on burned areas arid flood banks as well as
road cuts (Wagner et aI 1992) Rapid growth crown sprouting and prolific root
suckering favor the successful establishment ofNew Mexico locust on disturbed sites
(Simpson 1988 Gottfried 1980) These attributes also may make New Mexico locust
well suited for steep-slope revegetation where erosion is a problem
New Mexico locust is a nitrogen-fixing legume that tolerates and improves
nutrient-poor soils Stands ofNew Mexico locust increase Nitrogen (N) and Calcium
(Ca) in the forest floor Levels of Carbon (C) Phosphorous (P) Sulfur (8) and
Potassium (K) aIso have been shown to increase in soil beneath New Mexico locust
(Klemmedson 1994)
The ability to colonize rapidly and ameliorate harsh sites contributes to New
Mexico locusts potential as a nurse plant A nurse plant colonizes an inhospitable
site and creates an environment suitable for successional plant establishment New
Mexico locust demonstrates this role in the ponderosa pine (Pinus ponderosa Douglas
ex Lawson amp C Lawson)Gambel oak (Quercus gambelii Nutt) community After a
disturbance such as wildfire New Mexico locust and Gambel oak colonize and
dominate the site until shaded out by ponderosa pine Once shaded these species
become understory shrubs a process taking an average of 15-20 years (Dick-Peddie
1993 Hanks and Dick-Peddie 1974) New Mexico locust gradually declines with
small dense patches averaging about 005 ha beneath pine until the next disturbance
once again offers it a competitive advantage (Klemmedson 1994)
Gottfried (1980) expecting ponderosa pine regeneration to be greater where
New Mexico locust had been eradicated found that survival of planted pine seedlings
was greater where locust had not been removed In a follow-up study Gottfried
(1980) noted that soil moisture in the top 57 cm was highest in 5-year old locust sites
as compared to grass or 20-year old locust sites He concluded that managing an
appropriate cover ofNew Mexico locust could help regenerate pine the later
successional species
The ability ofNew Mexico locust to improve harsh sites also makes it a
candidate for reclaiming disturbances associated with mining Black locust (Robinia
pseudoacacia 1) has been used for decades to reclaim mine spoils and other
disturbed sites in the eastern US and around the world (Keresztesi 1988 Zimmerman
and Carpenter 1980) Ashby et al (1985) described similar positive attributes of
black locust also mentioned previously for New Mexico locust quick cover for
stabilization supply ofN and nutrient-rich litter to the soil and site improvement for
establishment of later successional trees Referring specifically to mine reclamation
the authors note the ability of black locust to grow on a wide range ofmine soil
conditions including extremely acid soils Black locust also shows some tolerance to
soils compacted by grading and topsoiling practices (Ashby et aL 1985) New
2
Mexico locust holds promise as a native southwe~tem counterpart to black locust in
mine reclamation but it has been used infrequently to date
Natural invasion and succession occur slowly on most mine sites (Monsen
1984) While New Mexico locust often colonizes sites naturally there is no
assurance a seed will reach a particular site and establish in a reasonable time frame
The challenge is to introduce New Mexico locust as mother plants to facilitate
colonization Seedling production for outplanting on disturbed sites has been
hampered by poor germination (Lin et al 1996) The goal then is to develop
techniques to overcome poor germination in New Mexico locust thereby facilitating
macropropagation
LITERATURE REVIEW
Seed Dormancy
Low gennination in New Mexico locust is primarily the result of seed
donnancy the failure of an intact viable seed to genninate under favorable
conditions (Bewley 1997) Seed donnancy also can be defined as the inability of the
embryo to genninate because of some inherent constraint (Bewley and Black 1994)
The constraint may originate within the embryo referred to as endogenous or embryo
donnancy or from the tissues surrounding the embryo known as exogenous or coatshy
imposed donnancy (Baskin and Baskin 1998 Leadem 1997 Bewley and Black
1994)
New Mexico locust seed is donnant when the impenneable seed coat does not
allow movement ofwater to the embryo This specific fonn of coat-imposed
donnancy where the restraining tissue involved is an impenneable seed coat is
known as physical donnancy (Baskin and Baskin 1998 Bewley and Black 1994)
Physical donnancy is relatively uncommon in seeds less than 10 of all woody
species exhibit this condition (USDA Forest Service 1948) However it is
characteristic of many genera of the Fabaceae (bean) family For example all
successional trees in the genera Gleditsia and Robinia (of which New Mexico locust
is a member) produce seeds that exhibit physical donnancy (Baskin and Baskin
4
Physical Dormancy
Seeds that exhibit physical dormancy are said to be hard seeded referring to
the thick impermeable seed coat (Leadem 1997) The first line of protection of the
testa in leguminous seeds is the outermost waxy cuticle followed by a subcuticle
consisting ofsuberin Next is a palisade layer consisting of sclereid cells that have
thick lignified secondary walls The most common type of sclereid cell in palisade
layers is the macrosclereid The macrosclereids contain water-repellent substances
including cutin lignin suberin and wax (Rolston 1978) This palisade layer is the
primary barrier to water uptake (Serrato-Valenti et al 1993 Tran and Cavanagh
1984)
All seed coat openings must be blocked to effectively prevent water uptake
The natural opening in a seed where it was attached to the mother plant is the weak
link where physical dormancy is usually broken (Hopkinson 1993 Kuo and Tam
1988) Anatomical structures associated with the seed coat opening vary from
family to family In the Papilionoideae the subfamily of the Fabaceae family
containing New Mexico locust the site ofwater entry is the strophiole The
strophiole is a swelling on the seed coat close to the stalk scar (Hopkinson 1997)
Kelly and van Staden (1987) refer to the strophiole site as the lens where
macrosclereid cells must separate and raise before water can pass through Since
middot6germination ofdormant seeds of the Papilionoideae takes place when the strophiole
permeable (Hopkinson 1993 Kuo and Tam 1988) a logical approach to
5
genninating New Mexico locust artificially would be to loosen the strophiolar cells
and allow water to pass through to the embryo
Evolutionary Role of Physical Dormancy
Why is physical dormancy a mechanism that blocks germination
ecologically advantageous When a plant produc~s seeds with different degrees of
dormancy seeds that are dispersed at one time will germinate over a range of time
(Bewley and Black 1994) Through variability of dormancy within a seed crop seeds
are exposed to changing environmental conditions improving the overall chances of
survival of the seed line (Bewley and Black 1994) Varying expression of dormancy
is under genetic control which indicates that this may be an evolved survival strategy
(Kigel and Galili 1995) A range of dormancy levels may be a particular advantage in
the southwestern US where extreme environmental variations exist
Methods for Breaking Dormancy
There are three common approaches to breaking dormancy in New Mexico
locust seed acid scarification hot water soaks and mechanical treatments In each
case the objective of the treatment is to scarify or abrade the seed coat to make it
permeable to water
Acid Scarification
Acid scarification consists of soaking seeds in concentrated sulfuric acid
rinsing thoroughly with water upon removal followed by subsequent drying before
PlaJlltmg Olson (1974) reports that this scarification method produces the highest
middot~~jllalUll results for all Robinia species Most literature refers to a seminal 1937
6
study conducted by HG Meginnis This stud~ t~ough conducted on black locust
seed is regularly cited in the suggested use of acid to treat New Mexico locust seed
as well (Olson 1974 Vines 1960)
Thee published studies document the specific use of sulfuric acid to treat
New Mexico locust seed Cox and Klett (1984) report that soaking seeds in
concentrated sulfuric acid for thirty minutes improved germination to 34 over an
untreated control of4 Lin et al (1996) found that for two seed lots acid
scarification for twenty-five to eighty minutes produced germination of 80 A third
lot however resulted in less than 20 germination at all durations of 10 to 80
minutes Hine et al (1997) showed that acid treatment levels of 1 2 5 and 10
minutes resulted in less than 20 germination across five sources However there
was an increasing trend in germination with acid soak duration and acid treatments
were not imposed for as long a time as the previous two studies (30 to 80 minutes)
The mixed results obtained by these investigators were probably due to the
widely varying germination response of seed lots to acid treatment For this reason
Olson (1974) recommends correlating duration of acid treatment to each seed lot If
treatment duration is too long the embryo will be permanently damaged If soak
duration is too short the seed will remain impermeable to water and fail to germinate
Compounding the problem of treatment duration is the negative effect acid
have on seedling growth Any type of scarification can injure seeds to some
by disrupting cells essential for growth leading to fungal invasion and
middot-uuu~w injury (Copeland and MacDonald 1995) Some legume seeds subjected
7
to 60 minutes of sulfuric acid scarification (to achieve maximum gennination results)
showed more than 50 seedling mortality after gennination (Kelly and van Staden
1987) Bewley and Black (1994) noted that fungal growth on seeds is an indication
of leakage of sugars organic acids amino acids and proteins
Perhaps the greatest drawback to acid scarification is the dangerous nature of
the treatment itself Difficulties associated with storage and trained use of
concentrated acid may discourage many propagators from using this option (Dreesen
and Harrington 1997) Consequently hot water and to a lesser extent mechanical
scarification are more commonly used and practical treatments for legume seeds
Most southwestern container growers use either of these two treatments to scarify
New Mexico locust seed in particular (Hine et al 1997)
Hot Water Scarification
The widespread use ofhot water scarification to treat locust seed is probably
due to its ease of application and efficacy in comparison to other scarification
treatments Although this practice had been in use for some time Wilson (1944) was
one of the first to report on its systematic application for black locust The treatment
consists of placing seeds in boiling water (4 parts water tomiddot 1 part seed by volume)
removing the vessel from the heat source and allowing the water to cool and seeds to
soak for 24 hours
Hine et al (1997) Lin et al (1996) and Cox and Klett (1984) have examined
the specific application ofhot water scarification on New Mexico locust seed Each
these authors compared hot water to acid scarification Lin et aL (1996) reported
8
60 gennination for two hot water-treated seed lots while a third lot germinated
around 50 (as reported above acid scarification germination ranged from 20 to
80 for these lots) It should be noted that the authors only soaked the seeds in water
from 10 to 80 minutes for this experiment as opposed to the standard 24 hours Cox
and Klett (1984) stated that using the standard 24-hour hot water treatment improved
germination over an acid pre-treatment (which produced less than 20 germination)
but they did not give specific gennination numbers for the hot water treatment Hine
et al (1997) found that across five seed lots germination was 66 for the standard
hot water treatment (as compared to less than 20 germination for acid scarification)
Variations in treatment intensity and duration and their subsequent effects on seed lot
appear to contribute to conflicting conclusions as to whether acid scarification or hot
water treatment is a more successful treatment
Given mixed results ease ofapplication is an important argument for use of
hot water application over acid soaks Safety concerns and material costs for hot
water treatments are low (Dreesen and Harrington 1997) An extension of this is the
practicality ofhot water for treating large batches of seed where a relative increase in
water volume is all that is needed However increases in water and seed even when
inthe same 4 1 proportion can result in dramatically different rates of cooling than in
atest tube experiment Different cooling rates can lead to contrasting results for the
treatment temperature duration and seed lot (USDA Forest Service 1948)
As with acid scarification parameters ofhot water treatment application may
to be optimized to maximize germination Hine et al (1997) found that as
9
temperature was increased from 70degC to 100degC overall germination in New Mexico
locust seed increased The 100degC treatment maximized germination for each of five
sources tested with germination usually above 60
Prolonged exposure of seeds ofmany woody legumes to boiling temperatures
also may improve germination (Briscoe 1996) This approach has not been tested on
New Mexico locust seed Exposing seed to prolonged boiling temperatures may
result in fungal infection and subsequent seedling mortality Marunda (1990) noted
severe fungal infections on both acid and hot water treated Acacia holoserica Cunn
ex G Don seeds attributing the infections to seed damage resulting from the
treatments Identifying an optimal hot water treatment level that does not result in a
high rate of seedling mortality is a logical goal
With hot water treatments as with acid there is also a danger of overtreating
Seeds and reducing germination (Gosling et al 1995) The legume Leucaena
leucocephala (Lam) de Wit germinated equally well after 7 seconds of exposure to
water at 100degC 7 seconds at 90degC 30 seconds at 80degC and 4 minutes at 70degC In
addition acceptably high germination was obtained from treatment at 70degC from 3
minutes to 1 hour whereas no seeds germinated following treatment at 100degC for 2
minutes Perhaps along with a greater margin of error for treatment duration treating
~ew Mexico locust seeds at a lower temperature may result in a comparatively lower
of seedling mortality In practice however nursery growers tend to use the
and quickest treatment available The short treatment length at near-boiling
whether recommended or not may be what is used most frequently
10
Fine-tuning a treatment in this range of treatment temperature and duration may be
the most logical approach
Mechanical Scarification
This treatment involves scratching the seed coat with an abrasive material
(USDA Forest Service 1948) One system employs a rotating drum lined with
sandpaper A paddle spinning at a given number ofrevolutions per minute throws the
seed against the sandpaper (Hine et al 1997) A second method dry tumbling
tumbles seed in a mixer with pea gravel or sharp sand for 5 to 7 days (Dreesen 2001)
These approaches have the advantage that control of temperature is not required as in
water treatments nor is safety an issue as with add scarification
Hine et al (1997) mechanically treated New Mexico locust seed using 100shy
grit sandpaper in a Forsberg (rotating drum) scarifier Gennination was less than
10 They concluded that the seed coats were too thin for their apparatus and
proposed that a finer grit sandpaper would produce more even abrasion Although
germination varied by seed lot Dreesen (2001) reported that using a slow-speed dry
tumbling technique for 5 to 7 days produced gennination rates as high as 90
As with acid and hot water treatments treatment duration must be specifically
shy
correlated to seed lot Treated seeds are also susceptible to fungal infection
iHopkinson 1997) In addition equipment is specialized and may not always be
ltU~CUJl~ to a nursery (USDA Forest Service 1948) Several studies have confinned
degrading ofstrophiolar cells due to acid and hot water scarification (Miklas et al
11
1987 Dell 1980 Hanna 1984) but the effect ofmechanical scarification on the
strophiole has not been studied
Alternative Method for Breaking Dormancy Percussion
Strophiole degradation has been examined in depth using an impaction
treatment more commonly known as percussion Hamly (1932) first proposed that
shaking or impacting seeds could split the macrosc1ereid cells at the strophiole
Hamly successfully released tension ofbent strophiolar cells of impermeable
Melilotus alba Medik (White sweet clover) seeds by delivering physical blows
resulting in cells that had split apart and were only slightly bent Ninety-two percent
oftreated seeds became permeable as compared to 05 of control seeds Barton
(1947) showed that percussion significantly increases permeability in black locust
seed Ballard (1976) reasoned that the integrated nature of the strophiole within the
seed coat means a blow on any part of the seed weakens the strophiole There are no
published studies on the use of percussion on New Mexico locust
Percussion methods vary and often seem SUbjective or ill-defined Hamly
(1932) placed seeds in a glass bottle and shook them three times a second for 20
minutes resulting in 180 oscillations per minute Ballard (1973) describes shaking
seeds in a closed metal container with at least fifty seeds per container at 850-900
JS(~l11alIOIlS per minute for 1 minute Kelly and van Staden (1987) placed seeds into
sealed glass flasks Flasks were shaken by a wrist-shaker at a rate of 250
~UUVJ~ per minute Subsequent germination rates were 45 after 2 hours of
55 after 3 hours 60 after 6 hours and 100 after 12 hours
12
There are several potential advantages of percussion over acid hot water and
mechanical scarification Kelly and van Staden (1987) note that a percussion
treatment only alters macrosclereid cells of the strophiole by loosening separating
and raising them They emphasize that minimal damage is done to the overall
anatomical structure of the seed In acid scarification of the same seed lot radicle
and cotyledon damage occurred Based on photographic evidence the authors
believe that this morphological damage was due to uneven pressure during
imbibition with water entering haphazardly wherever the acid had sufficiently
dissolved the coat This ultimately led to uneven hydration of the embryo In
percussed seeds water only entered through the s~ophiole when the entire seed coat
had been saturated resulting in controlled and even embryo hydration (Kelly and van
Staden 1987)
Although all successful scarification treatments may focus at least in part on
the strophiole for papilionoid legumes (Baskin and Baskin 1998) percussion may be
less damaging to seed coat structures particularly when compared with acid Kelly
and van Staden did not compare hot water or mechanical scarification methods
against percussion However the fungal infection associated with hot water and
mechanical scarification as with acid scarificatio~ have been directly associated with
seed damage (Rehman et al 1998 USDA Forest Service 1948) Embryo damage in
has been observed in hot-water scarified legume seeds (Rehman et al 1998
13
By limiting embryo damage percussion scarification reduces seedling
mortality compared to other treatments Kelly and van Staden (1987) noted that
seedlings from the percussion treatment were morphologically sound and vigorous
when compared to acid treatment seedlings The authors attribute this improved
seedling vigor to the controlled water uptake that limited anatomical damage
Despite a 12-hour percussion treatment morphological damage was not substantially
greater than untreated seeds In addition seedling mass measured 10 days following
germination was not significantly different between percussed and untreated seeds
A final advantage ofpercussion may be the ability to adequately treat the most
dormant seeds while avoiding harm to seeds that have already been made permeable
by the treatment or that were permeable to begin with Instead ofcorroding scalding
or scratching the seed coat percussion appears to be just a loosening of the
strophiole spring (Ballard 1973) Kelly and van Staden (1987) were able to
increase germination with a lengthy treatment (12 hours) Figuring out whether a
substantial cushion oftreatment durations exist for percussion ofNew Mexico locust
seed would be a significant improvement to current scarification methods where seed
damage and seedling health is an issue
The greatest challenge for applying percussion to New Mexico locust seeds is
standardizing a treatment that is both effective and readily reproducible by nurseries
with mechanical scarification this may require the development of specific
~UllDment to properly quantify and execute the treatment
14
f
Objectives
The first objective of the study is to detennine which scarification treatment
results in optimal gennination speed and percentage ofNew Mexico locust The
second objective is to describe treatment interactions with nine seed sources collected
in New Mexico
Hypothesis
A percussion treatment acts exclusively on the main area ofwater uptake in
New Mexico locust seed loosening the strophiole so that imbibition can occur This
direct mechanism ofdonnancy release will result in improved gennination rate and
overall gennination percentage for New Mexico locust seed compared to standard
treatments Standard treatments including acid hot water and mechanical
scarification while eventually weakening the strophiolar cleft cause excessive
morphological damage through generalized seed coat degradation These treatments
are therefore prone to inconsistency due to over and under-treatment
15
MATERIALS AND METHODS
Seed Collection and Cleaning
Seeds were collected from August 25 through September 15 2000 from nine
locations along a latitudinal gradient in New Mexico Seed source locations and
descriptive information are given in Figure 1 and Tables I and 2 Stands were
separated by at least 45 km
Pods were hand picked as they began to ripen and dehisce (open) Collections
were made from ground level to approximately 3 m from several trees at each site
Pods were dried in open-ended brown paper bags for two to four weeks at room
temperature (21-230C) to facilitate cleaning Dried pods were placed in a closed cloth
sack which was pounded against the ground to separate seeds from pods For pods
still containing seeds hand maceration (crunching pods with hands) completed seed
separation Debris was removed by threshing with the aid of a greenhouse fan A
South Dakota Seed Blower (Seedbureau Chicago Illinois) was used to remove
remaining chaff and light seeds by air density separation Seeds were placed in dry
storage in sealed quart-size zip-lock bags within a sealed plastic container Storage
temperatures ranged from 2-4oC Seeds remained in storage for approximately 7
months before the experiment
Experimental Design and Treatment Structure
The experiment compared the impact ofhot water bath and percussion
l11aLl11 on germination properties ofNew Mexico locust seed A single
16
-l
Figure 1 Seed source collection map All sources were collected within New Mexico USA separated by at least 4S lan
IIIRZltItW lIJPIirmiddot rllll1lbullbull_~~~_II
Table 1 Latitude longitude location elevation and collection date ofNew Mexico locust seed sources
Lot title Latitude (N) Longitude (W) Location Elevation (m) Collection Date
Raton 36deg5846 104deg2872 Raton Pass NM 2295 82500
Holman 36deg0306 105deg2429 HolmanNM 2476 911500
Jemez 35deg4500 106deg4100 Jemez Springs NM 1976 9900 00
Sandia 35deg1083 106deg2341 Cedar Crest NM 2432 82600
Manzano 34deg3689 106deg2494 ManzanoNM 2465 9200
Magdalena 33deg5953 107deg0903 Magdalena NM 2544 82900
Capitan 33deg2728 105deg4325 Alto NM 2189 911200
Sacramento 32deg5798 105deg4496 Cloudcroft NM 2630 91400 Gila 32deg5400 107deg4600 Kingston NM 2432 9700
f
Table 2 Mean seed weight and standard error by source
a
Source Mean seed weight (g) for Standard error 100 seeds n = 5
Raton Holman Jemez Sandia Manzano Magdalena Capitan Sacramento Gila
2872 b c 2522 d 1826 e 2753 c 2413 d 2559 d 2443 d 3130 a 3029 ab
00103 00403 00276 00301 00370 00374 00458 00279 00348
a Mean seed weights followed by the same letter are not significantly
different
19
experiment was conducted utilizing a randomized complete block design blocked by
time with a factorial treatment structure Experimental factors were seed source and
scarification treatment Nine seed sources from throughout New Mexico were used
(Figure 1 Table 1) Scarification treatments included a control three levels ofhot
water and four levels of percussion Hot water treatments included immersion at
100degC and boiling for periods of 30 seconds and 60 seconds Percussion treatment
levels were 1 2 4 and 8 minutes One hundred seeds per block were used to test
each treatment combination Four blocks of seed were tested one at a time (all seed
sources included in each run) in close chronological sequence (within 6 weeks) A
total of four 100-seed samples were used to test each source by scarification treatment
combination
Treatments
Hot water treatment of seeds utilized 50 ml test tubes filled with 30 ml
distilled water Each test tube was used to treat one 100-seed sample at a time
resulting in a ratio of4 parts water to 1 part seed by volume Test tubes were placed
in a beaker with the beaker filled with tap water to the same level as the water within
test tubes Nine test tubes (one per seed source) were placed in the beaker
simultaneously so that one replication of each seed sourcescarification treatment
cotnblnatlon was conducted simultaneously Hot water treatments include immersion
boiling For immersion treatments seeds were added to test tubes with the water
(boiling) and the test tubes were immediately removed from the beaker to
For the boiling treatments test tubes remained in the beaker with water at
20
boiling temperature for a set duration after the addition of seeds Following all
treatments test tubes with seeds cooled 24 hours in a test tube rack
Percussion treatment utilized a pneumatic paint shaker (Central Pneumatic
model 00422 Camarillo CA) Compressed air was maintained at 530 kPa plusmn30 kPa
(80 psi plusmn 5 psi) resulting in approximately 350 oscillations per minute Seeds were
placed in 118 ml (4 oz) soil tin cans (US Can Lobard IL) and the lids were secured
with duct tape Each can was used to treat one 100-seed sample To take advantage
ofgreatest lateral movement of the paint shaker a spacer (paint can) was used to
position the soil tin at the end of the shaking arm This allowed shaking to occur at
the greatest distance from the pivot point (fulcrum) Tins were placed perpendicular
to the direction of shaking motion allowing seeds to impact at greatest force by
hitting flat surfaces of the soil tin
Germination Testing
Before gennination testing all seeds were soaked 24 hours in reverse
osmosis-treated water (this was included in the process ofhot water treatment
jU~SCIlOea above) Following soaking seeds were placed in 100 cm Petri dishes lined
90 cm Whatman qualitative 1 filter paper circles (MG Scientific Pleasant
WI) One Petri dish was used for each treatment by source combination
papers were initially moistened with 5 mL reverse osmosis-treated water then
~VU1CUlJl moistened as needed to ensure adequate conditions for gennination
were monitored daily (at 300 PM) for gennination for 14 days in accordance
~UIUlnJlVUa Seed Testing Association (ISTA) standards for Robinia species
21
(1STA 1999) Tests were carried out at room temperature (22degC plusmnOSoC)
Germination was classified as protrusion of the radicle from the embryo by at least
1mm as viewed with the naked eye
X-ray Radiography
On August 132001 three lOO-seed samples from the Sacramento seed source
were treated for 14 or 8-minute percussion durations The seeds were sent
immediately by overnight delivery to the Faxitron X-Ray Corporation in Wheeling
Illinois where Dr Bob Karrfalt ofthe USDA Forest Service oversaw their imaging
Radiographs were made at SX with a Faxitron Specimen Radiography System Model
MX-20
Scanning Electron Microscope Photography
On February 192002 a Hitachi S-3200N Variable Pressure Scanning
Electron Microscope (Mountain View CA) was used to examine strophiolar cells of
untreated seeds and seeds percussed for 4 minutes Seeds were cut longitudinally
igt their entire length using a razor blade splitting the strophiolar region roughly in
Cross sections of seed samples magnifying the strophiolar region were viewed
600X under the microscope
Analysis
Categorical Analysis of Variance was used to determine treatment source and
(Le time) main effects and interactions The response variable was total
~vlI This procedure is a Chi-square test of homogeneity using the natural log
of germinated to non-germinated seed for each treatment Maximumshy
22
likelihood analysis was used to calculate Chi-square test statistics with their observed
significance levels A single contrast was conducted to cempare treatment means
against the control Observed significance levels less than or equal to 005 were
considered significant Pairwise comparisons were conducted between non-control
treatment mean percents using a conservative alp~a value of 005 divided by the
number of comparisons (Bonferroni approach) and approximate pairwise z statistics
Categorical analyses were performed using Proc Catmod (SAS Institute 1989)
RESULTS
Scarification seed source block and all interactions between factors
influenced germination (Table 3) The main effects are reported in the presence of
these interactions because the overall robustness of treatments and seed sources are of
interest Background levels ofnaturally occurring variation in seed sources are
seen in control germination percentages in Figure 2 In particular time variability
is unlikely to be under the control of the producer Appendix B details experimental
data for source by treatment gennination percents for each of the four blocks
Averaged over all treatments germination by source ranged from 40 to
79 (Figure 2) Averaged over all sources (Figure 3) and for each source (Figure 4
5) percussion scarification durations of4 or 8 minutes yielded greatest germination
Averaged over all sources germination increased with increasing durations of
percussion then leveled off for the 4- and 8-minute treatments (Figure 3) Immersion
and 30-second boiling scarification maximized germination for the hot water
treatments with a decrease in germination when seeds were boiled for 60 seconds
For all but two sources there is a similar ipcreasing trend in germination from
1to 4 minutes ofpercussion (Figure 4) The Holman source increased in germination
1 to 2 minutes then leveled off at 4 minutes The Jemez source changed little
1 to 4 minutes of treatment As treatment duration increased from 4 to 8
mutes five sources decreased in germination (Figure 4a) whereas three sources
24
Table 3 Categorical Analysis of Variance table for germination response to seed treatment seed source block and the interaction of the factors
Source of DF Chi-Square Observed Variability Significance Level
Source 8 98370 lt00001 Trt 7 233876 lt00001
contrast 1 16976 lt00001 SourceTrt 56 97077 lt00001 Block 3 5538 lt00001
SourceBlock 24 10162 lt00001 TrtBlock 21 7564 lt00001
SourceTrtBlock 168 34283 lt00001
S 4)
8 ~
0
sectg ~ 40 ~
N 0 0
20
o f0ltgt ~ ~4 jybshy ((iltgto ~~ K1ltgt 6~b-~ ~~ 0b-~~ ~~ ~b-~ ~b-~b
80
60 -I-- - ~- middotmiddotmiddotmiddotmiddot1
r -] Northern NM Sources
Central NM Sources
Southern NM Sources
sect-o ~
~b-v(i
SOURCE
Figure 2 Effect ofNew Mexico locust seed source on germination for data averaged over all scarification treatments Control germination is shown by solid line within bar
80
~ cd ~ ltl) () 60 ~ ~
N
t 0shyrti tshySltl)
40
0
20
o lmmersion Boiling 30 sec Boiling 60 sec Control 1 min 2min 4 min 8 min
Hot Water Treatments Percussion Treatments
Figure 3 Effect of scarification on germination ofNew Mexico locust for data averaged over all seed sources Means labelled with the same letter are not significantly different
100~----------------------~--~----------------~
80
60
ltU
~
40 IJ
sectgsI
l100
80
60
40
a I middotmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot middotImiddotmiddotmiddotmiddotmiddotmiddot E
_~iii-bull _ _ bull f --~--
t-- -~ ---- -shy ---- shyI ~ ---- - shy
L
fiII
~
b
- shy ---r
- Holman
Sacramento Sandia Gila Jemez
c ~
ltgt ~
Tmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot-I
j~=~=-~~-1
I middotmiddot ~ bull r
t tr
Y -------M-a-gd-a-Ie-n-a--
1 _ _
fCapitan
---- Manzano ~y -_ Raton
2 4 8
Time interval ofpercussion scarification (min)
Figure 4 Effect of seed source by treatment interaction on New Mexico locust germination for percussion scarification pattern a and pattern b
28
tv 0
~ d
~ A s o
1 c3
80
60
40
20
o
TIT
t------=middotfmiddotmiddot-middotmiddot-middotmiddot-middotmiddot 1- --- - - --=- - -shy--cE= - _--shy --shy ----shy ~ ~ ---shy ~ -~- _ --shy-------r 1
I
Raton Holman
-_ Jemez
--- Sandia Manzano Magdalena Capitan Sacramento Gila
Immersion Boiling 30 sec Boiling 60 sec
Hot Water Scarification
Figure 5 Effect of seed source by treatment interaction on New Mexico locust germination for hot water scarification
continued to increase in germination (Figure 4b) There was no difference between
the two treatment levels for the Manzano source (Figure 4b)
The effect ofhot water scarification also varied among sources with few
overall differences between hot water treatments (Figure 5) However boiling for 60
seconds yielded highest germination for the Jemez source whereas increasing
durations ofboiling decreased germination for the Raton source
In addition to consistently high germination 4- and 8-minute percussionshy
treated seeds germinated as fast or faster than all other treatments (Figure 6 Table 4)
050 measured as the number of days needed to complete 50 ofeventual
germination averaged 2 days for both ofthese treatments
X-ray radiographs were taken of seeds collected from the Sacramento source
percussed for 1 4 or 8 minutes (Figure 7) Seeds show signs of damage at 8 minutes
ofpercussion (Figure 8) Compared to an undamaged seed percussed for only 4
minutes (Figure 8a) damage at the 8-minute level includes fractured radicles pitting
of the seed coat and chips in seed coat structure (Figure 8b-d)
Scanning electron microscope (SEM) images at 600X reveal the direct effect
of 4 minutes of percussion on cells of the strophiolar region (Figure 9) Whereas
untreated seeds retain cellular organization in the strophiolar region strophiolar cells
seeds treated for 4 minutes ofpercussion separate and raise resulting in a
~-~e~U appearance
30
Umiddot-~middot--l-~---middotymiddotmiddotmiddot-----middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot--middot--middotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middotmiddotmiddotmiddotmiddotmiddotmiddot- -
050= gt1
-e-- Percussion 8 min -0- Percussion 4 min ---T- Percussion 2 min -----ltil- Percussion 1 min -- Immersion --0- Boiling 30 sec -+- Boiling 60 sec -ltgt- Control
2 4 6 8 10 12
Day Figure 6 Timecourse ofNew Mexico locust gennination following scarification
treatments G50 is the number of days to 50 of eventual germination
31
14
Table 4 Effect of seed source and treatment on G50 (days to 50 germination)
Seed Source Control Immersion B30sec B60sec Plmin P2min P4min P8min mean Raton 3 3 5 7 4 3 2 2 4 Holman 2 2 2 2 2 2 2 2 2
Jemez 4 6 6 5 3 2 2 2 4 Sandia 2 2 2 2 2 2 2 2 2
w tv Manzano 5 2 2 3 3 2 2 2 3
Magdalena 4 2 2 3 4 3 2 1 3
Capitan 5 4 2 2 3 2 2 2 3
Sacramento 3 2 2 2 4 3 2 2 3 Gila 3 2 2 2 2 2 2 2 2 mean 3 3 3 3 3 2 2 2 3 standard 11 14 16 18 09 05 0 03 08 deviation
Note Columns and rows may not agree due to rounding
Figure 7 X-ray radiographs ofSacramento seed source percussed for 1 4 or 8 minutes
33
Figure 8a-d Seed damage close-ups Figure a shows undamaged seed percussed for 4 minutes Figures b-d show seeds percussed for 8 minutes Damage includes fracturing ofradicle in b complete removal ofseed coat in c and pitting ofseed coat in d
34
Untreated Treated
Figure 9 SEM images ofuntreated vs 4-minute percussion-treated strophiolar cells ofNew Mexico locust seeds Treated cells are loosened separated and raised
DISCUSSION
Determining the Optimal Treatment
Averaging results of scarification treatmems across seed sources is a useful
tool to predict which treatment is most likely to be successful for an unknown seed
source Averaged over all sources four- and eight-minute percussion treatments
resulted in the highest germination 815 and 790 respectively and produced
germination that was as fast or faster than all other treatments Although there was no
difference between these two treatments on average an argument for 4 minutes of
percussion as a standard protocol for treating New Mexico locust seeds can be made
Descriptively 4 minutes of percussion improved germination over 8 minutes
ofpercussion for a majority of sources (five out of nine) 8 minutes of percussion
improved germination for three ofnine sources with no difference for a fmal source
For individual sources 4 minutes ofpercussion was always the best or secondmiddot best
treatment and was always equal to or better than all hot water treatments
Germination at 8 minutes ofpercussion was lower than all other percussion durations
for the Jemez and Gila sources and was lower than at least one hot water treatment for
these sources as well
The x-ray radiographs of the Sacramento seed source illustrate the excessive
internal and external seed damage that can occur with 8 minutes of percussion
germination counts 8-minute percussed seeds from Holman and Capitan
ClUll signs of damage that were visible to the naked eye (see below)
r~evertheless some of these seeds still fit the criteria for germination Neither x-ray
36
photography nor visible observations during seed counts revealed seed damage at the
4-minute percussion level In operational trials seeds percussed for 4 minutes
developed into healthy seedlings (Khadduri and Harrington unpublished data)
Strophiole and Seed Coat Behavior
Four minutes ofpercussion successfully breaks seed dormancy in a large
percentage ofNew Mexico locust seeds without excessively damaging the
surrounding seed coat or internal seed coat structures Most papilionoid legume
seeds including seeds ofNew Mexico locust have a specific region of the seed coat
known as the strophiole or lens (Hamly 1932) The strophiole is located on the
cotyledonary lobe of a locust seed (Figure 10 a-c) If seeds are not indiscriminately
damaged the strophiole is the first point of water entry to the embryo (Hopkinson
1997) When a papilionoid seed is percussed for the appropriate amount of time
repeated hits on the integrated seed coat loosen the constrained cells of the strophiolar
region without excessively damaging the rest of the seed coat (Ballard 1976) SEM
photos illuStrate that 4 minutes of percussion loosened and separated strophiolar cells
ofNew Mexico locust seeds when compared with constrained organized strophiolar
cells of untreated seeds (Figure 9) Research-has shown that water passes to the
embryo through the treated strophiole in a controlled manner and regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues
(Kelly and van Staden 1987)
37
10 Strophiole ofunimbibed New Mexico locust seed (a) and following uptake (bc)
38
In contrast hot-water treatments can excessively degrade the seed coat
(Rebman et al 1998 Marunda 1990) Random cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rebman et aI 1998 Marunda 1990) Along with a
proportionately large amount ofun-imbibed (thus under-treated) seeds (Figure l1a)
damage was observed in hot-water treated seeds from six sources during germination
counts Damage included seeds where the radicle failed to elongate (Figure 11 b)
seeds where the radicle elongated but was delayed in freeing itself from the seed coat
(Figure 11c) and seeds where the radicle completely broke off from the embryo
(Figure lId) In addition some seeds germinated in reverse (the cotyledon emerged
fIrst) If the radicle emerged completely from the seed coat they were counted as
germinated
While nearly all seeds treated for 8 minutes ofpercussion imbibed water
damage was evident in x-ray evaluations (Sacramento seed source) and in
germination counts (Holman and Capitan) at this treatment level Seed damage
during germination counts was similar to levels found in hot-water treated seeds
suggesting that over-treatment by both hot water and longer durations of percussion
may result in similar damage to seed structure
The Sacramento and Holman seed sources were two of the fIve sources that
opcrplltlpn in germination from the 4- to 8-minute durations of percussion
the benefIts ofloosening the strophiole as evidenced by imbibition of
all 8-minute treated seeds were outweighed by external and internal seed coat
39
a
b c d
11 Damage to New Mexico locust seeds observed during germination cotmts following hot water scarification 9a Hot water replicate including ~t seedlings and numerous unimbibed seeds 9b Close-up of seed where raallCle failed to elongate 9c Close-up ofseed where radicle elongated but was
-gtIn in freeing itself from the seed coat 9d Close-up ofseed where radicle broke off from embryo
40
damage (which also may be contributing to high imbibition) For the three sources
for which 8 minutes of percussion resulted in maximum germination extended
percussion of seeds necessary to loosen the strophiole may not have excessively
damaged the seed anatomy The Manzano source did not significantly change in
germination from 4 to 8 minutes of percussion suggesting that an increase in
permeable seeds at the 8-minute treatment duration was balanced by the number of
seeds that failed to germinate due to seed damage To summarize the 4-minute
percussion treatment improved germination over 8 minutes ofpercussion for a
majority of sources (five ofnine) the 8-minute percussion treatment improved
germination over 4-minutes ofpercussion for three sources and there was no
difference with the Manzano source Notably for the Raton source 8 minutes of
percussion increased germination by 235 percentage points over 4-minutes of
percussion (7875 vs 5525 respectively) the next best treatment Raton was the
only source to result in germination less than 75 for 4 minutes ofpercussion so a
grower may wish to apply longer percussion durations to lots that do not respond well
to this treatment level
Finally there was an interesting trend in the Jemez and Raton sources seed
that significantly contributed to the source by treatment interaction (Figure 12) A
~u test showed that the Jemez source had a significantly lighter seed weight than
other sources while the Raton source had an average seed weight For the Jemez
the longest duration of boiling increased germination Conversely only short
U4UVll) of percussion were needed to achieve high germination and the longest
41
80 _ Jemez a Raton
iD
~ C iD e iD
Q c 0 ~
c ~ iD 0
20
0------shy
Immersion Boiling 30 sec Boiling 60 sec
Hot Water Scarification
10degTr========~--------~------------------------------
80
60
40
20
_ Jemez Raton
0------shy
1 min 2 min 4 min 8 min
Percussion Scarification
Figure 12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources
42
b
--~----~~~~========~---- duration ofpercussion reduced germination For the Raton source longer durations
ofboiling consistently decreased germination whereas longer percussion treatments
consistently increased germination
A possible explanation is that a thick seed coat is associated with a loose
strophiole structure and that a thin seed coat is associated with a constrained
strophiole This contrast merits further study and may suggest an ecological
adaptation Further investigation ofthis phenomenon may contribute to successful
pre-screening ofunknown seed sources to determine optimal treatment leveL
43
CONCLUSION
Based on results obtained in pilot study (see Appendix) and thesis
experiments 4 minutes ofpercussion is recommended as a standard protocol for
treating an unknown source ofNew Mexico locust seed In addition to laboratory
results the 4-minute percussion treatment has resulted in germination greater than
80 and healthy seedling grow-outs for production purposes (unpublished data) In
laboratory experiments only one hundred seeds per 118 ml (40z) soil tin were
percussed at a time whereas for production we have treated up to five hundred seeds
per tin For large-scale production ofNew Mexico locust seedlings growers may
wish to experiment with larger quantities of seed and larger-size soil tins
Seed storage trials following percussion treatments were not conducted
However germination greater than 80 and healthy seedlings were obtained when
seeds were percussion-treated and subsequently stored at room-temperature (22degC)
for one week (unpublished data) Seed storage trials merit further study as the
standard hot water treatment leaves seeds in a softened condition that is not
conducive to storage
Hydroseeding is an effective method for direct seeding in reclamation
situations Percussion treatment may be conducive to hydro seeding as seeds remain
a dry relatively undamaged yet permeable condition following treatment Sulfuric
seeds also remain dry following treatment but extensive damage to seed coat
poor seedling vigor have been reported with this treatment (Kelly and van Staden
A grow-out study using a hydro seeder on a reclamation site testing percussion
44
against other treatments such as sulfuric acid and mechanical scarification also
merits further study
Papilionoids are the largest subfamily oflegumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust these legumes often play an integral role in the revegetation of
disturbed lands The pilot study (Appendix) indicates that percussion scarification
successfully treats the widely used black locust as well Developing superior
scarification methods to the standard hot water mechanical scarification and acid
treatments may facilitate the use ofpapilionoid legumes in reclamation projects
45
APPENDICES
APPENDIX A
PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR
NEW MEXICO LOCUST AND BLACK LOCUST SEEDS
Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4
Hot water and sulfuric acid soaks are traditional treatments for seeds of many
temperate woody legwnes including locusts However these scarification techniques
often produce inconsistent germination Percussion scarification where seeds are
repeatedly propelled against a hard surface was compared with hot water
scarification to evaluate treatment efficacy for New Mexico locust (Robinia
neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment
seeds were placed in a 98degC water bath which was immediately removed from the
heat source For percussion scarification seeds were placed in a soil sample tin and
agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the
control were followed by 24-hour water soaks Hot water baths resulted in 56 and
41 germination for New Mexico locust and black locust respectively For both
species nearly all durations ofpercussion increased germination over the hot water
treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3
4 and 5 minutes for black locust resulted in at least 90 germination Traditional
scarification treatments randomly degrade the entire seed coat which can lead to
tissue damage during water uptake Percussion scarification specifically weakens the
strophiole the natural source ofwater entry to the seed in papilionoid legwnes
INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom
2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031
48
Following percussion imbibition is controlled through the strophiole and underlying
tissue is protected
Keywords New Mexico locust black locust scarification percussion germination
Introduction
Locust trees (Robinia spp) are aggressive pioneer species that quickly
colonize disturbed land fix atmospheric nitrogen in the soil and stabilize slopes that
are prone to erosion (Klemmedson 1994 Ashby et al 1985) (Figure A1ab)
Recognition of these attributes has contributed to increased demand for locusts in
reclamation projects Locust seeds however exhibit physical dormancy where a
thick seed coat prevents the movement of water and gasses to the embryo (Leadem
1997)
Sulfuric acid or hot water soaks have traditionally been used to break seed
dormancy in macro-propagation of locusts Many growers are moving away from the
use of su1furic acid in treating locust seeds (Dreesen and Harrington 1997) In
addition to being dangerous acid soak durations must be specifically correlated to
seed lot (Olson 1974) Collections of native seed can vary tremendously in seed size
seed weight and hard seededness etc This variability limits the utility of sulfuric
acid soaks as even within a given lot some seeds may be damaged whereas others
are under-treated and fail to imbibe (take up water) A survey of Southwestern
container growers indicates that hot water baths are currently the most common
scarification method for locust seeds (Hine et al 1997) However hot water baths
also produce inconsistent germination (Lin et al 1996)
49
b
Figure AI New Mexico locust a primary invader following disturbance Ala Crown-sprouting at edge ofburn near Los Alamos NM--one year after Cerro Grande fire Alb Colonizing road cut Gila National Forest NM
50
Percussion scarification where seeds are repeatedly propelled against a hard
surface is an alternative dormancy-breaking method that has proven successful in
legumes related to locusts (Hamly 1932 Barton 1947) We compared this alternative
treatment to hot water scarification to evaluate treatment efficacy for New Mexico
locust and black locust seeds
Materials and Methods
Commercial New Mexico locust seeds (Western Native Seed Coaldale CO)
were collected fall 2000 in Huerfano County CO Black locust seeds were collected
September 2000 from Taos County NM by harvesting ripe pods from several trees at
a distance of up to 3 m from ground level Black locust seeds were allowed to air-dry
for 3 weeks and were threshed and separated from large chaffusing a greenhouse
fan A South Dakota Seed Blower (Chicago Illinltois) was used to remove fine chaff
from both seed lots Seeds were storedmiddotat 2-4C until the germination study was
conducted in March 2001
This study compared hot water scarification with percussion scarification
using treatment and species as experimental factors Along with a control
scarification treatments included immersion of seed in boiling water as well as
percussion durations of 1 2 3 45 or 10 minutes Four 100-seed samples were used
to test each species by scarification treatment combination
For the hot water treatment test tubes were filled with 30 mL water and
placed in a water-filled beaker (Figure A2a) The bath was raised to boiling (-98C at
51
b
c
Figure A2 Implementation ofhot water and percussion scarification A2a Hot water bath A2b Paint-shaker set-up A2c Placing soil tin containing seeds at end ofpaint shaker ann
52
Las Cruces NM elevation of 1300 meters) Each 100-seed replicate was placed in a
test tube and each test tube was immediately removed to cool at room temperature
Percussion scarification was implemented using a pneumatic paint shaker
(Central Pneumatic model 00422 Camarillo CA) (Figure A2b) This allowed
standardization of treatment intensity Compressed air pressure was maintained at
530kPa plusmn 30kPa (80psi plusmn 5psi) resulting in approximately 350 oscillations per
minute Each lOO-seed sample was placed in a 118 ml (4 oz) soil tin for shaking To
maximize lateral movement of paint shaker a paint can was used as a spacer to place
soil tins at the end of the shaking arm (Figure A2c) This allowed shaking to occur at
the greatest distance from the pivot point (fulcrum) Following all treatments
including the control seeds were soaked for twenty-four hours in distilled water prior
to plating out
Gennination was evaluated on lab benches at room temperature Each
replicate of seeds was placed in a 100 cm petri dish on moistened filter paper with
humidity maintained by enclosing the petri dishes in ziplock bags Gennination was
monitored daily for 14 days in accordance with International Seed Testing
Association standards for Robinia species (ISTA 1999) Gennination was defined as
protrusion of the radicle from the embryo by at least 1mm as viewed by the naked
eye
Categorical Analysis of Variance using Prpc Catmod (SAS Institute 1989)
was used to detennine treatment differences in germination percentages for each
source This procedure is an extension of a Chi-square test of homogeneity using the
53
natural log of the ratio of germinated to non-germinated seeds for each treatment Pshy
values less than 005 were considered significant A limited set of pairwiseshy
comparisons was conducted to compare treatment mean percents using a
conservative alpha value of 005 divided by the number of comparisons
Results
Percussion maximized germination for both New Mexico locust and black
locust There were three germination response levels for New Mexico locust (Figure
A3) First there was a low response in the control indicating a dormant seed lot
The hot water treatment and I-minute duration of percussion share a second
intermediate response Germination was highest at percussion levels from 2 to 10
minutes
A selected percussion treatment intermediate in time (5 minutes) produced the
steepest germination curve indicating the fastest germination rate (Figure A4) The
hot water treatment produced an intermediate curve or relatively slower germination
rate The control curve was very flat indicating the slowest germination rate for New
Mexico locust
Control seeds germinated poorly for black locust as well (Figure A5) Hot
water scarification improved germination over the control but all levels of percussion
improved germination over hot water The optimal durations of percussion treatment
were bracketed with a significant increase from the 1 to 2 minute percussion level a
high germination response from 2 to 5 minutes of percussion and finally a significant
54
~ d 25 CL)
p t1 0 ~ t1
g Vl ~ Vl
100~---------------------------------------------------------------
80
60
40
20
o Control Hot Water Percussion
I min Percussion
2 min Percussion
3 min
Scarification
Percussion 4 min
Figure A3 Effect of scarification on germination response for New Mexico locust
Percussion 5 min
=significant difference between adjacent treatment bars ns = no significant difference
Percussion 10 min
~ ~ () u agt
Il-t d o ~
~ ~ c3
100middotlr~~~------------------------------------e- Percussion 5min middot 0middot Hot Water -- Control
80-J=middot======
6b-middot-lmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middot--middotmiddot-middot~-middotmiddot -middotmiddot middot middotmiddot-middot 1
40
20
-----~-----------
0 0 0 middotmiddotmiddotmiddotmiddot0middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot0
0 --~-----y-----~------_---~--- T-----~
o shy -TDay 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A4 Effect of selected scarification treatments on germination speed for New Mexico locust First seven days of fourteen-day experiment are shown
Day 7
80
~ cd
5 60 ()
[) Cl d 0 1t1 d
40sect dUl
-J
20
o
ns
Scarification
Figure A5 Effect of scarification on germination response for black locust = significant difference between adjacent treatment bars ns = no significant difference
decrease at the 10-minute level This drop-off suggests that 10 minutes of percussion
over-treats seeds from this lot of-black locust
A selected percussion treatment intermediate in time (5 minutes) also had the
fastest germination rate for black locust (Figure A6)_ The hot water curve is
somewhat flatter indicating a slower germination rate The control curve is very flat
indicating a slow germination rate
A qualitative difference between hot water and percussion-treated seeds was
also observed Along with healthy germinants the hot water treatment produced
over-treated seeds (Figure A 7a) Examples of damaged germinants include seeds
where the radicle did not elongate (Figure A 7b) seeds where the radicle elongated
but was delayed in freeing itself from the seed coat (Figure A7c) and seeds where the
radicle broke off completely from the embryo (Figure A7d) In addition to these
examples of over-treatment there were numerous small dark seeds that failed to
imbibe water For percussion treatment nearly all seeds took up water (Figure A8a)
Germination was rapid and uniform with little damage evident (Figure A8b)
To summarize both New Mexico locust and black locust seed lots were
dormant The standard hot water scarification treatment improved germination but
only to 56 and 41 for New Mexico locust and black locust respectively Nearly
all percussion durations improved total germination as well as germination rate over
the hot water treatment Germination was greater than 90 for percussion durations
of 4 5 and 10 minutes for New Mexico locust and 3 4 and 5 minutes for black locust
58
CI)
sgt t CI) 0
igt 0
sect ttl t E
Vl 0
c3
100r==~==~~=r------------------------------------~ ___ Percussion 5 min
middotmiddotmiddot0middotmiddotmiddot Hot Water -lIf- Control
80r=========~--~~----------------------------~
60
40
20 -~-~---~---------- ---~------------I
omiddot
O
_~_----------lIf-----~bull -----_ shy---shy I
Day 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A6 Effect of selected scarification treatments on germination speed for black locust First seven days of fourteen-day experiment are shown
Day 7
a
b c d
Figure A7 Damage to New Mexico locust seeds observed during germination counts following hot water scarification A7a Hot water replicate including damaged seedlings and numerous unimbibed seeds A7b Close-up ofseed where radicle failed to elongate A7c Close-up ofseed where radicle elongated but was delayed in freeing itself from the seed coat A7d Close-up ofseed where radicle completely broke offfrom embryo
60
a
b
Figure A8 New Mexico locust seeds following percussion treatment at time of germination counts A8a Percussion replicate with uniformly healthy seedlings and near complete imbibition of seeds A8b Close-up ofpercussion-treated seedling
61
Discussion
Papilionoid seeds have a specific anatomical feature known as the strophiole
or lens-the natural site of water entry to the seed (Hamly 1932) The strophiole is
located on the cotyledonary lobe of a locust seed (Figure A9) When a papilionoid
legume seed is percussed for the appropriate amount of time repeated hits on the
integrated seed coat loosen the constrained cells of the strophiolar region without
excessively damaging the rest of the seed coat (Ballard 1976)
When a percussed seed is soaked water enters exclusively through the
strophiole in a controlled manner (Kelly and Staden 1987) This regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues This
contrasts with seeds that have been hot water or acid scarified treatments that can
randomly degrade the seed coat Unlocalized cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rehman et al 1998 Marunda 1990)
Papilionoids are the largest subfamily of legumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust and black locust these legumes often play an integral role in the
revegetation ofdisturbed lands Developing superior scarification methods to the
standard hot water and acid treatments should fadlitate the use ofthese legumes in
restoration projects
62
Figure A9 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (bc)
63
APPENDIXB
GERMINATION PERCENTS AND STANDARD ERRORS FOR SOURCE
BLOCK AND TREATMENT FOR NEW MEXICO LOCUST
Table B 1 Gennination percents and standard errors for source block and treatment for New Mexico locust
Source Block Treatment Germination Percentage Standard Error
Capitan 1 Immersion 74 437 Capitan 2 Immersion 69 462 Capitan 3 Immersion 71 454 Capitan 4 Immersion 67 470 Capitan Boiling 30 sec 79 407 Capitan 2 Boiling 30 sec 76 427 Capitan 3 Boiling 30 sec 72 449 Capitan 4 Boiling 30 sec 76 427 Capitan Boiling 60 sec 71 454 Capitan 2 Boiling 60 sec 54 498 Capitan 3 Boiling 60 sec 67 470 Capitan 4 Boiling 60 sec 76 427 Capitan I Control 5 218 Capitan 2 Control 7 255 Capitan 3 Control 4 196 Capitan 4 Control 5 218 Capitan 1 Percussion I min 47 499 Capitan 2 Percussion I min
470
Capitan Percussion I min 39 488
Capitan 4 Percussion I min 38 485 Capitan I Percussion 2 min 55 497 Capitan Capitan
2
Percussion 2 min Percussion 2 min
71 65
454 477
Capitan 4 Percussion 2 min 56 496 Capitan I Percussion 4 min 86 347 Capitan 2 Percussion 4 min 79 407 Capitan 3 Percussion 4 min 81 392 Capitan 4 Percussion 4 min 82 384 Capitan I Percussion 8 min 90 300 Capitan 2 Percussion 8 min 88 325 Capitan 3 Percussion 8 min 90 300 Capitan 4 Percussion 8 min 90 300 Gila I Immersion 71 454 Gila 2 Immersion 77 421 Gila
Immersion 75 433
Gila 4 Immersion 72 449
Gila I Boiling 30 sec 68 466
Gila 2 Boiling 30 sec 81 392
Gila 3 Boiling 30 sec 54 498
Gila 4 Boiling 30 sec 63 483
Gila I Boiling 60 sec 55 497
Gila 2 Boiling 60 sec 68 466
Gila 3 Boiling 60 sec 57 495
Gila 4 Boiling 60 sec 70 458
65
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Gila 2 Control 13 336 Gila 3 Control 13 336 Gila 4 Control 20 400 Gila 1 Percussion 1 min 66 474 Gila 2 Percussion 1 min 57 495 Gila 3 Percussion 1 min 73 444 Gila 4 Percussion 1 min 82 384 Gila 1 Percussion 2 min 81 392 Gila 2 Percussion 2 min 86 347 Gila 3 Percussion 2 min 85 357 Gila 4 Percussion 2 min 54 498 Gila 1 Percussion 4 min 79 407 Gila 2 Percussion 4 min 83 376 Gila 3 Percussion 4 min 84 367 Gila 4 Percussion 4 min 79 407 Gila 1 Percussion 8 min 51 500 Gila 2 Percussion 8 min 76 427 Gila 3 Percussion 8 min 52 500 Gila 4 Percussion 8 min 74 439 Holman 1 Immersion 83 376 Holman 2 Immersion 86 347 Holman 3 Immersion 81 392 Holman 4 Immersion 90 300 Holman 1 Boiling 30 sec 90 300 Holman 2 Boiling 30 sec 85 357 Holman 3 Boiling 30 sec 87 336 Holman 4 Boiling 30 sec 83 376 Holman 1 Boiling 60 sec 79 407 Holman 2 Boiling 60 sec 80 400 Holman 3 Boiling 60 sec 86 347 Holman 4 Boiling 60 sec 93 255 Holman Control 25 433 Holman 2 Control 19 392 Holman Control 21 407
Holman 4 Control 24 427
Holman Percussion 1 min 77 421
Holman 2 Percussion 1 min 85 357
Holman 3 Percussion 1 min 80 400
Holman 4 Percussion 1 min 89 313
Holman 1 Percussion 2 min 88 325
Holman 2 Percussion 2 min 96 196
Holman 3 Percussion 2 min 93 255
Holman 4 Percussion 2 min 96 196
Holman 1 Percussion 4 min 92 271
Holman 2 Percussion 4 min 97 171
66
I
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Holman 3 Percussion 4 min 91 286 Holman 4 Percussion 4 min 96 196 Holman 1 Percussion 8 min 88 325 Holman 2 Percussion 8 min 85 357 Holman 3 Percussion 8 min 83 376 Holman 4 Percussion 8 min 82 384 Jemez 1 Immersion 53 499 Jemez 2 Immersion 44 496 Jemez 3 Immersion 52 500 Jemez 4 Immersion 70 458 Jemez 1 Boiling 30 sec 58 494 Jemez 2 Boiling 30 sec 55 497 Jemez 3 Boiling 30 sec 48 500 Jemez 4 Boiling 30 sec 65 477 Jemez 1 Boiling 60 sec 68 466 Jemez 2 Boiling 60 sec 63 483 Jemez 3 Boiling 60 sec 73 444 Jemez 4 Boiling 60 sec 89 313 Jemez 1 Control 2 140 Jemez 2 Control 9 286 Jemez 3 Control 6 237 Jemez 4 Control 8 271 Jemez 1 Percussion 1 min 58 494 Jemez 2 Percussion 1 min 63 483 Jemez 3 Percussion 1 min 77 421 Jemez 4 Percussion 1 min 89 313 Jemez 1 Percussion 2 min 68 466 Jemez 2 Percussion 2 min 64 480 Jemez 3 Percussion 2 min 80 400 Jemez 4 Percussion 2 min 83 376 Jemez Percussion 4 min 68 466 Jemez 2 Percussion 4 min 79 407 Jemez 3 Percussion 4 min 70 458 Jemez 4 Percussion 4 min 83 376 Jemez 1 Percussion 8 min 52 500 Jemez 2 Percussion 8 min 59 492 Jemez 3 Percussion 8 min 61 488 Jemez 4 Percussion 8 min 70 458 Magdalena 1 Immersion 69 462 Magdalena 2 Immersion 69 462 Magdalena J Immersion 70 458 Magdalena 4 Immersion 85 357 Magdalena 1 Boiling 30 sec 65 477 Magdalena 2 Boiling 30 sec 71 454 Magdalena 3 Boiling 30 sec 68 466
67
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Magdalena 4 Boiling 30 sec 83 376 Magdalena 1 Boiling 60 sec 63 483 Magdalena 2 Boiling 60 sec 68 466 Magdalena 3 Boiling 60 sec 63 483 Magdalena 4 Boiling 60 sec 82 384 Magdalena 1 Control 11 313 Magdalena 2 Control 6 237 Magdalena 3 Control 5 218 Magdalena 4 Control 4 196 Magdalena Percussion 1 min 32 466 Magdalena 2 Percussion 1 min 45 497 Magdalena 3 Percussion 1 min 46 498 Magdalena 4 Percussion I min 36 480 Magdalena 1 Percussion 2 min 67 470 Magdalena 2 Percussion 2 min 68 466 Magdalena 3 Percussion 2 min 65 477 Magdalena 4 Percussion 2 min 81 392 Magdalena 1 Percussion 4 min 87 336 Magdalena 2 Percussion 4 min 97 171 Magdalena 3 Percussion 4 min 83 376 Magdalena 4 Percussion 4 min 97 171 Magdalena 1 Percussion 8 min 98 lAO Magdalena 2 Percussion 8 min 100 000 Magdalena 3 Percussion 8 min 96 196 Magdalena 4 Percussion 8 min 98 lAO Manzano 1 Immersion 67 470 Manzano 2 Immersion 57 495 Manzano 3 Immersion 72 4049 Manzano 4 Immersion 73 4044 Manzano 1 Boiling 30 sec 70 458 Manzano 2 Boiling 30 sec 64 480 Manzano 3 Boiling 30 sec 71 454 Manzano 4 Boiling 30 sec 80 400 Manzano I Boiling 60 sec 50 500 Manzano 2 Boiling 60 sec 53 499 Manzano 3 Boiling 60 sec 59 492 Manzano 4 Boiling 60 sec 74 439 Manzano I Control 4 196 Manzano 2 Control 8 271 Manzano 3 Control 1 099 Manzano 4 Control 6 237 Manzano 1 Percussion 1 min 52 500 Manzano 2 Percussion 1 min 39 488 Manzano 3 Percussion 1 min 36 480 Manzano 4 Percussion 1 min 51 500
68
Table B 1 Continued
Source Block Treatment Gerrni~ation Percentage Standard Error
Manzano Percussion 2 min 53 499 Manzano 2 Percussion 2 min 58 494 Manzano 3 Percussion 2 min 61 488 Manzano 4 Percussion 2 min 62 485 Manzano 1 Percussion 4 min 76 427 Manzano 2 Percussion 4 min 85 357 Manzano 3 Percussion 4 min 79 407 Manzano 4 Percussion 4 min 83 376 Manzano 1 Percussion 8 min 87 336 Manzano 2 Percussion 8 min 83 376 Manzano 3 Percussion 8 min 83 376 Manzano 4 Percussion 8 min 75 433 Raton 1 Immersion 54 498 Raton 2 Immersion 37 483 Raton 3 Immersion 39 488 Raton 4 Immersion 69 462 Raton 1 Boiling 30 sec 36 480 Raton 2 Boiling 30 sec 20 400 Raton 3 Boiling 30 sec 28 449 Raton 4 Boiling 30 sec 60 490 Raton 1 Boiling 60 sec 15 357 Raton 2 Boiling 60 sec 171 Raton 3 Boiling 60 sec 4 196 Raton 4 Boiling 60 sec 35 477 Raton 1 Control 4 196 Raton 2 Control 12 325 Raton 3 Control 2 140 Raton 4 Control 6 237 Raton 1 Percussion 1 min 52 500 Raton 2 Percussion I min 29 454 Raton 3 Percussion I min 35 477 Raton 4 Percussion 1 min 31 462 Raton 1 Percussion 2 min 55 497 Raton 2 Percussion 2 min 35 477 Raton 3 Percussion 2 min 33 470 Raton 4 Percussion 2 min 42 494 Raton 1 Percussion 4 min 65 477 Raton 2 Percussion 4 min 52 500 Raton Percussion 4 min 51 500 Raton 4 Percussion 4 min 53 499 Raton 1 Percussion 8 min 88 325 Raton 2 Percussion 8 min 80 400 Raton 3 Percussion 8 min 73 444 Raton 4 Percussion 8 min 74 439 Sacramento 1 Immersion 43 495
69
Table B 1 Continued
-Source Block Treatment Germination Percentage Standard Error
Sacramento 2 Immersion 30 458 Sacramento 3 Immersion 38 485 Sacramento 4 Immersion 56 496 Sacramento 1 Boiling 30 sec 47 499 Sacramento 2 Boiling 30 sec 48 500 Sacramento 3 Boiling 30 sec 48 500 Sacramento 4 Boiling 30 sec 56 496 Sacramento 1 Boiling 60 sec 52 500 Sacramento 2 Boiling 60 sec 67 470 Sacramento 3 Boiling 60 sec 43 495 Sacramento 4 Boiling 60 sec 48 500 Sacramento 1 Control 0 000 Sacramento 2 Control 3 171 Sacramento 3 Control 3 171 Sacramento 4 Control 099 Sacramento 1 Percussion 1 min 57 495 Sacramento 2 Percussion 1 min 61 488 Sacramento 3 Percussion 1 min 62 485 Sacramento 4 Percussion 1 min 55 497 Sacramento 1 Percussion 2 min 84 367 Sacramento 2 Percussion 2 min 70 458 Sacramento 3 Percussion 2 min 81 392 Sacramento 4 Percussion 2 min 69 462 Sacramento 1 Percussion 4 min 91 286 Sacramento 2 Percussion 4 min 84 367 Sacramento 3 Percussion 4 min 84 367 Sacramento 4 Percussion 4 min 94 237 Sacramento 1 Percussion 8 min 82 384 Sacramento 2 Percussion 8 min 81 392 Sacramento 3 Percussion 8 min 82 384 Sacramento 4 Percussion 8 min 81 392 Sandia 1 Immersion 76 427 Sandia 2 Immersion 70 458 Sandia 3 Immersion 70 458 Sandia 4 Immersion 81 392 Sandia 1 Boiling 30 sec 78 414 Sandia 2 Boiling 30 sec 71 454 Sandia 3 Boiling 30 sec 63 483 Sandia 4 Boiling 30 sec 78 414 Sandia 1 Boiling 60 sec 67 470 Sandia 2 Boiling 60 sec 65 477 Sandia 3 Boiling 60 sec 74 439 Sandia 4 Boiling 60 sec 66 474 Sandia 1 Control 16 367 Sandia 2 Control 7 255
70
Table B 1 Continued
Source Block Treatment Gennination Percentage Standard Error
Sandia 3 Control 8 271 Sandia 4 Control 16 367 Sandia 1 Percussion 1 min 67 470 Sandia 2 Percussion 1 min 61 488 Sandia 3 Percussion 1 min 66 474 Sandia 4 Percussion 1 min 59 492 Sandia I Percussion 2 min 82 384 Sandia 2 Percussion 2 min 83 376 Sandia 3 Percussion 2 min 81 392 Sandia 4 Percussion 2 min 84 367 Sandia 1 Percussion 4 min 89 313 Sandia 2 Percussion 4 min 86 347 Sandia 3 Percussion 4 min 89 313 Sandia 4 Percussion 4 min 85 357 Sandia 1 Percussion 8 min 80 400 Sandia 2 Percussion 8 min 77 421 Sandia 3 Percussion 8 min 69 462 Sandia 4 Percussion 8 min 66 474
LITERATURE CITED
Ashby WC Vogel WG Rogers NF 1985 Black locust in the reclamation equation General Technical Report Northeastern Forest Experiment Station USDA Forest Service NE-I05 12 p
Ballard LAT 1973 Physical barriers to gennination Seed Sci Technoll 285-303
___ 1976 Strophiolar water conduction in the seeds ofTrifolieae induced by action on the testa at nonstrophiolar sites Austr J Plant Physiol3 465-469
Barton LV 1947 Special studies on seed coat impenneability Contrib Boyce Thompson Inst 14 355-362
Baskin CC Baskin JM 1998 Seeds Ecology biogeography and evolution of donnancy and gennination Academic Press London UK 666 p
Bewley JD 1997 Seed germination and donnancy Plant Ce119 1055-1066
Bewley JD Black M 1994 Seeds physiology of development and germination 2nd ed Plenum Press New York NY 445 p
Briscoe CB 1996 Growing Nitrogen Fixing Trees in Acid Soils In MH Powell (ed) Nitrogen Fixing Trees for Acid Soils- A Field ManuaL Winrock International Morrilton AR 22 p
Copeland LO McDonald MB 1995 Principles of Seed Science and Technology 3rded Chapman amp Hall New York NY 409 p
Cox RE Klett JE 1984 Seed gennination requirements of native Colorado plants for use in the landscape Plant Propag 30(2) 6-10
Dell B 1980 Structure and function ofthe strophiolar plug in seeds ofAlbizia Lophantha Am J Bot 67 556-563
Dick-Peddie WA 1993 New Mexico Vegetation Past Present and Future University ofNew Mexico Press Albuquerque NM 244 p
Dreesen DR 2001 Propagation protocol for production ofcontainer Robinia neomexicana plants Los Lunas Plant Materials Center Los Lunas New Mexico In Native Plant Network URL httpwwwnativeplantnetworkorg (accessed 9 January 2002) Moscow (ID) University ofIdaho CoIl Natural Resources For Res Nursery
72
Dreesen DR Harrington JT 1997 Propagation of native plants for restoration projects in the Southwestern US- Preliminary Investigations In Landis TD Thompson JR tech coords National Proceedings Forest and Conservation Nursery Assocs Gen Tech Rep PNW-GTR-419 Portland OR USDA For Ser PNW Res Sta 77-88
Gottfried GJ 1980 Control ofNew Mexican locust and the effect on planted ponderosa pine in central Arizona US For Ser Res Note RM-386 Rocky Mountain For Range ExpStn Fort Collins CO
Gosling PG Samuel YK Jones SK 1995 A systematic examination of germination temperature chipping and water temperaturesoak duration pretreatments on the seeds ofLeucaena leucocephala Seed Sci amp Technol 23521-532
Hamly DH 1932 Softening of the seeds of Melilotus alba Bot Gazette 93 345shy375
Hanna PJ 1984 Anatomical features of the seed coat ofAcacia kempeana (Mueller) which relate to increased germination rate induced by heat treatment New Phytol 96 23-29
Hanks JP Dick-Peddie WA 1974 Vegetation patterns of the White Mountains New Mexico Southwest Naturalist 18(4) 371-382
Hine S Harrington JT Dreesen DR 1997 Seed scarification requirements for Robinia neomexicana In Harrington JTtech coord Proc Third SouthwestemContainer Growers Conf Feb 12-13 1997 Ft Collins CO
Hopkinson JM 1993 The strophiole of leguminous seeds Newsletter- IntI Herbage Seed Production Res Grp 18 7-8
---- 1997 Differences in hardseededness between Cunningham and Tarramba leucaena In Leucnet News The Newsletter of the International LeucaenaRes and Devlt Netwk No4
ISTA (International Seed Testing Association) 1999 International rules for seed testing rules Seed Sci amp Technol27 Supplement
Kelly KM van Staden J 1987 The lens as the site of permeability in the papilionoid seed Aspalathus linearis J Plant Physiol 128(45) 395-404
73
Keresztesi B 1988 Black locust the tree of agriculture Outlook on Agr 17(2) 77~85
Kigel 1 and Galili G 1995 Seed development and germination Marcel Dekker New York 853 p
Klemmedson 10 1994 New Mexico locust and parent material Influence on macronutrients of forest floor and soil Soil Sci Soc Am 1 58 974~980
Kuo WH Tam AW 1988 The pathway of water absorption of mung bean seeds Seed Sci amp Technol16(1) 139~144
Leadem CL 1997 Dormancy--unlocking seed secrets In Landis TD Thompson lR tech coords National Proceedings Forestand Conservation Nursery Assocs Gen Tech Rep PNW-G TR-419 Portland OR USDA For Ser PNW Res Sta 43-52
Lin Y Wagner MR Seweyestewa A Honahni MD DeGomez M McMillin JD 1996 Influence of cultural treatments on seed germination and seedling growth of Robinia neomexicana Forest Farm and Community Tree Res Reports Volume 1
Marunda CT 1990 Effects of seed pretreatment on the development ofAcacia auriculforinis and A holosericea seedlings ACIAR Proceedings Series No 28
Meginnis HG 1937 Sulphuric acid treatment to increase germination of black locust seed USDA Circ 453 34 p
Miklas PN Towensend CE Ladd SL 1987 Seedcoat anatomy and the scarification ofcicer milkvetch seed Crop Sci 27 766-772
Monsen SB 1984 Use of shrubs on mine spoils In The Challenge ofProducing Native Plants for the Intermountain Area Proceedings Intermountain Nurserymans Association 1983 conference Las Vegas NV compo PM Murphy 26-31 Ogden UT USDA For Ser Gen Tech Report INT-l68
Olson DF 1974 Robinia L Locust In Seeds of Woody Plants in the US tech coord CS Schopmeyer 728-731 USDA For Ser Agr Handbk No450
Rehman S Loescher RNl Harris P1C 1998 Dormancy breaking and germination ofAcacia salicina Lindl seeds Seed Sci amp Techno127 553-557
Rolston MP 1978 Water impermeable seed dormancy Bot Rev 44 365-396
74
SAS Institute Inc 1989 SASSTAT Users Guide Version 6 4th Edition Volume 1 Cary NC SAS Institute Inc 943 p
SerratoValenti G Conara L Ferrando M Modenesi P 1993 Structural and histochemical features ofStylosanthes scabra (Leguminosae Papilionoidae) seed coat as related to water entry Can J Bot 71 834-840
Simpson BJ 1988 A field guide to Texas trees Austin TX Texas Monthly Press 372p
Tran VN Cavanagh AK 1984 Structural aspects ofdormancy In Seed Physiology Vol 2 Murray DR (ed) p1-44 Academic Press Australia
USDA Forest Service 1948 Woody-plant seed manual USDA Misc Pub1654 416p
Vines RA 1960 Trees Shrubs and Woody Vines of the Southwest Austin Univ ofTexas Press 1104 p
Wagner MR Larson RE Craig TP 1992 Ecology and silvics ofNew Mexico locust (Robinia neomexicana) with an emphasis on the arboreal form p53-57 In JW Hanover K Miller and S Plesko (eds) Proc Intemat Confon Black Locust Biology Culture and Utilization Mich State Univ USA
Wilson JK 1944 Immersing seeds of species of Robinia in boiling water hastens germination J For 42 453-454
Zimmerman RW Carpenter SB 1980 First-year coppice production from a 5shyyear old black locust stand on surface mine spoil p309-314 Centr Hardwd For ConfProc University ofMissouri Columbia
2 4 and 8 minutes A total of four lOO-seed samples were used to test each source
by scarification treatment combination Categorical Analysis of Variance was used to
determine main effects and interactions Pairwise comparisons were conducted to
compare non-control treatment means using a conservative alpha value of 005
divided by the number of comparisons
Averaged over all sources and for each source percussion scarification
durations of4 or 8 minutes yielded greatest germination In addition to consistently
bigh germination 4- and 8-minute percussion-treated seeds germinated as fast or
faster than all other treatments Averaged over all sources germination increased
with increasing durations of percussion then leveled off for the 4- and 8-minute
treatments Immersion and 30-second boiling scarification maximized germination
for the hot water treatments with a decrease in germination when seeds were boiled
for 60 seconds
Percussion scarification specifically weakens the strophiole the natural
dormancy barrier in many papilionoid legumes without excessively damaging the
seed coat As with New Mexico locust papilionoid legumes often play an integral
role in the revegetation ofdisturbed lands The percussion scarification technique
developed in this research has the potential to facilitate the use of these valuable
species in reclamation projects
VI
CONTENTS
LIST OF TABLES ix
LIST OF FIGURES middot middotmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot x
INTRODUCTION 1
LITERATlTRE REVIEW 4
Seed Donnancy 4
Physical Donnancy 5
Evolutionary Role ofPhysical Donnancy 6
Methods for Breaking Donnancy 6
Acid Scarification 6
Hot Water Scarification 8
Mechanical Scarification 11
Alternative Method for Breaking Donnancy Percussion 12
Objectives 15
Hypothesis 15
MATERIALS AND METHODS 16
Seed Collection and Cleaining 16
Experimental Design and Treatment Structure 16
Treatments ~ bullbull bullbullbullbull - bullbull ~ ~ 20
G T ennlnatlon estmg 21
X-ray Radiography 22
Scanning Electron Microscope Photography 22
Analysis 22
RESULTS 24
DISCUSSION 36
Detennining the Optimal Treatment 36
Strophiole and Seed Coat Behavior 37
CONCLUSION 44
Appendices
A PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR NEW MEXICO LOCUST AND BLACK LOCUST SEEDS 47
B GERMINATION PERCENTS AND STANDARD ERRORS FOR SOURCE BLOCK AND TREATMENT FOR NEW MEXICO LOCUST 64
LITERATURE CITED 72
Vlll
LIST OF TABLES
Table Page
1 Latitude longitude location elevation and collection date ofNew Mexico locust seed sources 18
2 Mean seed weight and standard error by source 19
3 Categorical Analysis ofVariance table for germination response to seed treatment seed source block and the interaction of the factors 25
4 Effect of seed source and treatment on G50 (days to 50 germination) 32
B1 Germination percents and standard errors for source block and treatment for New Mexico locust 65
LIST OF FIGURES
Figures Page
1 Seedsourcecollectionmap 17
2 Effect ofNew Mexico locust seed source on germination for data averaged over scarification treatments 26
3 Effect of scarification on germination for New Mexico locust for data averaged over all seed sources 27
4 Effect of seed source by treatment interaction on New Mexico locust germination for percussion scarification pattern a and pattern b 28
5 Effect of source by treatment interaction on New Mexico locust germination for hot water scarification 29
6 Timecourse of New Mexico locust germination following scarification treatments 31
7 X-ray radiographs of Sacramento seed source perc ussed for 1 4 or 8 minutes 33
8 Seed damage close-ups 34
9 SEM images of untreated vs 4-minute percussion-treated strophiolar cells ofNew Mexico locust seeds 35
10 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (b c) 38
11 Damage to New Mexico locust seeds observed during germination counts following hot water scarification 40
12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources 42
New Mexico locust a primary invader following disturbance 50
Implementation of hot water and percussion scarification 52
Effect of scarification on germination response for New Mexico locust 55
x
A4 Effect of selected scarification treatments on germination speed for New Mexico locust 56
AS Effect of scarification on germination response for black locust 57
A6 Effect of selected scarification treatments on germination speed for black locust 59
A7 Damage to New Mexico locust seeds observed during germination counts following hot water scarification 60
AS New Mexico locust seeds following percussion treatment at time of germination counts 61
A9 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (bc) 63
INTRODUCTION
New Mexico locust (Robinia neomexicana A Gray) a small tree native to the
southwestern United States occurs at elevations from 1200 to 2800 meters This
species fills a successional role in post-disturbance situations As a primary invader
New Mexico locust quickly establishes on burned areas arid flood banks as well as
road cuts (Wagner et aI 1992) Rapid growth crown sprouting and prolific root
suckering favor the successful establishment ofNew Mexico locust on disturbed sites
(Simpson 1988 Gottfried 1980) These attributes also may make New Mexico locust
well suited for steep-slope revegetation where erosion is a problem
New Mexico locust is a nitrogen-fixing legume that tolerates and improves
nutrient-poor soils Stands ofNew Mexico locust increase Nitrogen (N) and Calcium
(Ca) in the forest floor Levels of Carbon (C) Phosphorous (P) Sulfur (8) and
Potassium (K) aIso have been shown to increase in soil beneath New Mexico locust
(Klemmedson 1994)
The ability to colonize rapidly and ameliorate harsh sites contributes to New
Mexico locusts potential as a nurse plant A nurse plant colonizes an inhospitable
site and creates an environment suitable for successional plant establishment New
Mexico locust demonstrates this role in the ponderosa pine (Pinus ponderosa Douglas
ex Lawson amp C Lawson)Gambel oak (Quercus gambelii Nutt) community After a
disturbance such as wildfire New Mexico locust and Gambel oak colonize and
dominate the site until shaded out by ponderosa pine Once shaded these species
become understory shrubs a process taking an average of 15-20 years (Dick-Peddie
1993 Hanks and Dick-Peddie 1974) New Mexico locust gradually declines with
small dense patches averaging about 005 ha beneath pine until the next disturbance
once again offers it a competitive advantage (Klemmedson 1994)
Gottfried (1980) expecting ponderosa pine regeneration to be greater where
New Mexico locust had been eradicated found that survival of planted pine seedlings
was greater where locust had not been removed In a follow-up study Gottfried
(1980) noted that soil moisture in the top 57 cm was highest in 5-year old locust sites
as compared to grass or 20-year old locust sites He concluded that managing an
appropriate cover ofNew Mexico locust could help regenerate pine the later
successional species
The ability ofNew Mexico locust to improve harsh sites also makes it a
candidate for reclaiming disturbances associated with mining Black locust (Robinia
pseudoacacia 1) has been used for decades to reclaim mine spoils and other
disturbed sites in the eastern US and around the world (Keresztesi 1988 Zimmerman
and Carpenter 1980) Ashby et al (1985) described similar positive attributes of
black locust also mentioned previously for New Mexico locust quick cover for
stabilization supply ofN and nutrient-rich litter to the soil and site improvement for
establishment of later successional trees Referring specifically to mine reclamation
the authors note the ability of black locust to grow on a wide range ofmine soil
conditions including extremely acid soils Black locust also shows some tolerance to
soils compacted by grading and topsoiling practices (Ashby et aL 1985) New
2
Mexico locust holds promise as a native southwe~tem counterpart to black locust in
mine reclamation but it has been used infrequently to date
Natural invasion and succession occur slowly on most mine sites (Monsen
1984) While New Mexico locust often colonizes sites naturally there is no
assurance a seed will reach a particular site and establish in a reasonable time frame
The challenge is to introduce New Mexico locust as mother plants to facilitate
colonization Seedling production for outplanting on disturbed sites has been
hampered by poor germination (Lin et al 1996) The goal then is to develop
techniques to overcome poor germination in New Mexico locust thereby facilitating
macropropagation
LITERATURE REVIEW
Seed Dormancy
Low gennination in New Mexico locust is primarily the result of seed
donnancy the failure of an intact viable seed to genninate under favorable
conditions (Bewley 1997) Seed donnancy also can be defined as the inability of the
embryo to genninate because of some inherent constraint (Bewley and Black 1994)
The constraint may originate within the embryo referred to as endogenous or embryo
donnancy or from the tissues surrounding the embryo known as exogenous or coatshy
imposed donnancy (Baskin and Baskin 1998 Leadem 1997 Bewley and Black
1994)
New Mexico locust seed is donnant when the impenneable seed coat does not
allow movement ofwater to the embryo This specific fonn of coat-imposed
donnancy where the restraining tissue involved is an impenneable seed coat is
known as physical donnancy (Baskin and Baskin 1998 Bewley and Black 1994)
Physical donnancy is relatively uncommon in seeds less than 10 of all woody
species exhibit this condition (USDA Forest Service 1948) However it is
characteristic of many genera of the Fabaceae (bean) family For example all
successional trees in the genera Gleditsia and Robinia (of which New Mexico locust
is a member) produce seeds that exhibit physical donnancy (Baskin and Baskin
4
Physical Dormancy
Seeds that exhibit physical dormancy are said to be hard seeded referring to
the thick impermeable seed coat (Leadem 1997) The first line of protection of the
testa in leguminous seeds is the outermost waxy cuticle followed by a subcuticle
consisting ofsuberin Next is a palisade layer consisting of sclereid cells that have
thick lignified secondary walls The most common type of sclereid cell in palisade
layers is the macrosclereid The macrosclereids contain water-repellent substances
including cutin lignin suberin and wax (Rolston 1978) This palisade layer is the
primary barrier to water uptake (Serrato-Valenti et al 1993 Tran and Cavanagh
1984)
All seed coat openings must be blocked to effectively prevent water uptake
The natural opening in a seed where it was attached to the mother plant is the weak
link where physical dormancy is usually broken (Hopkinson 1993 Kuo and Tam
1988) Anatomical structures associated with the seed coat opening vary from
family to family In the Papilionoideae the subfamily of the Fabaceae family
containing New Mexico locust the site ofwater entry is the strophiole The
strophiole is a swelling on the seed coat close to the stalk scar (Hopkinson 1997)
Kelly and van Staden (1987) refer to the strophiole site as the lens where
macrosclereid cells must separate and raise before water can pass through Since
middot6germination ofdormant seeds of the Papilionoideae takes place when the strophiole
permeable (Hopkinson 1993 Kuo and Tam 1988) a logical approach to
5
genninating New Mexico locust artificially would be to loosen the strophiolar cells
and allow water to pass through to the embryo
Evolutionary Role of Physical Dormancy
Why is physical dormancy a mechanism that blocks germination
ecologically advantageous When a plant produc~s seeds with different degrees of
dormancy seeds that are dispersed at one time will germinate over a range of time
(Bewley and Black 1994) Through variability of dormancy within a seed crop seeds
are exposed to changing environmental conditions improving the overall chances of
survival of the seed line (Bewley and Black 1994) Varying expression of dormancy
is under genetic control which indicates that this may be an evolved survival strategy
(Kigel and Galili 1995) A range of dormancy levels may be a particular advantage in
the southwestern US where extreme environmental variations exist
Methods for Breaking Dormancy
There are three common approaches to breaking dormancy in New Mexico
locust seed acid scarification hot water soaks and mechanical treatments In each
case the objective of the treatment is to scarify or abrade the seed coat to make it
permeable to water
Acid Scarification
Acid scarification consists of soaking seeds in concentrated sulfuric acid
rinsing thoroughly with water upon removal followed by subsequent drying before
PlaJlltmg Olson (1974) reports that this scarification method produces the highest
middot~~jllalUll results for all Robinia species Most literature refers to a seminal 1937
6
study conducted by HG Meginnis This stud~ t~ough conducted on black locust
seed is regularly cited in the suggested use of acid to treat New Mexico locust seed
as well (Olson 1974 Vines 1960)
Thee published studies document the specific use of sulfuric acid to treat
New Mexico locust seed Cox and Klett (1984) report that soaking seeds in
concentrated sulfuric acid for thirty minutes improved germination to 34 over an
untreated control of4 Lin et al (1996) found that for two seed lots acid
scarification for twenty-five to eighty minutes produced germination of 80 A third
lot however resulted in less than 20 germination at all durations of 10 to 80
minutes Hine et al (1997) showed that acid treatment levels of 1 2 5 and 10
minutes resulted in less than 20 germination across five sources However there
was an increasing trend in germination with acid soak duration and acid treatments
were not imposed for as long a time as the previous two studies (30 to 80 minutes)
The mixed results obtained by these investigators were probably due to the
widely varying germination response of seed lots to acid treatment For this reason
Olson (1974) recommends correlating duration of acid treatment to each seed lot If
treatment duration is too long the embryo will be permanently damaged If soak
duration is too short the seed will remain impermeable to water and fail to germinate
Compounding the problem of treatment duration is the negative effect acid
have on seedling growth Any type of scarification can injure seeds to some
by disrupting cells essential for growth leading to fungal invasion and
middot-uuu~w injury (Copeland and MacDonald 1995) Some legume seeds subjected
7
to 60 minutes of sulfuric acid scarification (to achieve maximum gennination results)
showed more than 50 seedling mortality after gennination (Kelly and van Staden
1987) Bewley and Black (1994) noted that fungal growth on seeds is an indication
of leakage of sugars organic acids amino acids and proteins
Perhaps the greatest drawback to acid scarification is the dangerous nature of
the treatment itself Difficulties associated with storage and trained use of
concentrated acid may discourage many propagators from using this option (Dreesen
and Harrington 1997) Consequently hot water and to a lesser extent mechanical
scarification are more commonly used and practical treatments for legume seeds
Most southwestern container growers use either of these two treatments to scarify
New Mexico locust seed in particular (Hine et al 1997)
Hot Water Scarification
The widespread use ofhot water scarification to treat locust seed is probably
due to its ease of application and efficacy in comparison to other scarification
treatments Although this practice had been in use for some time Wilson (1944) was
one of the first to report on its systematic application for black locust The treatment
consists of placing seeds in boiling water (4 parts water tomiddot 1 part seed by volume)
removing the vessel from the heat source and allowing the water to cool and seeds to
soak for 24 hours
Hine et al (1997) Lin et al (1996) and Cox and Klett (1984) have examined
the specific application ofhot water scarification on New Mexico locust seed Each
these authors compared hot water to acid scarification Lin et aL (1996) reported
8
60 gennination for two hot water-treated seed lots while a third lot germinated
around 50 (as reported above acid scarification germination ranged from 20 to
80 for these lots) It should be noted that the authors only soaked the seeds in water
from 10 to 80 minutes for this experiment as opposed to the standard 24 hours Cox
and Klett (1984) stated that using the standard 24-hour hot water treatment improved
germination over an acid pre-treatment (which produced less than 20 germination)
but they did not give specific gennination numbers for the hot water treatment Hine
et al (1997) found that across five seed lots germination was 66 for the standard
hot water treatment (as compared to less than 20 germination for acid scarification)
Variations in treatment intensity and duration and their subsequent effects on seed lot
appear to contribute to conflicting conclusions as to whether acid scarification or hot
water treatment is a more successful treatment
Given mixed results ease ofapplication is an important argument for use of
hot water application over acid soaks Safety concerns and material costs for hot
water treatments are low (Dreesen and Harrington 1997) An extension of this is the
practicality ofhot water for treating large batches of seed where a relative increase in
water volume is all that is needed However increases in water and seed even when
inthe same 4 1 proportion can result in dramatically different rates of cooling than in
atest tube experiment Different cooling rates can lead to contrasting results for the
treatment temperature duration and seed lot (USDA Forest Service 1948)
As with acid scarification parameters ofhot water treatment application may
to be optimized to maximize germination Hine et al (1997) found that as
9
temperature was increased from 70degC to 100degC overall germination in New Mexico
locust seed increased The 100degC treatment maximized germination for each of five
sources tested with germination usually above 60
Prolonged exposure of seeds ofmany woody legumes to boiling temperatures
also may improve germination (Briscoe 1996) This approach has not been tested on
New Mexico locust seed Exposing seed to prolonged boiling temperatures may
result in fungal infection and subsequent seedling mortality Marunda (1990) noted
severe fungal infections on both acid and hot water treated Acacia holoserica Cunn
ex G Don seeds attributing the infections to seed damage resulting from the
treatments Identifying an optimal hot water treatment level that does not result in a
high rate of seedling mortality is a logical goal
With hot water treatments as with acid there is also a danger of overtreating
Seeds and reducing germination (Gosling et al 1995) The legume Leucaena
leucocephala (Lam) de Wit germinated equally well after 7 seconds of exposure to
water at 100degC 7 seconds at 90degC 30 seconds at 80degC and 4 minutes at 70degC In
addition acceptably high germination was obtained from treatment at 70degC from 3
minutes to 1 hour whereas no seeds germinated following treatment at 100degC for 2
minutes Perhaps along with a greater margin of error for treatment duration treating
~ew Mexico locust seeds at a lower temperature may result in a comparatively lower
of seedling mortality In practice however nursery growers tend to use the
and quickest treatment available The short treatment length at near-boiling
whether recommended or not may be what is used most frequently
10
Fine-tuning a treatment in this range of treatment temperature and duration may be
the most logical approach
Mechanical Scarification
This treatment involves scratching the seed coat with an abrasive material
(USDA Forest Service 1948) One system employs a rotating drum lined with
sandpaper A paddle spinning at a given number ofrevolutions per minute throws the
seed against the sandpaper (Hine et al 1997) A second method dry tumbling
tumbles seed in a mixer with pea gravel or sharp sand for 5 to 7 days (Dreesen 2001)
These approaches have the advantage that control of temperature is not required as in
water treatments nor is safety an issue as with add scarification
Hine et al (1997) mechanically treated New Mexico locust seed using 100shy
grit sandpaper in a Forsberg (rotating drum) scarifier Gennination was less than
10 They concluded that the seed coats were too thin for their apparatus and
proposed that a finer grit sandpaper would produce more even abrasion Although
germination varied by seed lot Dreesen (2001) reported that using a slow-speed dry
tumbling technique for 5 to 7 days produced gennination rates as high as 90
As with acid and hot water treatments treatment duration must be specifically
shy
correlated to seed lot Treated seeds are also susceptible to fungal infection
iHopkinson 1997) In addition equipment is specialized and may not always be
ltU~CUJl~ to a nursery (USDA Forest Service 1948) Several studies have confinned
degrading ofstrophiolar cells due to acid and hot water scarification (Miklas et al
11
1987 Dell 1980 Hanna 1984) but the effect ofmechanical scarification on the
strophiole has not been studied
Alternative Method for Breaking Dormancy Percussion
Strophiole degradation has been examined in depth using an impaction
treatment more commonly known as percussion Hamly (1932) first proposed that
shaking or impacting seeds could split the macrosc1ereid cells at the strophiole
Hamly successfully released tension ofbent strophiolar cells of impermeable
Melilotus alba Medik (White sweet clover) seeds by delivering physical blows
resulting in cells that had split apart and were only slightly bent Ninety-two percent
oftreated seeds became permeable as compared to 05 of control seeds Barton
(1947) showed that percussion significantly increases permeability in black locust
seed Ballard (1976) reasoned that the integrated nature of the strophiole within the
seed coat means a blow on any part of the seed weakens the strophiole There are no
published studies on the use of percussion on New Mexico locust
Percussion methods vary and often seem SUbjective or ill-defined Hamly
(1932) placed seeds in a glass bottle and shook them three times a second for 20
minutes resulting in 180 oscillations per minute Ballard (1973) describes shaking
seeds in a closed metal container with at least fifty seeds per container at 850-900
JS(~l11alIOIlS per minute for 1 minute Kelly and van Staden (1987) placed seeds into
sealed glass flasks Flasks were shaken by a wrist-shaker at a rate of 250
~UUVJ~ per minute Subsequent germination rates were 45 after 2 hours of
55 after 3 hours 60 after 6 hours and 100 after 12 hours
12
There are several potential advantages of percussion over acid hot water and
mechanical scarification Kelly and van Staden (1987) note that a percussion
treatment only alters macrosclereid cells of the strophiole by loosening separating
and raising them They emphasize that minimal damage is done to the overall
anatomical structure of the seed In acid scarification of the same seed lot radicle
and cotyledon damage occurred Based on photographic evidence the authors
believe that this morphological damage was due to uneven pressure during
imbibition with water entering haphazardly wherever the acid had sufficiently
dissolved the coat This ultimately led to uneven hydration of the embryo In
percussed seeds water only entered through the s~ophiole when the entire seed coat
had been saturated resulting in controlled and even embryo hydration (Kelly and van
Staden 1987)
Although all successful scarification treatments may focus at least in part on
the strophiole for papilionoid legumes (Baskin and Baskin 1998) percussion may be
less damaging to seed coat structures particularly when compared with acid Kelly
and van Staden did not compare hot water or mechanical scarification methods
against percussion However the fungal infection associated with hot water and
mechanical scarification as with acid scarificatio~ have been directly associated with
seed damage (Rehman et al 1998 USDA Forest Service 1948) Embryo damage in
has been observed in hot-water scarified legume seeds (Rehman et al 1998
13
By limiting embryo damage percussion scarification reduces seedling
mortality compared to other treatments Kelly and van Staden (1987) noted that
seedlings from the percussion treatment were morphologically sound and vigorous
when compared to acid treatment seedlings The authors attribute this improved
seedling vigor to the controlled water uptake that limited anatomical damage
Despite a 12-hour percussion treatment morphological damage was not substantially
greater than untreated seeds In addition seedling mass measured 10 days following
germination was not significantly different between percussed and untreated seeds
A final advantage ofpercussion may be the ability to adequately treat the most
dormant seeds while avoiding harm to seeds that have already been made permeable
by the treatment or that were permeable to begin with Instead ofcorroding scalding
or scratching the seed coat percussion appears to be just a loosening of the
strophiole spring (Ballard 1973) Kelly and van Staden (1987) were able to
increase germination with a lengthy treatment (12 hours) Figuring out whether a
substantial cushion oftreatment durations exist for percussion ofNew Mexico locust
seed would be a significant improvement to current scarification methods where seed
damage and seedling health is an issue
The greatest challenge for applying percussion to New Mexico locust seeds is
standardizing a treatment that is both effective and readily reproducible by nurseries
with mechanical scarification this may require the development of specific
~UllDment to properly quantify and execute the treatment
14
f
Objectives
The first objective of the study is to detennine which scarification treatment
results in optimal gennination speed and percentage ofNew Mexico locust The
second objective is to describe treatment interactions with nine seed sources collected
in New Mexico
Hypothesis
A percussion treatment acts exclusively on the main area ofwater uptake in
New Mexico locust seed loosening the strophiole so that imbibition can occur This
direct mechanism ofdonnancy release will result in improved gennination rate and
overall gennination percentage for New Mexico locust seed compared to standard
treatments Standard treatments including acid hot water and mechanical
scarification while eventually weakening the strophiolar cleft cause excessive
morphological damage through generalized seed coat degradation These treatments
are therefore prone to inconsistency due to over and under-treatment
15
MATERIALS AND METHODS
Seed Collection and Cleaning
Seeds were collected from August 25 through September 15 2000 from nine
locations along a latitudinal gradient in New Mexico Seed source locations and
descriptive information are given in Figure 1 and Tables I and 2 Stands were
separated by at least 45 km
Pods were hand picked as they began to ripen and dehisce (open) Collections
were made from ground level to approximately 3 m from several trees at each site
Pods were dried in open-ended brown paper bags for two to four weeks at room
temperature (21-230C) to facilitate cleaning Dried pods were placed in a closed cloth
sack which was pounded against the ground to separate seeds from pods For pods
still containing seeds hand maceration (crunching pods with hands) completed seed
separation Debris was removed by threshing with the aid of a greenhouse fan A
South Dakota Seed Blower (Seedbureau Chicago Illinois) was used to remove
remaining chaff and light seeds by air density separation Seeds were placed in dry
storage in sealed quart-size zip-lock bags within a sealed plastic container Storage
temperatures ranged from 2-4oC Seeds remained in storage for approximately 7
months before the experiment
Experimental Design and Treatment Structure
The experiment compared the impact ofhot water bath and percussion
l11aLl11 on germination properties ofNew Mexico locust seed A single
16
-l
Figure 1 Seed source collection map All sources were collected within New Mexico USA separated by at least 4S lan
IIIRZltItW lIJPIirmiddot rllll1lbullbull_~~~_II
Table 1 Latitude longitude location elevation and collection date ofNew Mexico locust seed sources
Lot title Latitude (N) Longitude (W) Location Elevation (m) Collection Date
Raton 36deg5846 104deg2872 Raton Pass NM 2295 82500
Holman 36deg0306 105deg2429 HolmanNM 2476 911500
Jemez 35deg4500 106deg4100 Jemez Springs NM 1976 9900 00
Sandia 35deg1083 106deg2341 Cedar Crest NM 2432 82600
Manzano 34deg3689 106deg2494 ManzanoNM 2465 9200
Magdalena 33deg5953 107deg0903 Magdalena NM 2544 82900
Capitan 33deg2728 105deg4325 Alto NM 2189 911200
Sacramento 32deg5798 105deg4496 Cloudcroft NM 2630 91400 Gila 32deg5400 107deg4600 Kingston NM 2432 9700
f
Table 2 Mean seed weight and standard error by source
a
Source Mean seed weight (g) for Standard error 100 seeds n = 5
Raton Holman Jemez Sandia Manzano Magdalena Capitan Sacramento Gila
2872 b c 2522 d 1826 e 2753 c 2413 d 2559 d 2443 d 3130 a 3029 ab
00103 00403 00276 00301 00370 00374 00458 00279 00348
a Mean seed weights followed by the same letter are not significantly
different
19
experiment was conducted utilizing a randomized complete block design blocked by
time with a factorial treatment structure Experimental factors were seed source and
scarification treatment Nine seed sources from throughout New Mexico were used
(Figure 1 Table 1) Scarification treatments included a control three levels ofhot
water and four levels of percussion Hot water treatments included immersion at
100degC and boiling for periods of 30 seconds and 60 seconds Percussion treatment
levels were 1 2 4 and 8 minutes One hundred seeds per block were used to test
each treatment combination Four blocks of seed were tested one at a time (all seed
sources included in each run) in close chronological sequence (within 6 weeks) A
total of four 100-seed samples were used to test each source by scarification treatment
combination
Treatments
Hot water treatment of seeds utilized 50 ml test tubes filled with 30 ml
distilled water Each test tube was used to treat one 100-seed sample at a time
resulting in a ratio of4 parts water to 1 part seed by volume Test tubes were placed
in a beaker with the beaker filled with tap water to the same level as the water within
test tubes Nine test tubes (one per seed source) were placed in the beaker
simultaneously so that one replication of each seed sourcescarification treatment
cotnblnatlon was conducted simultaneously Hot water treatments include immersion
boiling For immersion treatments seeds were added to test tubes with the water
(boiling) and the test tubes were immediately removed from the beaker to
For the boiling treatments test tubes remained in the beaker with water at
20
boiling temperature for a set duration after the addition of seeds Following all
treatments test tubes with seeds cooled 24 hours in a test tube rack
Percussion treatment utilized a pneumatic paint shaker (Central Pneumatic
model 00422 Camarillo CA) Compressed air was maintained at 530 kPa plusmn30 kPa
(80 psi plusmn 5 psi) resulting in approximately 350 oscillations per minute Seeds were
placed in 118 ml (4 oz) soil tin cans (US Can Lobard IL) and the lids were secured
with duct tape Each can was used to treat one 100-seed sample To take advantage
ofgreatest lateral movement of the paint shaker a spacer (paint can) was used to
position the soil tin at the end of the shaking arm This allowed shaking to occur at
the greatest distance from the pivot point (fulcrum) Tins were placed perpendicular
to the direction of shaking motion allowing seeds to impact at greatest force by
hitting flat surfaces of the soil tin
Germination Testing
Before gennination testing all seeds were soaked 24 hours in reverse
osmosis-treated water (this was included in the process ofhot water treatment
jU~SCIlOea above) Following soaking seeds were placed in 100 cm Petri dishes lined
90 cm Whatman qualitative 1 filter paper circles (MG Scientific Pleasant
WI) One Petri dish was used for each treatment by source combination
papers were initially moistened with 5 mL reverse osmosis-treated water then
~VU1CUlJl moistened as needed to ensure adequate conditions for gennination
were monitored daily (at 300 PM) for gennination for 14 days in accordance
~UIUlnJlVUa Seed Testing Association (ISTA) standards for Robinia species
21
(1STA 1999) Tests were carried out at room temperature (22degC plusmnOSoC)
Germination was classified as protrusion of the radicle from the embryo by at least
1mm as viewed with the naked eye
X-ray Radiography
On August 132001 three lOO-seed samples from the Sacramento seed source
were treated for 14 or 8-minute percussion durations The seeds were sent
immediately by overnight delivery to the Faxitron X-Ray Corporation in Wheeling
Illinois where Dr Bob Karrfalt ofthe USDA Forest Service oversaw their imaging
Radiographs were made at SX with a Faxitron Specimen Radiography System Model
MX-20
Scanning Electron Microscope Photography
On February 192002 a Hitachi S-3200N Variable Pressure Scanning
Electron Microscope (Mountain View CA) was used to examine strophiolar cells of
untreated seeds and seeds percussed for 4 minutes Seeds were cut longitudinally
igt their entire length using a razor blade splitting the strophiolar region roughly in
Cross sections of seed samples magnifying the strophiolar region were viewed
600X under the microscope
Analysis
Categorical Analysis of Variance was used to determine treatment source and
(Le time) main effects and interactions The response variable was total
~vlI This procedure is a Chi-square test of homogeneity using the natural log
of germinated to non-germinated seed for each treatment Maximumshy
22
likelihood analysis was used to calculate Chi-square test statistics with their observed
significance levels A single contrast was conducted to cempare treatment means
against the control Observed significance levels less than or equal to 005 were
considered significant Pairwise comparisons were conducted between non-control
treatment mean percents using a conservative alp~a value of 005 divided by the
number of comparisons (Bonferroni approach) and approximate pairwise z statistics
Categorical analyses were performed using Proc Catmod (SAS Institute 1989)
RESULTS
Scarification seed source block and all interactions between factors
influenced germination (Table 3) The main effects are reported in the presence of
these interactions because the overall robustness of treatments and seed sources are of
interest Background levels ofnaturally occurring variation in seed sources are
seen in control germination percentages in Figure 2 In particular time variability
is unlikely to be under the control of the producer Appendix B details experimental
data for source by treatment gennination percents for each of the four blocks
Averaged over all treatments germination by source ranged from 40 to
79 (Figure 2) Averaged over all sources (Figure 3) and for each source (Figure 4
5) percussion scarification durations of4 or 8 minutes yielded greatest germination
Averaged over all sources germination increased with increasing durations of
percussion then leveled off for the 4- and 8-minute treatments (Figure 3) Immersion
and 30-second boiling scarification maximized germination for the hot water
treatments with a decrease in germination when seeds were boiled for 60 seconds
For all but two sources there is a similar ipcreasing trend in germination from
1to 4 minutes ofpercussion (Figure 4) The Holman source increased in germination
1 to 2 minutes then leveled off at 4 minutes The Jemez source changed little
1 to 4 minutes of treatment As treatment duration increased from 4 to 8
mutes five sources decreased in germination (Figure 4a) whereas three sources
24
Table 3 Categorical Analysis of Variance table for germination response to seed treatment seed source block and the interaction of the factors
Source of DF Chi-Square Observed Variability Significance Level
Source 8 98370 lt00001 Trt 7 233876 lt00001
contrast 1 16976 lt00001 SourceTrt 56 97077 lt00001 Block 3 5538 lt00001
SourceBlock 24 10162 lt00001 TrtBlock 21 7564 lt00001
SourceTrtBlock 168 34283 lt00001
S 4)
8 ~
0
sectg ~ 40 ~
N 0 0
20
o f0ltgt ~ ~4 jybshy ((iltgto ~~ K1ltgt 6~b-~ ~~ 0b-~~ ~~ ~b-~ ~b-~b
80
60 -I-- - ~- middotmiddotmiddotmiddotmiddot1
r -] Northern NM Sources
Central NM Sources
Southern NM Sources
sect-o ~
~b-v(i
SOURCE
Figure 2 Effect ofNew Mexico locust seed source on germination for data averaged over all scarification treatments Control germination is shown by solid line within bar
80
~ cd ~ ltl) () 60 ~ ~
N
t 0shyrti tshySltl)
40
0
20
o lmmersion Boiling 30 sec Boiling 60 sec Control 1 min 2min 4 min 8 min
Hot Water Treatments Percussion Treatments
Figure 3 Effect of scarification on germination ofNew Mexico locust for data averaged over all seed sources Means labelled with the same letter are not significantly different
100~----------------------~--~----------------~
80
60
ltU
~
40 IJ
sectgsI
l100
80
60
40
a I middotmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot middotImiddotmiddotmiddotmiddotmiddotmiddot E
_~iii-bull _ _ bull f --~--
t-- -~ ---- -shy ---- shyI ~ ---- - shy
L
fiII
~
b
- shy ---r
- Holman
Sacramento Sandia Gila Jemez
c ~
ltgt ~
Tmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot-I
j~=~=-~~-1
I middotmiddot ~ bull r
t tr
Y -------M-a-gd-a-Ie-n-a--
1 _ _
fCapitan
---- Manzano ~y -_ Raton
2 4 8
Time interval ofpercussion scarification (min)
Figure 4 Effect of seed source by treatment interaction on New Mexico locust germination for percussion scarification pattern a and pattern b
28
tv 0
~ d
~ A s o
1 c3
80
60
40
20
o
TIT
t------=middotfmiddotmiddot-middotmiddot-middotmiddot-middotmiddot 1- --- - - --=- - -shy--cE= - _--shy --shy ----shy ~ ~ ---shy ~ -~- _ --shy-------r 1
I
Raton Holman
-_ Jemez
--- Sandia Manzano Magdalena Capitan Sacramento Gila
Immersion Boiling 30 sec Boiling 60 sec
Hot Water Scarification
Figure 5 Effect of seed source by treatment interaction on New Mexico locust germination for hot water scarification
continued to increase in germination (Figure 4b) There was no difference between
the two treatment levels for the Manzano source (Figure 4b)
The effect ofhot water scarification also varied among sources with few
overall differences between hot water treatments (Figure 5) However boiling for 60
seconds yielded highest germination for the Jemez source whereas increasing
durations ofboiling decreased germination for the Raton source
In addition to consistently high germination 4- and 8-minute percussionshy
treated seeds germinated as fast or faster than all other treatments (Figure 6 Table 4)
050 measured as the number of days needed to complete 50 ofeventual
germination averaged 2 days for both ofthese treatments
X-ray radiographs were taken of seeds collected from the Sacramento source
percussed for 1 4 or 8 minutes (Figure 7) Seeds show signs of damage at 8 minutes
ofpercussion (Figure 8) Compared to an undamaged seed percussed for only 4
minutes (Figure 8a) damage at the 8-minute level includes fractured radicles pitting
of the seed coat and chips in seed coat structure (Figure 8b-d)
Scanning electron microscope (SEM) images at 600X reveal the direct effect
of 4 minutes of percussion on cells of the strophiolar region (Figure 9) Whereas
untreated seeds retain cellular organization in the strophiolar region strophiolar cells
seeds treated for 4 minutes ofpercussion separate and raise resulting in a
~-~e~U appearance
30
Umiddot-~middot--l-~---middotymiddotmiddotmiddot-----middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot--middot--middotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middotmiddotmiddotmiddotmiddotmiddotmiddot- -
050= gt1
-e-- Percussion 8 min -0- Percussion 4 min ---T- Percussion 2 min -----ltil- Percussion 1 min -- Immersion --0- Boiling 30 sec -+- Boiling 60 sec -ltgt- Control
2 4 6 8 10 12
Day Figure 6 Timecourse ofNew Mexico locust gennination following scarification
treatments G50 is the number of days to 50 of eventual germination
31
14
Table 4 Effect of seed source and treatment on G50 (days to 50 germination)
Seed Source Control Immersion B30sec B60sec Plmin P2min P4min P8min mean Raton 3 3 5 7 4 3 2 2 4 Holman 2 2 2 2 2 2 2 2 2
Jemez 4 6 6 5 3 2 2 2 4 Sandia 2 2 2 2 2 2 2 2 2
w tv Manzano 5 2 2 3 3 2 2 2 3
Magdalena 4 2 2 3 4 3 2 1 3
Capitan 5 4 2 2 3 2 2 2 3
Sacramento 3 2 2 2 4 3 2 2 3 Gila 3 2 2 2 2 2 2 2 2 mean 3 3 3 3 3 2 2 2 3 standard 11 14 16 18 09 05 0 03 08 deviation
Note Columns and rows may not agree due to rounding
Figure 7 X-ray radiographs ofSacramento seed source percussed for 1 4 or 8 minutes
33
Figure 8a-d Seed damage close-ups Figure a shows undamaged seed percussed for 4 minutes Figures b-d show seeds percussed for 8 minutes Damage includes fracturing ofradicle in b complete removal ofseed coat in c and pitting ofseed coat in d
34
Untreated Treated
Figure 9 SEM images ofuntreated vs 4-minute percussion-treated strophiolar cells ofNew Mexico locust seeds Treated cells are loosened separated and raised
DISCUSSION
Determining the Optimal Treatment
Averaging results of scarification treatmems across seed sources is a useful
tool to predict which treatment is most likely to be successful for an unknown seed
source Averaged over all sources four- and eight-minute percussion treatments
resulted in the highest germination 815 and 790 respectively and produced
germination that was as fast or faster than all other treatments Although there was no
difference between these two treatments on average an argument for 4 minutes of
percussion as a standard protocol for treating New Mexico locust seeds can be made
Descriptively 4 minutes of percussion improved germination over 8 minutes
ofpercussion for a majority of sources (five out of nine) 8 minutes of percussion
improved germination for three ofnine sources with no difference for a fmal source
For individual sources 4 minutes ofpercussion was always the best or secondmiddot best
treatment and was always equal to or better than all hot water treatments
Germination at 8 minutes ofpercussion was lower than all other percussion durations
for the Jemez and Gila sources and was lower than at least one hot water treatment for
these sources as well
The x-ray radiographs of the Sacramento seed source illustrate the excessive
internal and external seed damage that can occur with 8 minutes of percussion
germination counts 8-minute percussed seeds from Holman and Capitan
ClUll signs of damage that were visible to the naked eye (see below)
r~evertheless some of these seeds still fit the criteria for germination Neither x-ray
36
photography nor visible observations during seed counts revealed seed damage at the
4-minute percussion level In operational trials seeds percussed for 4 minutes
developed into healthy seedlings (Khadduri and Harrington unpublished data)
Strophiole and Seed Coat Behavior
Four minutes ofpercussion successfully breaks seed dormancy in a large
percentage ofNew Mexico locust seeds without excessively damaging the
surrounding seed coat or internal seed coat structures Most papilionoid legume
seeds including seeds ofNew Mexico locust have a specific region of the seed coat
known as the strophiole or lens (Hamly 1932) The strophiole is located on the
cotyledonary lobe of a locust seed (Figure 10 a-c) If seeds are not indiscriminately
damaged the strophiole is the first point of water entry to the embryo (Hopkinson
1997) When a papilionoid seed is percussed for the appropriate amount of time
repeated hits on the integrated seed coat loosen the constrained cells of the strophiolar
region without excessively damaging the rest of the seed coat (Ballard 1976) SEM
photos illuStrate that 4 minutes of percussion loosened and separated strophiolar cells
ofNew Mexico locust seeds when compared with constrained organized strophiolar
cells of untreated seeds (Figure 9) Research-has shown that water passes to the
embryo through the treated strophiole in a controlled manner and regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues
(Kelly and van Staden 1987)
37
10 Strophiole ofunimbibed New Mexico locust seed (a) and following uptake (bc)
38
In contrast hot-water treatments can excessively degrade the seed coat
(Rebman et al 1998 Marunda 1990) Random cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rebman et aI 1998 Marunda 1990) Along with a
proportionately large amount ofun-imbibed (thus under-treated) seeds (Figure l1a)
damage was observed in hot-water treated seeds from six sources during germination
counts Damage included seeds where the radicle failed to elongate (Figure 11 b)
seeds where the radicle elongated but was delayed in freeing itself from the seed coat
(Figure 11c) and seeds where the radicle completely broke off from the embryo
(Figure lId) In addition some seeds germinated in reverse (the cotyledon emerged
fIrst) If the radicle emerged completely from the seed coat they were counted as
germinated
While nearly all seeds treated for 8 minutes ofpercussion imbibed water
damage was evident in x-ray evaluations (Sacramento seed source) and in
germination counts (Holman and Capitan) at this treatment level Seed damage
during germination counts was similar to levels found in hot-water treated seeds
suggesting that over-treatment by both hot water and longer durations of percussion
may result in similar damage to seed structure
The Sacramento and Holman seed sources were two of the fIve sources that
opcrplltlpn in germination from the 4- to 8-minute durations of percussion
the benefIts ofloosening the strophiole as evidenced by imbibition of
all 8-minute treated seeds were outweighed by external and internal seed coat
39
a
b c d
11 Damage to New Mexico locust seeds observed during germination cotmts following hot water scarification 9a Hot water replicate including ~t seedlings and numerous unimbibed seeds 9b Close-up of seed where raallCle failed to elongate 9c Close-up ofseed where radicle elongated but was
-gtIn in freeing itself from the seed coat 9d Close-up ofseed where radicle broke off from embryo
40
damage (which also may be contributing to high imbibition) For the three sources
for which 8 minutes of percussion resulted in maximum germination extended
percussion of seeds necessary to loosen the strophiole may not have excessively
damaged the seed anatomy The Manzano source did not significantly change in
germination from 4 to 8 minutes of percussion suggesting that an increase in
permeable seeds at the 8-minute treatment duration was balanced by the number of
seeds that failed to germinate due to seed damage To summarize the 4-minute
percussion treatment improved germination over 8 minutes ofpercussion for a
majority of sources (five ofnine) the 8-minute percussion treatment improved
germination over 4-minutes ofpercussion for three sources and there was no
difference with the Manzano source Notably for the Raton source 8 minutes of
percussion increased germination by 235 percentage points over 4-minutes of
percussion (7875 vs 5525 respectively) the next best treatment Raton was the
only source to result in germination less than 75 for 4 minutes ofpercussion so a
grower may wish to apply longer percussion durations to lots that do not respond well
to this treatment level
Finally there was an interesting trend in the Jemez and Raton sources seed
that significantly contributed to the source by treatment interaction (Figure 12) A
~u test showed that the Jemez source had a significantly lighter seed weight than
other sources while the Raton source had an average seed weight For the Jemez
the longest duration of boiling increased germination Conversely only short
U4UVll) of percussion were needed to achieve high germination and the longest
41
80 _ Jemez a Raton
iD
~ C iD e iD
Q c 0 ~
c ~ iD 0
20
0------shy
Immersion Boiling 30 sec Boiling 60 sec
Hot Water Scarification
10degTr========~--------~------------------------------
80
60
40
20
_ Jemez Raton
0------shy
1 min 2 min 4 min 8 min
Percussion Scarification
Figure 12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources
42
b
--~----~~~~========~---- duration ofpercussion reduced germination For the Raton source longer durations
ofboiling consistently decreased germination whereas longer percussion treatments
consistently increased germination
A possible explanation is that a thick seed coat is associated with a loose
strophiole structure and that a thin seed coat is associated with a constrained
strophiole This contrast merits further study and may suggest an ecological
adaptation Further investigation ofthis phenomenon may contribute to successful
pre-screening ofunknown seed sources to determine optimal treatment leveL
43
CONCLUSION
Based on results obtained in pilot study (see Appendix) and thesis
experiments 4 minutes ofpercussion is recommended as a standard protocol for
treating an unknown source ofNew Mexico locust seed In addition to laboratory
results the 4-minute percussion treatment has resulted in germination greater than
80 and healthy seedling grow-outs for production purposes (unpublished data) In
laboratory experiments only one hundred seeds per 118 ml (40z) soil tin were
percussed at a time whereas for production we have treated up to five hundred seeds
per tin For large-scale production ofNew Mexico locust seedlings growers may
wish to experiment with larger quantities of seed and larger-size soil tins
Seed storage trials following percussion treatments were not conducted
However germination greater than 80 and healthy seedlings were obtained when
seeds were percussion-treated and subsequently stored at room-temperature (22degC)
for one week (unpublished data) Seed storage trials merit further study as the
standard hot water treatment leaves seeds in a softened condition that is not
conducive to storage
Hydroseeding is an effective method for direct seeding in reclamation
situations Percussion treatment may be conducive to hydro seeding as seeds remain
a dry relatively undamaged yet permeable condition following treatment Sulfuric
seeds also remain dry following treatment but extensive damage to seed coat
poor seedling vigor have been reported with this treatment (Kelly and van Staden
A grow-out study using a hydro seeder on a reclamation site testing percussion
44
against other treatments such as sulfuric acid and mechanical scarification also
merits further study
Papilionoids are the largest subfamily oflegumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust these legumes often play an integral role in the revegetation of
disturbed lands The pilot study (Appendix) indicates that percussion scarification
successfully treats the widely used black locust as well Developing superior
scarification methods to the standard hot water mechanical scarification and acid
treatments may facilitate the use ofpapilionoid legumes in reclamation projects
45
APPENDICES
APPENDIX A
PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR
NEW MEXICO LOCUST AND BLACK LOCUST SEEDS
Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4
Hot water and sulfuric acid soaks are traditional treatments for seeds of many
temperate woody legwnes including locusts However these scarification techniques
often produce inconsistent germination Percussion scarification where seeds are
repeatedly propelled against a hard surface was compared with hot water
scarification to evaluate treatment efficacy for New Mexico locust (Robinia
neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment
seeds were placed in a 98degC water bath which was immediately removed from the
heat source For percussion scarification seeds were placed in a soil sample tin and
agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the
control were followed by 24-hour water soaks Hot water baths resulted in 56 and
41 germination for New Mexico locust and black locust respectively For both
species nearly all durations ofpercussion increased germination over the hot water
treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3
4 and 5 minutes for black locust resulted in at least 90 germination Traditional
scarification treatments randomly degrade the entire seed coat which can lead to
tissue damage during water uptake Percussion scarification specifically weakens the
strophiole the natural source ofwater entry to the seed in papilionoid legwnes
INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom
2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031
48
Following percussion imbibition is controlled through the strophiole and underlying
tissue is protected
Keywords New Mexico locust black locust scarification percussion germination
Introduction
Locust trees (Robinia spp) are aggressive pioneer species that quickly
colonize disturbed land fix atmospheric nitrogen in the soil and stabilize slopes that
are prone to erosion (Klemmedson 1994 Ashby et al 1985) (Figure A1ab)
Recognition of these attributes has contributed to increased demand for locusts in
reclamation projects Locust seeds however exhibit physical dormancy where a
thick seed coat prevents the movement of water and gasses to the embryo (Leadem
1997)
Sulfuric acid or hot water soaks have traditionally been used to break seed
dormancy in macro-propagation of locusts Many growers are moving away from the
use of su1furic acid in treating locust seeds (Dreesen and Harrington 1997) In
addition to being dangerous acid soak durations must be specifically correlated to
seed lot (Olson 1974) Collections of native seed can vary tremendously in seed size
seed weight and hard seededness etc This variability limits the utility of sulfuric
acid soaks as even within a given lot some seeds may be damaged whereas others
are under-treated and fail to imbibe (take up water) A survey of Southwestern
container growers indicates that hot water baths are currently the most common
scarification method for locust seeds (Hine et al 1997) However hot water baths
also produce inconsistent germination (Lin et al 1996)
49
b
Figure AI New Mexico locust a primary invader following disturbance Ala Crown-sprouting at edge ofburn near Los Alamos NM--one year after Cerro Grande fire Alb Colonizing road cut Gila National Forest NM
50
Percussion scarification where seeds are repeatedly propelled against a hard
surface is an alternative dormancy-breaking method that has proven successful in
legumes related to locusts (Hamly 1932 Barton 1947) We compared this alternative
treatment to hot water scarification to evaluate treatment efficacy for New Mexico
locust and black locust seeds
Materials and Methods
Commercial New Mexico locust seeds (Western Native Seed Coaldale CO)
were collected fall 2000 in Huerfano County CO Black locust seeds were collected
September 2000 from Taos County NM by harvesting ripe pods from several trees at
a distance of up to 3 m from ground level Black locust seeds were allowed to air-dry
for 3 weeks and were threshed and separated from large chaffusing a greenhouse
fan A South Dakota Seed Blower (Chicago Illinltois) was used to remove fine chaff
from both seed lots Seeds were storedmiddotat 2-4C until the germination study was
conducted in March 2001
This study compared hot water scarification with percussion scarification
using treatment and species as experimental factors Along with a control
scarification treatments included immersion of seed in boiling water as well as
percussion durations of 1 2 3 45 or 10 minutes Four 100-seed samples were used
to test each species by scarification treatment combination
For the hot water treatment test tubes were filled with 30 mL water and
placed in a water-filled beaker (Figure A2a) The bath was raised to boiling (-98C at
51
b
c
Figure A2 Implementation ofhot water and percussion scarification A2a Hot water bath A2b Paint-shaker set-up A2c Placing soil tin containing seeds at end ofpaint shaker ann
52
Las Cruces NM elevation of 1300 meters) Each 100-seed replicate was placed in a
test tube and each test tube was immediately removed to cool at room temperature
Percussion scarification was implemented using a pneumatic paint shaker
(Central Pneumatic model 00422 Camarillo CA) (Figure A2b) This allowed
standardization of treatment intensity Compressed air pressure was maintained at
530kPa plusmn 30kPa (80psi plusmn 5psi) resulting in approximately 350 oscillations per
minute Each lOO-seed sample was placed in a 118 ml (4 oz) soil tin for shaking To
maximize lateral movement of paint shaker a paint can was used as a spacer to place
soil tins at the end of the shaking arm (Figure A2c) This allowed shaking to occur at
the greatest distance from the pivot point (fulcrum) Following all treatments
including the control seeds were soaked for twenty-four hours in distilled water prior
to plating out
Gennination was evaluated on lab benches at room temperature Each
replicate of seeds was placed in a 100 cm petri dish on moistened filter paper with
humidity maintained by enclosing the petri dishes in ziplock bags Gennination was
monitored daily for 14 days in accordance with International Seed Testing
Association standards for Robinia species (ISTA 1999) Gennination was defined as
protrusion of the radicle from the embryo by at least 1mm as viewed by the naked
eye
Categorical Analysis of Variance using Prpc Catmod (SAS Institute 1989)
was used to detennine treatment differences in germination percentages for each
source This procedure is an extension of a Chi-square test of homogeneity using the
53
natural log of the ratio of germinated to non-germinated seeds for each treatment Pshy
values less than 005 were considered significant A limited set of pairwiseshy
comparisons was conducted to compare treatment mean percents using a
conservative alpha value of 005 divided by the number of comparisons
Results
Percussion maximized germination for both New Mexico locust and black
locust There were three germination response levels for New Mexico locust (Figure
A3) First there was a low response in the control indicating a dormant seed lot
The hot water treatment and I-minute duration of percussion share a second
intermediate response Germination was highest at percussion levels from 2 to 10
minutes
A selected percussion treatment intermediate in time (5 minutes) produced the
steepest germination curve indicating the fastest germination rate (Figure A4) The
hot water treatment produced an intermediate curve or relatively slower germination
rate The control curve was very flat indicating the slowest germination rate for New
Mexico locust
Control seeds germinated poorly for black locust as well (Figure A5) Hot
water scarification improved germination over the control but all levels of percussion
improved germination over hot water The optimal durations of percussion treatment
were bracketed with a significant increase from the 1 to 2 minute percussion level a
high germination response from 2 to 5 minutes of percussion and finally a significant
54
~ d 25 CL)
p t1 0 ~ t1
g Vl ~ Vl
100~---------------------------------------------------------------
80
60
40
20
o Control Hot Water Percussion
I min Percussion
2 min Percussion
3 min
Scarification
Percussion 4 min
Figure A3 Effect of scarification on germination response for New Mexico locust
Percussion 5 min
=significant difference between adjacent treatment bars ns = no significant difference
Percussion 10 min
~ ~ () u agt
Il-t d o ~
~ ~ c3
100middotlr~~~------------------------------------e- Percussion 5min middot 0middot Hot Water -- Control
80-J=middot======
6b-middot-lmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middot--middotmiddot-middot~-middotmiddot -middotmiddot middot middotmiddot-middot 1
40
20
-----~-----------
0 0 0 middotmiddotmiddotmiddotmiddot0middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot0
0 --~-----y-----~------_---~--- T-----~
o shy -TDay 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A4 Effect of selected scarification treatments on germination speed for New Mexico locust First seven days of fourteen-day experiment are shown
Day 7
80
~ cd
5 60 ()
[) Cl d 0 1t1 d
40sect dUl
-J
20
o
ns
Scarification
Figure A5 Effect of scarification on germination response for black locust = significant difference between adjacent treatment bars ns = no significant difference
decrease at the 10-minute level This drop-off suggests that 10 minutes of percussion
over-treats seeds from this lot of-black locust
A selected percussion treatment intermediate in time (5 minutes) also had the
fastest germination rate for black locust (Figure A6)_ The hot water curve is
somewhat flatter indicating a slower germination rate The control curve is very flat
indicating a slow germination rate
A qualitative difference between hot water and percussion-treated seeds was
also observed Along with healthy germinants the hot water treatment produced
over-treated seeds (Figure A 7a) Examples of damaged germinants include seeds
where the radicle did not elongate (Figure A 7b) seeds where the radicle elongated
but was delayed in freeing itself from the seed coat (Figure A7c) and seeds where the
radicle broke off completely from the embryo (Figure A7d) In addition to these
examples of over-treatment there were numerous small dark seeds that failed to
imbibe water For percussion treatment nearly all seeds took up water (Figure A8a)
Germination was rapid and uniform with little damage evident (Figure A8b)
To summarize both New Mexico locust and black locust seed lots were
dormant The standard hot water scarification treatment improved germination but
only to 56 and 41 for New Mexico locust and black locust respectively Nearly
all percussion durations improved total germination as well as germination rate over
the hot water treatment Germination was greater than 90 for percussion durations
of 4 5 and 10 minutes for New Mexico locust and 3 4 and 5 minutes for black locust
58
CI)
sgt t CI) 0
igt 0
sect ttl t E
Vl 0
c3
100r==~==~~=r------------------------------------~ ___ Percussion 5 min
middotmiddotmiddot0middotmiddotmiddot Hot Water -lIf- Control
80r=========~--~~----------------------------~
60
40
20 -~-~---~---------- ---~------------I
omiddot
O
_~_----------lIf-----~bull -----_ shy---shy I
Day 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A6 Effect of selected scarification treatments on germination speed for black locust First seven days of fourteen-day experiment are shown
Day 7
a
b c d
Figure A7 Damage to New Mexico locust seeds observed during germination counts following hot water scarification A7a Hot water replicate including damaged seedlings and numerous unimbibed seeds A7b Close-up ofseed where radicle failed to elongate A7c Close-up ofseed where radicle elongated but was delayed in freeing itself from the seed coat A7d Close-up ofseed where radicle completely broke offfrom embryo
60
a
b
Figure A8 New Mexico locust seeds following percussion treatment at time of germination counts A8a Percussion replicate with uniformly healthy seedlings and near complete imbibition of seeds A8b Close-up ofpercussion-treated seedling
61
Discussion
Papilionoid seeds have a specific anatomical feature known as the strophiole
or lens-the natural site of water entry to the seed (Hamly 1932) The strophiole is
located on the cotyledonary lobe of a locust seed (Figure A9) When a papilionoid
legume seed is percussed for the appropriate amount of time repeated hits on the
integrated seed coat loosen the constrained cells of the strophiolar region without
excessively damaging the rest of the seed coat (Ballard 1976)
When a percussed seed is soaked water enters exclusively through the
strophiole in a controlled manner (Kelly and Staden 1987) This regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues This
contrasts with seeds that have been hot water or acid scarified treatments that can
randomly degrade the seed coat Unlocalized cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rehman et al 1998 Marunda 1990)
Papilionoids are the largest subfamily of legumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust and black locust these legumes often play an integral role in the
revegetation ofdisturbed lands Developing superior scarification methods to the
standard hot water and acid treatments should fadlitate the use ofthese legumes in
restoration projects
62
Figure A9 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (bc)
63
APPENDIXB
GERMINATION PERCENTS AND STANDARD ERRORS FOR SOURCE
BLOCK AND TREATMENT FOR NEW MEXICO LOCUST
Table B 1 Gennination percents and standard errors for source block and treatment for New Mexico locust
Source Block Treatment Germination Percentage Standard Error
Capitan 1 Immersion 74 437 Capitan 2 Immersion 69 462 Capitan 3 Immersion 71 454 Capitan 4 Immersion 67 470 Capitan Boiling 30 sec 79 407 Capitan 2 Boiling 30 sec 76 427 Capitan 3 Boiling 30 sec 72 449 Capitan 4 Boiling 30 sec 76 427 Capitan Boiling 60 sec 71 454 Capitan 2 Boiling 60 sec 54 498 Capitan 3 Boiling 60 sec 67 470 Capitan 4 Boiling 60 sec 76 427 Capitan I Control 5 218 Capitan 2 Control 7 255 Capitan 3 Control 4 196 Capitan 4 Control 5 218 Capitan 1 Percussion I min 47 499 Capitan 2 Percussion I min
470
Capitan Percussion I min 39 488
Capitan 4 Percussion I min 38 485 Capitan I Percussion 2 min 55 497 Capitan Capitan
2
Percussion 2 min Percussion 2 min
71 65
454 477
Capitan 4 Percussion 2 min 56 496 Capitan I Percussion 4 min 86 347 Capitan 2 Percussion 4 min 79 407 Capitan 3 Percussion 4 min 81 392 Capitan 4 Percussion 4 min 82 384 Capitan I Percussion 8 min 90 300 Capitan 2 Percussion 8 min 88 325 Capitan 3 Percussion 8 min 90 300 Capitan 4 Percussion 8 min 90 300 Gila I Immersion 71 454 Gila 2 Immersion 77 421 Gila
Immersion 75 433
Gila 4 Immersion 72 449
Gila I Boiling 30 sec 68 466
Gila 2 Boiling 30 sec 81 392
Gila 3 Boiling 30 sec 54 498
Gila 4 Boiling 30 sec 63 483
Gila I Boiling 60 sec 55 497
Gila 2 Boiling 60 sec 68 466
Gila 3 Boiling 60 sec 57 495
Gila 4 Boiling 60 sec 70 458
65
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Gila 2 Control 13 336 Gila 3 Control 13 336 Gila 4 Control 20 400 Gila 1 Percussion 1 min 66 474 Gila 2 Percussion 1 min 57 495 Gila 3 Percussion 1 min 73 444 Gila 4 Percussion 1 min 82 384 Gila 1 Percussion 2 min 81 392 Gila 2 Percussion 2 min 86 347 Gila 3 Percussion 2 min 85 357 Gila 4 Percussion 2 min 54 498 Gila 1 Percussion 4 min 79 407 Gila 2 Percussion 4 min 83 376 Gila 3 Percussion 4 min 84 367 Gila 4 Percussion 4 min 79 407 Gila 1 Percussion 8 min 51 500 Gila 2 Percussion 8 min 76 427 Gila 3 Percussion 8 min 52 500 Gila 4 Percussion 8 min 74 439 Holman 1 Immersion 83 376 Holman 2 Immersion 86 347 Holman 3 Immersion 81 392 Holman 4 Immersion 90 300 Holman 1 Boiling 30 sec 90 300 Holman 2 Boiling 30 sec 85 357 Holman 3 Boiling 30 sec 87 336 Holman 4 Boiling 30 sec 83 376 Holman 1 Boiling 60 sec 79 407 Holman 2 Boiling 60 sec 80 400 Holman 3 Boiling 60 sec 86 347 Holman 4 Boiling 60 sec 93 255 Holman Control 25 433 Holman 2 Control 19 392 Holman Control 21 407
Holman 4 Control 24 427
Holman Percussion 1 min 77 421
Holman 2 Percussion 1 min 85 357
Holman 3 Percussion 1 min 80 400
Holman 4 Percussion 1 min 89 313
Holman 1 Percussion 2 min 88 325
Holman 2 Percussion 2 min 96 196
Holman 3 Percussion 2 min 93 255
Holman 4 Percussion 2 min 96 196
Holman 1 Percussion 4 min 92 271
Holman 2 Percussion 4 min 97 171
66
I
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Holman 3 Percussion 4 min 91 286 Holman 4 Percussion 4 min 96 196 Holman 1 Percussion 8 min 88 325 Holman 2 Percussion 8 min 85 357 Holman 3 Percussion 8 min 83 376 Holman 4 Percussion 8 min 82 384 Jemez 1 Immersion 53 499 Jemez 2 Immersion 44 496 Jemez 3 Immersion 52 500 Jemez 4 Immersion 70 458 Jemez 1 Boiling 30 sec 58 494 Jemez 2 Boiling 30 sec 55 497 Jemez 3 Boiling 30 sec 48 500 Jemez 4 Boiling 30 sec 65 477 Jemez 1 Boiling 60 sec 68 466 Jemez 2 Boiling 60 sec 63 483 Jemez 3 Boiling 60 sec 73 444 Jemez 4 Boiling 60 sec 89 313 Jemez 1 Control 2 140 Jemez 2 Control 9 286 Jemez 3 Control 6 237 Jemez 4 Control 8 271 Jemez 1 Percussion 1 min 58 494 Jemez 2 Percussion 1 min 63 483 Jemez 3 Percussion 1 min 77 421 Jemez 4 Percussion 1 min 89 313 Jemez 1 Percussion 2 min 68 466 Jemez 2 Percussion 2 min 64 480 Jemez 3 Percussion 2 min 80 400 Jemez 4 Percussion 2 min 83 376 Jemez Percussion 4 min 68 466 Jemez 2 Percussion 4 min 79 407 Jemez 3 Percussion 4 min 70 458 Jemez 4 Percussion 4 min 83 376 Jemez 1 Percussion 8 min 52 500 Jemez 2 Percussion 8 min 59 492 Jemez 3 Percussion 8 min 61 488 Jemez 4 Percussion 8 min 70 458 Magdalena 1 Immersion 69 462 Magdalena 2 Immersion 69 462 Magdalena J Immersion 70 458 Magdalena 4 Immersion 85 357 Magdalena 1 Boiling 30 sec 65 477 Magdalena 2 Boiling 30 sec 71 454 Magdalena 3 Boiling 30 sec 68 466
67
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Magdalena 4 Boiling 30 sec 83 376 Magdalena 1 Boiling 60 sec 63 483 Magdalena 2 Boiling 60 sec 68 466 Magdalena 3 Boiling 60 sec 63 483 Magdalena 4 Boiling 60 sec 82 384 Magdalena 1 Control 11 313 Magdalena 2 Control 6 237 Magdalena 3 Control 5 218 Magdalena 4 Control 4 196 Magdalena Percussion 1 min 32 466 Magdalena 2 Percussion 1 min 45 497 Magdalena 3 Percussion 1 min 46 498 Magdalena 4 Percussion I min 36 480 Magdalena 1 Percussion 2 min 67 470 Magdalena 2 Percussion 2 min 68 466 Magdalena 3 Percussion 2 min 65 477 Magdalena 4 Percussion 2 min 81 392 Magdalena 1 Percussion 4 min 87 336 Magdalena 2 Percussion 4 min 97 171 Magdalena 3 Percussion 4 min 83 376 Magdalena 4 Percussion 4 min 97 171 Magdalena 1 Percussion 8 min 98 lAO Magdalena 2 Percussion 8 min 100 000 Magdalena 3 Percussion 8 min 96 196 Magdalena 4 Percussion 8 min 98 lAO Manzano 1 Immersion 67 470 Manzano 2 Immersion 57 495 Manzano 3 Immersion 72 4049 Manzano 4 Immersion 73 4044 Manzano 1 Boiling 30 sec 70 458 Manzano 2 Boiling 30 sec 64 480 Manzano 3 Boiling 30 sec 71 454 Manzano 4 Boiling 30 sec 80 400 Manzano I Boiling 60 sec 50 500 Manzano 2 Boiling 60 sec 53 499 Manzano 3 Boiling 60 sec 59 492 Manzano 4 Boiling 60 sec 74 439 Manzano I Control 4 196 Manzano 2 Control 8 271 Manzano 3 Control 1 099 Manzano 4 Control 6 237 Manzano 1 Percussion 1 min 52 500 Manzano 2 Percussion 1 min 39 488 Manzano 3 Percussion 1 min 36 480 Manzano 4 Percussion 1 min 51 500
68
Table B 1 Continued
Source Block Treatment Gerrni~ation Percentage Standard Error
Manzano Percussion 2 min 53 499 Manzano 2 Percussion 2 min 58 494 Manzano 3 Percussion 2 min 61 488 Manzano 4 Percussion 2 min 62 485 Manzano 1 Percussion 4 min 76 427 Manzano 2 Percussion 4 min 85 357 Manzano 3 Percussion 4 min 79 407 Manzano 4 Percussion 4 min 83 376 Manzano 1 Percussion 8 min 87 336 Manzano 2 Percussion 8 min 83 376 Manzano 3 Percussion 8 min 83 376 Manzano 4 Percussion 8 min 75 433 Raton 1 Immersion 54 498 Raton 2 Immersion 37 483 Raton 3 Immersion 39 488 Raton 4 Immersion 69 462 Raton 1 Boiling 30 sec 36 480 Raton 2 Boiling 30 sec 20 400 Raton 3 Boiling 30 sec 28 449 Raton 4 Boiling 30 sec 60 490 Raton 1 Boiling 60 sec 15 357 Raton 2 Boiling 60 sec 171 Raton 3 Boiling 60 sec 4 196 Raton 4 Boiling 60 sec 35 477 Raton 1 Control 4 196 Raton 2 Control 12 325 Raton 3 Control 2 140 Raton 4 Control 6 237 Raton 1 Percussion 1 min 52 500 Raton 2 Percussion I min 29 454 Raton 3 Percussion I min 35 477 Raton 4 Percussion 1 min 31 462 Raton 1 Percussion 2 min 55 497 Raton 2 Percussion 2 min 35 477 Raton 3 Percussion 2 min 33 470 Raton 4 Percussion 2 min 42 494 Raton 1 Percussion 4 min 65 477 Raton 2 Percussion 4 min 52 500 Raton Percussion 4 min 51 500 Raton 4 Percussion 4 min 53 499 Raton 1 Percussion 8 min 88 325 Raton 2 Percussion 8 min 80 400 Raton 3 Percussion 8 min 73 444 Raton 4 Percussion 8 min 74 439 Sacramento 1 Immersion 43 495
69
Table B 1 Continued
-Source Block Treatment Germination Percentage Standard Error
Sacramento 2 Immersion 30 458 Sacramento 3 Immersion 38 485 Sacramento 4 Immersion 56 496 Sacramento 1 Boiling 30 sec 47 499 Sacramento 2 Boiling 30 sec 48 500 Sacramento 3 Boiling 30 sec 48 500 Sacramento 4 Boiling 30 sec 56 496 Sacramento 1 Boiling 60 sec 52 500 Sacramento 2 Boiling 60 sec 67 470 Sacramento 3 Boiling 60 sec 43 495 Sacramento 4 Boiling 60 sec 48 500 Sacramento 1 Control 0 000 Sacramento 2 Control 3 171 Sacramento 3 Control 3 171 Sacramento 4 Control 099 Sacramento 1 Percussion 1 min 57 495 Sacramento 2 Percussion 1 min 61 488 Sacramento 3 Percussion 1 min 62 485 Sacramento 4 Percussion 1 min 55 497 Sacramento 1 Percussion 2 min 84 367 Sacramento 2 Percussion 2 min 70 458 Sacramento 3 Percussion 2 min 81 392 Sacramento 4 Percussion 2 min 69 462 Sacramento 1 Percussion 4 min 91 286 Sacramento 2 Percussion 4 min 84 367 Sacramento 3 Percussion 4 min 84 367 Sacramento 4 Percussion 4 min 94 237 Sacramento 1 Percussion 8 min 82 384 Sacramento 2 Percussion 8 min 81 392 Sacramento 3 Percussion 8 min 82 384 Sacramento 4 Percussion 8 min 81 392 Sandia 1 Immersion 76 427 Sandia 2 Immersion 70 458 Sandia 3 Immersion 70 458 Sandia 4 Immersion 81 392 Sandia 1 Boiling 30 sec 78 414 Sandia 2 Boiling 30 sec 71 454 Sandia 3 Boiling 30 sec 63 483 Sandia 4 Boiling 30 sec 78 414 Sandia 1 Boiling 60 sec 67 470 Sandia 2 Boiling 60 sec 65 477 Sandia 3 Boiling 60 sec 74 439 Sandia 4 Boiling 60 sec 66 474 Sandia 1 Control 16 367 Sandia 2 Control 7 255
70
Table B 1 Continued
Source Block Treatment Gennination Percentage Standard Error
Sandia 3 Control 8 271 Sandia 4 Control 16 367 Sandia 1 Percussion 1 min 67 470 Sandia 2 Percussion 1 min 61 488 Sandia 3 Percussion 1 min 66 474 Sandia 4 Percussion 1 min 59 492 Sandia I Percussion 2 min 82 384 Sandia 2 Percussion 2 min 83 376 Sandia 3 Percussion 2 min 81 392 Sandia 4 Percussion 2 min 84 367 Sandia 1 Percussion 4 min 89 313 Sandia 2 Percussion 4 min 86 347 Sandia 3 Percussion 4 min 89 313 Sandia 4 Percussion 4 min 85 357 Sandia 1 Percussion 8 min 80 400 Sandia 2 Percussion 8 min 77 421 Sandia 3 Percussion 8 min 69 462 Sandia 4 Percussion 8 min 66 474
LITERATURE CITED
Ashby WC Vogel WG Rogers NF 1985 Black locust in the reclamation equation General Technical Report Northeastern Forest Experiment Station USDA Forest Service NE-I05 12 p
Ballard LAT 1973 Physical barriers to gennination Seed Sci Technoll 285-303
___ 1976 Strophiolar water conduction in the seeds ofTrifolieae induced by action on the testa at nonstrophiolar sites Austr J Plant Physiol3 465-469
Barton LV 1947 Special studies on seed coat impenneability Contrib Boyce Thompson Inst 14 355-362
Baskin CC Baskin JM 1998 Seeds Ecology biogeography and evolution of donnancy and gennination Academic Press London UK 666 p
Bewley JD 1997 Seed germination and donnancy Plant Ce119 1055-1066
Bewley JD Black M 1994 Seeds physiology of development and germination 2nd ed Plenum Press New York NY 445 p
Briscoe CB 1996 Growing Nitrogen Fixing Trees in Acid Soils In MH Powell (ed) Nitrogen Fixing Trees for Acid Soils- A Field ManuaL Winrock International Morrilton AR 22 p
Copeland LO McDonald MB 1995 Principles of Seed Science and Technology 3rded Chapman amp Hall New York NY 409 p
Cox RE Klett JE 1984 Seed gennination requirements of native Colorado plants for use in the landscape Plant Propag 30(2) 6-10
Dell B 1980 Structure and function ofthe strophiolar plug in seeds ofAlbizia Lophantha Am J Bot 67 556-563
Dick-Peddie WA 1993 New Mexico Vegetation Past Present and Future University ofNew Mexico Press Albuquerque NM 244 p
Dreesen DR 2001 Propagation protocol for production ofcontainer Robinia neomexicana plants Los Lunas Plant Materials Center Los Lunas New Mexico In Native Plant Network URL httpwwwnativeplantnetworkorg (accessed 9 January 2002) Moscow (ID) University ofIdaho CoIl Natural Resources For Res Nursery
72
Dreesen DR Harrington JT 1997 Propagation of native plants for restoration projects in the Southwestern US- Preliminary Investigations In Landis TD Thompson JR tech coords National Proceedings Forest and Conservation Nursery Assocs Gen Tech Rep PNW-GTR-419 Portland OR USDA For Ser PNW Res Sta 77-88
Gottfried GJ 1980 Control ofNew Mexican locust and the effect on planted ponderosa pine in central Arizona US For Ser Res Note RM-386 Rocky Mountain For Range ExpStn Fort Collins CO
Gosling PG Samuel YK Jones SK 1995 A systematic examination of germination temperature chipping and water temperaturesoak duration pretreatments on the seeds ofLeucaena leucocephala Seed Sci amp Technol 23521-532
Hamly DH 1932 Softening of the seeds of Melilotus alba Bot Gazette 93 345shy375
Hanna PJ 1984 Anatomical features of the seed coat ofAcacia kempeana (Mueller) which relate to increased germination rate induced by heat treatment New Phytol 96 23-29
Hanks JP Dick-Peddie WA 1974 Vegetation patterns of the White Mountains New Mexico Southwest Naturalist 18(4) 371-382
Hine S Harrington JT Dreesen DR 1997 Seed scarification requirements for Robinia neomexicana In Harrington JTtech coord Proc Third SouthwestemContainer Growers Conf Feb 12-13 1997 Ft Collins CO
Hopkinson JM 1993 The strophiole of leguminous seeds Newsletter- IntI Herbage Seed Production Res Grp 18 7-8
---- 1997 Differences in hardseededness between Cunningham and Tarramba leucaena In Leucnet News The Newsletter of the International LeucaenaRes and Devlt Netwk No4
ISTA (International Seed Testing Association) 1999 International rules for seed testing rules Seed Sci amp Technol27 Supplement
Kelly KM van Staden J 1987 The lens as the site of permeability in the papilionoid seed Aspalathus linearis J Plant Physiol 128(45) 395-404
73
Keresztesi B 1988 Black locust the tree of agriculture Outlook on Agr 17(2) 77~85
Kigel 1 and Galili G 1995 Seed development and germination Marcel Dekker New York 853 p
Klemmedson 10 1994 New Mexico locust and parent material Influence on macronutrients of forest floor and soil Soil Sci Soc Am 1 58 974~980
Kuo WH Tam AW 1988 The pathway of water absorption of mung bean seeds Seed Sci amp Technol16(1) 139~144
Leadem CL 1997 Dormancy--unlocking seed secrets In Landis TD Thompson lR tech coords National Proceedings Forestand Conservation Nursery Assocs Gen Tech Rep PNW-G TR-419 Portland OR USDA For Ser PNW Res Sta 43-52
Lin Y Wagner MR Seweyestewa A Honahni MD DeGomez M McMillin JD 1996 Influence of cultural treatments on seed germination and seedling growth of Robinia neomexicana Forest Farm and Community Tree Res Reports Volume 1
Marunda CT 1990 Effects of seed pretreatment on the development ofAcacia auriculforinis and A holosericea seedlings ACIAR Proceedings Series No 28
Meginnis HG 1937 Sulphuric acid treatment to increase germination of black locust seed USDA Circ 453 34 p
Miklas PN Towensend CE Ladd SL 1987 Seedcoat anatomy and the scarification ofcicer milkvetch seed Crop Sci 27 766-772
Monsen SB 1984 Use of shrubs on mine spoils In The Challenge ofProducing Native Plants for the Intermountain Area Proceedings Intermountain Nurserymans Association 1983 conference Las Vegas NV compo PM Murphy 26-31 Ogden UT USDA For Ser Gen Tech Report INT-l68
Olson DF 1974 Robinia L Locust In Seeds of Woody Plants in the US tech coord CS Schopmeyer 728-731 USDA For Ser Agr Handbk No450
Rehman S Loescher RNl Harris P1C 1998 Dormancy breaking and germination ofAcacia salicina Lindl seeds Seed Sci amp Techno127 553-557
Rolston MP 1978 Water impermeable seed dormancy Bot Rev 44 365-396
74
SAS Institute Inc 1989 SASSTAT Users Guide Version 6 4th Edition Volume 1 Cary NC SAS Institute Inc 943 p
SerratoValenti G Conara L Ferrando M Modenesi P 1993 Structural and histochemical features ofStylosanthes scabra (Leguminosae Papilionoidae) seed coat as related to water entry Can J Bot 71 834-840
Simpson BJ 1988 A field guide to Texas trees Austin TX Texas Monthly Press 372p
Tran VN Cavanagh AK 1984 Structural aspects ofdormancy In Seed Physiology Vol 2 Murray DR (ed) p1-44 Academic Press Australia
USDA Forest Service 1948 Woody-plant seed manual USDA Misc Pub1654 416p
Vines RA 1960 Trees Shrubs and Woody Vines of the Southwest Austin Univ ofTexas Press 1104 p
Wagner MR Larson RE Craig TP 1992 Ecology and silvics ofNew Mexico locust (Robinia neomexicana) with an emphasis on the arboreal form p53-57 In JW Hanover K Miller and S Plesko (eds) Proc Intemat Confon Black Locust Biology Culture and Utilization Mich State Univ USA
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CONTENTS
LIST OF TABLES ix
LIST OF FIGURES middot middotmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot x
INTRODUCTION 1
LITERATlTRE REVIEW 4
Seed Donnancy 4
Physical Donnancy 5
Evolutionary Role ofPhysical Donnancy 6
Methods for Breaking Donnancy 6
Acid Scarification 6
Hot Water Scarification 8
Mechanical Scarification 11
Alternative Method for Breaking Donnancy Percussion 12
Objectives 15
Hypothesis 15
MATERIALS AND METHODS 16
Seed Collection and Cleaining 16
Experimental Design and Treatment Structure 16
Treatments ~ bullbull bullbullbullbull - bullbull ~ ~ 20
G T ennlnatlon estmg 21
X-ray Radiography 22
Scanning Electron Microscope Photography 22
Analysis 22
RESULTS 24
DISCUSSION 36
Detennining the Optimal Treatment 36
Strophiole and Seed Coat Behavior 37
CONCLUSION 44
Appendices
A PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR NEW MEXICO LOCUST AND BLACK LOCUST SEEDS 47
B GERMINATION PERCENTS AND STANDARD ERRORS FOR SOURCE BLOCK AND TREATMENT FOR NEW MEXICO LOCUST 64
LITERATURE CITED 72
Vlll
LIST OF TABLES
Table Page
1 Latitude longitude location elevation and collection date ofNew Mexico locust seed sources 18
2 Mean seed weight and standard error by source 19
3 Categorical Analysis ofVariance table for germination response to seed treatment seed source block and the interaction of the factors 25
4 Effect of seed source and treatment on G50 (days to 50 germination) 32
B1 Germination percents and standard errors for source block and treatment for New Mexico locust 65
LIST OF FIGURES
Figures Page
1 Seedsourcecollectionmap 17
2 Effect ofNew Mexico locust seed source on germination for data averaged over scarification treatments 26
3 Effect of scarification on germination for New Mexico locust for data averaged over all seed sources 27
4 Effect of seed source by treatment interaction on New Mexico locust germination for percussion scarification pattern a and pattern b 28
5 Effect of source by treatment interaction on New Mexico locust germination for hot water scarification 29
6 Timecourse of New Mexico locust germination following scarification treatments 31
7 X-ray radiographs of Sacramento seed source perc ussed for 1 4 or 8 minutes 33
8 Seed damage close-ups 34
9 SEM images of untreated vs 4-minute percussion-treated strophiolar cells ofNew Mexico locust seeds 35
10 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (b c) 38
11 Damage to New Mexico locust seeds observed during germination counts following hot water scarification 40
12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources 42
New Mexico locust a primary invader following disturbance 50
Implementation of hot water and percussion scarification 52
Effect of scarification on germination response for New Mexico locust 55
x
A4 Effect of selected scarification treatments on germination speed for New Mexico locust 56
AS Effect of scarification on germination response for black locust 57
A6 Effect of selected scarification treatments on germination speed for black locust 59
A7 Damage to New Mexico locust seeds observed during germination counts following hot water scarification 60
AS New Mexico locust seeds following percussion treatment at time of germination counts 61
A9 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (bc) 63
INTRODUCTION
New Mexico locust (Robinia neomexicana A Gray) a small tree native to the
southwestern United States occurs at elevations from 1200 to 2800 meters This
species fills a successional role in post-disturbance situations As a primary invader
New Mexico locust quickly establishes on burned areas arid flood banks as well as
road cuts (Wagner et aI 1992) Rapid growth crown sprouting and prolific root
suckering favor the successful establishment ofNew Mexico locust on disturbed sites
(Simpson 1988 Gottfried 1980) These attributes also may make New Mexico locust
well suited for steep-slope revegetation where erosion is a problem
New Mexico locust is a nitrogen-fixing legume that tolerates and improves
nutrient-poor soils Stands ofNew Mexico locust increase Nitrogen (N) and Calcium
(Ca) in the forest floor Levels of Carbon (C) Phosphorous (P) Sulfur (8) and
Potassium (K) aIso have been shown to increase in soil beneath New Mexico locust
(Klemmedson 1994)
The ability to colonize rapidly and ameliorate harsh sites contributes to New
Mexico locusts potential as a nurse plant A nurse plant colonizes an inhospitable
site and creates an environment suitable for successional plant establishment New
Mexico locust demonstrates this role in the ponderosa pine (Pinus ponderosa Douglas
ex Lawson amp C Lawson)Gambel oak (Quercus gambelii Nutt) community After a
disturbance such as wildfire New Mexico locust and Gambel oak colonize and
dominate the site until shaded out by ponderosa pine Once shaded these species
become understory shrubs a process taking an average of 15-20 years (Dick-Peddie
1993 Hanks and Dick-Peddie 1974) New Mexico locust gradually declines with
small dense patches averaging about 005 ha beneath pine until the next disturbance
once again offers it a competitive advantage (Klemmedson 1994)
Gottfried (1980) expecting ponderosa pine regeneration to be greater where
New Mexico locust had been eradicated found that survival of planted pine seedlings
was greater where locust had not been removed In a follow-up study Gottfried
(1980) noted that soil moisture in the top 57 cm was highest in 5-year old locust sites
as compared to grass or 20-year old locust sites He concluded that managing an
appropriate cover ofNew Mexico locust could help regenerate pine the later
successional species
The ability ofNew Mexico locust to improve harsh sites also makes it a
candidate for reclaiming disturbances associated with mining Black locust (Robinia
pseudoacacia 1) has been used for decades to reclaim mine spoils and other
disturbed sites in the eastern US and around the world (Keresztesi 1988 Zimmerman
and Carpenter 1980) Ashby et al (1985) described similar positive attributes of
black locust also mentioned previously for New Mexico locust quick cover for
stabilization supply ofN and nutrient-rich litter to the soil and site improvement for
establishment of later successional trees Referring specifically to mine reclamation
the authors note the ability of black locust to grow on a wide range ofmine soil
conditions including extremely acid soils Black locust also shows some tolerance to
soils compacted by grading and topsoiling practices (Ashby et aL 1985) New
2
Mexico locust holds promise as a native southwe~tem counterpart to black locust in
mine reclamation but it has been used infrequently to date
Natural invasion and succession occur slowly on most mine sites (Monsen
1984) While New Mexico locust often colonizes sites naturally there is no
assurance a seed will reach a particular site and establish in a reasonable time frame
The challenge is to introduce New Mexico locust as mother plants to facilitate
colonization Seedling production for outplanting on disturbed sites has been
hampered by poor germination (Lin et al 1996) The goal then is to develop
techniques to overcome poor germination in New Mexico locust thereby facilitating
macropropagation
LITERATURE REVIEW
Seed Dormancy
Low gennination in New Mexico locust is primarily the result of seed
donnancy the failure of an intact viable seed to genninate under favorable
conditions (Bewley 1997) Seed donnancy also can be defined as the inability of the
embryo to genninate because of some inherent constraint (Bewley and Black 1994)
The constraint may originate within the embryo referred to as endogenous or embryo
donnancy or from the tissues surrounding the embryo known as exogenous or coatshy
imposed donnancy (Baskin and Baskin 1998 Leadem 1997 Bewley and Black
1994)
New Mexico locust seed is donnant when the impenneable seed coat does not
allow movement ofwater to the embryo This specific fonn of coat-imposed
donnancy where the restraining tissue involved is an impenneable seed coat is
known as physical donnancy (Baskin and Baskin 1998 Bewley and Black 1994)
Physical donnancy is relatively uncommon in seeds less than 10 of all woody
species exhibit this condition (USDA Forest Service 1948) However it is
characteristic of many genera of the Fabaceae (bean) family For example all
successional trees in the genera Gleditsia and Robinia (of which New Mexico locust
is a member) produce seeds that exhibit physical donnancy (Baskin and Baskin
4
Physical Dormancy
Seeds that exhibit physical dormancy are said to be hard seeded referring to
the thick impermeable seed coat (Leadem 1997) The first line of protection of the
testa in leguminous seeds is the outermost waxy cuticle followed by a subcuticle
consisting ofsuberin Next is a palisade layer consisting of sclereid cells that have
thick lignified secondary walls The most common type of sclereid cell in palisade
layers is the macrosclereid The macrosclereids contain water-repellent substances
including cutin lignin suberin and wax (Rolston 1978) This palisade layer is the
primary barrier to water uptake (Serrato-Valenti et al 1993 Tran and Cavanagh
1984)
All seed coat openings must be blocked to effectively prevent water uptake
The natural opening in a seed where it was attached to the mother plant is the weak
link where physical dormancy is usually broken (Hopkinson 1993 Kuo and Tam
1988) Anatomical structures associated with the seed coat opening vary from
family to family In the Papilionoideae the subfamily of the Fabaceae family
containing New Mexico locust the site ofwater entry is the strophiole The
strophiole is a swelling on the seed coat close to the stalk scar (Hopkinson 1997)
Kelly and van Staden (1987) refer to the strophiole site as the lens where
macrosclereid cells must separate and raise before water can pass through Since
middot6germination ofdormant seeds of the Papilionoideae takes place when the strophiole
permeable (Hopkinson 1993 Kuo and Tam 1988) a logical approach to
5
genninating New Mexico locust artificially would be to loosen the strophiolar cells
and allow water to pass through to the embryo
Evolutionary Role of Physical Dormancy
Why is physical dormancy a mechanism that blocks germination
ecologically advantageous When a plant produc~s seeds with different degrees of
dormancy seeds that are dispersed at one time will germinate over a range of time
(Bewley and Black 1994) Through variability of dormancy within a seed crop seeds
are exposed to changing environmental conditions improving the overall chances of
survival of the seed line (Bewley and Black 1994) Varying expression of dormancy
is under genetic control which indicates that this may be an evolved survival strategy
(Kigel and Galili 1995) A range of dormancy levels may be a particular advantage in
the southwestern US where extreme environmental variations exist
Methods for Breaking Dormancy
There are three common approaches to breaking dormancy in New Mexico
locust seed acid scarification hot water soaks and mechanical treatments In each
case the objective of the treatment is to scarify or abrade the seed coat to make it
permeable to water
Acid Scarification
Acid scarification consists of soaking seeds in concentrated sulfuric acid
rinsing thoroughly with water upon removal followed by subsequent drying before
PlaJlltmg Olson (1974) reports that this scarification method produces the highest
middot~~jllalUll results for all Robinia species Most literature refers to a seminal 1937
6
study conducted by HG Meginnis This stud~ t~ough conducted on black locust
seed is regularly cited in the suggested use of acid to treat New Mexico locust seed
as well (Olson 1974 Vines 1960)
Thee published studies document the specific use of sulfuric acid to treat
New Mexico locust seed Cox and Klett (1984) report that soaking seeds in
concentrated sulfuric acid for thirty minutes improved germination to 34 over an
untreated control of4 Lin et al (1996) found that for two seed lots acid
scarification for twenty-five to eighty minutes produced germination of 80 A third
lot however resulted in less than 20 germination at all durations of 10 to 80
minutes Hine et al (1997) showed that acid treatment levels of 1 2 5 and 10
minutes resulted in less than 20 germination across five sources However there
was an increasing trend in germination with acid soak duration and acid treatments
were not imposed for as long a time as the previous two studies (30 to 80 minutes)
The mixed results obtained by these investigators were probably due to the
widely varying germination response of seed lots to acid treatment For this reason
Olson (1974) recommends correlating duration of acid treatment to each seed lot If
treatment duration is too long the embryo will be permanently damaged If soak
duration is too short the seed will remain impermeable to water and fail to germinate
Compounding the problem of treatment duration is the negative effect acid
have on seedling growth Any type of scarification can injure seeds to some
by disrupting cells essential for growth leading to fungal invasion and
middot-uuu~w injury (Copeland and MacDonald 1995) Some legume seeds subjected
7
to 60 minutes of sulfuric acid scarification (to achieve maximum gennination results)
showed more than 50 seedling mortality after gennination (Kelly and van Staden
1987) Bewley and Black (1994) noted that fungal growth on seeds is an indication
of leakage of sugars organic acids amino acids and proteins
Perhaps the greatest drawback to acid scarification is the dangerous nature of
the treatment itself Difficulties associated with storage and trained use of
concentrated acid may discourage many propagators from using this option (Dreesen
and Harrington 1997) Consequently hot water and to a lesser extent mechanical
scarification are more commonly used and practical treatments for legume seeds
Most southwestern container growers use either of these two treatments to scarify
New Mexico locust seed in particular (Hine et al 1997)
Hot Water Scarification
The widespread use ofhot water scarification to treat locust seed is probably
due to its ease of application and efficacy in comparison to other scarification
treatments Although this practice had been in use for some time Wilson (1944) was
one of the first to report on its systematic application for black locust The treatment
consists of placing seeds in boiling water (4 parts water tomiddot 1 part seed by volume)
removing the vessel from the heat source and allowing the water to cool and seeds to
soak for 24 hours
Hine et al (1997) Lin et al (1996) and Cox and Klett (1984) have examined
the specific application ofhot water scarification on New Mexico locust seed Each
these authors compared hot water to acid scarification Lin et aL (1996) reported
8
60 gennination for two hot water-treated seed lots while a third lot germinated
around 50 (as reported above acid scarification germination ranged from 20 to
80 for these lots) It should be noted that the authors only soaked the seeds in water
from 10 to 80 minutes for this experiment as opposed to the standard 24 hours Cox
and Klett (1984) stated that using the standard 24-hour hot water treatment improved
germination over an acid pre-treatment (which produced less than 20 germination)
but they did not give specific gennination numbers for the hot water treatment Hine
et al (1997) found that across five seed lots germination was 66 for the standard
hot water treatment (as compared to less than 20 germination for acid scarification)
Variations in treatment intensity and duration and their subsequent effects on seed lot
appear to contribute to conflicting conclusions as to whether acid scarification or hot
water treatment is a more successful treatment
Given mixed results ease ofapplication is an important argument for use of
hot water application over acid soaks Safety concerns and material costs for hot
water treatments are low (Dreesen and Harrington 1997) An extension of this is the
practicality ofhot water for treating large batches of seed where a relative increase in
water volume is all that is needed However increases in water and seed even when
inthe same 4 1 proportion can result in dramatically different rates of cooling than in
atest tube experiment Different cooling rates can lead to contrasting results for the
treatment temperature duration and seed lot (USDA Forest Service 1948)
As with acid scarification parameters ofhot water treatment application may
to be optimized to maximize germination Hine et al (1997) found that as
9
temperature was increased from 70degC to 100degC overall germination in New Mexico
locust seed increased The 100degC treatment maximized germination for each of five
sources tested with germination usually above 60
Prolonged exposure of seeds ofmany woody legumes to boiling temperatures
also may improve germination (Briscoe 1996) This approach has not been tested on
New Mexico locust seed Exposing seed to prolonged boiling temperatures may
result in fungal infection and subsequent seedling mortality Marunda (1990) noted
severe fungal infections on both acid and hot water treated Acacia holoserica Cunn
ex G Don seeds attributing the infections to seed damage resulting from the
treatments Identifying an optimal hot water treatment level that does not result in a
high rate of seedling mortality is a logical goal
With hot water treatments as with acid there is also a danger of overtreating
Seeds and reducing germination (Gosling et al 1995) The legume Leucaena
leucocephala (Lam) de Wit germinated equally well after 7 seconds of exposure to
water at 100degC 7 seconds at 90degC 30 seconds at 80degC and 4 minutes at 70degC In
addition acceptably high germination was obtained from treatment at 70degC from 3
minutes to 1 hour whereas no seeds germinated following treatment at 100degC for 2
minutes Perhaps along with a greater margin of error for treatment duration treating
~ew Mexico locust seeds at a lower temperature may result in a comparatively lower
of seedling mortality In practice however nursery growers tend to use the
and quickest treatment available The short treatment length at near-boiling
whether recommended or not may be what is used most frequently
10
Fine-tuning a treatment in this range of treatment temperature and duration may be
the most logical approach
Mechanical Scarification
This treatment involves scratching the seed coat with an abrasive material
(USDA Forest Service 1948) One system employs a rotating drum lined with
sandpaper A paddle spinning at a given number ofrevolutions per minute throws the
seed against the sandpaper (Hine et al 1997) A second method dry tumbling
tumbles seed in a mixer with pea gravel or sharp sand for 5 to 7 days (Dreesen 2001)
These approaches have the advantage that control of temperature is not required as in
water treatments nor is safety an issue as with add scarification
Hine et al (1997) mechanically treated New Mexico locust seed using 100shy
grit sandpaper in a Forsberg (rotating drum) scarifier Gennination was less than
10 They concluded that the seed coats were too thin for their apparatus and
proposed that a finer grit sandpaper would produce more even abrasion Although
germination varied by seed lot Dreesen (2001) reported that using a slow-speed dry
tumbling technique for 5 to 7 days produced gennination rates as high as 90
As with acid and hot water treatments treatment duration must be specifically
shy
correlated to seed lot Treated seeds are also susceptible to fungal infection
iHopkinson 1997) In addition equipment is specialized and may not always be
ltU~CUJl~ to a nursery (USDA Forest Service 1948) Several studies have confinned
degrading ofstrophiolar cells due to acid and hot water scarification (Miklas et al
11
1987 Dell 1980 Hanna 1984) but the effect ofmechanical scarification on the
strophiole has not been studied
Alternative Method for Breaking Dormancy Percussion
Strophiole degradation has been examined in depth using an impaction
treatment more commonly known as percussion Hamly (1932) first proposed that
shaking or impacting seeds could split the macrosc1ereid cells at the strophiole
Hamly successfully released tension ofbent strophiolar cells of impermeable
Melilotus alba Medik (White sweet clover) seeds by delivering physical blows
resulting in cells that had split apart and were only slightly bent Ninety-two percent
oftreated seeds became permeable as compared to 05 of control seeds Barton
(1947) showed that percussion significantly increases permeability in black locust
seed Ballard (1976) reasoned that the integrated nature of the strophiole within the
seed coat means a blow on any part of the seed weakens the strophiole There are no
published studies on the use of percussion on New Mexico locust
Percussion methods vary and often seem SUbjective or ill-defined Hamly
(1932) placed seeds in a glass bottle and shook them three times a second for 20
minutes resulting in 180 oscillations per minute Ballard (1973) describes shaking
seeds in a closed metal container with at least fifty seeds per container at 850-900
JS(~l11alIOIlS per minute for 1 minute Kelly and van Staden (1987) placed seeds into
sealed glass flasks Flasks were shaken by a wrist-shaker at a rate of 250
~UUVJ~ per minute Subsequent germination rates were 45 after 2 hours of
55 after 3 hours 60 after 6 hours and 100 after 12 hours
12
There are several potential advantages of percussion over acid hot water and
mechanical scarification Kelly and van Staden (1987) note that a percussion
treatment only alters macrosclereid cells of the strophiole by loosening separating
and raising them They emphasize that minimal damage is done to the overall
anatomical structure of the seed In acid scarification of the same seed lot radicle
and cotyledon damage occurred Based on photographic evidence the authors
believe that this morphological damage was due to uneven pressure during
imbibition with water entering haphazardly wherever the acid had sufficiently
dissolved the coat This ultimately led to uneven hydration of the embryo In
percussed seeds water only entered through the s~ophiole when the entire seed coat
had been saturated resulting in controlled and even embryo hydration (Kelly and van
Staden 1987)
Although all successful scarification treatments may focus at least in part on
the strophiole for papilionoid legumes (Baskin and Baskin 1998) percussion may be
less damaging to seed coat structures particularly when compared with acid Kelly
and van Staden did not compare hot water or mechanical scarification methods
against percussion However the fungal infection associated with hot water and
mechanical scarification as with acid scarificatio~ have been directly associated with
seed damage (Rehman et al 1998 USDA Forest Service 1948) Embryo damage in
has been observed in hot-water scarified legume seeds (Rehman et al 1998
13
By limiting embryo damage percussion scarification reduces seedling
mortality compared to other treatments Kelly and van Staden (1987) noted that
seedlings from the percussion treatment were morphologically sound and vigorous
when compared to acid treatment seedlings The authors attribute this improved
seedling vigor to the controlled water uptake that limited anatomical damage
Despite a 12-hour percussion treatment morphological damage was not substantially
greater than untreated seeds In addition seedling mass measured 10 days following
germination was not significantly different between percussed and untreated seeds
A final advantage ofpercussion may be the ability to adequately treat the most
dormant seeds while avoiding harm to seeds that have already been made permeable
by the treatment or that were permeable to begin with Instead ofcorroding scalding
or scratching the seed coat percussion appears to be just a loosening of the
strophiole spring (Ballard 1973) Kelly and van Staden (1987) were able to
increase germination with a lengthy treatment (12 hours) Figuring out whether a
substantial cushion oftreatment durations exist for percussion ofNew Mexico locust
seed would be a significant improvement to current scarification methods where seed
damage and seedling health is an issue
The greatest challenge for applying percussion to New Mexico locust seeds is
standardizing a treatment that is both effective and readily reproducible by nurseries
with mechanical scarification this may require the development of specific
~UllDment to properly quantify and execute the treatment
14
f
Objectives
The first objective of the study is to detennine which scarification treatment
results in optimal gennination speed and percentage ofNew Mexico locust The
second objective is to describe treatment interactions with nine seed sources collected
in New Mexico
Hypothesis
A percussion treatment acts exclusively on the main area ofwater uptake in
New Mexico locust seed loosening the strophiole so that imbibition can occur This
direct mechanism ofdonnancy release will result in improved gennination rate and
overall gennination percentage for New Mexico locust seed compared to standard
treatments Standard treatments including acid hot water and mechanical
scarification while eventually weakening the strophiolar cleft cause excessive
morphological damage through generalized seed coat degradation These treatments
are therefore prone to inconsistency due to over and under-treatment
15
MATERIALS AND METHODS
Seed Collection and Cleaning
Seeds were collected from August 25 through September 15 2000 from nine
locations along a latitudinal gradient in New Mexico Seed source locations and
descriptive information are given in Figure 1 and Tables I and 2 Stands were
separated by at least 45 km
Pods were hand picked as they began to ripen and dehisce (open) Collections
were made from ground level to approximately 3 m from several trees at each site
Pods were dried in open-ended brown paper bags for two to four weeks at room
temperature (21-230C) to facilitate cleaning Dried pods were placed in a closed cloth
sack which was pounded against the ground to separate seeds from pods For pods
still containing seeds hand maceration (crunching pods with hands) completed seed
separation Debris was removed by threshing with the aid of a greenhouse fan A
South Dakota Seed Blower (Seedbureau Chicago Illinois) was used to remove
remaining chaff and light seeds by air density separation Seeds were placed in dry
storage in sealed quart-size zip-lock bags within a sealed plastic container Storage
temperatures ranged from 2-4oC Seeds remained in storage for approximately 7
months before the experiment
Experimental Design and Treatment Structure
The experiment compared the impact ofhot water bath and percussion
l11aLl11 on germination properties ofNew Mexico locust seed A single
16
-l
Figure 1 Seed source collection map All sources were collected within New Mexico USA separated by at least 4S lan
IIIRZltItW lIJPIirmiddot rllll1lbullbull_~~~_II
Table 1 Latitude longitude location elevation and collection date ofNew Mexico locust seed sources
Lot title Latitude (N) Longitude (W) Location Elevation (m) Collection Date
Raton 36deg5846 104deg2872 Raton Pass NM 2295 82500
Holman 36deg0306 105deg2429 HolmanNM 2476 911500
Jemez 35deg4500 106deg4100 Jemez Springs NM 1976 9900 00
Sandia 35deg1083 106deg2341 Cedar Crest NM 2432 82600
Manzano 34deg3689 106deg2494 ManzanoNM 2465 9200
Magdalena 33deg5953 107deg0903 Magdalena NM 2544 82900
Capitan 33deg2728 105deg4325 Alto NM 2189 911200
Sacramento 32deg5798 105deg4496 Cloudcroft NM 2630 91400 Gila 32deg5400 107deg4600 Kingston NM 2432 9700
f
Table 2 Mean seed weight and standard error by source
a
Source Mean seed weight (g) for Standard error 100 seeds n = 5
Raton Holman Jemez Sandia Manzano Magdalena Capitan Sacramento Gila
2872 b c 2522 d 1826 e 2753 c 2413 d 2559 d 2443 d 3130 a 3029 ab
00103 00403 00276 00301 00370 00374 00458 00279 00348
a Mean seed weights followed by the same letter are not significantly
different
19
experiment was conducted utilizing a randomized complete block design blocked by
time with a factorial treatment structure Experimental factors were seed source and
scarification treatment Nine seed sources from throughout New Mexico were used
(Figure 1 Table 1) Scarification treatments included a control three levels ofhot
water and four levels of percussion Hot water treatments included immersion at
100degC and boiling for periods of 30 seconds and 60 seconds Percussion treatment
levels were 1 2 4 and 8 minutes One hundred seeds per block were used to test
each treatment combination Four blocks of seed were tested one at a time (all seed
sources included in each run) in close chronological sequence (within 6 weeks) A
total of four 100-seed samples were used to test each source by scarification treatment
combination
Treatments
Hot water treatment of seeds utilized 50 ml test tubes filled with 30 ml
distilled water Each test tube was used to treat one 100-seed sample at a time
resulting in a ratio of4 parts water to 1 part seed by volume Test tubes were placed
in a beaker with the beaker filled with tap water to the same level as the water within
test tubes Nine test tubes (one per seed source) were placed in the beaker
simultaneously so that one replication of each seed sourcescarification treatment
cotnblnatlon was conducted simultaneously Hot water treatments include immersion
boiling For immersion treatments seeds were added to test tubes with the water
(boiling) and the test tubes were immediately removed from the beaker to
For the boiling treatments test tubes remained in the beaker with water at
20
boiling temperature for a set duration after the addition of seeds Following all
treatments test tubes with seeds cooled 24 hours in a test tube rack
Percussion treatment utilized a pneumatic paint shaker (Central Pneumatic
model 00422 Camarillo CA) Compressed air was maintained at 530 kPa plusmn30 kPa
(80 psi plusmn 5 psi) resulting in approximately 350 oscillations per minute Seeds were
placed in 118 ml (4 oz) soil tin cans (US Can Lobard IL) and the lids were secured
with duct tape Each can was used to treat one 100-seed sample To take advantage
ofgreatest lateral movement of the paint shaker a spacer (paint can) was used to
position the soil tin at the end of the shaking arm This allowed shaking to occur at
the greatest distance from the pivot point (fulcrum) Tins were placed perpendicular
to the direction of shaking motion allowing seeds to impact at greatest force by
hitting flat surfaces of the soil tin
Germination Testing
Before gennination testing all seeds were soaked 24 hours in reverse
osmosis-treated water (this was included in the process ofhot water treatment
jU~SCIlOea above) Following soaking seeds were placed in 100 cm Petri dishes lined
90 cm Whatman qualitative 1 filter paper circles (MG Scientific Pleasant
WI) One Petri dish was used for each treatment by source combination
papers were initially moistened with 5 mL reverse osmosis-treated water then
~VU1CUlJl moistened as needed to ensure adequate conditions for gennination
were monitored daily (at 300 PM) for gennination for 14 days in accordance
~UIUlnJlVUa Seed Testing Association (ISTA) standards for Robinia species
21
(1STA 1999) Tests were carried out at room temperature (22degC plusmnOSoC)
Germination was classified as protrusion of the radicle from the embryo by at least
1mm as viewed with the naked eye
X-ray Radiography
On August 132001 three lOO-seed samples from the Sacramento seed source
were treated for 14 or 8-minute percussion durations The seeds were sent
immediately by overnight delivery to the Faxitron X-Ray Corporation in Wheeling
Illinois where Dr Bob Karrfalt ofthe USDA Forest Service oversaw their imaging
Radiographs were made at SX with a Faxitron Specimen Radiography System Model
MX-20
Scanning Electron Microscope Photography
On February 192002 a Hitachi S-3200N Variable Pressure Scanning
Electron Microscope (Mountain View CA) was used to examine strophiolar cells of
untreated seeds and seeds percussed for 4 minutes Seeds were cut longitudinally
igt their entire length using a razor blade splitting the strophiolar region roughly in
Cross sections of seed samples magnifying the strophiolar region were viewed
600X under the microscope
Analysis
Categorical Analysis of Variance was used to determine treatment source and
(Le time) main effects and interactions The response variable was total
~vlI This procedure is a Chi-square test of homogeneity using the natural log
of germinated to non-germinated seed for each treatment Maximumshy
22
likelihood analysis was used to calculate Chi-square test statistics with their observed
significance levels A single contrast was conducted to cempare treatment means
against the control Observed significance levels less than or equal to 005 were
considered significant Pairwise comparisons were conducted between non-control
treatment mean percents using a conservative alp~a value of 005 divided by the
number of comparisons (Bonferroni approach) and approximate pairwise z statistics
Categorical analyses were performed using Proc Catmod (SAS Institute 1989)
RESULTS
Scarification seed source block and all interactions between factors
influenced germination (Table 3) The main effects are reported in the presence of
these interactions because the overall robustness of treatments and seed sources are of
interest Background levels ofnaturally occurring variation in seed sources are
seen in control germination percentages in Figure 2 In particular time variability
is unlikely to be under the control of the producer Appendix B details experimental
data for source by treatment gennination percents for each of the four blocks
Averaged over all treatments germination by source ranged from 40 to
79 (Figure 2) Averaged over all sources (Figure 3) and for each source (Figure 4
5) percussion scarification durations of4 or 8 minutes yielded greatest germination
Averaged over all sources germination increased with increasing durations of
percussion then leveled off for the 4- and 8-minute treatments (Figure 3) Immersion
and 30-second boiling scarification maximized germination for the hot water
treatments with a decrease in germination when seeds were boiled for 60 seconds
For all but two sources there is a similar ipcreasing trend in germination from
1to 4 minutes ofpercussion (Figure 4) The Holman source increased in germination
1 to 2 minutes then leveled off at 4 minutes The Jemez source changed little
1 to 4 minutes of treatment As treatment duration increased from 4 to 8
mutes five sources decreased in germination (Figure 4a) whereas three sources
24
Table 3 Categorical Analysis of Variance table for germination response to seed treatment seed source block and the interaction of the factors
Source of DF Chi-Square Observed Variability Significance Level
Source 8 98370 lt00001 Trt 7 233876 lt00001
contrast 1 16976 lt00001 SourceTrt 56 97077 lt00001 Block 3 5538 lt00001
SourceBlock 24 10162 lt00001 TrtBlock 21 7564 lt00001
SourceTrtBlock 168 34283 lt00001
S 4)
8 ~
0
sectg ~ 40 ~
N 0 0
20
o f0ltgt ~ ~4 jybshy ((iltgto ~~ K1ltgt 6~b-~ ~~ 0b-~~ ~~ ~b-~ ~b-~b
80
60 -I-- - ~- middotmiddotmiddotmiddotmiddot1
r -] Northern NM Sources
Central NM Sources
Southern NM Sources
sect-o ~
~b-v(i
SOURCE
Figure 2 Effect ofNew Mexico locust seed source on germination for data averaged over all scarification treatments Control germination is shown by solid line within bar
80
~ cd ~ ltl) () 60 ~ ~
N
t 0shyrti tshySltl)
40
0
20
o lmmersion Boiling 30 sec Boiling 60 sec Control 1 min 2min 4 min 8 min
Hot Water Treatments Percussion Treatments
Figure 3 Effect of scarification on germination ofNew Mexico locust for data averaged over all seed sources Means labelled with the same letter are not significantly different
100~----------------------~--~----------------~
80
60
ltU
~
40 IJ
sectgsI
l100
80
60
40
a I middotmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot middotImiddotmiddotmiddotmiddotmiddotmiddot E
_~iii-bull _ _ bull f --~--
t-- -~ ---- -shy ---- shyI ~ ---- - shy
L
fiII
~
b
- shy ---r
- Holman
Sacramento Sandia Gila Jemez
c ~
ltgt ~
Tmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot-I
j~=~=-~~-1
I middotmiddot ~ bull r
t tr
Y -------M-a-gd-a-Ie-n-a--
1 _ _
fCapitan
---- Manzano ~y -_ Raton
2 4 8
Time interval ofpercussion scarification (min)
Figure 4 Effect of seed source by treatment interaction on New Mexico locust germination for percussion scarification pattern a and pattern b
28
tv 0
~ d
~ A s o
1 c3
80
60
40
20
o
TIT
t------=middotfmiddotmiddot-middotmiddot-middotmiddot-middotmiddot 1- --- - - --=- - -shy--cE= - _--shy --shy ----shy ~ ~ ---shy ~ -~- _ --shy-------r 1
I
Raton Holman
-_ Jemez
--- Sandia Manzano Magdalena Capitan Sacramento Gila
Immersion Boiling 30 sec Boiling 60 sec
Hot Water Scarification
Figure 5 Effect of seed source by treatment interaction on New Mexico locust germination for hot water scarification
continued to increase in germination (Figure 4b) There was no difference between
the two treatment levels for the Manzano source (Figure 4b)
The effect ofhot water scarification also varied among sources with few
overall differences between hot water treatments (Figure 5) However boiling for 60
seconds yielded highest germination for the Jemez source whereas increasing
durations ofboiling decreased germination for the Raton source
In addition to consistently high germination 4- and 8-minute percussionshy
treated seeds germinated as fast or faster than all other treatments (Figure 6 Table 4)
050 measured as the number of days needed to complete 50 ofeventual
germination averaged 2 days for both ofthese treatments
X-ray radiographs were taken of seeds collected from the Sacramento source
percussed for 1 4 or 8 minutes (Figure 7) Seeds show signs of damage at 8 minutes
ofpercussion (Figure 8) Compared to an undamaged seed percussed for only 4
minutes (Figure 8a) damage at the 8-minute level includes fractured radicles pitting
of the seed coat and chips in seed coat structure (Figure 8b-d)
Scanning electron microscope (SEM) images at 600X reveal the direct effect
of 4 minutes of percussion on cells of the strophiolar region (Figure 9) Whereas
untreated seeds retain cellular organization in the strophiolar region strophiolar cells
seeds treated for 4 minutes ofpercussion separate and raise resulting in a
~-~e~U appearance
30
Umiddot-~middot--l-~---middotymiddotmiddotmiddot-----middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot--middot--middotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middotmiddotmiddotmiddotmiddotmiddotmiddot- -
050= gt1
-e-- Percussion 8 min -0- Percussion 4 min ---T- Percussion 2 min -----ltil- Percussion 1 min -- Immersion --0- Boiling 30 sec -+- Boiling 60 sec -ltgt- Control
2 4 6 8 10 12
Day Figure 6 Timecourse ofNew Mexico locust gennination following scarification
treatments G50 is the number of days to 50 of eventual germination
31
14
Table 4 Effect of seed source and treatment on G50 (days to 50 germination)
Seed Source Control Immersion B30sec B60sec Plmin P2min P4min P8min mean Raton 3 3 5 7 4 3 2 2 4 Holman 2 2 2 2 2 2 2 2 2
Jemez 4 6 6 5 3 2 2 2 4 Sandia 2 2 2 2 2 2 2 2 2
w tv Manzano 5 2 2 3 3 2 2 2 3
Magdalena 4 2 2 3 4 3 2 1 3
Capitan 5 4 2 2 3 2 2 2 3
Sacramento 3 2 2 2 4 3 2 2 3 Gila 3 2 2 2 2 2 2 2 2 mean 3 3 3 3 3 2 2 2 3 standard 11 14 16 18 09 05 0 03 08 deviation
Note Columns and rows may not agree due to rounding
Figure 7 X-ray radiographs ofSacramento seed source percussed for 1 4 or 8 minutes
33
Figure 8a-d Seed damage close-ups Figure a shows undamaged seed percussed for 4 minutes Figures b-d show seeds percussed for 8 minutes Damage includes fracturing ofradicle in b complete removal ofseed coat in c and pitting ofseed coat in d
34
Untreated Treated
Figure 9 SEM images ofuntreated vs 4-minute percussion-treated strophiolar cells ofNew Mexico locust seeds Treated cells are loosened separated and raised
DISCUSSION
Determining the Optimal Treatment
Averaging results of scarification treatmems across seed sources is a useful
tool to predict which treatment is most likely to be successful for an unknown seed
source Averaged over all sources four- and eight-minute percussion treatments
resulted in the highest germination 815 and 790 respectively and produced
germination that was as fast or faster than all other treatments Although there was no
difference between these two treatments on average an argument for 4 minutes of
percussion as a standard protocol for treating New Mexico locust seeds can be made
Descriptively 4 minutes of percussion improved germination over 8 minutes
ofpercussion for a majority of sources (five out of nine) 8 minutes of percussion
improved germination for three ofnine sources with no difference for a fmal source
For individual sources 4 minutes ofpercussion was always the best or secondmiddot best
treatment and was always equal to or better than all hot water treatments
Germination at 8 minutes ofpercussion was lower than all other percussion durations
for the Jemez and Gila sources and was lower than at least one hot water treatment for
these sources as well
The x-ray radiographs of the Sacramento seed source illustrate the excessive
internal and external seed damage that can occur with 8 minutes of percussion
germination counts 8-minute percussed seeds from Holman and Capitan
ClUll signs of damage that were visible to the naked eye (see below)
r~evertheless some of these seeds still fit the criteria for germination Neither x-ray
36
photography nor visible observations during seed counts revealed seed damage at the
4-minute percussion level In operational trials seeds percussed for 4 minutes
developed into healthy seedlings (Khadduri and Harrington unpublished data)
Strophiole and Seed Coat Behavior
Four minutes ofpercussion successfully breaks seed dormancy in a large
percentage ofNew Mexico locust seeds without excessively damaging the
surrounding seed coat or internal seed coat structures Most papilionoid legume
seeds including seeds ofNew Mexico locust have a specific region of the seed coat
known as the strophiole or lens (Hamly 1932) The strophiole is located on the
cotyledonary lobe of a locust seed (Figure 10 a-c) If seeds are not indiscriminately
damaged the strophiole is the first point of water entry to the embryo (Hopkinson
1997) When a papilionoid seed is percussed for the appropriate amount of time
repeated hits on the integrated seed coat loosen the constrained cells of the strophiolar
region without excessively damaging the rest of the seed coat (Ballard 1976) SEM
photos illuStrate that 4 minutes of percussion loosened and separated strophiolar cells
ofNew Mexico locust seeds when compared with constrained organized strophiolar
cells of untreated seeds (Figure 9) Research-has shown that water passes to the
embryo through the treated strophiole in a controlled manner and regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues
(Kelly and van Staden 1987)
37
10 Strophiole ofunimbibed New Mexico locust seed (a) and following uptake (bc)
38
In contrast hot-water treatments can excessively degrade the seed coat
(Rebman et al 1998 Marunda 1990) Random cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rebman et aI 1998 Marunda 1990) Along with a
proportionately large amount ofun-imbibed (thus under-treated) seeds (Figure l1a)
damage was observed in hot-water treated seeds from six sources during germination
counts Damage included seeds where the radicle failed to elongate (Figure 11 b)
seeds where the radicle elongated but was delayed in freeing itself from the seed coat
(Figure 11c) and seeds where the radicle completely broke off from the embryo
(Figure lId) In addition some seeds germinated in reverse (the cotyledon emerged
fIrst) If the radicle emerged completely from the seed coat they were counted as
germinated
While nearly all seeds treated for 8 minutes ofpercussion imbibed water
damage was evident in x-ray evaluations (Sacramento seed source) and in
germination counts (Holman and Capitan) at this treatment level Seed damage
during germination counts was similar to levels found in hot-water treated seeds
suggesting that over-treatment by both hot water and longer durations of percussion
may result in similar damage to seed structure
The Sacramento and Holman seed sources were two of the fIve sources that
opcrplltlpn in germination from the 4- to 8-minute durations of percussion
the benefIts ofloosening the strophiole as evidenced by imbibition of
all 8-minute treated seeds were outweighed by external and internal seed coat
39
a
b c d
11 Damage to New Mexico locust seeds observed during germination cotmts following hot water scarification 9a Hot water replicate including ~t seedlings and numerous unimbibed seeds 9b Close-up of seed where raallCle failed to elongate 9c Close-up ofseed where radicle elongated but was
-gtIn in freeing itself from the seed coat 9d Close-up ofseed where radicle broke off from embryo
40
damage (which also may be contributing to high imbibition) For the three sources
for which 8 minutes of percussion resulted in maximum germination extended
percussion of seeds necessary to loosen the strophiole may not have excessively
damaged the seed anatomy The Manzano source did not significantly change in
germination from 4 to 8 minutes of percussion suggesting that an increase in
permeable seeds at the 8-minute treatment duration was balanced by the number of
seeds that failed to germinate due to seed damage To summarize the 4-minute
percussion treatment improved germination over 8 minutes ofpercussion for a
majority of sources (five ofnine) the 8-minute percussion treatment improved
germination over 4-minutes ofpercussion for three sources and there was no
difference with the Manzano source Notably for the Raton source 8 minutes of
percussion increased germination by 235 percentage points over 4-minutes of
percussion (7875 vs 5525 respectively) the next best treatment Raton was the
only source to result in germination less than 75 for 4 minutes ofpercussion so a
grower may wish to apply longer percussion durations to lots that do not respond well
to this treatment level
Finally there was an interesting trend in the Jemez and Raton sources seed
that significantly contributed to the source by treatment interaction (Figure 12) A
~u test showed that the Jemez source had a significantly lighter seed weight than
other sources while the Raton source had an average seed weight For the Jemez
the longest duration of boiling increased germination Conversely only short
U4UVll) of percussion were needed to achieve high germination and the longest
41
80 _ Jemez a Raton
iD
~ C iD e iD
Q c 0 ~
c ~ iD 0
20
0------shy
Immersion Boiling 30 sec Boiling 60 sec
Hot Water Scarification
10degTr========~--------~------------------------------
80
60
40
20
_ Jemez Raton
0------shy
1 min 2 min 4 min 8 min
Percussion Scarification
Figure 12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources
42
b
--~----~~~~========~---- duration ofpercussion reduced germination For the Raton source longer durations
ofboiling consistently decreased germination whereas longer percussion treatments
consistently increased germination
A possible explanation is that a thick seed coat is associated with a loose
strophiole structure and that a thin seed coat is associated with a constrained
strophiole This contrast merits further study and may suggest an ecological
adaptation Further investigation ofthis phenomenon may contribute to successful
pre-screening ofunknown seed sources to determine optimal treatment leveL
43
CONCLUSION
Based on results obtained in pilot study (see Appendix) and thesis
experiments 4 minutes ofpercussion is recommended as a standard protocol for
treating an unknown source ofNew Mexico locust seed In addition to laboratory
results the 4-minute percussion treatment has resulted in germination greater than
80 and healthy seedling grow-outs for production purposes (unpublished data) In
laboratory experiments only one hundred seeds per 118 ml (40z) soil tin were
percussed at a time whereas for production we have treated up to five hundred seeds
per tin For large-scale production ofNew Mexico locust seedlings growers may
wish to experiment with larger quantities of seed and larger-size soil tins
Seed storage trials following percussion treatments were not conducted
However germination greater than 80 and healthy seedlings were obtained when
seeds were percussion-treated and subsequently stored at room-temperature (22degC)
for one week (unpublished data) Seed storage trials merit further study as the
standard hot water treatment leaves seeds in a softened condition that is not
conducive to storage
Hydroseeding is an effective method for direct seeding in reclamation
situations Percussion treatment may be conducive to hydro seeding as seeds remain
a dry relatively undamaged yet permeable condition following treatment Sulfuric
seeds also remain dry following treatment but extensive damage to seed coat
poor seedling vigor have been reported with this treatment (Kelly and van Staden
A grow-out study using a hydro seeder on a reclamation site testing percussion
44
against other treatments such as sulfuric acid and mechanical scarification also
merits further study
Papilionoids are the largest subfamily oflegumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust these legumes often play an integral role in the revegetation of
disturbed lands The pilot study (Appendix) indicates that percussion scarification
successfully treats the widely used black locust as well Developing superior
scarification methods to the standard hot water mechanical scarification and acid
treatments may facilitate the use ofpapilionoid legumes in reclamation projects
45
APPENDICES
APPENDIX A
PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR
NEW MEXICO LOCUST AND BLACK LOCUST SEEDS
Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4
Hot water and sulfuric acid soaks are traditional treatments for seeds of many
temperate woody legwnes including locusts However these scarification techniques
often produce inconsistent germination Percussion scarification where seeds are
repeatedly propelled against a hard surface was compared with hot water
scarification to evaluate treatment efficacy for New Mexico locust (Robinia
neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment
seeds were placed in a 98degC water bath which was immediately removed from the
heat source For percussion scarification seeds were placed in a soil sample tin and
agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the
control were followed by 24-hour water soaks Hot water baths resulted in 56 and
41 germination for New Mexico locust and black locust respectively For both
species nearly all durations ofpercussion increased germination over the hot water
treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3
4 and 5 minutes for black locust resulted in at least 90 germination Traditional
scarification treatments randomly degrade the entire seed coat which can lead to
tissue damage during water uptake Percussion scarification specifically weakens the
strophiole the natural source ofwater entry to the seed in papilionoid legwnes
INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom
2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031
48
Following percussion imbibition is controlled through the strophiole and underlying
tissue is protected
Keywords New Mexico locust black locust scarification percussion germination
Introduction
Locust trees (Robinia spp) are aggressive pioneer species that quickly
colonize disturbed land fix atmospheric nitrogen in the soil and stabilize slopes that
are prone to erosion (Klemmedson 1994 Ashby et al 1985) (Figure A1ab)
Recognition of these attributes has contributed to increased demand for locusts in
reclamation projects Locust seeds however exhibit physical dormancy where a
thick seed coat prevents the movement of water and gasses to the embryo (Leadem
1997)
Sulfuric acid or hot water soaks have traditionally been used to break seed
dormancy in macro-propagation of locusts Many growers are moving away from the
use of su1furic acid in treating locust seeds (Dreesen and Harrington 1997) In
addition to being dangerous acid soak durations must be specifically correlated to
seed lot (Olson 1974) Collections of native seed can vary tremendously in seed size
seed weight and hard seededness etc This variability limits the utility of sulfuric
acid soaks as even within a given lot some seeds may be damaged whereas others
are under-treated and fail to imbibe (take up water) A survey of Southwestern
container growers indicates that hot water baths are currently the most common
scarification method for locust seeds (Hine et al 1997) However hot water baths
also produce inconsistent germination (Lin et al 1996)
49
b
Figure AI New Mexico locust a primary invader following disturbance Ala Crown-sprouting at edge ofburn near Los Alamos NM--one year after Cerro Grande fire Alb Colonizing road cut Gila National Forest NM
50
Percussion scarification where seeds are repeatedly propelled against a hard
surface is an alternative dormancy-breaking method that has proven successful in
legumes related to locusts (Hamly 1932 Barton 1947) We compared this alternative
treatment to hot water scarification to evaluate treatment efficacy for New Mexico
locust and black locust seeds
Materials and Methods
Commercial New Mexico locust seeds (Western Native Seed Coaldale CO)
were collected fall 2000 in Huerfano County CO Black locust seeds were collected
September 2000 from Taos County NM by harvesting ripe pods from several trees at
a distance of up to 3 m from ground level Black locust seeds were allowed to air-dry
for 3 weeks and were threshed and separated from large chaffusing a greenhouse
fan A South Dakota Seed Blower (Chicago Illinltois) was used to remove fine chaff
from both seed lots Seeds were storedmiddotat 2-4C until the germination study was
conducted in March 2001
This study compared hot water scarification with percussion scarification
using treatment and species as experimental factors Along with a control
scarification treatments included immersion of seed in boiling water as well as
percussion durations of 1 2 3 45 or 10 minutes Four 100-seed samples were used
to test each species by scarification treatment combination
For the hot water treatment test tubes were filled with 30 mL water and
placed in a water-filled beaker (Figure A2a) The bath was raised to boiling (-98C at
51
b
c
Figure A2 Implementation ofhot water and percussion scarification A2a Hot water bath A2b Paint-shaker set-up A2c Placing soil tin containing seeds at end ofpaint shaker ann
52
Las Cruces NM elevation of 1300 meters) Each 100-seed replicate was placed in a
test tube and each test tube was immediately removed to cool at room temperature
Percussion scarification was implemented using a pneumatic paint shaker
(Central Pneumatic model 00422 Camarillo CA) (Figure A2b) This allowed
standardization of treatment intensity Compressed air pressure was maintained at
530kPa plusmn 30kPa (80psi plusmn 5psi) resulting in approximately 350 oscillations per
minute Each lOO-seed sample was placed in a 118 ml (4 oz) soil tin for shaking To
maximize lateral movement of paint shaker a paint can was used as a spacer to place
soil tins at the end of the shaking arm (Figure A2c) This allowed shaking to occur at
the greatest distance from the pivot point (fulcrum) Following all treatments
including the control seeds were soaked for twenty-four hours in distilled water prior
to plating out
Gennination was evaluated on lab benches at room temperature Each
replicate of seeds was placed in a 100 cm petri dish on moistened filter paper with
humidity maintained by enclosing the petri dishes in ziplock bags Gennination was
monitored daily for 14 days in accordance with International Seed Testing
Association standards for Robinia species (ISTA 1999) Gennination was defined as
protrusion of the radicle from the embryo by at least 1mm as viewed by the naked
eye
Categorical Analysis of Variance using Prpc Catmod (SAS Institute 1989)
was used to detennine treatment differences in germination percentages for each
source This procedure is an extension of a Chi-square test of homogeneity using the
53
natural log of the ratio of germinated to non-germinated seeds for each treatment Pshy
values less than 005 were considered significant A limited set of pairwiseshy
comparisons was conducted to compare treatment mean percents using a
conservative alpha value of 005 divided by the number of comparisons
Results
Percussion maximized germination for both New Mexico locust and black
locust There were three germination response levels for New Mexico locust (Figure
A3) First there was a low response in the control indicating a dormant seed lot
The hot water treatment and I-minute duration of percussion share a second
intermediate response Germination was highest at percussion levels from 2 to 10
minutes
A selected percussion treatment intermediate in time (5 minutes) produced the
steepest germination curve indicating the fastest germination rate (Figure A4) The
hot water treatment produced an intermediate curve or relatively slower germination
rate The control curve was very flat indicating the slowest germination rate for New
Mexico locust
Control seeds germinated poorly for black locust as well (Figure A5) Hot
water scarification improved germination over the control but all levels of percussion
improved germination over hot water The optimal durations of percussion treatment
were bracketed with a significant increase from the 1 to 2 minute percussion level a
high germination response from 2 to 5 minutes of percussion and finally a significant
54
~ d 25 CL)
p t1 0 ~ t1
g Vl ~ Vl
100~---------------------------------------------------------------
80
60
40
20
o Control Hot Water Percussion
I min Percussion
2 min Percussion
3 min
Scarification
Percussion 4 min
Figure A3 Effect of scarification on germination response for New Mexico locust
Percussion 5 min
=significant difference between adjacent treatment bars ns = no significant difference
Percussion 10 min
~ ~ () u agt
Il-t d o ~
~ ~ c3
100middotlr~~~------------------------------------e- Percussion 5min middot 0middot Hot Water -- Control
80-J=middot======
6b-middot-lmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middot--middotmiddot-middot~-middotmiddot -middotmiddot middot middotmiddot-middot 1
40
20
-----~-----------
0 0 0 middotmiddotmiddotmiddotmiddot0middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot0
0 --~-----y-----~------_---~--- T-----~
o shy -TDay 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A4 Effect of selected scarification treatments on germination speed for New Mexico locust First seven days of fourteen-day experiment are shown
Day 7
80
~ cd
5 60 ()
[) Cl d 0 1t1 d
40sect dUl
-J
20
o
ns
Scarification
Figure A5 Effect of scarification on germination response for black locust = significant difference between adjacent treatment bars ns = no significant difference
decrease at the 10-minute level This drop-off suggests that 10 minutes of percussion
over-treats seeds from this lot of-black locust
A selected percussion treatment intermediate in time (5 minutes) also had the
fastest germination rate for black locust (Figure A6)_ The hot water curve is
somewhat flatter indicating a slower germination rate The control curve is very flat
indicating a slow germination rate
A qualitative difference between hot water and percussion-treated seeds was
also observed Along with healthy germinants the hot water treatment produced
over-treated seeds (Figure A 7a) Examples of damaged germinants include seeds
where the radicle did not elongate (Figure A 7b) seeds where the radicle elongated
but was delayed in freeing itself from the seed coat (Figure A7c) and seeds where the
radicle broke off completely from the embryo (Figure A7d) In addition to these
examples of over-treatment there were numerous small dark seeds that failed to
imbibe water For percussion treatment nearly all seeds took up water (Figure A8a)
Germination was rapid and uniform with little damage evident (Figure A8b)
To summarize both New Mexico locust and black locust seed lots were
dormant The standard hot water scarification treatment improved germination but
only to 56 and 41 for New Mexico locust and black locust respectively Nearly
all percussion durations improved total germination as well as germination rate over
the hot water treatment Germination was greater than 90 for percussion durations
of 4 5 and 10 minutes for New Mexico locust and 3 4 and 5 minutes for black locust
58
CI)
sgt t CI) 0
igt 0
sect ttl t E
Vl 0
c3
100r==~==~~=r------------------------------------~ ___ Percussion 5 min
middotmiddotmiddot0middotmiddotmiddot Hot Water -lIf- Control
80r=========~--~~----------------------------~
60
40
20 -~-~---~---------- ---~------------I
omiddot
O
_~_----------lIf-----~bull -----_ shy---shy I
Day 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A6 Effect of selected scarification treatments on germination speed for black locust First seven days of fourteen-day experiment are shown
Day 7
a
b c d
Figure A7 Damage to New Mexico locust seeds observed during germination counts following hot water scarification A7a Hot water replicate including damaged seedlings and numerous unimbibed seeds A7b Close-up ofseed where radicle failed to elongate A7c Close-up ofseed where radicle elongated but was delayed in freeing itself from the seed coat A7d Close-up ofseed where radicle completely broke offfrom embryo
60
a
b
Figure A8 New Mexico locust seeds following percussion treatment at time of germination counts A8a Percussion replicate with uniformly healthy seedlings and near complete imbibition of seeds A8b Close-up ofpercussion-treated seedling
61
Discussion
Papilionoid seeds have a specific anatomical feature known as the strophiole
or lens-the natural site of water entry to the seed (Hamly 1932) The strophiole is
located on the cotyledonary lobe of a locust seed (Figure A9) When a papilionoid
legume seed is percussed for the appropriate amount of time repeated hits on the
integrated seed coat loosen the constrained cells of the strophiolar region without
excessively damaging the rest of the seed coat (Ballard 1976)
When a percussed seed is soaked water enters exclusively through the
strophiole in a controlled manner (Kelly and Staden 1987) This regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues This
contrasts with seeds that have been hot water or acid scarified treatments that can
randomly degrade the seed coat Unlocalized cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rehman et al 1998 Marunda 1990)
Papilionoids are the largest subfamily of legumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust and black locust these legumes often play an integral role in the
revegetation ofdisturbed lands Developing superior scarification methods to the
standard hot water and acid treatments should fadlitate the use ofthese legumes in
restoration projects
62
Figure A9 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (bc)
63
APPENDIXB
GERMINATION PERCENTS AND STANDARD ERRORS FOR SOURCE
BLOCK AND TREATMENT FOR NEW MEXICO LOCUST
Table B 1 Gennination percents and standard errors for source block and treatment for New Mexico locust
Source Block Treatment Germination Percentage Standard Error
Capitan 1 Immersion 74 437 Capitan 2 Immersion 69 462 Capitan 3 Immersion 71 454 Capitan 4 Immersion 67 470 Capitan Boiling 30 sec 79 407 Capitan 2 Boiling 30 sec 76 427 Capitan 3 Boiling 30 sec 72 449 Capitan 4 Boiling 30 sec 76 427 Capitan Boiling 60 sec 71 454 Capitan 2 Boiling 60 sec 54 498 Capitan 3 Boiling 60 sec 67 470 Capitan 4 Boiling 60 sec 76 427 Capitan I Control 5 218 Capitan 2 Control 7 255 Capitan 3 Control 4 196 Capitan 4 Control 5 218 Capitan 1 Percussion I min 47 499 Capitan 2 Percussion I min
470
Capitan Percussion I min 39 488
Capitan 4 Percussion I min 38 485 Capitan I Percussion 2 min 55 497 Capitan Capitan
2
Percussion 2 min Percussion 2 min
71 65
454 477
Capitan 4 Percussion 2 min 56 496 Capitan I Percussion 4 min 86 347 Capitan 2 Percussion 4 min 79 407 Capitan 3 Percussion 4 min 81 392 Capitan 4 Percussion 4 min 82 384 Capitan I Percussion 8 min 90 300 Capitan 2 Percussion 8 min 88 325 Capitan 3 Percussion 8 min 90 300 Capitan 4 Percussion 8 min 90 300 Gila I Immersion 71 454 Gila 2 Immersion 77 421 Gila
Immersion 75 433
Gila 4 Immersion 72 449
Gila I Boiling 30 sec 68 466
Gila 2 Boiling 30 sec 81 392
Gila 3 Boiling 30 sec 54 498
Gila 4 Boiling 30 sec 63 483
Gila I Boiling 60 sec 55 497
Gila 2 Boiling 60 sec 68 466
Gila 3 Boiling 60 sec 57 495
Gila 4 Boiling 60 sec 70 458
65
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Gila 2 Control 13 336 Gila 3 Control 13 336 Gila 4 Control 20 400 Gila 1 Percussion 1 min 66 474 Gila 2 Percussion 1 min 57 495 Gila 3 Percussion 1 min 73 444 Gila 4 Percussion 1 min 82 384 Gila 1 Percussion 2 min 81 392 Gila 2 Percussion 2 min 86 347 Gila 3 Percussion 2 min 85 357 Gila 4 Percussion 2 min 54 498 Gila 1 Percussion 4 min 79 407 Gila 2 Percussion 4 min 83 376 Gila 3 Percussion 4 min 84 367 Gila 4 Percussion 4 min 79 407 Gila 1 Percussion 8 min 51 500 Gila 2 Percussion 8 min 76 427 Gila 3 Percussion 8 min 52 500 Gila 4 Percussion 8 min 74 439 Holman 1 Immersion 83 376 Holman 2 Immersion 86 347 Holman 3 Immersion 81 392 Holman 4 Immersion 90 300 Holman 1 Boiling 30 sec 90 300 Holman 2 Boiling 30 sec 85 357 Holman 3 Boiling 30 sec 87 336 Holman 4 Boiling 30 sec 83 376 Holman 1 Boiling 60 sec 79 407 Holman 2 Boiling 60 sec 80 400 Holman 3 Boiling 60 sec 86 347 Holman 4 Boiling 60 sec 93 255 Holman Control 25 433 Holman 2 Control 19 392 Holman Control 21 407
Holman 4 Control 24 427
Holman Percussion 1 min 77 421
Holman 2 Percussion 1 min 85 357
Holman 3 Percussion 1 min 80 400
Holman 4 Percussion 1 min 89 313
Holman 1 Percussion 2 min 88 325
Holman 2 Percussion 2 min 96 196
Holman 3 Percussion 2 min 93 255
Holman 4 Percussion 2 min 96 196
Holman 1 Percussion 4 min 92 271
Holman 2 Percussion 4 min 97 171
66
I
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Holman 3 Percussion 4 min 91 286 Holman 4 Percussion 4 min 96 196 Holman 1 Percussion 8 min 88 325 Holman 2 Percussion 8 min 85 357 Holman 3 Percussion 8 min 83 376 Holman 4 Percussion 8 min 82 384 Jemez 1 Immersion 53 499 Jemez 2 Immersion 44 496 Jemez 3 Immersion 52 500 Jemez 4 Immersion 70 458 Jemez 1 Boiling 30 sec 58 494 Jemez 2 Boiling 30 sec 55 497 Jemez 3 Boiling 30 sec 48 500 Jemez 4 Boiling 30 sec 65 477 Jemez 1 Boiling 60 sec 68 466 Jemez 2 Boiling 60 sec 63 483 Jemez 3 Boiling 60 sec 73 444 Jemez 4 Boiling 60 sec 89 313 Jemez 1 Control 2 140 Jemez 2 Control 9 286 Jemez 3 Control 6 237 Jemez 4 Control 8 271 Jemez 1 Percussion 1 min 58 494 Jemez 2 Percussion 1 min 63 483 Jemez 3 Percussion 1 min 77 421 Jemez 4 Percussion 1 min 89 313 Jemez 1 Percussion 2 min 68 466 Jemez 2 Percussion 2 min 64 480 Jemez 3 Percussion 2 min 80 400 Jemez 4 Percussion 2 min 83 376 Jemez Percussion 4 min 68 466 Jemez 2 Percussion 4 min 79 407 Jemez 3 Percussion 4 min 70 458 Jemez 4 Percussion 4 min 83 376 Jemez 1 Percussion 8 min 52 500 Jemez 2 Percussion 8 min 59 492 Jemez 3 Percussion 8 min 61 488 Jemez 4 Percussion 8 min 70 458 Magdalena 1 Immersion 69 462 Magdalena 2 Immersion 69 462 Magdalena J Immersion 70 458 Magdalena 4 Immersion 85 357 Magdalena 1 Boiling 30 sec 65 477 Magdalena 2 Boiling 30 sec 71 454 Magdalena 3 Boiling 30 sec 68 466
67
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Magdalena 4 Boiling 30 sec 83 376 Magdalena 1 Boiling 60 sec 63 483 Magdalena 2 Boiling 60 sec 68 466 Magdalena 3 Boiling 60 sec 63 483 Magdalena 4 Boiling 60 sec 82 384 Magdalena 1 Control 11 313 Magdalena 2 Control 6 237 Magdalena 3 Control 5 218 Magdalena 4 Control 4 196 Magdalena Percussion 1 min 32 466 Magdalena 2 Percussion 1 min 45 497 Magdalena 3 Percussion 1 min 46 498 Magdalena 4 Percussion I min 36 480 Magdalena 1 Percussion 2 min 67 470 Magdalena 2 Percussion 2 min 68 466 Magdalena 3 Percussion 2 min 65 477 Magdalena 4 Percussion 2 min 81 392 Magdalena 1 Percussion 4 min 87 336 Magdalena 2 Percussion 4 min 97 171 Magdalena 3 Percussion 4 min 83 376 Magdalena 4 Percussion 4 min 97 171 Magdalena 1 Percussion 8 min 98 lAO Magdalena 2 Percussion 8 min 100 000 Magdalena 3 Percussion 8 min 96 196 Magdalena 4 Percussion 8 min 98 lAO Manzano 1 Immersion 67 470 Manzano 2 Immersion 57 495 Manzano 3 Immersion 72 4049 Manzano 4 Immersion 73 4044 Manzano 1 Boiling 30 sec 70 458 Manzano 2 Boiling 30 sec 64 480 Manzano 3 Boiling 30 sec 71 454 Manzano 4 Boiling 30 sec 80 400 Manzano I Boiling 60 sec 50 500 Manzano 2 Boiling 60 sec 53 499 Manzano 3 Boiling 60 sec 59 492 Manzano 4 Boiling 60 sec 74 439 Manzano I Control 4 196 Manzano 2 Control 8 271 Manzano 3 Control 1 099 Manzano 4 Control 6 237 Manzano 1 Percussion 1 min 52 500 Manzano 2 Percussion 1 min 39 488 Manzano 3 Percussion 1 min 36 480 Manzano 4 Percussion 1 min 51 500
68
Table B 1 Continued
Source Block Treatment Gerrni~ation Percentage Standard Error
Manzano Percussion 2 min 53 499 Manzano 2 Percussion 2 min 58 494 Manzano 3 Percussion 2 min 61 488 Manzano 4 Percussion 2 min 62 485 Manzano 1 Percussion 4 min 76 427 Manzano 2 Percussion 4 min 85 357 Manzano 3 Percussion 4 min 79 407 Manzano 4 Percussion 4 min 83 376 Manzano 1 Percussion 8 min 87 336 Manzano 2 Percussion 8 min 83 376 Manzano 3 Percussion 8 min 83 376 Manzano 4 Percussion 8 min 75 433 Raton 1 Immersion 54 498 Raton 2 Immersion 37 483 Raton 3 Immersion 39 488 Raton 4 Immersion 69 462 Raton 1 Boiling 30 sec 36 480 Raton 2 Boiling 30 sec 20 400 Raton 3 Boiling 30 sec 28 449 Raton 4 Boiling 30 sec 60 490 Raton 1 Boiling 60 sec 15 357 Raton 2 Boiling 60 sec 171 Raton 3 Boiling 60 sec 4 196 Raton 4 Boiling 60 sec 35 477 Raton 1 Control 4 196 Raton 2 Control 12 325 Raton 3 Control 2 140 Raton 4 Control 6 237 Raton 1 Percussion 1 min 52 500 Raton 2 Percussion I min 29 454 Raton 3 Percussion I min 35 477 Raton 4 Percussion 1 min 31 462 Raton 1 Percussion 2 min 55 497 Raton 2 Percussion 2 min 35 477 Raton 3 Percussion 2 min 33 470 Raton 4 Percussion 2 min 42 494 Raton 1 Percussion 4 min 65 477 Raton 2 Percussion 4 min 52 500 Raton Percussion 4 min 51 500 Raton 4 Percussion 4 min 53 499 Raton 1 Percussion 8 min 88 325 Raton 2 Percussion 8 min 80 400 Raton 3 Percussion 8 min 73 444 Raton 4 Percussion 8 min 74 439 Sacramento 1 Immersion 43 495
69
Table B 1 Continued
-Source Block Treatment Germination Percentage Standard Error
Sacramento 2 Immersion 30 458 Sacramento 3 Immersion 38 485 Sacramento 4 Immersion 56 496 Sacramento 1 Boiling 30 sec 47 499 Sacramento 2 Boiling 30 sec 48 500 Sacramento 3 Boiling 30 sec 48 500 Sacramento 4 Boiling 30 sec 56 496 Sacramento 1 Boiling 60 sec 52 500 Sacramento 2 Boiling 60 sec 67 470 Sacramento 3 Boiling 60 sec 43 495 Sacramento 4 Boiling 60 sec 48 500 Sacramento 1 Control 0 000 Sacramento 2 Control 3 171 Sacramento 3 Control 3 171 Sacramento 4 Control 099 Sacramento 1 Percussion 1 min 57 495 Sacramento 2 Percussion 1 min 61 488 Sacramento 3 Percussion 1 min 62 485 Sacramento 4 Percussion 1 min 55 497 Sacramento 1 Percussion 2 min 84 367 Sacramento 2 Percussion 2 min 70 458 Sacramento 3 Percussion 2 min 81 392 Sacramento 4 Percussion 2 min 69 462 Sacramento 1 Percussion 4 min 91 286 Sacramento 2 Percussion 4 min 84 367 Sacramento 3 Percussion 4 min 84 367 Sacramento 4 Percussion 4 min 94 237 Sacramento 1 Percussion 8 min 82 384 Sacramento 2 Percussion 8 min 81 392 Sacramento 3 Percussion 8 min 82 384 Sacramento 4 Percussion 8 min 81 392 Sandia 1 Immersion 76 427 Sandia 2 Immersion 70 458 Sandia 3 Immersion 70 458 Sandia 4 Immersion 81 392 Sandia 1 Boiling 30 sec 78 414 Sandia 2 Boiling 30 sec 71 454 Sandia 3 Boiling 30 sec 63 483 Sandia 4 Boiling 30 sec 78 414 Sandia 1 Boiling 60 sec 67 470 Sandia 2 Boiling 60 sec 65 477 Sandia 3 Boiling 60 sec 74 439 Sandia 4 Boiling 60 sec 66 474 Sandia 1 Control 16 367 Sandia 2 Control 7 255
70
Table B 1 Continued
Source Block Treatment Gennination Percentage Standard Error
Sandia 3 Control 8 271 Sandia 4 Control 16 367 Sandia 1 Percussion 1 min 67 470 Sandia 2 Percussion 1 min 61 488 Sandia 3 Percussion 1 min 66 474 Sandia 4 Percussion 1 min 59 492 Sandia I Percussion 2 min 82 384 Sandia 2 Percussion 2 min 83 376 Sandia 3 Percussion 2 min 81 392 Sandia 4 Percussion 2 min 84 367 Sandia 1 Percussion 4 min 89 313 Sandia 2 Percussion 4 min 86 347 Sandia 3 Percussion 4 min 89 313 Sandia 4 Percussion 4 min 85 357 Sandia 1 Percussion 8 min 80 400 Sandia 2 Percussion 8 min 77 421 Sandia 3 Percussion 8 min 69 462 Sandia 4 Percussion 8 min 66 474
LITERATURE CITED
Ashby WC Vogel WG Rogers NF 1985 Black locust in the reclamation equation General Technical Report Northeastern Forest Experiment Station USDA Forest Service NE-I05 12 p
Ballard LAT 1973 Physical barriers to gennination Seed Sci Technoll 285-303
___ 1976 Strophiolar water conduction in the seeds ofTrifolieae induced by action on the testa at nonstrophiolar sites Austr J Plant Physiol3 465-469
Barton LV 1947 Special studies on seed coat impenneability Contrib Boyce Thompson Inst 14 355-362
Baskin CC Baskin JM 1998 Seeds Ecology biogeography and evolution of donnancy and gennination Academic Press London UK 666 p
Bewley JD 1997 Seed germination and donnancy Plant Ce119 1055-1066
Bewley JD Black M 1994 Seeds physiology of development and germination 2nd ed Plenum Press New York NY 445 p
Briscoe CB 1996 Growing Nitrogen Fixing Trees in Acid Soils In MH Powell (ed) Nitrogen Fixing Trees for Acid Soils- A Field ManuaL Winrock International Morrilton AR 22 p
Copeland LO McDonald MB 1995 Principles of Seed Science and Technology 3rded Chapman amp Hall New York NY 409 p
Cox RE Klett JE 1984 Seed gennination requirements of native Colorado plants for use in the landscape Plant Propag 30(2) 6-10
Dell B 1980 Structure and function ofthe strophiolar plug in seeds ofAlbizia Lophantha Am J Bot 67 556-563
Dick-Peddie WA 1993 New Mexico Vegetation Past Present and Future University ofNew Mexico Press Albuquerque NM 244 p
Dreesen DR 2001 Propagation protocol for production ofcontainer Robinia neomexicana plants Los Lunas Plant Materials Center Los Lunas New Mexico In Native Plant Network URL httpwwwnativeplantnetworkorg (accessed 9 January 2002) Moscow (ID) University ofIdaho CoIl Natural Resources For Res Nursery
72
Dreesen DR Harrington JT 1997 Propagation of native plants for restoration projects in the Southwestern US- Preliminary Investigations In Landis TD Thompson JR tech coords National Proceedings Forest and Conservation Nursery Assocs Gen Tech Rep PNW-GTR-419 Portland OR USDA For Ser PNW Res Sta 77-88
Gottfried GJ 1980 Control ofNew Mexican locust and the effect on planted ponderosa pine in central Arizona US For Ser Res Note RM-386 Rocky Mountain For Range ExpStn Fort Collins CO
Gosling PG Samuel YK Jones SK 1995 A systematic examination of germination temperature chipping and water temperaturesoak duration pretreatments on the seeds ofLeucaena leucocephala Seed Sci amp Technol 23521-532
Hamly DH 1932 Softening of the seeds of Melilotus alba Bot Gazette 93 345shy375
Hanna PJ 1984 Anatomical features of the seed coat ofAcacia kempeana (Mueller) which relate to increased germination rate induced by heat treatment New Phytol 96 23-29
Hanks JP Dick-Peddie WA 1974 Vegetation patterns of the White Mountains New Mexico Southwest Naturalist 18(4) 371-382
Hine S Harrington JT Dreesen DR 1997 Seed scarification requirements for Robinia neomexicana In Harrington JTtech coord Proc Third SouthwestemContainer Growers Conf Feb 12-13 1997 Ft Collins CO
Hopkinson JM 1993 The strophiole of leguminous seeds Newsletter- IntI Herbage Seed Production Res Grp 18 7-8
---- 1997 Differences in hardseededness between Cunningham and Tarramba leucaena In Leucnet News The Newsletter of the International LeucaenaRes and Devlt Netwk No4
ISTA (International Seed Testing Association) 1999 International rules for seed testing rules Seed Sci amp Technol27 Supplement
Kelly KM van Staden J 1987 The lens as the site of permeability in the papilionoid seed Aspalathus linearis J Plant Physiol 128(45) 395-404
73
Keresztesi B 1988 Black locust the tree of agriculture Outlook on Agr 17(2) 77~85
Kigel 1 and Galili G 1995 Seed development and germination Marcel Dekker New York 853 p
Klemmedson 10 1994 New Mexico locust and parent material Influence on macronutrients of forest floor and soil Soil Sci Soc Am 1 58 974~980
Kuo WH Tam AW 1988 The pathway of water absorption of mung bean seeds Seed Sci amp Technol16(1) 139~144
Leadem CL 1997 Dormancy--unlocking seed secrets In Landis TD Thompson lR tech coords National Proceedings Forestand Conservation Nursery Assocs Gen Tech Rep PNW-G TR-419 Portland OR USDA For Ser PNW Res Sta 43-52
Lin Y Wagner MR Seweyestewa A Honahni MD DeGomez M McMillin JD 1996 Influence of cultural treatments on seed germination and seedling growth of Robinia neomexicana Forest Farm and Community Tree Res Reports Volume 1
Marunda CT 1990 Effects of seed pretreatment on the development ofAcacia auriculforinis and A holosericea seedlings ACIAR Proceedings Series No 28
Meginnis HG 1937 Sulphuric acid treatment to increase germination of black locust seed USDA Circ 453 34 p
Miklas PN Towensend CE Ladd SL 1987 Seedcoat anatomy and the scarification ofcicer milkvetch seed Crop Sci 27 766-772
Monsen SB 1984 Use of shrubs on mine spoils In The Challenge ofProducing Native Plants for the Intermountain Area Proceedings Intermountain Nurserymans Association 1983 conference Las Vegas NV compo PM Murphy 26-31 Ogden UT USDA For Ser Gen Tech Report INT-l68
Olson DF 1974 Robinia L Locust In Seeds of Woody Plants in the US tech coord CS Schopmeyer 728-731 USDA For Ser Agr Handbk No450
Rehman S Loescher RNl Harris P1C 1998 Dormancy breaking and germination ofAcacia salicina Lindl seeds Seed Sci amp Techno127 553-557
Rolston MP 1978 Water impermeable seed dormancy Bot Rev 44 365-396
74
SAS Institute Inc 1989 SASSTAT Users Guide Version 6 4th Edition Volume 1 Cary NC SAS Institute Inc 943 p
SerratoValenti G Conara L Ferrando M Modenesi P 1993 Structural and histochemical features ofStylosanthes scabra (Leguminosae Papilionoidae) seed coat as related to water entry Can J Bot 71 834-840
Simpson BJ 1988 A field guide to Texas trees Austin TX Texas Monthly Press 372p
Tran VN Cavanagh AK 1984 Structural aspects ofdormancy In Seed Physiology Vol 2 Murray DR (ed) p1-44 Academic Press Australia
USDA Forest Service 1948 Woody-plant seed manual USDA Misc Pub1654 416p
Vines RA 1960 Trees Shrubs and Woody Vines of the Southwest Austin Univ ofTexas Press 1104 p
Wagner MR Larson RE Craig TP 1992 Ecology and silvics ofNew Mexico locust (Robinia neomexicana) with an emphasis on the arboreal form p53-57 In JW Hanover K Miller and S Plesko (eds) Proc Intemat Confon Black Locust Biology Culture and Utilization Mich State Univ USA
Wilson JK 1944 Immersing seeds of species of Robinia in boiling water hastens germination J For 42 453-454
Zimmerman RW Carpenter SB 1980 First-year coppice production from a 5shyyear old black locust stand on surface mine spoil p309-314 Centr Hardwd For ConfProc University ofMissouri Columbia
Analysis 22
RESULTS 24
DISCUSSION 36
Detennining the Optimal Treatment 36
Strophiole and Seed Coat Behavior 37
CONCLUSION 44
Appendices
A PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR NEW MEXICO LOCUST AND BLACK LOCUST SEEDS 47
B GERMINATION PERCENTS AND STANDARD ERRORS FOR SOURCE BLOCK AND TREATMENT FOR NEW MEXICO LOCUST 64
LITERATURE CITED 72
Vlll
LIST OF TABLES
Table Page
1 Latitude longitude location elevation and collection date ofNew Mexico locust seed sources 18
2 Mean seed weight and standard error by source 19
3 Categorical Analysis ofVariance table for germination response to seed treatment seed source block and the interaction of the factors 25
4 Effect of seed source and treatment on G50 (days to 50 germination) 32
B1 Germination percents and standard errors for source block and treatment for New Mexico locust 65
LIST OF FIGURES
Figures Page
1 Seedsourcecollectionmap 17
2 Effect ofNew Mexico locust seed source on germination for data averaged over scarification treatments 26
3 Effect of scarification on germination for New Mexico locust for data averaged over all seed sources 27
4 Effect of seed source by treatment interaction on New Mexico locust germination for percussion scarification pattern a and pattern b 28
5 Effect of source by treatment interaction on New Mexico locust germination for hot water scarification 29
6 Timecourse of New Mexico locust germination following scarification treatments 31
7 X-ray radiographs of Sacramento seed source perc ussed for 1 4 or 8 minutes 33
8 Seed damage close-ups 34
9 SEM images of untreated vs 4-minute percussion-treated strophiolar cells ofNew Mexico locust seeds 35
10 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (b c) 38
11 Damage to New Mexico locust seeds observed during germination counts following hot water scarification 40
12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources 42
New Mexico locust a primary invader following disturbance 50
Implementation of hot water and percussion scarification 52
Effect of scarification on germination response for New Mexico locust 55
x
A4 Effect of selected scarification treatments on germination speed for New Mexico locust 56
AS Effect of scarification on germination response for black locust 57
A6 Effect of selected scarification treatments on germination speed for black locust 59
A7 Damage to New Mexico locust seeds observed during germination counts following hot water scarification 60
AS New Mexico locust seeds following percussion treatment at time of germination counts 61
A9 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (bc) 63
INTRODUCTION
New Mexico locust (Robinia neomexicana A Gray) a small tree native to the
southwestern United States occurs at elevations from 1200 to 2800 meters This
species fills a successional role in post-disturbance situations As a primary invader
New Mexico locust quickly establishes on burned areas arid flood banks as well as
road cuts (Wagner et aI 1992) Rapid growth crown sprouting and prolific root
suckering favor the successful establishment ofNew Mexico locust on disturbed sites
(Simpson 1988 Gottfried 1980) These attributes also may make New Mexico locust
well suited for steep-slope revegetation where erosion is a problem
New Mexico locust is a nitrogen-fixing legume that tolerates and improves
nutrient-poor soils Stands ofNew Mexico locust increase Nitrogen (N) and Calcium
(Ca) in the forest floor Levels of Carbon (C) Phosphorous (P) Sulfur (8) and
Potassium (K) aIso have been shown to increase in soil beneath New Mexico locust
(Klemmedson 1994)
The ability to colonize rapidly and ameliorate harsh sites contributes to New
Mexico locusts potential as a nurse plant A nurse plant colonizes an inhospitable
site and creates an environment suitable for successional plant establishment New
Mexico locust demonstrates this role in the ponderosa pine (Pinus ponderosa Douglas
ex Lawson amp C Lawson)Gambel oak (Quercus gambelii Nutt) community After a
disturbance such as wildfire New Mexico locust and Gambel oak colonize and
dominate the site until shaded out by ponderosa pine Once shaded these species
become understory shrubs a process taking an average of 15-20 years (Dick-Peddie
1993 Hanks and Dick-Peddie 1974) New Mexico locust gradually declines with
small dense patches averaging about 005 ha beneath pine until the next disturbance
once again offers it a competitive advantage (Klemmedson 1994)
Gottfried (1980) expecting ponderosa pine regeneration to be greater where
New Mexico locust had been eradicated found that survival of planted pine seedlings
was greater where locust had not been removed In a follow-up study Gottfried
(1980) noted that soil moisture in the top 57 cm was highest in 5-year old locust sites
as compared to grass or 20-year old locust sites He concluded that managing an
appropriate cover ofNew Mexico locust could help regenerate pine the later
successional species
The ability ofNew Mexico locust to improve harsh sites also makes it a
candidate for reclaiming disturbances associated with mining Black locust (Robinia
pseudoacacia 1) has been used for decades to reclaim mine spoils and other
disturbed sites in the eastern US and around the world (Keresztesi 1988 Zimmerman
and Carpenter 1980) Ashby et al (1985) described similar positive attributes of
black locust also mentioned previously for New Mexico locust quick cover for
stabilization supply ofN and nutrient-rich litter to the soil and site improvement for
establishment of later successional trees Referring specifically to mine reclamation
the authors note the ability of black locust to grow on a wide range ofmine soil
conditions including extremely acid soils Black locust also shows some tolerance to
soils compacted by grading and topsoiling practices (Ashby et aL 1985) New
2
Mexico locust holds promise as a native southwe~tem counterpart to black locust in
mine reclamation but it has been used infrequently to date
Natural invasion and succession occur slowly on most mine sites (Monsen
1984) While New Mexico locust often colonizes sites naturally there is no
assurance a seed will reach a particular site and establish in a reasonable time frame
The challenge is to introduce New Mexico locust as mother plants to facilitate
colonization Seedling production for outplanting on disturbed sites has been
hampered by poor germination (Lin et al 1996) The goal then is to develop
techniques to overcome poor germination in New Mexico locust thereby facilitating
macropropagation
LITERATURE REVIEW
Seed Dormancy
Low gennination in New Mexico locust is primarily the result of seed
donnancy the failure of an intact viable seed to genninate under favorable
conditions (Bewley 1997) Seed donnancy also can be defined as the inability of the
embryo to genninate because of some inherent constraint (Bewley and Black 1994)
The constraint may originate within the embryo referred to as endogenous or embryo
donnancy or from the tissues surrounding the embryo known as exogenous or coatshy
imposed donnancy (Baskin and Baskin 1998 Leadem 1997 Bewley and Black
1994)
New Mexico locust seed is donnant when the impenneable seed coat does not
allow movement ofwater to the embryo This specific fonn of coat-imposed
donnancy where the restraining tissue involved is an impenneable seed coat is
known as physical donnancy (Baskin and Baskin 1998 Bewley and Black 1994)
Physical donnancy is relatively uncommon in seeds less than 10 of all woody
species exhibit this condition (USDA Forest Service 1948) However it is
characteristic of many genera of the Fabaceae (bean) family For example all
successional trees in the genera Gleditsia and Robinia (of which New Mexico locust
is a member) produce seeds that exhibit physical donnancy (Baskin and Baskin
4
Physical Dormancy
Seeds that exhibit physical dormancy are said to be hard seeded referring to
the thick impermeable seed coat (Leadem 1997) The first line of protection of the
testa in leguminous seeds is the outermost waxy cuticle followed by a subcuticle
consisting ofsuberin Next is a palisade layer consisting of sclereid cells that have
thick lignified secondary walls The most common type of sclereid cell in palisade
layers is the macrosclereid The macrosclereids contain water-repellent substances
including cutin lignin suberin and wax (Rolston 1978) This palisade layer is the
primary barrier to water uptake (Serrato-Valenti et al 1993 Tran and Cavanagh
1984)
All seed coat openings must be blocked to effectively prevent water uptake
The natural opening in a seed where it was attached to the mother plant is the weak
link where physical dormancy is usually broken (Hopkinson 1993 Kuo and Tam
1988) Anatomical structures associated with the seed coat opening vary from
family to family In the Papilionoideae the subfamily of the Fabaceae family
containing New Mexico locust the site ofwater entry is the strophiole The
strophiole is a swelling on the seed coat close to the stalk scar (Hopkinson 1997)
Kelly and van Staden (1987) refer to the strophiole site as the lens where
macrosclereid cells must separate and raise before water can pass through Since
middot6germination ofdormant seeds of the Papilionoideae takes place when the strophiole
permeable (Hopkinson 1993 Kuo and Tam 1988) a logical approach to
5
genninating New Mexico locust artificially would be to loosen the strophiolar cells
and allow water to pass through to the embryo
Evolutionary Role of Physical Dormancy
Why is physical dormancy a mechanism that blocks germination
ecologically advantageous When a plant produc~s seeds with different degrees of
dormancy seeds that are dispersed at one time will germinate over a range of time
(Bewley and Black 1994) Through variability of dormancy within a seed crop seeds
are exposed to changing environmental conditions improving the overall chances of
survival of the seed line (Bewley and Black 1994) Varying expression of dormancy
is under genetic control which indicates that this may be an evolved survival strategy
(Kigel and Galili 1995) A range of dormancy levels may be a particular advantage in
the southwestern US where extreme environmental variations exist
Methods for Breaking Dormancy
There are three common approaches to breaking dormancy in New Mexico
locust seed acid scarification hot water soaks and mechanical treatments In each
case the objective of the treatment is to scarify or abrade the seed coat to make it
permeable to water
Acid Scarification
Acid scarification consists of soaking seeds in concentrated sulfuric acid
rinsing thoroughly with water upon removal followed by subsequent drying before
PlaJlltmg Olson (1974) reports that this scarification method produces the highest
middot~~jllalUll results for all Robinia species Most literature refers to a seminal 1937
6
study conducted by HG Meginnis This stud~ t~ough conducted on black locust
seed is regularly cited in the suggested use of acid to treat New Mexico locust seed
as well (Olson 1974 Vines 1960)
Thee published studies document the specific use of sulfuric acid to treat
New Mexico locust seed Cox and Klett (1984) report that soaking seeds in
concentrated sulfuric acid for thirty minutes improved germination to 34 over an
untreated control of4 Lin et al (1996) found that for two seed lots acid
scarification for twenty-five to eighty minutes produced germination of 80 A third
lot however resulted in less than 20 germination at all durations of 10 to 80
minutes Hine et al (1997) showed that acid treatment levels of 1 2 5 and 10
minutes resulted in less than 20 germination across five sources However there
was an increasing trend in germination with acid soak duration and acid treatments
were not imposed for as long a time as the previous two studies (30 to 80 minutes)
The mixed results obtained by these investigators were probably due to the
widely varying germination response of seed lots to acid treatment For this reason
Olson (1974) recommends correlating duration of acid treatment to each seed lot If
treatment duration is too long the embryo will be permanently damaged If soak
duration is too short the seed will remain impermeable to water and fail to germinate
Compounding the problem of treatment duration is the negative effect acid
have on seedling growth Any type of scarification can injure seeds to some
by disrupting cells essential for growth leading to fungal invasion and
middot-uuu~w injury (Copeland and MacDonald 1995) Some legume seeds subjected
7
to 60 minutes of sulfuric acid scarification (to achieve maximum gennination results)
showed more than 50 seedling mortality after gennination (Kelly and van Staden
1987) Bewley and Black (1994) noted that fungal growth on seeds is an indication
of leakage of sugars organic acids amino acids and proteins
Perhaps the greatest drawback to acid scarification is the dangerous nature of
the treatment itself Difficulties associated with storage and trained use of
concentrated acid may discourage many propagators from using this option (Dreesen
and Harrington 1997) Consequently hot water and to a lesser extent mechanical
scarification are more commonly used and practical treatments for legume seeds
Most southwestern container growers use either of these two treatments to scarify
New Mexico locust seed in particular (Hine et al 1997)
Hot Water Scarification
The widespread use ofhot water scarification to treat locust seed is probably
due to its ease of application and efficacy in comparison to other scarification
treatments Although this practice had been in use for some time Wilson (1944) was
one of the first to report on its systematic application for black locust The treatment
consists of placing seeds in boiling water (4 parts water tomiddot 1 part seed by volume)
removing the vessel from the heat source and allowing the water to cool and seeds to
soak for 24 hours
Hine et al (1997) Lin et al (1996) and Cox and Klett (1984) have examined
the specific application ofhot water scarification on New Mexico locust seed Each
these authors compared hot water to acid scarification Lin et aL (1996) reported
8
60 gennination for two hot water-treated seed lots while a third lot germinated
around 50 (as reported above acid scarification germination ranged from 20 to
80 for these lots) It should be noted that the authors only soaked the seeds in water
from 10 to 80 minutes for this experiment as opposed to the standard 24 hours Cox
and Klett (1984) stated that using the standard 24-hour hot water treatment improved
germination over an acid pre-treatment (which produced less than 20 germination)
but they did not give specific gennination numbers for the hot water treatment Hine
et al (1997) found that across five seed lots germination was 66 for the standard
hot water treatment (as compared to less than 20 germination for acid scarification)
Variations in treatment intensity and duration and their subsequent effects on seed lot
appear to contribute to conflicting conclusions as to whether acid scarification or hot
water treatment is a more successful treatment
Given mixed results ease ofapplication is an important argument for use of
hot water application over acid soaks Safety concerns and material costs for hot
water treatments are low (Dreesen and Harrington 1997) An extension of this is the
practicality ofhot water for treating large batches of seed where a relative increase in
water volume is all that is needed However increases in water and seed even when
inthe same 4 1 proportion can result in dramatically different rates of cooling than in
atest tube experiment Different cooling rates can lead to contrasting results for the
treatment temperature duration and seed lot (USDA Forest Service 1948)
As with acid scarification parameters ofhot water treatment application may
to be optimized to maximize germination Hine et al (1997) found that as
9
temperature was increased from 70degC to 100degC overall germination in New Mexico
locust seed increased The 100degC treatment maximized germination for each of five
sources tested with germination usually above 60
Prolonged exposure of seeds ofmany woody legumes to boiling temperatures
also may improve germination (Briscoe 1996) This approach has not been tested on
New Mexico locust seed Exposing seed to prolonged boiling temperatures may
result in fungal infection and subsequent seedling mortality Marunda (1990) noted
severe fungal infections on both acid and hot water treated Acacia holoserica Cunn
ex G Don seeds attributing the infections to seed damage resulting from the
treatments Identifying an optimal hot water treatment level that does not result in a
high rate of seedling mortality is a logical goal
With hot water treatments as with acid there is also a danger of overtreating
Seeds and reducing germination (Gosling et al 1995) The legume Leucaena
leucocephala (Lam) de Wit germinated equally well after 7 seconds of exposure to
water at 100degC 7 seconds at 90degC 30 seconds at 80degC and 4 minutes at 70degC In
addition acceptably high germination was obtained from treatment at 70degC from 3
minutes to 1 hour whereas no seeds germinated following treatment at 100degC for 2
minutes Perhaps along with a greater margin of error for treatment duration treating
~ew Mexico locust seeds at a lower temperature may result in a comparatively lower
of seedling mortality In practice however nursery growers tend to use the
and quickest treatment available The short treatment length at near-boiling
whether recommended or not may be what is used most frequently
10
Fine-tuning a treatment in this range of treatment temperature and duration may be
the most logical approach
Mechanical Scarification
This treatment involves scratching the seed coat with an abrasive material
(USDA Forest Service 1948) One system employs a rotating drum lined with
sandpaper A paddle spinning at a given number ofrevolutions per minute throws the
seed against the sandpaper (Hine et al 1997) A second method dry tumbling
tumbles seed in a mixer with pea gravel or sharp sand for 5 to 7 days (Dreesen 2001)
These approaches have the advantage that control of temperature is not required as in
water treatments nor is safety an issue as with add scarification
Hine et al (1997) mechanically treated New Mexico locust seed using 100shy
grit sandpaper in a Forsberg (rotating drum) scarifier Gennination was less than
10 They concluded that the seed coats were too thin for their apparatus and
proposed that a finer grit sandpaper would produce more even abrasion Although
germination varied by seed lot Dreesen (2001) reported that using a slow-speed dry
tumbling technique for 5 to 7 days produced gennination rates as high as 90
As with acid and hot water treatments treatment duration must be specifically
shy
correlated to seed lot Treated seeds are also susceptible to fungal infection
iHopkinson 1997) In addition equipment is specialized and may not always be
ltU~CUJl~ to a nursery (USDA Forest Service 1948) Several studies have confinned
degrading ofstrophiolar cells due to acid and hot water scarification (Miklas et al
11
1987 Dell 1980 Hanna 1984) but the effect ofmechanical scarification on the
strophiole has not been studied
Alternative Method for Breaking Dormancy Percussion
Strophiole degradation has been examined in depth using an impaction
treatment more commonly known as percussion Hamly (1932) first proposed that
shaking or impacting seeds could split the macrosc1ereid cells at the strophiole
Hamly successfully released tension ofbent strophiolar cells of impermeable
Melilotus alba Medik (White sweet clover) seeds by delivering physical blows
resulting in cells that had split apart and were only slightly bent Ninety-two percent
oftreated seeds became permeable as compared to 05 of control seeds Barton
(1947) showed that percussion significantly increases permeability in black locust
seed Ballard (1976) reasoned that the integrated nature of the strophiole within the
seed coat means a blow on any part of the seed weakens the strophiole There are no
published studies on the use of percussion on New Mexico locust
Percussion methods vary and often seem SUbjective or ill-defined Hamly
(1932) placed seeds in a glass bottle and shook them three times a second for 20
minutes resulting in 180 oscillations per minute Ballard (1973) describes shaking
seeds in a closed metal container with at least fifty seeds per container at 850-900
JS(~l11alIOIlS per minute for 1 minute Kelly and van Staden (1987) placed seeds into
sealed glass flasks Flasks were shaken by a wrist-shaker at a rate of 250
~UUVJ~ per minute Subsequent germination rates were 45 after 2 hours of
55 after 3 hours 60 after 6 hours and 100 after 12 hours
12
There are several potential advantages of percussion over acid hot water and
mechanical scarification Kelly and van Staden (1987) note that a percussion
treatment only alters macrosclereid cells of the strophiole by loosening separating
and raising them They emphasize that minimal damage is done to the overall
anatomical structure of the seed In acid scarification of the same seed lot radicle
and cotyledon damage occurred Based on photographic evidence the authors
believe that this morphological damage was due to uneven pressure during
imbibition with water entering haphazardly wherever the acid had sufficiently
dissolved the coat This ultimately led to uneven hydration of the embryo In
percussed seeds water only entered through the s~ophiole when the entire seed coat
had been saturated resulting in controlled and even embryo hydration (Kelly and van
Staden 1987)
Although all successful scarification treatments may focus at least in part on
the strophiole for papilionoid legumes (Baskin and Baskin 1998) percussion may be
less damaging to seed coat structures particularly when compared with acid Kelly
and van Staden did not compare hot water or mechanical scarification methods
against percussion However the fungal infection associated with hot water and
mechanical scarification as with acid scarificatio~ have been directly associated with
seed damage (Rehman et al 1998 USDA Forest Service 1948) Embryo damage in
has been observed in hot-water scarified legume seeds (Rehman et al 1998
13
By limiting embryo damage percussion scarification reduces seedling
mortality compared to other treatments Kelly and van Staden (1987) noted that
seedlings from the percussion treatment were morphologically sound and vigorous
when compared to acid treatment seedlings The authors attribute this improved
seedling vigor to the controlled water uptake that limited anatomical damage
Despite a 12-hour percussion treatment morphological damage was not substantially
greater than untreated seeds In addition seedling mass measured 10 days following
germination was not significantly different between percussed and untreated seeds
A final advantage ofpercussion may be the ability to adequately treat the most
dormant seeds while avoiding harm to seeds that have already been made permeable
by the treatment or that were permeable to begin with Instead ofcorroding scalding
or scratching the seed coat percussion appears to be just a loosening of the
strophiole spring (Ballard 1973) Kelly and van Staden (1987) were able to
increase germination with a lengthy treatment (12 hours) Figuring out whether a
substantial cushion oftreatment durations exist for percussion ofNew Mexico locust
seed would be a significant improvement to current scarification methods where seed
damage and seedling health is an issue
The greatest challenge for applying percussion to New Mexico locust seeds is
standardizing a treatment that is both effective and readily reproducible by nurseries
with mechanical scarification this may require the development of specific
~UllDment to properly quantify and execute the treatment
14
f
Objectives
The first objective of the study is to detennine which scarification treatment
results in optimal gennination speed and percentage ofNew Mexico locust The
second objective is to describe treatment interactions with nine seed sources collected
in New Mexico
Hypothesis
A percussion treatment acts exclusively on the main area ofwater uptake in
New Mexico locust seed loosening the strophiole so that imbibition can occur This
direct mechanism ofdonnancy release will result in improved gennination rate and
overall gennination percentage for New Mexico locust seed compared to standard
treatments Standard treatments including acid hot water and mechanical
scarification while eventually weakening the strophiolar cleft cause excessive
morphological damage through generalized seed coat degradation These treatments
are therefore prone to inconsistency due to over and under-treatment
15
MATERIALS AND METHODS
Seed Collection and Cleaning
Seeds were collected from August 25 through September 15 2000 from nine
locations along a latitudinal gradient in New Mexico Seed source locations and
descriptive information are given in Figure 1 and Tables I and 2 Stands were
separated by at least 45 km
Pods were hand picked as they began to ripen and dehisce (open) Collections
were made from ground level to approximately 3 m from several trees at each site
Pods were dried in open-ended brown paper bags for two to four weeks at room
temperature (21-230C) to facilitate cleaning Dried pods were placed in a closed cloth
sack which was pounded against the ground to separate seeds from pods For pods
still containing seeds hand maceration (crunching pods with hands) completed seed
separation Debris was removed by threshing with the aid of a greenhouse fan A
South Dakota Seed Blower (Seedbureau Chicago Illinois) was used to remove
remaining chaff and light seeds by air density separation Seeds were placed in dry
storage in sealed quart-size zip-lock bags within a sealed plastic container Storage
temperatures ranged from 2-4oC Seeds remained in storage for approximately 7
months before the experiment
Experimental Design and Treatment Structure
The experiment compared the impact ofhot water bath and percussion
l11aLl11 on germination properties ofNew Mexico locust seed A single
16
-l
Figure 1 Seed source collection map All sources were collected within New Mexico USA separated by at least 4S lan
IIIRZltItW lIJPIirmiddot rllll1lbullbull_~~~_II
Table 1 Latitude longitude location elevation and collection date ofNew Mexico locust seed sources
Lot title Latitude (N) Longitude (W) Location Elevation (m) Collection Date
Raton 36deg5846 104deg2872 Raton Pass NM 2295 82500
Holman 36deg0306 105deg2429 HolmanNM 2476 911500
Jemez 35deg4500 106deg4100 Jemez Springs NM 1976 9900 00
Sandia 35deg1083 106deg2341 Cedar Crest NM 2432 82600
Manzano 34deg3689 106deg2494 ManzanoNM 2465 9200
Magdalena 33deg5953 107deg0903 Magdalena NM 2544 82900
Capitan 33deg2728 105deg4325 Alto NM 2189 911200
Sacramento 32deg5798 105deg4496 Cloudcroft NM 2630 91400 Gila 32deg5400 107deg4600 Kingston NM 2432 9700
f
Table 2 Mean seed weight and standard error by source
a
Source Mean seed weight (g) for Standard error 100 seeds n = 5
Raton Holman Jemez Sandia Manzano Magdalena Capitan Sacramento Gila
2872 b c 2522 d 1826 e 2753 c 2413 d 2559 d 2443 d 3130 a 3029 ab
00103 00403 00276 00301 00370 00374 00458 00279 00348
a Mean seed weights followed by the same letter are not significantly
different
19
experiment was conducted utilizing a randomized complete block design blocked by
time with a factorial treatment structure Experimental factors were seed source and
scarification treatment Nine seed sources from throughout New Mexico were used
(Figure 1 Table 1) Scarification treatments included a control three levels ofhot
water and four levels of percussion Hot water treatments included immersion at
100degC and boiling for periods of 30 seconds and 60 seconds Percussion treatment
levels were 1 2 4 and 8 minutes One hundred seeds per block were used to test
each treatment combination Four blocks of seed were tested one at a time (all seed
sources included in each run) in close chronological sequence (within 6 weeks) A
total of four 100-seed samples were used to test each source by scarification treatment
combination
Treatments
Hot water treatment of seeds utilized 50 ml test tubes filled with 30 ml
distilled water Each test tube was used to treat one 100-seed sample at a time
resulting in a ratio of4 parts water to 1 part seed by volume Test tubes were placed
in a beaker with the beaker filled with tap water to the same level as the water within
test tubes Nine test tubes (one per seed source) were placed in the beaker
simultaneously so that one replication of each seed sourcescarification treatment
cotnblnatlon was conducted simultaneously Hot water treatments include immersion
boiling For immersion treatments seeds were added to test tubes with the water
(boiling) and the test tubes were immediately removed from the beaker to
For the boiling treatments test tubes remained in the beaker with water at
20
boiling temperature for a set duration after the addition of seeds Following all
treatments test tubes with seeds cooled 24 hours in a test tube rack
Percussion treatment utilized a pneumatic paint shaker (Central Pneumatic
model 00422 Camarillo CA) Compressed air was maintained at 530 kPa plusmn30 kPa
(80 psi plusmn 5 psi) resulting in approximately 350 oscillations per minute Seeds were
placed in 118 ml (4 oz) soil tin cans (US Can Lobard IL) and the lids were secured
with duct tape Each can was used to treat one 100-seed sample To take advantage
ofgreatest lateral movement of the paint shaker a spacer (paint can) was used to
position the soil tin at the end of the shaking arm This allowed shaking to occur at
the greatest distance from the pivot point (fulcrum) Tins were placed perpendicular
to the direction of shaking motion allowing seeds to impact at greatest force by
hitting flat surfaces of the soil tin
Germination Testing
Before gennination testing all seeds were soaked 24 hours in reverse
osmosis-treated water (this was included in the process ofhot water treatment
jU~SCIlOea above) Following soaking seeds were placed in 100 cm Petri dishes lined
90 cm Whatman qualitative 1 filter paper circles (MG Scientific Pleasant
WI) One Petri dish was used for each treatment by source combination
papers were initially moistened with 5 mL reverse osmosis-treated water then
~VU1CUlJl moistened as needed to ensure adequate conditions for gennination
were monitored daily (at 300 PM) for gennination for 14 days in accordance
~UIUlnJlVUa Seed Testing Association (ISTA) standards for Robinia species
21
(1STA 1999) Tests were carried out at room temperature (22degC plusmnOSoC)
Germination was classified as protrusion of the radicle from the embryo by at least
1mm as viewed with the naked eye
X-ray Radiography
On August 132001 three lOO-seed samples from the Sacramento seed source
were treated for 14 or 8-minute percussion durations The seeds were sent
immediately by overnight delivery to the Faxitron X-Ray Corporation in Wheeling
Illinois where Dr Bob Karrfalt ofthe USDA Forest Service oversaw their imaging
Radiographs were made at SX with a Faxitron Specimen Radiography System Model
MX-20
Scanning Electron Microscope Photography
On February 192002 a Hitachi S-3200N Variable Pressure Scanning
Electron Microscope (Mountain View CA) was used to examine strophiolar cells of
untreated seeds and seeds percussed for 4 minutes Seeds were cut longitudinally
igt their entire length using a razor blade splitting the strophiolar region roughly in
Cross sections of seed samples magnifying the strophiolar region were viewed
600X under the microscope
Analysis
Categorical Analysis of Variance was used to determine treatment source and
(Le time) main effects and interactions The response variable was total
~vlI This procedure is a Chi-square test of homogeneity using the natural log
of germinated to non-germinated seed for each treatment Maximumshy
22
likelihood analysis was used to calculate Chi-square test statistics with their observed
significance levels A single contrast was conducted to cempare treatment means
against the control Observed significance levels less than or equal to 005 were
considered significant Pairwise comparisons were conducted between non-control
treatment mean percents using a conservative alp~a value of 005 divided by the
number of comparisons (Bonferroni approach) and approximate pairwise z statistics
Categorical analyses were performed using Proc Catmod (SAS Institute 1989)
RESULTS
Scarification seed source block and all interactions between factors
influenced germination (Table 3) The main effects are reported in the presence of
these interactions because the overall robustness of treatments and seed sources are of
interest Background levels ofnaturally occurring variation in seed sources are
seen in control germination percentages in Figure 2 In particular time variability
is unlikely to be under the control of the producer Appendix B details experimental
data for source by treatment gennination percents for each of the four blocks
Averaged over all treatments germination by source ranged from 40 to
79 (Figure 2) Averaged over all sources (Figure 3) and for each source (Figure 4
5) percussion scarification durations of4 or 8 minutes yielded greatest germination
Averaged over all sources germination increased with increasing durations of
percussion then leveled off for the 4- and 8-minute treatments (Figure 3) Immersion
and 30-second boiling scarification maximized germination for the hot water
treatments with a decrease in germination when seeds were boiled for 60 seconds
For all but two sources there is a similar ipcreasing trend in germination from
1to 4 minutes ofpercussion (Figure 4) The Holman source increased in germination
1 to 2 minutes then leveled off at 4 minutes The Jemez source changed little
1 to 4 minutes of treatment As treatment duration increased from 4 to 8
mutes five sources decreased in germination (Figure 4a) whereas three sources
24
Table 3 Categorical Analysis of Variance table for germination response to seed treatment seed source block and the interaction of the factors
Source of DF Chi-Square Observed Variability Significance Level
Source 8 98370 lt00001 Trt 7 233876 lt00001
contrast 1 16976 lt00001 SourceTrt 56 97077 lt00001 Block 3 5538 lt00001
SourceBlock 24 10162 lt00001 TrtBlock 21 7564 lt00001
SourceTrtBlock 168 34283 lt00001
S 4)
8 ~
0
sectg ~ 40 ~
N 0 0
20
o f0ltgt ~ ~4 jybshy ((iltgto ~~ K1ltgt 6~b-~ ~~ 0b-~~ ~~ ~b-~ ~b-~b
80
60 -I-- - ~- middotmiddotmiddotmiddotmiddot1
r -] Northern NM Sources
Central NM Sources
Southern NM Sources
sect-o ~
~b-v(i
SOURCE
Figure 2 Effect ofNew Mexico locust seed source on germination for data averaged over all scarification treatments Control germination is shown by solid line within bar
80
~ cd ~ ltl) () 60 ~ ~
N
t 0shyrti tshySltl)
40
0
20
o lmmersion Boiling 30 sec Boiling 60 sec Control 1 min 2min 4 min 8 min
Hot Water Treatments Percussion Treatments
Figure 3 Effect of scarification on germination ofNew Mexico locust for data averaged over all seed sources Means labelled with the same letter are not significantly different
100~----------------------~--~----------------~
80
60
ltU
~
40 IJ
sectgsI
l100
80
60
40
a I middotmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot middotImiddotmiddotmiddotmiddotmiddotmiddot E
_~iii-bull _ _ bull f --~--
t-- -~ ---- -shy ---- shyI ~ ---- - shy
L
fiII
~
b
- shy ---r
- Holman
Sacramento Sandia Gila Jemez
c ~
ltgt ~
Tmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot-I
j~=~=-~~-1
I middotmiddot ~ bull r
t tr
Y -------M-a-gd-a-Ie-n-a--
1 _ _
fCapitan
---- Manzano ~y -_ Raton
2 4 8
Time interval ofpercussion scarification (min)
Figure 4 Effect of seed source by treatment interaction on New Mexico locust germination for percussion scarification pattern a and pattern b
28
tv 0
~ d
~ A s o
1 c3
80
60
40
20
o
TIT
t------=middotfmiddotmiddot-middotmiddot-middotmiddot-middotmiddot 1- --- - - --=- - -shy--cE= - _--shy --shy ----shy ~ ~ ---shy ~ -~- _ --shy-------r 1
I
Raton Holman
-_ Jemez
--- Sandia Manzano Magdalena Capitan Sacramento Gila
Immersion Boiling 30 sec Boiling 60 sec
Hot Water Scarification
Figure 5 Effect of seed source by treatment interaction on New Mexico locust germination for hot water scarification
continued to increase in germination (Figure 4b) There was no difference between
the two treatment levels for the Manzano source (Figure 4b)
The effect ofhot water scarification also varied among sources with few
overall differences between hot water treatments (Figure 5) However boiling for 60
seconds yielded highest germination for the Jemez source whereas increasing
durations ofboiling decreased germination for the Raton source
In addition to consistently high germination 4- and 8-minute percussionshy
treated seeds germinated as fast or faster than all other treatments (Figure 6 Table 4)
050 measured as the number of days needed to complete 50 ofeventual
germination averaged 2 days for both ofthese treatments
X-ray radiographs were taken of seeds collected from the Sacramento source
percussed for 1 4 or 8 minutes (Figure 7) Seeds show signs of damage at 8 minutes
ofpercussion (Figure 8) Compared to an undamaged seed percussed for only 4
minutes (Figure 8a) damage at the 8-minute level includes fractured radicles pitting
of the seed coat and chips in seed coat structure (Figure 8b-d)
Scanning electron microscope (SEM) images at 600X reveal the direct effect
of 4 minutes of percussion on cells of the strophiolar region (Figure 9) Whereas
untreated seeds retain cellular organization in the strophiolar region strophiolar cells
seeds treated for 4 minutes ofpercussion separate and raise resulting in a
~-~e~U appearance
30
Umiddot-~middot--l-~---middotymiddotmiddotmiddot-----middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot--middot--middotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middotmiddotmiddotmiddotmiddotmiddotmiddot- -
050= gt1
-e-- Percussion 8 min -0- Percussion 4 min ---T- Percussion 2 min -----ltil- Percussion 1 min -- Immersion --0- Boiling 30 sec -+- Boiling 60 sec -ltgt- Control
2 4 6 8 10 12
Day Figure 6 Timecourse ofNew Mexico locust gennination following scarification
treatments G50 is the number of days to 50 of eventual germination
31
14
Table 4 Effect of seed source and treatment on G50 (days to 50 germination)
Seed Source Control Immersion B30sec B60sec Plmin P2min P4min P8min mean Raton 3 3 5 7 4 3 2 2 4 Holman 2 2 2 2 2 2 2 2 2
Jemez 4 6 6 5 3 2 2 2 4 Sandia 2 2 2 2 2 2 2 2 2
w tv Manzano 5 2 2 3 3 2 2 2 3
Magdalena 4 2 2 3 4 3 2 1 3
Capitan 5 4 2 2 3 2 2 2 3
Sacramento 3 2 2 2 4 3 2 2 3 Gila 3 2 2 2 2 2 2 2 2 mean 3 3 3 3 3 2 2 2 3 standard 11 14 16 18 09 05 0 03 08 deviation
Note Columns and rows may not agree due to rounding
Figure 7 X-ray radiographs ofSacramento seed source percussed for 1 4 or 8 minutes
33
Figure 8a-d Seed damage close-ups Figure a shows undamaged seed percussed for 4 minutes Figures b-d show seeds percussed for 8 minutes Damage includes fracturing ofradicle in b complete removal ofseed coat in c and pitting ofseed coat in d
34
Untreated Treated
Figure 9 SEM images ofuntreated vs 4-minute percussion-treated strophiolar cells ofNew Mexico locust seeds Treated cells are loosened separated and raised
DISCUSSION
Determining the Optimal Treatment
Averaging results of scarification treatmems across seed sources is a useful
tool to predict which treatment is most likely to be successful for an unknown seed
source Averaged over all sources four- and eight-minute percussion treatments
resulted in the highest germination 815 and 790 respectively and produced
germination that was as fast or faster than all other treatments Although there was no
difference between these two treatments on average an argument for 4 minutes of
percussion as a standard protocol for treating New Mexico locust seeds can be made
Descriptively 4 minutes of percussion improved germination over 8 minutes
ofpercussion for a majority of sources (five out of nine) 8 minutes of percussion
improved germination for three ofnine sources with no difference for a fmal source
For individual sources 4 minutes ofpercussion was always the best or secondmiddot best
treatment and was always equal to or better than all hot water treatments
Germination at 8 minutes ofpercussion was lower than all other percussion durations
for the Jemez and Gila sources and was lower than at least one hot water treatment for
these sources as well
The x-ray radiographs of the Sacramento seed source illustrate the excessive
internal and external seed damage that can occur with 8 minutes of percussion
germination counts 8-minute percussed seeds from Holman and Capitan
ClUll signs of damage that were visible to the naked eye (see below)
r~evertheless some of these seeds still fit the criteria for germination Neither x-ray
36
photography nor visible observations during seed counts revealed seed damage at the
4-minute percussion level In operational trials seeds percussed for 4 minutes
developed into healthy seedlings (Khadduri and Harrington unpublished data)
Strophiole and Seed Coat Behavior
Four minutes ofpercussion successfully breaks seed dormancy in a large
percentage ofNew Mexico locust seeds without excessively damaging the
surrounding seed coat or internal seed coat structures Most papilionoid legume
seeds including seeds ofNew Mexico locust have a specific region of the seed coat
known as the strophiole or lens (Hamly 1932) The strophiole is located on the
cotyledonary lobe of a locust seed (Figure 10 a-c) If seeds are not indiscriminately
damaged the strophiole is the first point of water entry to the embryo (Hopkinson
1997) When a papilionoid seed is percussed for the appropriate amount of time
repeated hits on the integrated seed coat loosen the constrained cells of the strophiolar
region without excessively damaging the rest of the seed coat (Ballard 1976) SEM
photos illuStrate that 4 minutes of percussion loosened and separated strophiolar cells
ofNew Mexico locust seeds when compared with constrained organized strophiolar
cells of untreated seeds (Figure 9) Research-has shown that water passes to the
embryo through the treated strophiole in a controlled manner and regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues
(Kelly and van Staden 1987)
37
10 Strophiole ofunimbibed New Mexico locust seed (a) and following uptake (bc)
38
In contrast hot-water treatments can excessively degrade the seed coat
(Rebman et al 1998 Marunda 1990) Random cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rebman et aI 1998 Marunda 1990) Along with a
proportionately large amount ofun-imbibed (thus under-treated) seeds (Figure l1a)
damage was observed in hot-water treated seeds from six sources during germination
counts Damage included seeds where the radicle failed to elongate (Figure 11 b)
seeds where the radicle elongated but was delayed in freeing itself from the seed coat
(Figure 11c) and seeds where the radicle completely broke off from the embryo
(Figure lId) In addition some seeds germinated in reverse (the cotyledon emerged
fIrst) If the radicle emerged completely from the seed coat they were counted as
germinated
While nearly all seeds treated for 8 minutes ofpercussion imbibed water
damage was evident in x-ray evaluations (Sacramento seed source) and in
germination counts (Holman and Capitan) at this treatment level Seed damage
during germination counts was similar to levels found in hot-water treated seeds
suggesting that over-treatment by both hot water and longer durations of percussion
may result in similar damage to seed structure
The Sacramento and Holman seed sources were two of the fIve sources that
opcrplltlpn in germination from the 4- to 8-minute durations of percussion
the benefIts ofloosening the strophiole as evidenced by imbibition of
all 8-minute treated seeds were outweighed by external and internal seed coat
39
a
b c d
11 Damage to New Mexico locust seeds observed during germination cotmts following hot water scarification 9a Hot water replicate including ~t seedlings and numerous unimbibed seeds 9b Close-up of seed where raallCle failed to elongate 9c Close-up ofseed where radicle elongated but was
-gtIn in freeing itself from the seed coat 9d Close-up ofseed where radicle broke off from embryo
40
damage (which also may be contributing to high imbibition) For the three sources
for which 8 minutes of percussion resulted in maximum germination extended
percussion of seeds necessary to loosen the strophiole may not have excessively
damaged the seed anatomy The Manzano source did not significantly change in
germination from 4 to 8 minutes of percussion suggesting that an increase in
permeable seeds at the 8-minute treatment duration was balanced by the number of
seeds that failed to germinate due to seed damage To summarize the 4-minute
percussion treatment improved germination over 8 minutes ofpercussion for a
majority of sources (five ofnine) the 8-minute percussion treatment improved
germination over 4-minutes ofpercussion for three sources and there was no
difference with the Manzano source Notably for the Raton source 8 minutes of
percussion increased germination by 235 percentage points over 4-minutes of
percussion (7875 vs 5525 respectively) the next best treatment Raton was the
only source to result in germination less than 75 for 4 minutes ofpercussion so a
grower may wish to apply longer percussion durations to lots that do not respond well
to this treatment level
Finally there was an interesting trend in the Jemez and Raton sources seed
that significantly contributed to the source by treatment interaction (Figure 12) A
~u test showed that the Jemez source had a significantly lighter seed weight than
other sources while the Raton source had an average seed weight For the Jemez
the longest duration of boiling increased germination Conversely only short
U4UVll) of percussion were needed to achieve high germination and the longest
41
80 _ Jemez a Raton
iD
~ C iD e iD
Q c 0 ~
c ~ iD 0
20
0------shy
Immersion Boiling 30 sec Boiling 60 sec
Hot Water Scarification
10degTr========~--------~------------------------------
80
60
40
20
_ Jemez Raton
0------shy
1 min 2 min 4 min 8 min
Percussion Scarification
Figure 12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources
42
b
--~----~~~~========~---- duration ofpercussion reduced germination For the Raton source longer durations
ofboiling consistently decreased germination whereas longer percussion treatments
consistently increased germination
A possible explanation is that a thick seed coat is associated with a loose
strophiole structure and that a thin seed coat is associated with a constrained
strophiole This contrast merits further study and may suggest an ecological
adaptation Further investigation ofthis phenomenon may contribute to successful
pre-screening ofunknown seed sources to determine optimal treatment leveL
43
CONCLUSION
Based on results obtained in pilot study (see Appendix) and thesis
experiments 4 minutes ofpercussion is recommended as a standard protocol for
treating an unknown source ofNew Mexico locust seed In addition to laboratory
results the 4-minute percussion treatment has resulted in germination greater than
80 and healthy seedling grow-outs for production purposes (unpublished data) In
laboratory experiments only one hundred seeds per 118 ml (40z) soil tin were
percussed at a time whereas for production we have treated up to five hundred seeds
per tin For large-scale production ofNew Mexico locust seedlings growers may
wish to experiment with larger quantities of seed and larger-size soil tins
Seed storage trials following percussion treatments were not conducted
However germination greater than 80 and healthy seedlings were obtained when
seeds were percussion-treated and subsequently stored at room-temperature (22degC)
for one week (unpublished data) Seed storage trials merit further study as the
standard hot water treatment leaves seeds in a softened condition that is not
conducive to storage
Hydroseeding is an effective method for direct seeding in reclamation
situations Percussion treatment may be conducive to hydro seeding as seeds remain
a dry relatively undamaged yet permeable condition following treatment Sulfuric
seeds also remain dry following treatment but extensive damage to seed coat
poor seedling vigor have been reported with this treatment (Kelly and van Staden
A grow-out study using a hydro seeder on a reclamation site testing percussion
44
against other treatments such as sulfuric acid and mechanical scarification also
merits further study
Papilionoids are the largest subfamily oflegumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust these legumes often play an integral role in the revegetation of
disturbed lands The pilot study (Appendix) indicates that percussion scarification
successfully treats the widely used black locust as well Developing superior
scarification methods to the standard hot water mechanical scarification and acid
treatments may facilitate the use ofpapilionoid legumes in reclamation projects
45
APPENDICES
APPENDIX A
PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR
NEW MEXICO LOCUST AND BLACK LOCUST SEEDS
Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4
Hot water and sulfuric acid soaks are traditional treatments for seeds of many
temperate woody legwnes including locusts However these scarification techniques
often produce inconsistent germination Percussion scarification where seeds are
repeatedly propelled against a hard surface was compared with hot water
scarification to evaluate treatment efficacy for New Mexico locust (Robinia
neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment
seeds were placed in a 98degC water bath which was immediately removed from the
heat source For percussion scarification seeds were placed in a soil sample tin and
agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the
control were followed by 24-hour water soaks Hot water baths resulted in 56 and
41 germination for New Mexico locust and black locust respectively For both
species nearly all durations ofpercussion increased germination over the hot water
treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3
4 and 5 minutes for black locust resulted in at least 90 germination Traditional
scarification treatments randomly degrade the entire seed coat which can lead to
tissue damage during water uptake Percussion scarification specifically weakens the
strophiole the natural source ofwater entry to the seed in papilionoid legwnes
INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom
2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031
48
Following percussion imbibition is controlled through the strophiole and underlying
tissue is protected
Keywords New Mexico locust black locust scarification percussion germination
Introduction
Locust trees (Robinia spp) are aggressive pioneer species that quickly
colonize disturbed land fix atmospheric nitrogen in the soil and stabilize slopes that
are prone to erosion (Klemmedson 1994 Ashby et al 1985) (Figure A1ab)
Recognition of these attributes has contributed to increased demand for locusts in
reclamation projects Locust seeds however exhibit physical dormancy where a
thick seed coat prevents the movement of water and gasses to the embryo (Leadem
1997)
Sulfuric acid or hot water soaks have traditionally been used to break seed
dormancy in macro-propagation of locusts Many growers are moving away from the
use of su1furic acid in treating locust seeds (Dreesen and Harrington 1997) In
addition to being dangerous acid soak durations must be specifically correlated to
seed lot (Olson 1974) Collections of native seed can vary tremendously in seed size
seed weight and hard seededness etc This variability limits the utility of sulfuric
acid soaks as even within a given lot some seeds may be damaged whereas others
are under-treated and fail to imbibe (take up water) A survey of Southwestern
container growers indicates that hot water baths are currently the most common
scarification method for locust seeds (Hine et al 1997) However hot water baths
also produce inconsistent germination (Lin et al 1996)
49
b
Figure AI New Mexico locust a primary invader following disturbance Ala Crown-sprouting at edge ofburn near Los Alamos NM--one year after Cerro Grande fire Alb Colonizing road cut Gila National Forest NM
50
Percussion scarification where seeds are repeatedly propelled against a hard
surface is an alternative dormancy-breaking method that has proven successful in
legumes related to locusts (Hamly 1932 Barton 1947) We compared this alternative
treatment to hot water scarification to evaluate treatment efficacy for New Mexico
locust and black locust seeds
Materials and Methods
Commercial New Mexico locust seeds (Western Native Seed Coaldale CO)
were collected fall 2000 in Huerfano County CO Black locust seeds were collected
September 2000 from Taos County NM by harvesting ripe pods from several trees at
a distance of up to 3 m from ground level Black locust seeds were allowed to air-dry
for 3 weeks and were threshed and separated from large chaffusing a greenhouse
fan A South Dakota Seed Blower (Chicago Illinltois) was used to remove fine chaff
from both seed lots Seeds were storedmiddotat 2-4C until the germination study was
conducted in March 2001
This study compared hot water scarification with percussion scarification
using treatment and species as experimental factors Along with a control
scarification treatments included immersion of seed in boiling water as well as
percussion durations of 1 2 3 45 or 10 minutes Four 100-seed samples were used
to test each species by scarification treatment combination
For the hot water treatment test tubes were filled with 30 mL water and
placed in a water-filled beaker (Figure A2a) The bath was raised to boiling (-98C at
51
b
c
Figure A2 Implementation ofhot water and percussion scarification A2a Hot water bath A2b Paint-shaker set-up A2c Placing soil tin containing seeds at end ofpaint shaker ann
52
Las Cruces NM elevation of 1300 meters) Each 100-seed replicate was placed in a
test tube and each test tube was immediately removed to cool at room temperature
Percussion scarification was implemented using a pneumatic paint shaker
(Central Pneumatic model 00422 Camarillo CA) (Figure A2b) This allowed
standardization of treatment intensity Compressed air pressure was maintained at
530kPa plusmn 30kPa (80psi plusmn 5psi) resulting in approximately 350 oscillations per
minute Each lOO-seed sample was placed in a 118 ml (4 oz) soil tin for shaking To
maximize lateral movement of paint shaker a paint can was used as a spacer to place
soil tins at the end of the shaking arm (Figure A2c) This allowed shaking to occur at
the greatest distance from the pivot point (fulcrum) Following all treatments
including the control seeds were soaked for twenty-four hours in distilled water prior
to plating out
Gennination was evaluated on lab benches at room temperature Each
replicate of seeds was placed in a 100 cm petri dish on moistened filter paper with
humidity maintained by enclosing the petri dishes in ziplock bags Gennination was
monitored daily for 14 days in accordance with International Seed Testing
Association standards for Robinia species (ISTA 1999) Gennination was defined as
protrusion of the radicle from the embryo by at least 1mm as viewed by the naked
eye
Categorical Analysis of Variance using Prpc Catmod (SAS Institute 1989)
was used to detennine treatment differences in germination percentages for each
source This procedure is an extension of a Chi-square test of homogeneity using the
53
natural log of the ratio of germinated to non-germinated seeds for each treatment Pshy
values less than 005 were considered significant A limited set of pairwiseshy
comparisons was conducted to compare treatment mean percents using a
conservative alpha value of 005 divided by the number of comparisons
Results
Percussion maximized germination for both New Mexico locust and black
locust There were three germination response levels for New Mexico locust (Figure
A3) First there was a low response in the control indicating a dormant seed lot
The hot water treatment and I-minute duration of percussion share a second
intermediate response Germination was highest at percussion levels from 2 to 10
minutes
A selected percussion treatment intermediate in time (5 minutes) produced the
steepest germination curve indicating the fastest germination rate (Figure A4) The
hot water treatment produced an intermediate curve or relatively slower germination
rate The control curve was very flat indicating the slowest germination rate for New
Mexico locust
Control seeds germinated poorly for black locust as well (Figure A5) Hot
water scarification improved germination over the control but all levels of percussion
improved germination over hot water The optimal durations of percussion treatment
were bracketed with a significant increase from the 1 to 2 minute percussion level a
high germination response from 2 to 5 minutes of percussion and finally a significant
54
~ d 25 CL)
p t1 0 ~ t1
g Vl ~ Vl
100~---------------------------------------------------------------
80
60
40
20
o Control Hot Water Percussion
I min Percussion
2 min Percussion
3 min
Scarification
Percussion 4 min
Figure A3 Effect of scarification on germination response for New Mexico locust
Percussion 5 min
=significant difference between adjacent treatment bars ns = no significant difference
Percussion 10 min
~ ~ () u agt
Il-t d o ~
~ ~ c3
100middotlr~~~------------------------------------e- Percussion 5min middot 0middot Hot Water -- Control
80-J=middot======
6b-middot-lmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middot--middotmiddot-middot~-middotmiddot -middotmiddot middot middotmiddot-middot 1
40
20
-----~-----------
0 0 0 middotmiddotmiddotmiddotmiddot0middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot0
0 --~-----y-----~------_---~--- T-----~
o shy -TDay 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A4 Effect of selected scarification treatments on germination speed for New Mexico locust First seven days of fourteen-day experiment are shown
Day 7
80
~ cd
5 60 ()
[) Cl d 0 1t1 d
40sect dUl
-J
20
o
ns
Scarification
Figure A5 Effect of scarification on germination response for black locust = significant difference between adjacent treatment bars ns = no significant difference
decrease at the 10-minute level This drop-off suggests that 10 minutes of percussion
over-treats seeds from this lot of-black locust
A selected percussion treatment intermediate in time (5 minutes) also had the
fastest germination rate for black locust (Figure A6)_ The hot water curve is
somewhat flatter indicating a slower germination rate The control curve is very flat
indicating a slow germination rate
A qualitative difference between hot water and percussion-treated seeds was
also observed Along with healthy germinants the hot water treatment produced
over-treated seeds (Figure A 7a) Examples of damaged germinants include seeds
where the radicle did not elongate (Figure A 7b) seeds where the radicle elongated
but was delayed in freeing itself from the seed coat (Figure A7c) and seeds where the
radicle broke off completely from the embryo (Figure A7d) In addition to these
examples of over-treatment there were numerous small dark seeds that failed to
imbibe water For percussion treatment nearly all seeds took up water (Figure A8a)
Germination was rapid and uniform with little damage evident (Figure A8b)
To summarize both New Mexico locust and black locust seed lots were
dormant The standard hot water scarification treatment improved germination but
only to 56 and 41 for New Mexico locust and black locust respectively Nearly
all percussion durations improved total germination as well as germination rate over
the hot water treatment Germination was greater than 90 for percussion durations
of 4 5 and 10 minutes for New Mexico locust and 3 4 and 5 minutes for black locust
58
CI)
sgt t CI) 0
igt 0
sect ttl t E
Vl 0
c3
100r==~==~~=r------------------------------------~ ___ Percussion 5 min
middotmiddotmiddot0middotmiddotmiddot Hot Water -lIf- Control
80r=========~--~~----------------------------~
60
40
20 -~-~---~---------- ---~------------I
omiddot
O
_~_----------lIf-----~bull -----_ shy---shy I
Day 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A6 Effect of selected scarification treatments on germination speed for black locust First seven days of fourteen-day experiment are shown
Day 7
a
b c d
Figure A7 Damage to New Mexico locust seeds observed during germination counts following hot water scarification A7a Hot water replicate including damaged seedlings and numerous unimbibed seeds A7b Close-up ofseed where radicle failed to elongate A7c Close-up ofseed where radicle elongated but was delayed in freeing itself from the seed coat A7d Close-up ofseed where radicle completely broke offfrom embryo
60
a
b
Figure A8 New Mexico locust seeds following percussion treatment at time of germination counts A8a Percussion replicate with uniformly healthy seedlings and near complete imbibition of seeds A8b Close-up ofpercussion-treated seedling
61
Discussion
Papilionoid seeds have a specific anatomical feature known as the strophiole
or lens-the natural site of water entry to the seed (Hamly 1932) The strophiole is
located on the cotyledonary lobe of a locust seed (Figure A9) When a papilionoid
legume seed is percussed for the appropriate amount of time repeated hits on the
integrated seed coat loosen the constrained cells of the strophiolar region without
excessively damaging the rest of the seed coat (Ballard 1976)
When a percussed seed is soaked water enters exclusively through the
strophiole in a controlled manner (Kelly and Staden 1987) This regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues This
contrasts with seeds that have been hot water or acid scarified treatments that can
randomly degrade the seed coat Unlocalized cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rehman et al 1998 Marunda 1990)
Papilionoids are the largest subfamily of legumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust and black locust these legumes often play an integral role in the
revegetation ofdisturbed lands Developing superior scarification methods to the
standard hot water and acid treatments should fadlitate the use ofthese legumes in
restoration projects
62
Figure A9 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (bc)
63
APPENDIXB
GERMINATION PERCENTS AND STANDARD ERRORS FOR SOURCE
BLOCK AND TREATMENT FOR NEW MEXICO LOCUST
Table B 1 Gennination percents and standard errors for source block and treatment for New Mexico locust
Source Block Treatment Germination Percentage Standard Error
Capitan 1 Immersion 74 437 Capitan 2 Immersion 69 462 Capitan 3 Immersion 71 454 Capitan 4 Immersion 67 470 Capitan Boiling 30 sec 79 407 Capitan 2 Boiling 30 sec 76 427 Capitan 3 Boiling 30 sec 72 449 Capitan 4 Boiling 30 sec 76 427 Capitan Boiling 60 sec 71 454 Capitan 2 Boiling 60 sec 54 498 Capitan 3 Boiling 60 sec 67 470 Capitan 4 Boiling 60 sec 76 427 Capitan I Control 5 218 Capitan 2 Control 7 255 Capitan 3 Control 4 196 Capitan 4 Control 5 218 Capitan 1 Percussion I min 47 499 Capitan 2 Percussion I min
470
Capitan Percussion I min 39 488
Capitan 4 Percussion I min 38 485 Capitan I Percussion 2 min 55 497 Capitan Capitan
2
Percussion 2 min Percussion 2 min
71 65
454 477
Capitan 4 Percussion 2 min 56 496 Capitan I Percussion 4 min 86 347 Capitan 2 Percussion 4 min 79 407 Capitan 3 Percussion 4 min 81 392 Capitan 4 Percussion 4 min 82 384 Capitan I Percussion 8 min 90 300 Capitan 2 Percussion 8 min 88 325 Capitan 3 Percussion 8 min 90 300 Capitan 4 Percussion 8 min 90 300 Gila I Immersion 71 454 Gila 2 Immersion 77 421 Gila
Immersion 75 433
Gila 4 Immersion 72 449
Gila I Boiling 30 sec 68 466
Gila 2 Boiling 30 sec 81 392
Gila 3 Boiling 30 sec 54 498
Gila 4 Boiling 30 sec 63 483
Gila I Boiling 60 sec 55 497
Gila 2 Boiling 60 sec 68 466
Gila 3 Boiling 60 sec 57 495
Gila 4 Boiling 60 sec 70 458
65
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Gila 2 Control 13 336 Gila 3 Control 13 336 Gila 4 Control 20 400 Gila 1 Percussion 1 min 66 474 Gila 2 Percussion 1 min 57 495 Gila 3 Percussion 1 min 73 444 Gila 4 Percussion 1 min 82 384 Gila 1 Percussion 2 min 81 392 Gila 2 Percussion 2 min 86 347 Gila 3 Percussion 2 min 85 357 Gila 4 Percussion 2 min 54 498 Gila 1 Percussion 4 min 79 407 Gila 2 Percussion 4 min 83 376 Gila 3 Percussion 4 min 84 367 Gila 4 Percussion 4 min 79 407 Gila 1 Percussion 8 min 51 500 Gila 2 Percussion 8 min 76 427 Gila 3 Percussion 8 min 52 500 Gila 4 Percussion 8 min 74 439 Holman 1 Immersion 83 376 Holman 2 Immersion 86 347 Holman 3 Immersion 81 392 Holman 4 Immersion 90 300 Holman 1 Boiling 30 sec 90 300 Holman 2 Boiling 30 sec 85 357 Holman 3 Boiling 30 sec 87 336 Holman 4 Boiling 30 sec 83 376 Holman 1 Boiling 60 sec 79 407 Holman 2 Boiling 60 sec 80 400 Holman 3 Boiling 60 sec 86 347 Holman 4 Boiling 60 sec 93 255 Holman Control 25 433 Holman 2 Control 19 392 Holman Control 21 407
Holman 4 Control 24 427
Holman Percussion 1 min 77 421
Holman 2 Percussion 1 min 85 357
Holman 3 Percussion 1 min 80 400
Holman 4 Percussion 1 min 89 313
Holman 1 Percussion 2 min 88 325
Holman 2 Percussion 2 min 96 196
Holman 3 Percussion 2 min 93 255
Holman 4 Percussion 2 min 96 196
Holman 1 Percussion 4 min 92 271
Holman 2 Percussion 4 min 97 171
66
I
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Holman 3 Percussion 4 min 91 286 Holman 4 Percussion 4 min 96 196 Holman 1 Percussion 8 min 88 325 Holman 2 Percussion 8 min 85 357 Holman 3 Percussion 8 min 83 376 Holman 4 Percussion 8 min 82 384 Jemez 1 Immersion 53 499 Jemez 2 Immersion 44 496 Jemez 3 Immersion 52 500 Jemez 4 Immersion 70 458 Jemez 1 Boiling 30 sec 58 494 Jemez 2 Boiling 30 sec 55 497 Jemez 3 Boiling 30 sec 48 500 Jemez 4 Boiling 30 sec 65 477 Jemez 1 Boiling 60 sec 68 466 Jemez 2 Boiling 60 sec 63 483 Jemez 3 Boiling 60 sec 73 444 Jemez 4 Boiling 60 sec 89 313 Jemez 1 Control 2 140 Jemez 2 Control 9 286 Jemez 3 Control 6 237 Jemez 4 Control 8 271 Jemez 1 Percussion 1 min 58 494 Jemez 2 Percussion 1 min 63 483 Jemez 3 Percussion 1 min 77 421 Jemez 4 Percussion 1 min 89 313 Jemez 1 Percussion 2 min 68 466 Jemez 2 Percussion 2 min 64 480 Jemez 3 Percussion 2 min 80 400 Jemez 4 Percussion 2 min 83 376 Jemez Percussion 4 min 68 466 Jemez 2 Percussion 4 min 79 407 Jemez 3 Percussion 4 min 70 458 Jemez 4 Percussion 4 min 83 376 Jemez 1 Percussion 8 min 52 500 Jemez 2 Percussion 8 min 59 492 Jemez 3 Percussion 8 min 61 488 Jemez 4 Percussion 8 min 70 458 Magdalena 1 Immersion 69 462 Magdalena 2 Immersion 69 462 Magdalena J Immersion 70 458 Magdalena 4 Immersion 85 357 Magdalena 1 Boiling 30 sec 65 477 Magdalena 2 Boiling 30 sec 71 454 Magdalena 3 Boiling 30 sec 68 466
67
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Magdalena 4 Boiling 30 sec 83 376 Magdalena 1 Boiling 60 sec 63 483 Magdalena 2 Boiling 60 sec 68 466 Magdalena 3 Boiling 60 sec 63 483 Magdalena 4 Boiling 60 sec 82 384 Magdalena 1 Control 11 313 Magdalena 2 Control 6 237 Magdalena 3 Control 5 218 Magdalena 4 Control 4 196 Magdalena Percussion 1 min 32 466 Magdalena 2 Percussion 1 min 45 497 Magdalena 3 Percussion 1 min 46 498 Magdalena 4 Percussion I min 36 480 Magdalena 1 Percussion 2 min 67 470 Magdalena 2 Percussion 2 min 68 466 Magdalena 3 Percussion 2 min 65 477 Magdalena 4 Percussion 2 min 81 392 Magdalena 1 Percussion 4 min 87 336 Magdalena 2 Percussion 4 min 97 171 Magdalena 3 Percussion 4 min 83 376 Magdalena 4 Percussion 4 min 97 171 Magdalena 1 Percussion 8 min 98 lAO Magdalena 2 Percussion 8 min 100 000 Magdalena 3 Percussion 8 min 96 196 Magdalena 4 Percussion 8 min 98 lAO Manzano 1 Immersion 67 470 Manzano 2 Immersion 57 495 Manzano 3 Immersion 72 4049 Manzano 4 Immersion 73 4044 Manzano 1 Boiling 30 sec 70 458 Manzano 2 Boiling 30 sec 64 480 Manzano 3 Boiling 30 sec 71 454 Manzano 4 Boiling 30 sec 80 400 Manzano I Boiling 60 sec 50 500 Manzano 2 Boiling 60 sec 53 499 Manzano 3 Boiling 60 sec 59 492 Manzano 4 Boiling 60 sec 74 439 Manzano I Control 4 196 Manzano 2 Control 8 271 Manzano 3 Control 1 099 Manzano 4 Control 6 237 Manzano 1 Percussion 1 min 52 500 Manzano 2 Percussion 1 min 39 488 Manzano 3 Percussion 1 min 36 480 Manzano 4 Percussion 1 min 51 500
68
Table B 1 Continued
Source Block Treatment Gerrni~ation Percentage Standard Error
Manzano Percussion 2 min 53 499 Manzano 2 Percussion 2 min 58 494 Manzano 3 Percussion 2 min 61 488 Manzano 4 Percussion 2 min 62 485 Manzano 1 Percussion 4 min 76 427 Manzano 2 Percussion 4 min 85 357 Manzano 3 Percussion 4 min 79 407 Manzano 4 Percussion 4 min 83 376 Manzano 1 Percussion 8 min 87 336 Manzano 2 Percussion 8 min 83 376 Manzano 3 Percussion 8 min 83 376 Manzano 4 Percussion 8 min 75 433 Raton 1 Immersion 54 498 Raton 2 Immersion 37 483 Raton 3 Immersion 39 488 Raton 4 Immersion 69 462 Raton 1 Boiling 30 sec 36 480 Raton 2 Boiling 30 sec 20 400 Raton 3 Boiling 30 sec 28 449 Raton 4 Boiling 30 sec 60 490 Raton 1 Boiling 60 sec 15 357 Raton 2 Boiling 60 sec 171 Raton 3 Boiling 60 sec 4 196 Raton 4 Boiling 60 sec 35 477 Raton 1 Control 4 196 Raton 2 Control 12 325 Raton 3 Control 2 140 Raton 4 Control 6 237 Raton 1 Percussion 1 min 52 500 Raton 2 Percussion I min 29 454 Raton 3 Percussion I min 35 477 Raton 4 Percussion 1 min 31 462 Raton 1 Percussion 2 min 55 497 Raton 2 Percussion 2 min 35 477 Raton 3 Percussion 2 min 33 470 Raton 4 Percussion 2 min 42 494 Raton 1 Percussion 4 min 65 477 Raton 2 Percussion 4 min 52 500 Raton Percussion 4 min 51 500 Raton 4 Percussion 4 min 53 499 Raton 1 Percussion 8 min 88 325 Raton 2 Percussion 8 min 80 400 Raton 3 Percussion 8 min 73 444 Raton 4 Percussion 8 min 74 439 Sacramento 1 Immersion 43 495
69
Table B 1 Continued
-Source Block Treatment Germination Percentage Standard Error
Sacramento 2 Immersion 30 458 Sacramento 3 Immersion 38 485 Sacramento 4 Immersion 56 496 Sacramento 1 Boiling 30 sec 47 499 Sacramento 2 Boiling 30 sec 48 500 Sacramento 3 Boiling 30 sec 48 500 Sacramento 4 Boiling 30 sec 56 496 Sacramento 1 Boiling 60 sec 52 500 Sacramento 2 Boiling 60 sec 67 470 Sacramento 3 Boiling 60 sec 43 495 Sacramento 4 Boiling 60 sec 48 500 Sacramento 1 Control 0 000 Sacramento 2 Control 3 171 Sacramento 3 Control 3 171 Sacramento 4 Control 099 Sacramento 1 Percussion 1 min 57 495 Sacramento 2 Percussion 1 min 61 488 Sacramento 3 Percussion 1 min 62 485 Sacramento 4 Percussion 1 min 55 497 Sacramento 1 Percussion 2 min 84 367 Sacramento 2 Percussion 2 min 70 458 Sacramento 3 Percussion 2 min 81 392 Sacramento 4 Percussion 2 min 69 462 Sacramento 1 Percussion 4 min 91 286 Sacramento 2 Percussion 4 min 84 367 Sacramento 3 Percussion 4 min 84 367 Sacramento 4 Percussion 4 min 94 237 Sacramento 1 Percussion 8 min 82 384 Sacramento 2 Percussion 8 min 81 392 Sacramento 3 Percussion 8 min 82 384 Sacramento 4 Percussion 8 min 81 392 Sandia 1 Immersion 76 427 Sandia 2 Immersion 70 458 Sandia 3 Immersion 70 458 Sandia 4 Immersion 81 392 Sandia 1 Boiling 30 sec 78 414 Sandia 2 Boiling 30 sec 71 454 Sandia 3 Boiling 30 sec 63 483 Sandia 4 Boiling 30 sec 78 414 Sandia 1 Boiling 60 sec 67 470 Sandia 2 Boiling 60 sec 65 477 Sandia 3 Boiling 60 sec 74 439 Sandia 4 Boiling 60 sec 66 474 Sandia 1 Control 16 367 Sandia 2 Control 7 255
70
Table B 1 Continued
Source Block Treatment Gennination Percentage Standard Error
Sandia 3 Control 8 271 Sandia 4 Control 16 367 Sandia 1 Percussion 1 min 67 470 Sandia 2 Percussion 1 min 61 488 Sandia 3 Percussion 1 min 66 474 Sandia 4 Percussion 1 min 59 492 Sandia I Percussion 2 min 82 384 Sandia 2 Percussion 2 min 83 376 Sandia 3 Percussion 2 min 81 392 Sandia 4 Percussion 2 min 84 367 Sandia 1 Percussion 4 min 89 313 Sandia 2 Percussion 4 min 86 347 Sandia 3 Percussion 4 min 89 313 Sandia 4 Percussion 4 min 85 357 Sandia 1 Percussion 8 min 80 400 Sandia 2 Percussion 8 min 77 421 Sandia 3 Percussion 8 min 69 462 Sandia 4 Percussion 8 min 66 474
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Simpson BJ 1988 A field guide to Texas trees Austin TX Texas Monthly Press 372p
Tran VN Cavanagh AK 1984 Structural aspects ofdormancy In Seed Physiology Vol 2 Murray DR (ed) p1-44 Academic Press Australia
USDA Forest Service 1948 Woody-plant seed manual USDA Misc Pub1654 416p
Vines RA 1960 Trees Shrubs and Woody Vines of the Southwest Austin Univ ofTexas Press 1104 p
Wagner MR Larson RE Craig TP 1992 Ecology and silvics ofNew Mexico locust (Robinia neomexicana) with an emphasis on the arboreal form p53-57 In JW Hanover K Miller and S Plesko (eds) Proc Intemat Confon Black Locust Biology Culture and Utilization Mich State Univ USA
Wilson JK 1944 Immersing seeds of species of Robinia in boiling water hastens germination J For 42 453-454
Zimmerman RW Carpenter SB 1980 First-year coppice production from a 5shyyear old black locust stand on surface mine spoil p309-314 Centr Hardwd For ConfProc University ofMissouri Columbia
LIST OF TABLES
Table Page
1 Latitude longitude location elevation and collection date ofNew Mexico locust seed sources 18
2 Mean seed weight and standard error by source 19
3 Categorical Analysis ofVariance table for germination response to seed treatment seed source block and the interaction of the factors 25
4 Effect of seed source and treatment on G50 (days to 50 germination) 32
B1 Germination percents and standard errors for source block and treatment for New Mexico locust 65
LIST OF FIGURES
Figures Page
1 Seedsourcecollectionmap 17
2 Effect ofNew Mexico locust seed source on germination for data averaged over scarification treatments 26
3 Effect of scarification on germination for New Mexico locust for data averaged over all seed sources 27
4 Effect of seed source by treatment interaction on New Mexico locust germination for percussion scarification pattern a and pattern b 28
5 Effect of source by treatment interaction on New Mexico locust germination for hot water scarification 29
6 Timecourse of New Mexico locust germination following scarification treatments 31
7 X-ray radiographs of Sacramento seed source perc ussed for 1 4 or 8 minutes 33
8 Seed damage close-ups 34
9 SEM images of untreated vs 4-minute percussion-treated strophiolar cells ofNew Mexico locust seeds 35
10 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (b c) 38
11 Damage to New Mexico locust seeds observed during germination counts following hot water scarification 40
12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources 42
New Mexico locust a primary invader following disturbance 50
Implementation of hot water and percussion scarification 52
Effect of scarification on germination response for New Mexico locust 55
x
A4 Effect of selected scarification treatments on germination speed for New Mexico locust 56
AS Effect of scarification on germination response for black locust 57
A6 Effect of selected scarification treatments on germination speed for black locust 59
A7 Damage to New Mexico locust seeds observed during germination counts following hot water scarification 60
AS New Mexico locust seeds following percussion treatment at time of germination counts 61
A9 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (bc) 63
INTRODUCTION
New Mexico locust (Robinia neomexicana A Gray) a small tree native to the
southwestern United States occurs at elevations from 1200 to 2800 meters This
species fills a successional role in post-disturbance situations As a primary invader
New Mexico locust quickly establishes on burned areas arid flood banks as well as
road cuts (Wagner et aI 1992) Rapid growth crown sprouting and prolific root
suckering favor the successful establishment ofNew Mexico locust on disturbed sites
(Simpson 1988 Gottfried 1980) These attributes also may make New Mexico locust
well suited for steep-slope revegetation where erosion is a problem
New Mexico locust is a nitrogen-fixing legume that tolerates and improves
nutrient-poor soils Stands ofNew Mexico locust increase Nitrogen (N) and Calcium
(Ca) in the forest floor Levels of Carbon (C) Phosphorous (P) Sulfur (8) and
Potassium (K) aIso have been shown to increase in soil beneath New Mexico locust
(Klemmedson 1994)
The ability to colonize rapidly and ameliorate harsh sites contributes to New
Mexico locusts potential as a nurse plant A nurse plant colonizes an inhospitable
site and creates an environment suitable for successional plant establishment New
Mexico locust demonstrates this role in the ponderosa pine (Pinus ponderosa Douglas
ex Lawson amp C Lawson)Gambel oak (Quercus gambelii Nutt) community After a
disturbance such as wildfire New Mexico locust and Gambel oak colonize and
dominate the site until shaded out by ponderosa pine Once shaded these species
become understory shrubs a process taking an average of 15-20 years (Dick-Peddie
1993 Hanks and Dick-Peddie 1974) New Mexico locust gradually declines with
small dense patches averaging about 005 ha beneath pine until the next disturbance
once again offers it a competitive advantage (Klemmedson 1994)
Gottfried (1980) expecting ponderosa pine regeneration to be greater where
New Mexico locust had been eradicated found that survival of planted pine seedlings
was greater where locust had not been removed In a follow-up study Gottfried
(1980) noted that soil moisture in the top 57 cm was highest in 5-year old locust sites
as compared to grass or 20-year old locust sites He concluded that managing an
appropriate cover ofNew Mexico locust could help regenerate pine the later
successional species
The ability ofNew Mexico locust to improve harsh sites also makes it a
candidate for reclaiming disturbances associated with mining Black locust (Robinia
pseudoacacia 1) has been used for decades to reclaim mine spoils and other
disturbed sites in the eastern US and around the world (Keresztesi 1988 Zimmerman
and Carpenter 1980) Ashby et al (1985) described similar positive attributes of
black locust also mentioned previously for New Mexico locust quick cover for
stabilization supply ofN and nutrient-rich litter to the soil and site improvement for
establishment of later successional trees Referring specifically to mine reclamation
the authors note the ability of black locust to grow on a wide range ofmine soil
conditions including extremely acid soils Black locust also shows some tolerance to
soils compacted by grading and topsoiling practices (Ashby et aL 1985) New
2
Mexico locust holds promise as a native southwe~tem counterpart to black locust in
mine reclamation but it has been used infrequently to date
Natural invasion and succession occur slowly on most mine sites (Monsen
1984) While New Mexico locust often colonizes sites naturally there is no
assurance a seed will reach a particular site and establish in a reasonable time frame
The challenge is to introduce New Mexico locust as mother plants to facilitate
colonization Seedling production for outplanting on disturbed sites has been
hampered by poor germination (Lin et al 1996) The goal then is to develop
techniques to overcome poor germination in New Mexico locust thereby facilitating
macropropagation
LITERATURE REVIEW
Seed Dormancy
Low gennination in New Mexico locust is primarily the result of seed
donnancy the failure of an intact viable seed to genninate under favorable
conditions (Bewley 1997) Seed donnancy also can be defined as the inability of the
embryo to genninate because of some inherent constraint (Bewley and Black 1994)
The constraint may originate within the embryo referred to as endogenous or embryo
donnancy or from the tissues surrounding the embryo known as exogenous or coatshy
imposed donnancy (Baskin and Baskin 1998 Leadem 1997 Bewley and Black
1994)
New Mexico locust seed is donnant when the impenneable seed coat does not
allow movement ofwater to the embryo This specific fonn of coat-imposed
donnancy where the restraining tissue involved is an impenneable seed coat is
known as physical donnancy (Baskin and Baskin 1998 Bewley and Black 1994)
Physical donnancy is relatively uncommon in seeds less than 10 of all woody
species exhibit this condition (USDA Forest Service 1948) However it is
characteristic of many genera of the Fabaceae (bean) family For example all
successional trees in the genera Gleditsia and Robinia (of which New Mexico locust
is a member) produce seeds that exhibit physical donnancy (Baskin and Baskin
4
Physical Dormancy
Seeds that exhibit physical dormancy are said to be hard seeded referring to
the thick impermeable seed coat (Leadem 1997) The first line of protection of the
testa in leguminous seeds is the outermost waxy cuticle followed by a subcuticle
consisting ofsuberin Next is a palisade layer consisting of sclereid cells that have
thick lignified secondary walls The most common type of sclereid cell in palisade
layers is the macrosclereid The macrosclereids contain water-repellent substances
including cutin lignin suberin and wax (Rolston 1978) This palisade layer is the
primary barrier to water uptake (Serrato-Valenti et al 1993 Tran and Cavanagh
1984)
All seed coat openings must be blocked to effectively prevent water uptake
The natural opening in a seed where it was attached to the mother plant is the weak
link where physical dormancy is usually broken (Hopkinson 1993 Kuo and Tam
1988) Anatomical structures associated with the seed coat opening vary from
family to family In the Papilionoideae the subfamily of the Fabaceae family
containing New Mexico locust the site ofwater entry is the strophiole The
strophiole is a swelling on the seed coat close to the stalk scar (Hopkinson 1997)
Kelly and van Staden (1987) refer to the strophiole site as the lens where
macrosclereid cells must separate and raise before water can pass through Since
middot6germination ofdormant seeds of the Papilionoideae takes place when the strophiole
permeable (Hopkinson 1993 Kuo and Tam 1988) a logical approach to
5
genninating New Mexico locust artificially would be to loosen the strophiolar cells
and allow water to pass through to the embryo
Evolutionary Role of Physical Dormancy
Why is physical dormancy a mechanism that blocks germination
ecologically advantageous When a plant produc~s seeds with different degrees of
dormancy seeds that are dispersed at one time will germinate over a range of time
(Bewley and Black 1994) Through variability of dormancy within a seed crop seeds
are exposed to changing environmental conditions improving the overall chances of
survival of the seed line (Bewley and Black 1994) Varying expression of dormancy
is under genetic control which indicates that this may be an evolved survival strategy
(Kigel and Galili 1995) A range of dormancy levels may be a particular advantage in
the southwestern US where extreme environmental variations exist
Methods for Breaking Dormancy
There are three common approaches to breaking dormancy in New Mexico
locust seed acid scarification hot water soaks and mechanical treatments In each
case the objective of the treatment is to scarify or abrade the seed coat to make it
permeable to water
Acid Scarification
Acid scarification consists of soaking seeds in concentrated sulfuric acid
rinsing thoroughly with water upon removal followed by subsequent drying before
PlaJlltmg Olson (1974) reports that this scarification method produces the highest
middot~~jllalUll results for all Robinia species Most literature refers to a seminal 1937
6
study conducted by HG Meginnis This stud~ t~ough conducted on black locust
seed is regularly cited in the suggested use of acid to treat New Mexico locust seed
as well (Olson 1974 Vines 1960)
Thee published studies document the specific use of sulfuric acid to treat
New Mexico locust seed Cox and Klett (1984) report that soaking seeds in
concentrated sulfuric acid for thirty minutes improved germination to 34 over an
untreated control of4 Lin et al (1996) found that for two seed lots acid
scarification for twenty-five to eighty minutes produced germination of 80 A third
lot however resulted in less than 20 germination at all durations of 10 to 80
minutes Hine et al (1997) showed that acid treatment levels of 1 2 5 and 10
minutes resulted in less than 20 germination across five sources However there
was an increasing trend in germination with acid soak duration and acid treatments
were not imposed for as long a time as the previous two studies (30 to 80 minutes)
The mixed results obtained by these investigators were probably due to the
widely varying germination response of seed lots to acid treatment For this reason
Olson (1974) recommends correlating duration of acid treatment to each seed lot If
treatment duration is too long the embryo will be permanently damaged If soak
duration is too short the seed will remain impermeable to water and fail to germinate
Compounding the problem of treatment duration is the negative effect acid
have on seedling growth Any type of scarification can injure seeds to some
by disrupting cells essential for growth leading to fungal invasion and
middot-uuu~w injury (Copeland and MacDonald 1995) Some legume seeds subjected
7
to 60 minutes of sulfuric acid scarification (to achieve maximum gennination results)
showed more than 50 seedling mortality after gennination (Kelly and van Staden
1987) Bewley and Black (1994) noted that fungal growth on seeds is an indication
of leakage of sugars organic acids amino acids and proteins
Perhaps the greatest drawback to acid scarification is the dangerous nature of
the treatment itself Difficulties associated with storage and trained use of
concentrated acid may discourage many propagators from using this option (Dreesen
and Harrington 1997) Consequently hot water and to a lesser extent mechanical
scarification are more commonly used and practical treatments for legume seeds
Most southwestern container growers use either of these two treatments to scarify
New Mexico locust seed in particular (Hine et al 1997)
Hot Water Scarification
The widespread use ofhot water scarification to treat locust seed is probably
due to its ease of application and efficacy in comparison to other scarification
treatments Although this practice had been in use for some time Wilson (1944) was
one of the first to report on its systematic application for black locust The treatment
consists of placing seeds in boiling water (4 parts water tomiddot 1 part seed by volume)
removing the vessel from the heat source and allowing the water to cool and seeds to
soak for 24 hours
Hine et al (1997) Lin et al (1996) and Cox and Klett (1984) have examined
the specific application ofhot water scarification on New Mexico locust seed Each
these authors compared hot water to acid scarification Lin et aL (1996) reported
8
60 gennination for two hot water-treated seed lots while a third lot germinated
around 50 (as reported above acid scarification germination ranged from 20 to
80 for these lots) It should be noted that the authors only soaked the seeds in water
from 10 to 80 minutes for this experiment as opposed to the standard 24 hours Cox
and Klett (1984) stated that using the standard 24-hour hot water treatment improved
germination over an acid pre-treatment (which produced less than 20 germination)
but they did not give specific gennination numbers for the hot water treatment Hine
et al (1997) found that across five seed lots germination was 66 for the standard
hot water treatment (as compared to less than 20 germination for acid scarification)
Variations in treatment intensity and duration and their subsequent effects on seed lot
appear to contribute to conflicting conclusions as to whether acid scarification or hot
water treatment is a more successful treatment
Given mixed results ease ofapplication is an important argument for use of
hot water application over acid soaks Safety concerns and material costs for hot
water treatments are low (Dreesen and Harrington 1997) An extension of this is the
practicality ofhot water for treating large batches of seed where a relative increase in
water volume is all that is needed However increases in water and seed even when
inthe same 4 1 proportion can result in dramatically different rates of cooling than in
atest tube experiment Different cooling rates can lead to contrasting results for the
treatment temperature duration and seed lot (USDA Forest Service 1948)
As with acid scarification parameters ofhot water treatment application may
to be optimized to maximize germination Hine et al (1997) found that as
9
temperature was increased from 70degC to 100degC overall germination in New Mexico
locust seed increased The 100degC treatment maximized germination for each of five
sources tested with germination usually above 60
Prolonged exposure of seeds ofmany woody legumes to boiling temperatures
also may improve germination (Briscoe 1996) This approach has not been tested on
New Mexico locust seed Exposing seed to prolonged boiling temperatures may
result in fungal infection and subsequent seedling mortality Marunda (1990) noted
severe fungal infections on both acid and hot water treated Acacia holoserica Cunn
ex G Don seeds attributing the infections to seed damage resulting from the
treatments Identifying an optimal hot water treatment level that does not result in a
high rate of seedling mortality is a logical goal
With hot water treatments as with acid there is also a danger of overtreating
Seeds and reducing germination (Gosling et al 1995) The legume Leucaena
leucocephala (Lam) de Wit germinated equally well after 7 seconds of exposure to
water at 100degC 7 seconds at 90degC 30 seconds at 80degC and 4 minutes at 70degC In
addition acceptably high germination was obtained from treatment at 70degC from 3
minutes to 1 hour whereas no seeds germinated following treatment at 100degC for 2
minutes Perhaps along with a greater margin of error for treatment duration treating
~ew Mexico locust seeds at a lower temperature may result in a comparatively lower
of seedling mortality In practice however nursery growers tend to use the
and quickest treatment available The short treatment length at near-boiling
whether recommended or not may be what is used most frequently
10
Fine-tuning a treatment in this range of treatment temperature and duration may be
the most logical approach
Mechanical Scarification
This treatment involves scratching the seed coat with an abrasive material
(USDA Forest Service 1948) One system employs a rotating drum lined with
sandpaper A paddle spinning at a given number ofrevolutions per minute throws the
seed against the sandpaper (Hine et al 1997) A second method dry tumbling
tumbles seed in a mixer with pea gravel or sharp sand for 5 to 7 days (Dreesen 2001)
These approaches have the advantage that control of temperature is not required as in
water treatments nor is safety an issue as with add scarification
Hine et al (1997) mechanically treated New Mexico locust seed using 100shy
grit sandpaper in a Forsberg (rotating drum) scarifier Gennination was less than
10 They concluded that the seed coats were too thin for their apparatus and
proposed that a finer grit sandpaper would produce more even abrasion Although
germination varied by seed lot Dreesen (2001) reported that using a slow-speed dry
tumbling technique for 5 to 7 days produced gennination rates as high as 90
As with acid and hot water treatments treatment duration must be specifically
shy
correlated to seed lot Treated seeds are also susceptible to fungal infection
iHopkinson 1997) In addition equipment is specialized and may not always be
ltU~CUJl~ to a nursery (USDA Forest Service 1948) Several studies have confinned
degrading ofstrophiolar cells due to acid and hot water scarification (Miklas et al
11
1987 Dell 1980 Hanna 1984) but the effect ofmechanical scarification on the
strophiole has not been studied
Alternative Method for Breaking Dormancy Percussion
Strophiole degradation has been examined in depth using an impaction
treatment more commonly known as percussion Hamly (1932) first proposed that
shaking or impacting seeds could split the macrosc1ereid cells at the strophiole
Hamly successfully released tension ofbent strophiolar cells of impermeable
Melilotus alba Medik (White sweet clover) seeds by delivering physical blows
resulting in cells that had split apart and were only slightly bent Ninety-two percent
oftreated seeds became permeable as compared to 05 of control seeds Barton
(1947) showed that percussion significantly increases permeability in black locust
seed Ballard (1976) reasoned that the integrated nature of the strophiole within the
seed coat means a blow on any part of the seed weakens the strophiole There are no
published studies on the use of percussion on New Mexico locust
Percussion methods vary and often seem SUbjective or ill-defined Hamly
(1932) placed seeds in a glass bottle and shook them three times a second for 20
minutes resulting in 180 oscillations per minute Ballard (1973) describes shaking
seeds in a closed metal container with at least fifty seeds per container at 850-900
JS(~l11alIOIlS per minute for 1 minute Kelly and van Staden (1987) placed seeds into
sealed glass flasks Flasks were shaken by a wrist-shaker at a rate of 250
~UUVJ~ per minute Subsequent germination rates were 45 after 2 hours of
55 after 3 hours 60 after 6 hours and 100 after 12 hours
12
There are several potential advantages of percussion over acid hot water and
mechanical scarification Kelly and van Staden (1987) note that a percussion
treatment only alters macrosclereid cells of the strophiole by loosening separating
and raising them They emphasize that minimal damage is done to the overall
anatomical structure of the seed In acid scarification of the same seed lot radicle
and cotyledon damage occurred Based on photographic evidence the authors
believe that this morphological damage was due to uneven pressure during
imbibition with water entering haphazardly wherever the acid had sufficiently
dissolved the coat This ultimately led to uneven hydration of the embryo In
percussed seeds water only entered through the s~ophiole when the entire seed coat
had been saturated resulting in controlled and even embryo hydration (Kelly and van
Staden 1987)
Although all successful scarification treatments may focus at least in part on
the strophiole for papilionoid legumes (Baskin and Baskin 1998) percussion may be
less damaging to seed coat structures particularly when compared with acid Kelly
and van Staden did not compare hot water or mechanical scarification methods
against percussion However the fungal infection associated with hot water and
mechanical scarification as with acid scarificatio~ have been directly associated with
seed damage (Rehman et al 1998 USDA Forest Service 1948) Embryo damage in
has been observed in hot-water scarified legume seeds (Rehman et al 1998
13
By limiting embryo damage percussion scarification reduces seedling
mortality compared to other treatments Kelly and van Staden (1987) noted that
seedlings from the percussion treatment were morphologically sound and vigorous
when compared to acid treatment seedlings The authors attribute this improved
seedling vigor to the controlled water uptake that limited anatomical damage
Despite a 12-hour percussion treatment morphological damage was not substantially
greater than untreated seeds In addition seedling mass measured 10 days following
germination was not significantly different between percussed and untreated seeds
A final advantage ofpercussion may be the ability to adequately treat the most
dormant seeds while avoiding harm to seeds that have already been made permeable
by the treatment or that were permeable to begin with Instead ofcorroding scalding
or scratching the seed coat percussion appears to be just a loosening of the
strophiole spring (Ballard 1973) Kelly and van Staden (1987) were able to
increase germination with a lengthy treatment (12 hours) Figuring out whether a
substantial cushion oftreatment durations exist for percussion ofNew Mexico locust
seed would be a significant improvement to current scarification methods where seed
damage and seedling health is an issue
The greatest challenge for applying percussion to New Mexico locust seeds is
standardizing a treatment that is both effective and readily reproducible by nurseries
with mechanical scarification this may require the development of specific
~UllDment to properly quantify and execute the treatment
14
f
Objectives
The first objective of the study is to detennine which scarification treatment
results in optimal gennination speed and percentage ofNew Mexico locust The
second objective is to describe treatment interactions with nine seed sources collected
in New Mexico
Hypothesis
A percussion treatment acts exclusively on the main area ofwater uptake in
New Mexico locust seed loosening the strophiole so that imbibition can occur This
direct mechanism ofdonnancy release will result in improved gennination rate and
overall gennination percentage for New Mexico locust seed compared to standard
treatments Standard treatments including acid hot water and mechanical
scarification while eventually weakening the strophiolar cleft cause excessive
morphological damage through generalized seed coat degradation These treatments
are therefore prone to inconsistency due to over and under-treatment
15
MATERIALS AND METHODS
Seed Collection and Cleaning
Seeds were collected from August 25 through September 15 2000 from nine
locations along a latitudinal gradient in New Mexico Seed source locations and
descriptive information are given in Figure 1 and Tables I and 2 Stands were
separated by at least 45 km
Pods were hand picked as they began to ripen and dehisce (open) Collections
were made from ground level to approximately 3 m from several trees at each site
Pods were dried in open-ended brown paper bags for two to four weeks at room
temperature (21-230C) to facilitate cleaning Dried pods were placed in a closed cloth
sack which was pounded against the ground to separate seeds from pods For pods
still containing seeds hand maceration (crunching pods with hands) completed seed
separation Debris was removed by threshing with the aid of a greenhouse fan A
South Dakota Seed Blower (Seedbureau Chicago Illinois) was used to remove
remaining chaff and light seeds by air density separation Seeds were placed in dry
storage in sealed quart-size zip-lock bags within a sealed plastic container Storage
temperatures ranged from 2-4oC Seeds remained in storage for approximately 7
months before the experiment
Experimental Design and Treatment Structure
The experiment compared the impact ofhot water bath and percussion
l11aLl11 on germination properties ofNew Mexico locust seed A single
16
-l
Figure 1 Seed source collection map All sources were collected within New Mexico USA separated by at least 4S lan
IIIRZltItW lIJPIirmiddot rllll1lbullbull_~~~_II
Table 1 Latitude longitude location elevation and collection date ofNew Mexico locust seed sources
Lot title Latitude (N) Longitude (W) Location Elevation (m) Collection Date
Raton 36deg5846 104deg2872 Raton Pass NM 2295 82500
Holman 36deg0306 105deg2429 HolmanNM 2476 911500
Jemez 35deg4500 106deg4100 Jemez Springs NM 1976 9900 00
Sandia 35deg1083 106deg2341 Cedar Crest NM 2432 82600
Manzano 34deg3689 106deg2494 ManzanoNM 2465 9200
Magdalena 33deg5953 107deg0903 Magdalena NM 2544 82900
Capitan 33deg2728 105deg4325 Alto NM 2189 911200
Sacramento 32deg5798 105deg4496 Cloudcroft NM 2630 91400 Gila 32deg5400 107deg4600 Kingston NM 2432 9700
f
Table 2 Mean seed weight and standard error by source
a
Source Mean seed weight (g) for Standard error 100 seeds n = 5
Raton Holman Jemez Sandia Manzano Magdalena Capitan Sacramento Gila
2872 b c 2522 d 1826 e 2753 c 2413 d 2559 d 2443 d 3130 a 3029 ab
00103 00403 00276 00301 00370 00374 00458 00279 00348
a Mean seed weights followed by the same letter are not significantly
different
19
experiment was conducted utilizing a randomized complete block design blocked by
time with a factorial treatment structure Experimental factors were seed source and
scarification treatment Nine seed sources from throughout New Mexico were used
(Figure 1 Table 1) Scarification treatments included a control three levels ofhot
water and four levels of percussion Hot water treatments included immersion at
100degC and boiling for periods of 30 seconds and 60 seconds Percussion treatment
levels were 1 2 4 and 8 minutes One hundred seeds per block were used to test
each treatment combination Four blocks of seed were tested one at a time (all seed
sources included in each run) in close chronological sequence (within 6 weeks) A
total of four 100-seed samples were used to test each source by scarification treatment
combination
Treatments
Hot water treatment of seeds utilized 50 ml test tubes filled with 30 ml
distilled water Each test tube was used to treat one 100-seed sample at a time
resulting in a ratio of4 parts water to 1 part seed by volume Test tubes were placed
in a beaker with the beaker filled with tap water to the same level as the water within
test tubes Nine test tubes (one per seed source) were placed in the beaker
simultaneously so that one replication of each seed sourcescarification treatment
cotnblnatlon was conducted simultaneously Hot water treatments include immersion
boiling For immersion treatments seeds were added to test tubes with the water
(boiling) and the test tubes were immediately removed from the beaker to
For the boiling treatments test tubes remained in the beaker with water at
20
boiling temperature for a set duration after the addition of seeds Following all
treatments test tubes with seeds cooled 24 hours in a test tube rack
Percussion treatment utilized a pneumatic paint shaker (Central Pneumatic
model 00422 Camarillo CA) Compressed air was maintained at 530 kPa plusmn30 kPa
(80 psi plusmn 5 psi) resulting in approximately 350 oscillations per minute Seeds were
placed in 118 ml (4 oz) soil tin cans (US Can Lobard IL) and the lids were secured
with duct tape Each can was used to treat one 100-seed sample To take advantage
ofgreatest lateral movement of the paint shaker a spacer (paint can) was used to
position the soil tin at the end of the shaking arm This allowed shaking to occur at
the greatest distance from the pivot point (fulcrum) Tins were placed perpendicular
to the direction of shaking motion allowing seeds to impact at greatest force by
hitting flat surfaces of the soil tin
Germination Testing
Before gennination testing all seeds were soaked 24 hours in reverse
osmosis-treated water (this was included in the process ofhot water treatment
jU~SCIlOea above) Following soaking seeds were placed in 100 cm Petri dishes lined
90 cm Whatman qualitative 1 filter paper circles (MG Scientific Pleasant
WI) One Petri dish was used for each treatment by source combination
papers were initially moistened with 5 mL reverse osmosis-treated water then
~VU1CUlJl moistened as needed to ensure adequate conditions for gennination
were monitored daily (at 300 PM) for gennination for 14 days in accordance
~UIUlnJlVUa Seed Testing Association (ISTA) standards for Robinia species
21
(1STA 1999) Tests were carried out at room temperature (22degC plusmnOSoC)
Germination was classified as protrusion of the radicle from the embryo by at least
1mm as viewed with the naked eye
X-ray Radiography
On August 132001 three lOO-seed samples from the Sacramento seed source
were treated for 14 or 8-minute percussion durations The seeds were sent
immediately by overnight delivery to the Faxitron X-Ray Corporation in Wheeling
Illinois where Dr Bob Karrfalt ofthe USDA Forest Service oversaw their imaging
Radiographs were made at SX with a Faxitron Specimen Radiography System Model
MX-20
Scanning Electron Microscope Photography
On February 192002 a Hitachi S-3200N Variable Pressure Scanning
Electron Microscope (Mountain View CA) was used to examine strophiolar cells of
untreated seeds and seeds percussed for 4 minutes Seeds were cut longitudinally
igt their entire length using a razor blade splitting the strophiolar region roughly in
Cross sections of seed samples magnifying the strophiolar region were viewed
600X under the microscope
Analysis
Categorical Analysis of Variance was used to determine treatment source and
(Le time) main effects and interactions The response variable was total
~vlI This procedure is a Chi-square test of homogeneity using the natural log
of germinated to non-germinated seed for each treatment Maximumshy
22
likelihood analysis was used to calculate Chi-square test statistics with their observed
significance levels A single contrast was conducted to cempare treatment means
against the control Observed significance levels less than or equal to 005 were
considered significant Pairwise comparisons were conducted between non-control
treatment mean percents using a conservative alp~a value of 005 divided by the
number of comparisons (Bonferroni approach) and approximate pairwise z statistics
Categorical analyses were performed using Proc Catmod (SAS Institute 1989)
RESULTS
Scarification seed source block and all interactions between factors
influenced germination (Table 3) The main effects are reported in the presence of
these interactions because the overall robustness of treatments and seed sources are of
interest Background levels ofnaturally occurring variation in seed sources are
seen in control germination percentages in Figure 2 In particular time variability
is unlikely to be under the control of the producer Appendix B details experimental
data for source by treatment gennination percents for each of the four blocks
Averaged over all treatments germination by source ranged from 40 to
79 (Figure 2) Averaged over all sources (Figure 3) and for each source (Figure 4
5) percussion scarification durations of4 or 8 minutes yielded greatest germination
Averaged over all sources germination increased with increasing durations of
percussion then leveled off for the 4- and 8-minute treatments (Figure 3) Immersion
and 30-second boiling scarification maximized germination for the hot water
treatments with a decrease in germination when seeds were boiled for 60 seconds
For all but two sources there is a similar ipcreasing trend in germination from
1to 4 minutes ofpercussion (Figure 4) The Holman source increased in germination
1 to 2 minutes then leveled off at 4 minutes The Jemez source changed little
1 to 4 minutes of treatment As treatment duration increased from 4 to 8
mutes five sources decreased in germination (Figure 4a) whereas three sources
24
Table 3 Categorical Analysis of Variance table for germination response to seed treatment seed source block and the interaction of the factors
Source of DF Chi-Square Observed Variability Significance Level
Source 8 98370 lt00001 Trt 7 233876 lt00001
contrast 1 16976 lt00001 SourceTrt 56 97077 lt00001 Block 3 5538 lt00001
SourceBlock 24 10162 lt00001 TrtBlock 21 7564 lt00001
SourceTrtBlock 168 34283 lt00001
S 4)
8 ~
0
sectg ~ 40 ~
N 0 0
20
o f0ltgt ~ ~4 jybshy ((iltgto ~~ K1ltgt 6~b-~ ~~ 0b-~~ ~~ ~b-~ ~b-~b
80
60 -I-- - ~- middotmiddotmiddotmiddotmiddot1
r -] Northern NM Sources
Central NM Sources
Southern NM Sources
sect-o ~
~b-v(i
SOURCE
Figure 2 Effect ofNew Mexico locust seed source on germination for data averaged over all scarification treatments Control germination is shown by solid line within bar
80
~ cd ~ ltl) () 60 ~ ~
N
t 0shyrti tshySltl)
40
0
20
o lmmersion Boiling 30 sec Boiling 60 sec Control 1 min 2min 4 min 8 min
Hot Water Treatments Percussion Treatments
Figure 3 Effect of scarification on germination ofNew Mexico locust for data averaged over all seed sources Means labelled with the same letter are not significantly different
100~----------------------~--~----------------~
80
60
ltU
~
40 IJ
sectgsI
l100
80
60
40
a I middotmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot middotImiddotmiddotmiddotmiddotmiddotmiddot E
_~iii-bull _ _ bull f --~--
t-- -~ ---- -shy ---- shyI ~ ---- - shy
L
fiII
~
b
- shy ---r
- Holman
Sacramento Sandia Gila Jemez
c ~
ltgt ~
Tmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot-I
j~=~=-~~-1
I middotmiddot ~ bull r
t tr
Y -------M-a-gd-a-Ie-n-a--
1 _ _
fCapitan
---- Manzano ~y -_ Raton
2 4 8
Time interval ofpercussion scarification (min)
Figure 4 Effect of seed source by treatment interaction on New Mexico locust germination for percussion scarification pattern a and pattern b
28
tv 0
~ d
~ A s o
1 c3
80
60
40
20
o
TIT
t------=middotfmiddotmiddot-middotmiddot-middotmiddot-middotmiddot 1- --- - - --=- - -shy--cE= - _--shy --shy ----shy ~ ~ ---shy ~ -~- _ --shy-------r 1
I
Raton Holman
-_ Jemez
--- Sandia Manzano Magdalena Capitan Sacramento Gila
Immersion Boiling 30 sec Boiling 60 sec
Hot Water Scarification
Figure 5 Effect of seed source by treatment interaction on New Mexico locust germination for hot water scarification
continued to increase in germination (Figure 4b) There was no difference between
the two treatment levels for the Manzano source (Figure 4b)
The effect ofhot water scarification also varied among sources with few
overall differences between hot water treatments (Figure 5) However boiling for 60
seconds yielded highest germination for the Jemez source whereas increasing
durations ofboiling decreased germination for the Raton source
In addition to consistently high germination 4- and 8-minute percussionshy
treated seeds germinated as fast or faster than all other treatments (Figure 6 Table 4)
050 measured as the number of days needed to complete 50 ofeventual
germination averaged 2 days for both ofthese treatments
X-ray radiographs were taken of seeds collected from the Sacramento source
percussed for 1 4 or 8 minutes (Figure 7) Seeds show signs of damage at 8 minutes
ofpercussion (Figure 8) Compared to an undamaged seed percussed for only 4
minutes (Figure 8a) damage at the 8-minute level includes fractured radicles pitting
of the seed coat and chips in seed coat structure (Figure 8b-d)
Scanning electron microscope (SEM) images at 600X reveal the direct effect
of 4 minutes of percussion on cells of the strophiolar region (Figure 9) Whereas
untreated seeds retain cellular organization in the strophiolar region strophiolar cells
seeds treated for 4 minutes ofpercussion separate and raise resulting in a
~-~e~U appearance
30
Umiddot-~middot--l-~---middotymiddotmiddotmiddot-----middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot--middot--middotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middotmiddotmiddotmiddotmiddotmiddotmiddot- -
050= gt1
-e-- Percussion 8 min -0- Percussion 4 min ---T- Percussion 2 min -----ltil- Percussion 1 min -- Immersion --0- Boiling 30 sec -+- Boiling 60 sec -ltgt- Control
2 4 6 8 10 12
Day Figure 6 Timecourse ofNew Mexico locust gennination following scarification
treatments G50 is the number of days to 50 of eventual germination
31
14
Table 4 Effect of seed source and treatment on G50 (days to 50 germination)
Seed Source Control Immersion B30sec B60sec Plmin P2min P4min P8min mean Raton 3 3 5 7 4 3 2 2 4 Holman 2 2 2 2 2 2 2 2 2
Jemez 4 6 6 5 3 2 2 2 4 Sandia 2 2 2 2 2 2 2 2 2
w tv Manzano 5 2 2 3 3 2 2 2 3
Magdalena 4 2 2 3 4 3 2 1 3
Capitan 5 4 2 2 3 2 2 2 3
Sacramento 3 2 2 2 4 3 2 2 3 Gila 3 2 2 2 2 2 2 2 2 mean 3 3 3 3 3 2 2 2 3 standard 11 14 16 18 09 05 0 03 08 deviation
Note Columns and rows may not agree due to rounding
Figure 7 X-ray radiographs ofSacramento seed source percussed for 1 4 or 8 minutes
33
Figure 8a-d Seed damage close-ups Figure a shows undamaged seed percussed for 4 minutes Figures b-d show seeds percussed for 8 minutes Damage includes fracturing ofradicle in b complete removal ofseed coat in c and pitting ofseed coat in d
34
Untreated Treated
Figure 9 SEM images ofuntreated vs 4-minute percussion-treated strophiolar cells ofNew Mexico locust seeds Treated cells are loosened separated and raised
DISCUSSION
Determining the Optimal Treatment
Averaging results of scarification treatmems across seed sources is a useful
tool to predict which treatment is most likely to be successful for an unknown seed
source Averaged over all sources four- and eight-minute percussion treatments
resulted in the highest germination 815 and 790 respectively and produced
germination that was as fast or faster than all other treatments Although there was no
difference between these two treatments on average an argument for 4 minutes of
percussion as a standard protocol for treating New Mexico locust seeds can be made
Descriptively 4 minutes of percussion improved germination over 8 minutes
ofpercussion for a majority of sources (five out of nine) 8 minutes of percussion
improved germination for three ofnine sources with no difference for a fmal source
For individual sources 4 minutes ofpercussion was always the best or secondmiddot best
treatment and was always equal to or better than all hot water treatments
Germination at 8 minutes ofpercussion was lower than all other percussion durations
for the Jemez and Gila sources and was lower than at least one hot water treatment for
these sources as well
The x-ray radiographs of the Sacramento seed source illustrate the excessive
internal and external seed damage that can occur with 8 minutes of percussion
germination counts 8-minute percussed seeds from Holman and Capitan
ClUll signs of damage that were visible to the naked eye (see below)
r~evertheless some of these seeds still fit the criteria for germination Neither x-ray
36
photography nor visible observations during seed counts revealed seed damage at the
4-minute percussion level In operational trials seeds percussed for 4 minutes
developed into healthy seedlings (Khadduri and Harrington unpublished data)
Strophiole and Seed Coat Behavior
Four minutes ofpercussion successfully breaks seed dormancy in a large
percentage ofNew Mexico locust seeds without excessively damaging the
surrounding seed coat or internal seed coat structures Most papilionoid legume
seeds including seeds ofNew Mexico locust have a specific region of the seed coat
known as the strophiole or lens (Hamly 1932) The strophiole is located on the
cotyledonary lobe of a locust seed (Figure 10 a-c) If seeds are not indiscriminately
damaged the strophiole is the first point of water entry to the embryo (Hopkinson
1997) When a papilionoid seed is percussed for the appropriate amount of time
repeated hits on the integrated seed coat loosen the constrained cells of the strophiolar
region without excessively damaging the rest of the seed coat (Ballard 1976) SEM
photos illuStrate that 4 minutes of percussion loosened and separated strophiolar cells
ofNew Mexico locust seeds when compared with constrained organized strophiolar
cells of untreated seeds (Figure 9) Research-has shown that water passes to the
embryo through the treated strophiole in a controlled manner and regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues
(Kelly and van Staden 1987)
37
10 Strophiole ofunimbibed New Mexico locust seed (a) and following uptake (bc)
38
In contrast hot-water treatments can excessively degrade the seed coat
(Rebman et al 1998 Marunda 1990) Random cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rebman et aI 1998 Marunda 1990) Along with a
proportionately large amount ofun-imbibed (thus under-treated) seeds (Figure l1a)
damage was observed in hot-water treated seeds from six sources during germination
counts Damage included seeds where the radicle failed to elongate (Figure 11 b)
seeds where the radicle elongated but was delayed in freeing itself from the seed coat
(Figure 11c) and seeds where the radicle completely broke off from the embryo
(Figure lId) In addition some seeds germinated in reverse (the cotyledon emerged
fIrst) If the radicle emerged completely from the seed coat they were counted as
germinated
While nearly all seeds treated for 8 minutes ofpercussion imbibed water
damage was evident in x-ray evaluations (Sacramento seed source) and in
germination counts (Holman and Capitan) at this treatment level Seed damage
during germination counts was similar to levels found in hot-water treated seeds
suggesting that over-treatment by both hot water and longer durations of percussion
may result in similar damage to seed structure
The Sacramento and Holman seed sources were two of the fIve sources that
opcrplltlpn in germination from the 4- to 8-minute durations of percussion
the benefIts ofloosening the strophiole as evidenced by imbibition of
all 8-minute treated seeds were outweighed by external and internal seed coat
39
a
b c d
11 Damage to New Mexico locust seeds observed during germination cotmts following hot water scarification 9a Hot water replicate including ~t seedlings and numerous unimbibed seeds 9b Close-up of seed where raallCle failed to elongate 9c Close-up ofseed where radicle elongated but was
-gtIn in freeing itself from the seed coat 9d Close-up ofseed where radicle broke off from embryo
40
damage (which also may be contributing to high imbibition) For the three sources
for which 8 minutes of percussion resulted in maximum germination extended
percussion of seeds necessary to loosen the strophiole may not have excessively
damaged the seed anatomy The Manzano source did not significantly change in
germination from 4 to 8 minutes of percussion suggesting that an increase in
permeable seeds at the 8-minute treatment duration was balanced by the number of
seeds that failed to germinate due to seed damage To summarize the 4-minute
percussion treatment improved germination over 8 minutes ofpercussion for a
majority of sources (five ofnine) the 8-minute percussion treatment improved
germination over 4-minutes ofpercussion for three sources and there was no
difference with the Manzano source Notably for the Raton source 8 minutes of
percussion increased germination by 235 percentage points over 4-minutes of
percussion (7875 vs 5525 respectively) the next best treatment Raton was the
only source to result in germination less than 75 for 4 minutes ofpercussion so a
grower may wish to apply longer percussion durations to lots that do not respond well
to this treatment level
Finally there was an interesting trend in the Jemez and Raton sources seed
that significantly contributed to the source by treatment interaction (Figure 12) A
~u test showed that the Jemez source had a significantly lighter seed weight than
other sources while the Raton source had an average seed weight For the Jemez
the longest duration of boiling increased germination Conversely only short
U4UVll) of percussion were needed to achieve high germination and the longest
41
80 _ Jemez a Raton
iD
~ C iD e iD
Q c 0 ~
c ~ iD 0
20
0------shy
Immersion Boiling 30 sec Boiling 60 sec
Hot Water Scarification
10degTr========~--------~------------------------------
80
60
40
20
_ Jemez Raton
0------shy
1 min 2 min 4 min 8 min
Percussion Scarification
Figure 12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources
42
b
--~----~~~~========~---- duration ofpercussion reduced germination For the Raton source longer durations
ofboiling consistently decreased germination whereas longer percussion treatments
consistently increased germination
A possible explanation is that a thick seed coat is associated with a loose
strophiole structure and that a thin seed coat is associated with a constrained
strophiole This contrast merits further study and may suggest an ecological
adaptation Further investigation ofthis phenomenon may contribute to successful
pre-screening ofunknown seed sources to determine optimal treatment leveL
43
CONCLUSION
Based on results obtained in pilot study (see Appendix) and thesis
experiments 4 minutes ofpercussion is recommended as a standard protocol for
treating an unknown source ofNew Mexico locust seed In addition to laboratory
results the 4-minute percussion treatment has resulted in germination greater than
80 and healthy seedling grow-outs for production purposes (unpublished data) In
laboratory experiments only one hundred seeds per 118 ml (40z) soil tin were
percussed at a time whereas for production we have treated up to five hundred seeds
per tin For large-scale production ofNew Mexico locust seedlings growers may
wish to experiment with larger quantities of seed and larger-size soil tins
Seed storage trials following percussion treatments were not conducted
However germination greater than 80 and healthy seedlings were obtained when
seeds were percussion-treated and subsequently stored at room-temperature (22degC)
for one week (unpublished data) Seed storage trials merit further study as the
standard hot water treatment leaves seeds in a softened condition that is not
conducive to storage
Hydroseeding is an effective method for direct seeding in reclamation
situations Percussion treatment may be conducive to hydro seeding as seeds remain
a dry relatively undamaged yet permeable condition following treatment Sulfuric
seeds also remain dry following treatment but extensive damage to seed coat
poor seedling vigor have been reported with this treatment (Kelly and van Staden
A grow-out study using a hydro seeder on a reclamation site testing percussion
44
against other treatments such as sulfuric acid and mechanical scarification also
merits further study
Papilionoids are the largest subfamily oflegumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust these legumes often play an integral role in the revegetation of
disturbed lands The pilot study (Appendix) indicates that percussion scarification
successfully treats the widely used black locust as well Developing superior
scarification methods to the standard hot water mechanical scarification and acid
treatments may facilitate the use ofpapilionoid legumes in reclamation projects
45
APPENDICES
APPENDIX A
PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR
NEW MEXICO LOCUST AND BLACK LOCUST SEEDS
Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4
Hot water and sulfuric acid soaks are traditional treatments for seeds of many
temperate woody legwnes including locusts However these scarification techniques
often produce inconsistent germination Percussion scarification where seeds are
repeatedly propelled against a hard surface was compared with hot water
scarification to evaluate treatment efficacy for New Mexico locust (Robinia
neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment
seeds were placed in a 98degC water bath which was immediately removed from the
heat source For percussion scarification seeds were placed in a soil sample tin and
agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the
control were followed by 24-hour water soaks Hot water baths resulted in 56 and
41 germination for New Mexico locust and black locust respectively For both
species nearly all durations ofpercussion increased germination over the hot water
treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3
4 and 5 minutes for black locust resulted in at least 90 germination Traditional
scarification treatments randomly degrade the entire seed coat which can lead to
tissue damage during water uptake Percussion scarification specifically weakens the
strophiole the natural source ofwater entry to the seed in papilionoid legwnes
INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom
2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031
48
Following percussion imbibition is controlled through the strophiole and underlying
tissue is protected
Keywords New Mexico locust black locust scarification percussion germination
Introduction
Locust trees (Robinia spp) are aggressive pioneer species that quickly
colonize disturbed land fix atmospheric nitrogen in the soil and stabilize slopes that
are prone to erosion (Klemmedson 1994 Ashby et al 1985) (Figure A1ab)
Recognition of these attributes has contributed to increased demand for locusts in
reclamation projects Locust seeds however exhibit physical dormancy where a
thick seed coat prevents the movement of water and gasses to the embryo (Leadem
1997)
Sulfuric acid or hot water soaks have traditionally been used to break seed
dormancy in macro-propagation of locusts Many growers are moving away from the
use of su1furic acid in treating locust seeds (Dreesen and Harrington 1997) In
addition to being dangerous acid soak durations must be specifically correlated to
seed lot (Olson 1974) Collections of native seed can vary tremendously in seed size
seed weight and hard seededness etc This variability limits the utility of sulfuric
acid soaks as even within a given lot some seeds may be damaged whereas others
are under-treated and fail to imbibe (take up water) A survey of Southwestern
container growers indicates that hot water baths are currently the most common
scarification method for locust seeds (Hine et al 1997) However hot water baths
also produce inconsistent germination (Lin et al 1996)
49
b
Figure AI New Mexico locust a primary invader following disturbance Ala Crown-sprouting at edge ofburn near Los Alamos NM--one year after Cerro Grande fire Alb Colonizing road cut Gila National Forest NM
50
Percussion scarification where seeds are repeatedly propelled against a hard
surface is an alternative dormancy-breaking method that has proven successful in
legumes related to locusts (Hamly 1932 Barton 1947) We compared this alternative
treatment to hot water scarification to evaluate treatment efficacy for New Mexico
locust and black locust seeds
Materials and Methods
Commercial New Mexico locust seeds (Western Native Seed Coaldale CO)
were collected fall 2000 in Huerfano County CO Black locust seeds were collected
September 2000 from Taos County NM by harvesting ripe pods from several trees at
a distance of up to 3 m from ground level Black locust seeds were allowed to air-dry
for 3 weeks and were threshed and separated from large chaffusing a greenhouse
fan A South Dakota Seed Blower (Chicago Illinltois) was used to remove fine chaff
from both seed lots Seeds were storedmiddotat 2-4C until the germination study was
conducted in March 2001
This study compared hot water scarification with percussion scarification
using treatment and species as experimental factors Along with a control
scarification treatments included immersion of seed in boiling water as well as
percussion durations of 1 2 3 45 or 10 minutes Four 100-seed samples were used
to test each species by scarification treatment combination
For the hot water treatment test tubes were filled with 30 mL water and
placed in a water-filled beaker (Figure A2a) The bath was raised to boiling (-98C at
51
b
c
Figure A2 Implementation ofhot water and percussion scarification A2a Hot water bath A2b Paint-shaker set-up A2c Placing soil tin containing seeds at end ofpaint shaker ann
52
Las Cruces NM elevation of 1300 meters) Each 100-seed replicate was placed in a
test tube and each test tube was immediately removed to cool at room temperature
Percussion scarification was implemented using a pneumatic paint shaker
(Central Pneumatic model 00422 Camarillo CA) (Figure A2b) This allowed
standardization of treatment intensity Compressed air pressure was maintained at
530kPa plusmn 30kPa (80psi plusmn 5psi) resulting in approximately 350 oscillations per
minute Each lOO-seed sample was placed in a 118 ml (4 oz) soil tin for shaking To
maximize lateral movement of paint shaker a paint can was used as a spacer to place
soil tins at the end of the shaking arm (Figure A2c) This allowed shaking to occur at
the greatest distance from the pivot point (fulcrum) Following all treatments
including the control seeds were soaked for twenty-four hours in distilled water prior
to plating out
Gennination was evaluated on lab benches at room temperature Each
replicate of seeds was placed in a 100 cm petri dish on moistened filter paper with
humidity maintained by enclosing the petri dishes in ziplock bags Gennination was
monitored daily for 14 days in accordance with International Seed Testing
Association standards for Robinia species (ISTA 1999) Gennination was defined as
protrusion of the radicle from the embryo by at least 1mm as viewed by the naked
eye
Categorical Analysis of Variance using Prpc Catmod (SAS Institute 1989)
was used to detennine treatment differences in germination percentages for each
source This procedure is an extension of a Chi-square test of homogeneity using the
53
natural log of the ratio of germinated to non-germinated seeds for each treatment Pshy
values less than 005 were considered significant A limited set of pairwiseshy
comparisons was conducted to compare treatment mean percents using a
conservative alpha value of 005 divided by the number of comparisons
Results
Percussion maximized germination for both New Mexico locust and black
locust There were three germination response levels for New Mexico locust (Figure
A3) First there was a low response in the control indicating a dormant seed lot
The hot water treatment and I-minute duration of percussion share a second
intermediate response Germination was highest at percussion levels from 2 to 10
minutes
A selected percussion treatment intermediate in time (5 minutes) produced the
steepest germination curve indicating the fastest germination rate (Figure A4) The
hot water treatment produced an intermediate curve or relatively slower germination
rate The control curve was very flat indicating the slowest germination rate for New
Mexico locust
Control seeds germinated poorly for black locust as well (Figure A5) Hot
water scarification improved germination over the control but all levels of percussion
improved germination over hot water The optimal durations of percussion treatment
were bracketed with a significant increase from the 1 to 2 minute percussion level a
high germination response from 2 to 5 minutes of percussion and finally a significant
54
~ d 25 CL)
p t1 0 ~ t1
g Vl ~ Vl
100~---------------------------------------------------------------
80
60
40
20
o Control Hot Water Percussion
I min Percussion
2 min Percussion
3 min
Scarification
Percussion 4 min
Figure A3 Effect of scarification on germination response for New Mexico locust
Percussion 5 min
=significant difference between adjacent treatment bars ns = no significant difference
Percussion 10 min
~ ~ () u agt
Il-t d o ~
~ ~ c3
100middotlr~~~------------------------------------e- Percussion 5min middot 0middot Hot Water -- Control
80-J=middot======
6b-middot-lmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middot--middotmiddot-middot~-middotmiddot -middotmiddot middot middotmiddot-middot 1
40
20
-----~-----------
0 0 0 middotmiddotmiddotmiddotmiddot0middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot0
0 --~-----y-----~------_---~--- T-----~
o shy -TDay 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A4 Effect of selected scarification treatments on germination speed for New Mexico locust First seven days of fourteen-day experiment are shown
Day 7
80
~ cd
5 60 ()
[) Cl d 0 1t1 d
40sect dUl
-J
20
o
ns
Scarification
Figure A5 Effect of scarification on germination response for black locust = significant difference between adjacent treatment bars ns = no significant difference
decrease at the 10-minute level This drop-off suggests that 10 minutes of percussion
over-treats seeds from this lot of-black locust
A selected percussion treatment intermediate in time (5 minutes) also had the
fastest germination rate for black locust (Figure A6)_ The hot water curve is
somewhat flatter indicating a slower germination rate The control curve is very flat
indicating a slow germination rate
A qualitative difference between hot water and percussion-treated seeds was
also observed Along with healthy germinants the hot water treatment produced
over-treated seeds (Figure A 7a) Examples of damaged germinants include seeds
where the radicle did not elongate (Figure A 7b) seeds where the radicle elongated
but was delayed in freeing itself from the seed coat (Figure A7c) and seeds where the
radicle broke off completely from the embryo (Figure A7d) In addition to these
examples of over-treatment there were numerous small dark seeds that failed to
imbibe water For percussion treatment nearly all seeds took up water (Figure A8a)
Germination was rapid and uniform with little damage evident (Figure A8b)
To summarize both New Mexico locust and black locust seed lots were
dormant The standard hot water scarification treatment improved germination but
only to 56 and 41 for New Mexico locust and black locust respectively Nearly
all percussion durations improved total germination as well as germination rate over
the hot water treatment Germination was greater than 90 for percussion durations
of 4 5 and 10 minutes for New Mexico locust and 3 4 and 5 minutes for black locust
58
CI)
sgt t CI) 0
igt 0
sect ttl t E
Vl 0
c3
100r==~==~~=r------------------------------------~ ___ Percussion 5 min
middotmiddotmiddot0middotmiddotmiddot Hot Water -lIf- Control
80r=========~--~~----------------------------~
60
40
20 -~-~---~---------- ---~------------I
omiddot
O
_~_----------lIf-----~bull -----_ shy---shy I
Day 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A6 Effect of selected scarification treatments on germination speed for black locust First seven days of fourteen-day experiment are shown
Day 7
a
b c d
Figure A7 Damage to New Mexico locust seeds observed during germination counts following hot water scarification A7a Hot water replicate including damaged seedlings and numerous unimbibed seeds A7b Close-up ofseed where radicle failed to elongate A7c Close-up ofseed where radicle elongated but was delayed in freeing itself from the seed coat A7d Close-up ofseed where radicle completely broke offfrom embryo
60
a
b
Figure A8 New Mexico locust seeds following percussion treatment at time of germination counts A8a Percussion replicate with uniformly healthy seedlings and near complete imbibition of seeds A8b Close-up ofpercussion-treated seedling
61
Discussion
Papilionoid seeds have a specific anatomical feature known as the strophiole
or lens-the natural site of water entry to the seed (Hamly 1932) The strophiole is
located on the cotyledonary lobe of a locust seed (Figure A9) When a papilionoid
legume seed is percussed for the appropriate amount of time repeated hits on the
integrated seed coat loosen the constrained cells of the strophiolar region without
excessively damaging the rest of the seed coat (Ballard 1976)
When a percussed seed is soaked water enters exclusively through the
strophiole in a controlled manner (Kelly and Staden 1987) This regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues This
contrasts with seeds that have been hot water or acid scarified treatments that can
randomly degrade the seed coat Unlocalized cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rehman et al 1998 Marunda 1990)
Papilionoids are the largest subfamily of legumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust and black locust these legumes often play an integral role in the
revegetation ofdisturbed lands Developing superior scarification methods to the
standard hot water and acid treatments should fadlitate the use ofthese legumes in
restoration projects
62
Figure A9 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (bc)
63
APPENDIXB
GERMINATION PERCENTS AND STANDARD ERRORS FOR SOURCE
BLOCK AND TREATMENT FOR NEW MEXICO LOCUST
Table B 1 Gennination percents and standard errors for source block and treatment for New Mexico locust
Source Block Treatment Germination Percentage Standard Error
Capitan 1 Immersion 74 437 Capitan 2 Immersion 69 462 Capitan 3 Immersion 71 454 Capitan 4 Immersion 67 470 Capitan Boiling 30 sec 79 407 Capitan 2 Boiling 30 sec 76 427 Capitan 3 Boiling 30 sec 72 449 Capitan 4 Boiling 30 sec 76 427 Capitan Boiling 60 sec 71 454 Capitan 2 Boiling 60 sec 54 498 Capitan 3 Boiling 60 sec 67 470 Capitan 4 Boiling 60 sec 76 427 Capitan I Control 5 218 Capitan 2 Control 7 255 Capitan 3 Control 4 196 Capitan 4 Control 5 218 Capitan 1 Percussion I min 47 499 Capitan 2 Percussion I min
470
Capitan Percussion I min 39 488
Capitan 4 Percussion I min 38 485 Capitan I Percussion 2 min 55 497 Capitan Capitan
2
Percussion 2 min Percussion 2 min
71 65
454 477
Capitan 4 Percussion 2 min 56 496 Capitan I Percussion 4 min 86 347 Capitan 2 Percussion 4 min 79 407 Capitan 3 Percussion 4 min 81 392 Capitan 4 Percussion 4 min 82 384 Capitan I Percussion 8 min 90 300 Capitan 2 Percussion 8 min 88 325 Capitan 3 Percussion 8 min 90 300 Capitan 4 Percussion 8 min 90 300 Gila I Immersion 71 454 Gila 2 Immersion 77 421 Gila
Immersion 75 433
Gila 4 Immersion 72 449
Gila I Boiling 30 sec 68 466
Gila 2 Boiling 30 sec 81 392
Gila 3 Boiling 30 sec 54 498
Gila 4 Boiling 30 sec 63 483
Gila I Boiling 60 sec 55 497
Gila 2 Boiling 60 sec 68 466
Gila 3 Boiling 60 sec 57 495
Gila 4 Boiling 60 sec 70 458
65
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Gila 2 Control 13 336 Gila 3 Control 13 336 Gila 4 Control 20 400 Gila 1 Percussion 1 min 66 474 Gila 2 Percussion 1 min 57 495 Gila 3 Percussion 1 min 73 444 Gila 4 Percussion 1 min 82 384 Gila 1 Percussion 2 min 81 392 Gila 2 Percussion 2 min 86 347 Gila 3 Percussion 2 min 85 357 Gila 4 Percussion 2 min 54 498 Gila 1 Percussion 4 min 79 407 Gila 2 Percussion 4 min 83 376 Gila 3 Percussion 4 min 84 367 Gila 4 Percussion 4 min 79 407 Gila 1 Percussion 8 min 51 500 Gila 2 Percussion 8 min 76 427 Gila 3 Percussion 8 min 52 500 Gila 4 Percussion 8 min 74 439 Holman 1 Immersion 83 376 Holman 2 Immersion 86 347 Holman 3 Immersion 81 392 Holman 4 Immersion 90 300 Holman 1 Boiling 30 sec 90 300 Holman 2 Boiling 30 sec 85 357 Holman 3 Boiling 30 sec 87 336 Holman 4 Boiling 30 sec 83 376 Holman 1 Boiling 60 sec 79 407 Holman 2 Boiling 60 sec 80 400 Holman 3 Boiling 60 sec 86 347 Holman 4 Boiling 60 sec 93 255 Holman Control 25 433 Holman 2 Control 19 392 Holman Control 21 407
Holman 4 Control 24 427
Holman Percussion 1 min 77 421
Holman 2 Percussion 1 min 85 357
Holman 3 Percussion 1 min 80 400
Holman 4 Percussion 1 min 89 313
Holman 1 Percussion 2 min 88 325
Holman 2 Percussion 2 min 96 196
Holman 3 Percussion 2 min 93 255
Holman 4 Percussion 2 min 96 196
Holman 1 Percussion 4 min 92 271
Holman 2 Percussion 4 min 97 171
66
I
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Holman 3 Percussion 4 min 91 286 Holman 4 Percussion 4 min 96 196 Holman 1 Percussion 8 min 88 325 Holman 2 Percussion 8 min 85 357 Holman 3 Percussion 8 min 83 376 Holman 4 Percussion 8 min 82 384 Jemez 1 Immersion 53 499 Jemez 2 Immersion 44 496 Jemez 3 Immersion 52 500 Jemez 4 Immersion 70 458 Jemez 1 Boiling 30 sec 58 494 Jemez 2 Boiling 30 sec 55 497 Jemez 3 Boiling 30 sec 48 500 Jemez 4 Boiling 30 sec 65 477 Jemez 1 Boiling 60 sec 68 466 Jemez 2 Boiling 60 sec 63 483 Jemez 3 Boiling 60 sec 73 444 Jemez 4 Boiling 60 sec 89 313 Jemez 1 Control 2 140 Jemez 2 Control 9 286 Jemez 3 Control 6 237 Jemez 4 Control 8 271 Jemez 1 Percussion 1 min 58 494 Jemez 2 Percussion 1 min 63 483 Jemez 3 Percussion 1 min 77 421 Jemez 4 Percussion 1 min 89 313 Jemez 1 Percussion 2 min 68 466 Jemez 2 Percussion 2 min 64 480 Jemez 3 Percussion 2 min 80 400 Jemez 4 Percussion 2 min 83 376 Jemez Percussion 4 min 68 466 Jemez 2 Percussion 4 min 79 407 Jemez 3 Percussion 4 min 70 458 Jemez 4 Percussion 4 min 83 376 Jemez 1 Percussion 8 min 52 500 Jemez 2 Percussion 8 min 59 492 Jemez 3 Percussion 8 min 61 488 Jemez 4 Percussion 8 min 70 458 Magdalena 1 Immersion 69 462 Magdalena 2 Immersion 69 462 Magdalena J Immersion 70 458 Magdalena 4 Immersion 85 357 Magdalena 1 Boiling 30 sec 65 477 Magdalena 2 Boiling 30 sec 71 454 Magdalena 3 Boiling 30 sec 68 466
67
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Magdalena 4 Boiling 30 sec 83 376 Magdalena 1 Boiling 60 sec 63 483 Magdalena 2 Boiling 60 sec 68 466 Magdalena 3 Boiling 60 sec 63 483 Magdalena 4 Boiling 60 sec 82 384 Magdalena 1 Control 11 313 Magdalena 2 Control 6 237 Magdalena 3 Control 5 218 Magdalena 4 Control 4 196 Magdalena Percussion 1 min 32 466 Magdalena 2 Percussion 1 min 45 497 Magdalena 3 Percussion 1 min 46 498 Magdalena 4 Percussion I min 36 480 Magdalena 1 Percussion 2 min 67 470 Magdalena 2 Percussion 2 min 68 466 Magdalena 3 Percussion 2 min 65 477 Magdalena 4 Percussion 2 min 81 392 Magdalena 1 Percussion 4 min 87 336 Magdalena 2 Percussion 4 min 97 171 Magdalena 3 Percussion 4 min 83 376 Magdalena 4 Percussion 4 min 97 171 Magdalena 1 Percussion 8 min 98 lAO Magdalena 2 Percussion 8 min 100 000 Magdalena 3 Percussion 8 min 96 196 Magdalena 4 Percussion 8 min 98 lAO Manzano 1 Immersion 67 470 Manzano 2 Immersion 57 495 Manzano 3 Immersion 72 4049 Manzano 4 Immersion 73 4044 Manzano 1 Boiling 30 sec 70 458 Manzano 2 Boiling 30 sec 64 480 Manzano 3 Boiling 30 sec 71 454 Manzano 4 Boiling 30 sec 80 400 Manzano I Boiling 60 sec 50 500 Manzano 2 Boiling 60 sec 53 499 Manzano 3 Boiling 60 sec 59 492 Manzano 4 Boiling 60 sec 74 439 Manzano I Control 4 196 Manzano 2 Control 8 271 Manzano 3 Control 1 099 Manzano 4 Control 6 237 Manzano 1 Percussion 1 min 52 500 Manzano 2 Percussion 1 min 39 488 Manzano 3 Percussion 1 min 36 480 Manzano 4 Percussion 1 min 51 500
68
Table B 1 Continued
Source Block Treatment Gerrni~ation Percentage Standard Error
Manzano Percussion 2 min 53 499 Manzano 2 Percussion 2 min 58 494 Manzano 3 Percussion 2 min 61 488 Manzano 4 Percussion 2 min 62 485 Manzano 1 Percussion 4 min 76 427 Manzano 2 Percussion 4 min 85 357 Manzano 3 Percussion 4 min 79 407 Manzano 4 Percussion 4 min 83 376 Manzano 1 Percussion 8 min 87 336 Manzano 2 Percussion 8 min 83 376 Manzano 3 Percussion 8 min 83 376 Manzano 4 Percussion 8 min 75 433 Raton 1 Immersion 54 498 Raton 2 Immersion 37 483 Raton 3 Immersion 39 488 Raton 4 Immersion 69 462 Raton 1 Boiling 30 sec 36 480 Raton 2 Boiling 30 sec 20 400 Raton 3 Boiling 30 sec 28 449 Raton 4 Boiling 30 sec 60 490 Raton 1 Boiling 60 sec 15 357 Raton 2 Boiling 60 sec 171 Raton 3 Boiling 60 sec 4 196 Raton 4 Boiling 60 sec 35 477 Raton 1 Control 4 196 Raton 2 Control 12 325 Raton 3 Control 2 140 Raton 4 Control 6 237 Raton 1 Percussion 1 min 52 500 Raton 2 Percussion I min 29 454 Raton 3 Percussion I min 35 477 Raton 4 Percussion 1 min 31 462 Raton 1 Percussion 2 min 55 497 Raton 2 Percussion 2 min 35 477 Raton 3 Percussion 2 min 33 470 Raton 4 Percussion 2 min 42 494 Raton 1 Percussion 4 min 65 477 Raton 2 Percussion 4 min 52 500 Raton Percussion 4 min 51 500 Raton 4 Percussion 4 min 53 499 Raton 1 Percussion 8 min 88 325 Raton 2 Percussion 8 min 80 400 Raton 3 Percussion 8 min 73 444 Raton 4 Percussion 8 min 74 439 Sacramento 1 Immersion 43 495
69
Table B 1 Continued
-Source Block Treatment Germination Percentage Standard Error
Sacramento 2 Immersion 30 458 Sacramento 3 Immersion 38 485 Sacramento 4 Immersion 56 496 Sacramento 1 Boiling 30 sec 47 499 Sacramento 2 Boiling 30 sec 48 500 Sacramento 3 Boiling 30 sec 48 500 Sacramento 4 Boiling 30 sec 56 496 Sacramento 1 Boiling 60 sec 52 500 Sacramento 2 Boiling 60 sec 67 470 Sacramento 3 Boiling 60 sec 43 495 Sacramento 4 Boiling 60 sec 48 500 Sacramento 1 Control 0 000 Sacramento 2 Control 3 171 Sacramento 3 Control 3 171 Sacramento 4 Control 099 Sacramento 1 Percussion 1 min 57 495 Sacramento 2 Percussion 1 min 61 488 Sacramento 3 Percussion 1 min 62 485 Sacramento 4 Percussion 1 min 55 497 Sacramento 1 Percussion 2 min 84 367 Sacramento 2 Percussion 2 min 70 458 Sacramento 3 Percussion 2 min 81 392 Sacramento 4 Percussion 2 min 69 462 Sacramento 1 Percussion 4 min 91 286 Sacramento 2 Percussion 4 min 84 367 Sacramento 3 Percussion 4 min 84 367 Sacramento 4 Percussion 4 min 94 237 Sacramento 1 Percussion 8 min 82 384 Sacramento 2 Percussion 8 min 81 392 Sacramento 3 Percussion 8 min 82 384 Sacramento 4 Percussion 8 min 81 392 Sandia 1 Immersion 76 427 Sandia 2 Immersion 70 458 Sandia 3 Immersion 70 458 Sandia 4 Immersion 81 392 Sandia 1 Boiling 30 sec 78 414 Sandia 2 Boiling 30 sec 71 454 Sandia 3 Boiling 30 sec 63 483 Sandia 4 Boiling 30 sec 78 414 Sandia 1 Boiling 60 sec 67 470 Sandia 2 Boiling 60 sec 65 477 Sandia 3 Boiling 60 sec 74 439 Sandia 4 Boiling 60 sec 66 474 Sandia 1 Control 16 367 Sandia 2 Control 7 255
70
Table B 1 Continued
Source Block Treatment Gennination Percentage Standard Error
Sandia 3 Control 8 271 Sandia 4 Control 16 367 Sandia 1 Percussion 1 min 67 470 Sandia 2 Percussion 1 min 61 488 Sandia 3 Percussion 1 min 66 474 Sandia 4 Percussion 1 min 59 492 Sandia I Percussion 2 min 82 384 Sandia 2 Percussion 2 min 83 376 Sandia 3 Percussion 2 min 81 392 Sandia 4 Percussion 2 min 84 367 Sandia 1 Percussion 4 min 89 313 Sandia 2 Percussion 4 min 86 347 Sandia 3 Percussion 4 min 89 313 Sandia 4 Percussion 4 min 85 357 Sandia 1 Percussion 8 min 80 400 Sandia 2 Percussion 8 min 77 421 Sandia 3 Percussion 8 min 69 462 Sandia 4 Percussion 8 min 66 474
LITERATURE CITED
Ashby WC Vogel WG Rogers NF 1985 Black locust in the reclamation equation General Technical Report Northeastern Forest Experiment Station USDA Forest Service NE-I05 12 p
Ballard LAT 1973 Physical barriers to gennination Seed Sci Technoll 285-303
___ 1976 Strophiolar water conduction in the seeds ofTrifolieae induced by action on the testa at nonstrophiolar sites Austr J Plant Physiol3 465-469
Barton LV 1947 Special studies on seed coat impenneability Contrib Boyce Thompson Inst 14 355-362
Baskin CC Baskin JM 1998 Seeds Ecology biogeography and evolution of donnancy and gennination Academic Press London UK 666 p
Bewley JD 1997 Seed germination and donnancy Plant Ce119 1055-1066
Bewley JD Black M 1994 Seeds physiology of development and germination 2nd ed Plenum Press New York NY 445 p
Briscoe CB 1996 Growing Nitrogen Fixing Trees in Acid Soils In MH Powell (ed) Nitrogen Fixing Trees for Acid Soils- A Field ManuaL Winrock International Morrilton AR 22 p
Copeland LO McDonald MB 1995 Principles of Seed Science and Technology 3rded Chapman amp Hall New York NY 409 p
Cox RE Klett JE 1984 Seed gennination requirements of native Colorado plants for use in the landscape Plant Propag 30(2) 6-10
Dell B 1980 Structure and function ofthe strophiolar plug in seeds ofAlbizia Lophantha Am J Bot 67 556-563
Dick-Peddie WA 1993 New Mexico Vegetation Past Present and Future University ofNew Mexico Press Albuquerque NM 244 p
Dreesen DR 2001 Propagation protocol for production ofcontainer Robinia neomexicana plants Los Lunas Plant Materials Center Los Lunas New Mexico In Native Plant Network URL httpwwwnativeplantnetworkorg (accessed 9 January 2002) Moscow (ID) University ofIdaho CoIl Natural Resources For Res Nursery
72
Dreesen DR Harrington JT 1997 Propagation of native plants for restoration projects in the Southwestern US- Preliminary Investigations In Landis TD Thompson JR tech coords National Proceedings Forest and Conservation Nursery Assocs Gen Tech Rep PNW-GTR-419 Portland OR USDA For Ser PNW Res Sta 77-88
Gottfried GJ 1980 Control ofNew Mexican locust and the effect on planted ponderosa pine in central Arizona US For Ser Res Note RM-386 Rocky Mountain For Range ExpStn Fort Collins CO
Gosling PG Samuel YK Jones SK 1995 A systematic examination of germination temperature chipping and water temperaturesoak duration pretreatments on the seeds ofLeucaena leucocephala Seed Sci amp Technol 23521-532
Hamly DH 1932 Softening of the seeds of Melilotus alba Bot Gazette 93 345shy375
Hanna PJ 1984 Anatomical features of the seed coat ofAcacia kempeana (Mueller) which relate to increased germination rate induced by heat treatment New Phytol 96 23-29
Hanks JP Dick-Peddie WA 1974 Vegetation patterns of the White Mountains New Mexico Southwest Naturalist 18(4) 371-382
Hine S Harrington JT Dreesen DR 1997 Seed scarification requirements for Robinia neomexicana In Harrington JTtech coord Proc Third SouthwestemContainer Growers Conf Feb 12-13 1997 Ft Collins CO
Hopkinson JM 1993 The strophiole of leguminous seeds Newsletter- IntI Herbage Seed Production Res Grp 18 7-8
---- 1997 Differences in hardseededness between Cunningham and Tarramba leucaena In Leucnet News The Newsletter of the International LeucaenaRes and Devlt Netwk No4
ISTA (International Seed Testing Association) 1999 International rules for seed testing rules Seed Sci amp Technol27 Supplement
Kelly KM van Staden J 1987 The lens as the site of permeability in the papilionoid seed Aspalathus linearis J Plant Physiol 128(45) 395-404
73
Keresztesi B 1988 Black locust the tree of agriculture Outlook on Agr 17(2) 77~85
Kigel 1 and Galili G 1995 Seed development and germination Marcel Dekker New York 853 p
Klemmedson 10 1994 New Mexico locust and parent material Influence on macronutrients of forest floor and soil Soil Sci Soc Am 1 58 974~980
Kuo WH Tam AW 1988 The pathway of water absorption of mung bean seeds Seed Sci amp Technol16(1) 139~144
Leadem CL 1997 Dormancy--unlocking seed secrets In Landis TD Thompson lR tech coords National Proceedings Forestand Conservation Nursery Assocs Gen Tech Rep PNW-G TR-419 Portland OR USDA For Ser PNW Res Sta 43-52
Lin Y Wagner MR Seweyestewa A Honahni MD DeGomez M McMillin JD 1996 Influence of cultural treatments on seed germination and seedling growth of Robinia neomexicana Forest Farm and Community Tree Res Reports Volume 1
Marunda CT 1990 Effects of seed pretreatment on the development ofAcacia auriculforinis and A holosericea seedlings ACIAR Proceedings Series No 28
Meginnis HG 1937 Sulphuric acid treatment to increase germination of black locust seed USDA Circ 453 34 p
Miklas PN Towensend CE Ladd SL 1987 Seedcoat anatomy and the scarification ofcicer milkvetch seed Crop Sci 27 766-772
Monsen SB 1984 Use of shrubs on mine spoils In The Challenge ofProducing Native Plants for the Intermountain Area Proceedings Intermountain Nurserymans Association 1983 conference Las Vegas NV compo PM Murphy 26-31 Ogden UT USDA For Ser Gen Tech Report INT-l68
Olson DF 1974 Robinia L Locust In Seeds of Woody Plants in the US tech coord CS Schopmeyer 728-731 USDA For Ser Agr Handbk No450
Rehman S Loescher RNl Harris P1C 1998 Dormancy breaking and germination ofAcacia salicina Lindl seeds Seed Sci amp Techno127 553-557
Rolston MP 1978 Water impermeable seed dormancy Bot Rev 44 365-396
74
SAS Institute Inc 1989 SASSTAT Users Guide Version 6 4th Edition Volume 1 Cary NC SAS Institute Inc 943 p
SerratoValenti G Conara L Ferrando M Modenesi P 1993 Structural and histochemical features ofStylosanthes scabra (Leguminosae Papilionoidae) seed coat as related to water entry Can J Bot 71 834-840
Simpson BJ 1988 A field guide to Texas trees Austin TX Texas Monthly Press 372p
Tran VN Cavanagh AK 1984 Structural aspects ofdormancy In Seed Physiology Vol 2 Murray DR (ed) p1-44 Academic Press Australia
USDA Forest Service 1948 Woody-plant seed manual USDA Misc Pub1654 416p
Vines RA 1960 Trees Shrubs and Woody Vines of the Southwest Austin Univ ofTexas Press 1104 p
Wagner MR Larson RE Craig TP 1992 Ecology and silvics ofNew Mexico locust (Robinia neomexicana) with an emphasis on the arboreal form p53-57 In JW Hanover K Miller and S Plesko (eds) Proc Intemat Confon Black Locust Biology Culture and Utilization Mich State Univ USA
Wilson JK 1944 Immersing seeds of species of Robinia in boiling water hastens germination J For 42 453-454
Zimmerman RW Carpenter SB 1980 First-year coppice production from a 5shyyear old black locust stand on surface mine spoil p309-314 Centr Hardwd For ConfProc University ofMissouri Columbia
LIST OF FIGURES
Figures Page
1 Seedsourcecollectionmap 17
2 Effect ofNew Mexico locust seed source on germination for data averaged over scarification treatments 26
3 Effect of scarification on germination for New Mexico locust for data averaged over all seed sources 27
4 Effect of seed source by treatment interaction on New Mexico locust germination for percussion scarification pattern a and pattern b 28
5 Effect of source by treatment interaction on New Mexico locust germination for hot water scarification 29
6 Timecourse of New Mexico locust germination following scarification treatments 31
7 X-ray radiographs of Sacramento seed source perc ussed for 1 4 or 8 minutes 33
8 Seed damage close-ups 34
9 SEM images of untreated vs 4-minute percussion-treated strophiolar cells ofNew Mexico locust seeds 35
10 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (b c) 38
11 Damage to New Mexico locust seeds observed during germination counts following hot water scarification 40
12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources 42
New Mexico locust a primary invader following disturbance 50
Implementation of hot water and percussion scarification 52
Effect of scarification on germination response for New Mexico locust 55
x
A4 Effect of selected scarification treatments on germination speed for New Mexico locust 56
AS Effect of scarification on germination response for black locust 57
A6 Effect of selected scarification treatments on germination speed for black locust 59
A7 Damage to New Mexico locust seeds observed during germination counts following hot water scarification 60
AS New Mexico locust seeds following percussion treatment at time of germination counts 61
A9 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (bc) 63
INTRODUCTION
New Mexico locust (Robinia neomexicana A Gray) a small tree native to the
southwestern United States occurs at elevations from 1200 to 2800 meters This
species fills a successional role in post-disturbance situations As a primary invader
New Mexico locust quickly establishes on burned areas arid flood banks as well as
road cuts (Wagner et aI 1992) Rapid growth crown sprouting and prolific root
suckering favor the successful establishment ofNew Mexico locust on disturbed sites
(Simpson 1988 Gottfried 1980) These attributes also may make New Mexico locust
well suited for steep-slope revegetation where erosion is a problem
New Mexico locust is a nitrogen-fixing legume that tolerates and improves
nutrient-poor soils Stands ofNew Mexico locust increase Nitrogen (N) and Calcium
(Ca) in the forest floor Levels of Carbon (C) Phosphorous (P) Sulfur (8) and
Potassium (K) aIso have been shown to increase in soil beneath New Mexico locust
(Klemmedson 1994)
The ability to colonize rapidly and ameliorate harsh sites contributes to New
Mexico locusts potential as a nurse plant A nurse plant colonizes an inhospitable
site and creates an environment suitable for successional plant establishment New
Mexico locust demonstrates this role in the ponderosa pine (Pinus ponderosa Douglas
ex Lawson amp C Lawson)Gambel oak (Quercus gambelii Nutt) community After a
disturbance such as wildfire New Mexico locust and Gambel oak colonize and
dominate the site until shaded out by ponderosa pine Once shaded these species
become understory shrubs a process taking an average of 15-20 years (Dick-Peddie
1993 Hanks and Dick-Peddie 1974) New Mexico locust gradually declines with
small dense patches averaging about 005 ha beneath pine until the next disturbance
once again offers it a competitive advantage (Klemmedson 1994)
Gottfried (1980) expecting ponderosa pine regeneration to be greater where
New Mexico locust had been eradicated found that survival of planted pine seedlings
was greater where locust had not been removed In a follow-up study Gottfried
(1980) noted that soil moisture in the top 57 cm was highest in 5-year old locust sites
as compared to grass or 20-year old locust sites He concluded that managing an
appropriate cover ofNew Mexico locust could help regenerate pine the later
successional species
The ability ofNew Mexico locust to improve harsh sites also makes it a
candidate for reclaiming disturbances associated with mining Black locust (Robinia
pseudoacacia 1) has been used for decades to reclaim mine spoils and other
disturbed sites in the eastern US and around the world (Keresztesi 1988 Zimmerman
and Carpenter 1980) Ashby et al (1985) described similar positive attributes of
black locust also mentioned previously for New Mexico locust quick cover for
stabilization supply ofN and nutrient-rich litter to the soil and site improvement for
establishment of later successional trees Referring specifically to mine reclamation
the authors note the ability of black locust to grow on a wide range ofmine soil
conditions including extremely acid soils Black locust also shows some tolerance to
soils compacted by grading and topsoiling practices (Ashby et aL 1985) New
2
Mexico locust holds promise as a native southwe~tem counterpart to black locust in
mine reclamation but it has been used infrequently to date
Natural invasion and succession occur slowly on most mine sites (Monsen
1984) While New Mexico locust often colonizes sites naturally there is no
assurance a seed will reach a particular site and establish in a reasonable time frame
The challenge is to introduce New Mexico locust as mother plants to facilitate
colonization Seedling production for outplanting on disturbed sites has been
hampered by poor germination (Lin et al 1996) The goal then is to develop
techniques to overcome poor germination in New Mexico locust thereby facilitating
macropropagation
LITERATURE REVIEW
Seed Dormancy
Low gennination in New Mexico locust is primarily the result of seed
donnancy the failure of an intact viable seed to genninate under favorable
conditions (Bewley 1997) Seed donnancy also can be defined as the inability of the
embryo to genninate because of some inherent constraint (Bewley and Black 1994)
The constraint may originate within the embryo referred to as endogenous or embryo
donnancy or from the tissues surrounding the embryo known as exogenous or coatshy
imposed donnancy (Baskin and Baskin 1998 Leadem 1997 Bewley and Black
1994)
New Mexico locust seed is donnant when the impenneable seed coat does not
allow movement ofwater to the embryo This specific fonn of coat-imposed
donnancy where the restraining tissue involved is an impenneable seed coat is
known as physical donnancy (Baskin and Baskin 1998 Bewley and Black 1994)
Physical donnancy is relatively uncommon in seeds less than 10 of all woody
species exhibit this condition (USDA Forest Service 1948) However it is
characteristic of many genera of the Fabaceae (bean) family For example all
successional trees in the genera Gleditsia and Robinia (of which New Mexico locust
is a member) produce seeds that exhibit physical donnancy (Baskin and Baskin
4
Physical Dormancy
Seeds that exhibit physical dormancy are said to be hard seeded referring to
the thick impermeable seed coat (Leadem 1997) The first line of protection of the
testa in leguminous seeds is the outermost waxy cuticle followed by a subcuticle
consisting ofsuberin Next is a palisade layer consisting of sclereid cells that have
thick lignified secondary walls The most common type of sclereid cell in palisade
layers is the macrosclereid The macrosclereids contain water-repellent substances
including cutin lignin suberin and wax (Rolston 1978) This palisade layer is the
primary barrier to water uptake (Serrato-Valenti et al 1993 Tran and Cavanagh
1984)
All seed coat openings must be blocked to effectively prevent water uptake
The natural opening in a seed where it was attached to the mother plant is the weak
link where physical dormancy is usually broken (Hopkinson 1993 Kuo and Tam
1988) Anatomical structures associated with the seed coat opening vary from
family to family In the Papilionoideae the subfamily of the Fabaceae family
containing New Mexico locust the site ofwater entry is the strophiole The
strophiole is a swelling on the seed coat close to the stalk scar (Hopkinson 1997)
Kelly and van Staden (1987) refer to the strophiole site as the lens where
macrosclereid cells must separate and raise before water can pass through Since
middot6germination ofdormant seeds of the Papilionoideae takes place when the strophiole
permeable (Hopkinson 1993 Kuo and Tam 1988) a logical approach to
5
genninating New Mexico locust artificially would be to loosen the strophiolar cells
and allow water to pass through to the embryo
Evolutionary Role of Physical Dormancy
Why is physical dormancy a mechanism that blocks germination
ecologically advantageous When a plant produc~s seeds with different degrees of
dormancy seeds that are dispersed at one time will germinate over a range of time
(Bewley and Black 1994) Through variability of dormancy within a seed crop seeds
are exposed to changing environmental conditions improving the overall chances of
survival of the seed line (Bewley and Black 1994) Varying expression of dormancy
is under genetic control which indicates that this may be an evolved survival strategy
(Kigel and Galili 1995) A range of dormancy levels may be a particular advantage in
the southwestern US where extreme environmental variations exist
Methods for Breaking Dormancy
There are three common approaches to breaking dormancy in New Mexico
locust seed acid scarification hot water soaks and mechanical treatments In each
case the objective of the treatment is to scarify or abrade the seed coat to make it
permeable to water
Acid Scarification
Acid scarification consists of soaking seeds in concentrated sulfuric acid
rinsing thoroughly with water upon removal followed by subsequent drying before
PlaJlltmg Olson (1974) reports that this scarification method produces the highest
middot~~jllalUll results for all Robinia species Most literature refers to a seminal 1937
6
study conducted by HG Meginnis This stud~ t~ough conducted on black locust
seed is regularly cited in the suggested use of acid to treat New Mexico locust seed
as well (Olson 1974 Vines 1960)
Thee published studies document the specific use of sulfuric acid to treat
New Mexico locust seed Cox and Klett (1984) report that soaking seeds in
concentrated sulfuric acid for thirty minutes improved germination to 34 over an
untreated control of4 Lin et al (1996) found that for two seed lots acid
scarification for twenty-five to eighty minutes produced germination of 80 A third
lot however resulted in less than 20 germination at all durations of 10 to 80
minutes Hine et al (1997) showed that acid treatment levels of 1 2 5 and 10
minutes resulted in less than 20 germination across five sources However there
was an increasing trend in germination with acid soak duration and acid treatments
were not imposed for as long a time as the previous two studies (30 to 80 minutes)
The mixed results obtained by these investigators were probably due to the
widely varying germination response of seed lots to acid treatment For this reason
Olson (1974) recommends correlating duration of acid treatment to each seed lot If
treatment duration is too long the embryo will be permanently damaged If soak
duration is too short the seed will remain impermeable to water and fail to germinate
Compounding the problem of treatment duration is the negative effect acid
have on seedling growth Any type of scarification can injure seeds to some
by disrupting cells essential for growth leading to fungal invasion and
middot-uuu~w injury (Copeland and MacDonald 1995) Some legume seeds subjected
7
to 60 minutes of sulfuric acid scarification (to achieve maximum gennination results)
showed more than 50 seedling mortality after gennination (Kelly and van Staden
1987) Bewley and Black (1994) noted that fungal growth on seeds is an indication
of leakage of sugars organic acids amino acids and proteins
Perhaps the greatest drawback to acid scarification is the dangerous nature of
the treatment itself Difficulties associated with storage and trained use of
concentrated acid may discourage many propagators from using this option (Dreesen
and Harrington 1997) Consequently hot water and to a lesser extent mechanical
scarification are more commonly used and practical treatments for legume seeds
Most southwestern container growers use either of these two treatments to scarify
New Mexico locust seed in particular (Hine et al 1997)
Hot Water Scarification
The widespread use ofhot water scarification to treat locust seed is probably
due to its ease of application and efficacy in comparison to other scarification
treatments Although this practice had been in use for some time Wilson (1944) was
one of the first to report on its systematic application for black locust The treatment
consists of placing seeds in boiling water (4 parts water tomiddot 1 part seed by volume)
removing the vessel from the heat source and allowing the water to cool and seeds to
soak for 24 hours
Hine et al (1997) Lin et al (1996) and Cox and Klett (1984) have examined
the specific application ofhot water scarification on New Mexico locust seed Each
these authors compared hot water to acid scarification Lin et aL (1996) reported
8
60 gennination for two hot water-treated seed lots while a third lot germinated
around 50 (as reported above acid scarification germination ranged from 20 to
80 for these lots) It should be noted that the authors only soaked the seeds in water
from 10 to 80 minutes for this experiment as opposed to the standard 24 hours Cox
and Klett (1984) stated that using the standard 24-hour hot water treatment improved
germination over an acid pre-treatment (which produced less than 20 germination)
but they did not give specific gennination numbers for the hot water treatment Hine
et al (1997) found that across five seed lots germination was 66 for the standard
hot water treatment (as compared to less than 20 germination for acid scarification)
Variations in treatment intensity and duration and their subsequent effects on seed lot
appear to contribute to conflicting conclusions as to whether acid scarification or hot
water treatment is a more successful treatment
Given mixed results ease ofapplication is an important argument for use of
hot water application over acid soaks Safety concerns and material costs for hot
water treatments are low (Dreesen and Harrington 1997) An extension of this is the
practicality ofhot water for treating large batches of seed where a relative increase in
water volume is all that is needed However increases in water and seed even when
inthe same 4 1 proportion can result in dramatically different rates of cooling than in
atest tube experiment Different cooling rates can lead to contrasting results for the
treatment temperature duration and seed lot (USDA Forest Service 1948)
As with acid scarification parameters ofhot water treatment application may
to be optimized to maximize germination Hine et al (1997) found that as
9
temperature was increased from 70degC to 100degC overall germination in New Mexico
locust seed increased The 100degC treatment maximized germination for each of five
sources tested with germination usually above 60
Prolonged exposure of seeds ofmany woody legumes to boiling temperatures
also may improve germination (Briscoe 1996) This approach has not been tested on
New Mexico locust seed Exposing seed to prolonged boiling temperatures may
result in fungal infection and subsequent seedling mortality Marunda (1990) noted
severe fungal infections on both acid and hot water treated Acacia holoserica Cunn
ex G Don seeds attributing the infections to seed damage resulting from the
treatments Identifying an optimal hot water treatment level that does not result in a
high rate of seedling mortality is a logical goal
With hot water treatments as with acid there is also a danger of overtreating
Seeds and reducing germination (Gosling et al 1995) The legume Leucaena
leucocephala (Lam) de Wit germinated equally well after 7 seconds of exposure to
water at 100degC 7 seconds at 90degC 30 seconds at 80degC and 4 minutes at 70degC In
addition acceptably high germination was obtained from treatment at 70degC from 3
minutes to 1 hour whereas no seeds germinated following treatment at 100degC for 2
minutes Perhaps along with a greater margin of error for treatment duration treating
~ew Mexico locust seeds at a lower temperature may result in a comparatively lower
of seedling mortality In practice however nursery growers tend to use the
and quickest treatment available The short treatment length at near-boiling
whether recommended or not may be what is used most frequently
10
Fine-tuning a treatment in this range of treatment temperature and duration may be
the most logical approach
Mechanical Scarification
This treatment involves scratching the seed coat with an abrasive material
(USDA Forest Service 1948) One system employs a rotating drum lined with
sandpaper A paddle spinning at a given number ofrevolutions per minute throws the
seed against the sandpaper (Hine et al 1997) A second method dry tumbling
tumbles seed in a mixer with pea gravel or sharp sand for 5 to 7 days (Dreesen 2001)
These approaches have the advantage that control of temperature is not required as in
water treatments nor is safety an issue as with add scarification
Hine et al (1997) mechanically treated New Mexico locust seed using 100shy
grit sandpaper in a Forsberg (rotating drum) scarifier Gennination was less than
10 They concluded that the seed coats were too thin for their apparatus and
proposed that a finer grit sandpaper would produce more even abrasion Although
germination varied by seed lot Dreesen (2001) reported that using a slow-speed dry
tumbling technique for 5 to 7 days produced gennination rates as high as 90
As with acid and hot water treatments treatment duration must be specifically
shy
correlated to seed lot Treated seeds are also susceptible to fungal infection
iHopkinson 1997) In addition equipment is specialized and may not always be
ltU~CUJl~ to a nursery (USDA Forest Service 1948) Several studies have confinned
degrading ofstrophiolar cells due to acid and hot water scarification (Miklas et al
11
1987 Dell 1980 Hanna 1984) but the effect ofmechanical scarification on the
strophiole has not been studied
Alternative Method for Breaking Dormancy Percussion
Strophiole degradation has been examined in depth using an impaction
treatment more commonly known as percussion Hamly (1932) first proposed that
shaking or impacting seeds could split the macrosc1ereid cells at the strophiole
Hamly successfully released tension ofbent strophiolar cells of impermeable
Melilotus alba Medik (White sweet clover) seeds by delivering physical blows
resulting in cells that had split apart and were only slightly bent Ninety-two percent
oftreated seeds became permeable as compared to 05 of control seeds Barton
(1947) showed that percussion significantly increases permeability in black locust
seed Ballard (1976) reasoned that the integrated nature of the strophiole within the
seed coat means a blow on any part of the seed weakens the strophiole There are no
published studies on the use of percussion on New Mexico locust
Percussion methods vary and often seem SUbjective or ill-defined Hamly
(1932) placed seeds in a glass bottle and shook them three times a second for 20
minutes resulting in 180 oscillations per minute Ballard (1973) describes shaking
seeds in a closed metal container with at least fifty seeds per container at 850-900
JS(~l11alIOIlS per minute for 1 minute Kelly and van Staden (1987) placed seeds into
sealed glass flasks Flasks were shaken by a wrist-shaker at a rate of 250
~UUVJ~ per minute Subsequent germination rates were 45 after 2 hours of
55 after 3 hours 60 after 6 hours and 100 after 12 hours
12
There are several potential advantages of percussion over acid hot water and
mechanical scarification Kelly and van Staden (1987) note that a percussion
treatment only alters macrosclereid cells of the strophiole by loosening separating
and raising them They emphasize that minimal damage is done to the overall
anatomical structure of the seed In acid scarification of the same seed lot radicle
and cotyledon damage occurred Based on photographic evidence the authors
believe that this morphological damage was due to uneven pressure during
imbibition with water entering haphazardly wherever the acid had sufficiently
dissolved the coat This ultimately led to uneven hydration of the embryo In
percussed seeds water only entered through the s~ophiole when the entire seed coat
had been saturated resulting in controlled and even embryo hydration (Kelly and van
Staden 1987)
Although all successful scarification treatments may focus at least in part on
the strophiole for papilionoid legumes (Baskin and Baskin 1998) percussion may be
less damaging to seed coat structures particularly when compared with acid Kelly
and van Staden did not compare hot water or mechanical scarification methods
against percussion However the fungal infection associated with hot water and
mechanical scarification as with acid scarificatio~ have been directly associated with
seed damage (Rehman et al 1998 USDA Forest Service 1948) Embryo damage in
has been observed in hot-water scarified legume seeds (Rehman et al 1998
13
By limiting embryo damage percussion scarification reduces seedling
mortality compared to other treatments Kelly and van Staden (1987) noted that
seedlings from the percussion treatment were morphologically sound and vigorous
when compared to acid treatment seedlings The authors attribute this improved
seedling vigor to the controlled water uptake that limited anatomical damage
Despite a 12-hour percussion treatment morphological damage was not substantially
greater than untreated seeds In addition seedling mass measured 10 days following
germination was not significantly different between percussed and untreated seeds
A final advantage ofpercussion may be the ability to adequately treat the most
dormant seeds while avoiding harm to seeds that have already been made permeable
by the treatment or that were permeable to begin with Instead ofcorroding scalding
or scratching the seed coat percussion appears to be just a loosening of the
strophiole spring (Ballard 1973) Kelly and van Staden (1987) were able to
increase germination with a lengthy treatment (12 hours) Figuring out whether a
substantial cushion oftreatment durations exist for percussion ofNew Mexico locust
seed would be a significant improvement to current scarification methods where seed
damage and seedling health is an issue
The greatest challenge for applying percussion to New Mexico locust seeds is
standardizing a treatment that is both effective and readily reproducible by nurseries
with mechanical scarification this may require the development of specific
~UllDment to properly quantify and execute the treatment
14
f
Objectives
The first objective of the study is to detennine which scarification treatment
results in optimal gennination speed and percentage ofNew Mexico locust The
second objective is to describe treatment interactions with nine seed sources collected
in New Mexico
Hypothesis
A percussion treatment acts exclusively on the main area ofwater uptake in
New Mexico locust seed loosening the strophiole so that imbibition can occur This
direct mechanism ofdonnancy release will result in improved gennination rate and
overall gennination percentage for New Mexico locust seed compared to standard
treatments Standard treatments including acid hot water and mechanical
scarification while eventually weakening the strophiolar cleft cause excessive
morphological damage through generalized seed coat degradation These treatments
are therefore prone to inconsistency due to over and under-treatment
15
MATERIALS AND METHODS
Seed Collection and Cleaning
Seeds were collected from August 25 through September 15 2000 from nine
locations along a latitudinal gradient in New Mexico Seed source locations and
descriptive information are given in Figure 1 and Tables I and 2 Stands were
separated by at least 45 km
Pods were hand picked as they began to ripen and dehisce (open) Collections
were made from ground level to approximately 3 m from several trees at each site
Pods were dried in open-ended brown paper bags for two to four weeks at room
temperature (21-230C) to facilitate cleaning Dried pods were placed in a closed cloth
sack which was pounded against the ground to separate seeds from pods For pods
still containing seeds hand maceration (crunching pods with hands) completed seed
separation Debris was removed by threshing with the aid of a greenhouse fan A
South Dakota Seed Blower (Seedbureau Chicago Illinois) was used to remove
remaining chaff and light seeds by air density separation Seeds were placed in dry
storage in sealed quart-size zip-lock bags within a sealed plastic container Storage
temperatures ranged from 2-4oC Seeds remained in storage for approximately 7
months before the experiment
Experimental Design and Treatment Structure
The experiment compared the impact ofhot water bath and percussion
l11aLl11 on germination properties ofNew Mexico locust seed A single
16
-l
Figure 1 Seed source collection map All sources were collected within New Mexico USA separated by at least 4S lan
IIIRZltItW lIJPIirmiddot rllll1lbullbull_~~~_II
Table 1 Latitude longitude location elevation and collection date ofNew Mexico locust seed sources
Lot title Latitude (N) Longitude (W) Location Elevation (m) Collection Date
Raton 36deg5846 104deg2872 Raton Pass NM 2295 82500
Holman 36deg0306 105deg2429 HolmanNM 2476 911500
Jemez 35deg4500 106deg4100 Jemez Springs NM 1976 9900 00
Sandia 35deg1083 106deg2341 Cedar Crest NM 2432 82600
Manzano 34deg3689 106deg2494 ManzanoNM 2465 9200
Magdalena 33deg5953 107deg0903 Magdalena NM 2544 82900
Capitan 33deg2728 105deg4325 Alto NM 2189 911200
Sacramento 32deg5798 105deg4496 Cloudcroft NM 2630 91400 Gila 32deg5400 107deg4600 Kingston NM 2432 9700
f
Table 2 Mean seed weight and standard error by source
a
Source Mean seed weight (g) for Standard error 100 seeds n = 5
Raton Holman Jemez Sandia Manzano Magdalena Capitan Sacramento Gila
2872 b c 2522 d 1826 e 2753 c 2413 d 2559 d 2443 d 3130 a 3029 ab
00103 00403 00276 00301 00370 00374 00458 00279 00348
a Mean seed weights followed by the same letter are not significantly
different
19
experiment was conducted utilizing a randomized complete block design blocked by
time with a factorial treatment structure Experimental factors were seed source and
scarification treatment Nine seed sources from throughout New Mexico were used
(Figure 1 Table 1) Scarification treatments included a control three levels ofhot
water and four levels of percussion Hot water treatments included immersion at
100degC and boiling for periods of 30 seconds and 60 seconds Percussion treatment
levels were 1 2 4 and 8 minutes One hundred seeds per block were used to test
each treatment combination Four blocks of seed were tested one at a time (all seed
sources included in each run) in close chronological sequence (within 6 weeks) A
total of four 100-seed samples were used to test each source by scarification treatment
combination
Treatments
Hot water treatment of seeds utilized 50 ml test tubes filled with 30 ml
distilled water Each test tube was used to treat one 100-seed sample at a time
resulting in a ratio of4 parts water to 1 part seed by volume Test tubes were placed
in a beaker with the beaker filled with tap water to the same level as the water within
test tubes Nine test tubes (one per seed source) were placed in the beaker
simultaneously so that one replication of each seed sourcescarification treatment
cotnblnatlon was conducted simultaneously Hot water treatments include immersion
boiling For immersion treatments seeds were added to test tubes with the water
(boiling) and the test tubes were immediately removed from the beaker to
For the boiling treatments test tubes remained in the beaker with water at
20
boiling temperature for a set duration after the addition of seeds Following all
treatments test tubes with seeds cooled 24 hours in a test tube rack
Percussion treatment utilized a pneumatic paint shaker (Central Pneumatic
model 00422 Camarillo CA) Compressed air was maintained at 530 kPa plusmn30 kPa
(80 psi plusmn 5 psi) resulting in approximately 350 oscillations per minute Seeds were
placed in 118 ml (4 oz) soil tin cans (US Can Lobard IL) and the lids were secured
with duct tape Each can was used to treat one 100-seed sample To take advantage
ofgreatest lateral movement of the paint shaker a spacer (paint can) was used to
position the soil tin at the end of the shaking arm This allowed shaking to occur at
the greatest distance from the pivot point (fulcrum) Tins were placed perpendicular
to the direction of shaking motion allowing seeds to impact at greatest force by
hitting flat surfaces of the soil tin
Germination Testing
Before gennination testing all seeds were soaked 24 hours in reverse
osmosis-treated water (this was included in the process ofhot water treatment
jU~SCIlOea above) Following soaking seeds were placed in 100 cm Petri dishes lined
90 cm Whatman qualitative 1 filter paper circles (MG Scientific Pleasant
WI) One Petri dish was used for each treatment by source combination
papers were initially moistened with 5 mL reverse osmosis-treated water then
~VU1CUlJl moistened as needed to ensure adequate conditions for gennination
were monitored daily (at 300 PM) for gennination for 14 days in accordance
~UIUlnJlVUa Seed Testing Association (ISTA) standards for Robinia species
21
(1STA 1999) Tests were carried out at room temperature (22degC plusmnOSoC)
Germination was classified as protrusion of the radicle from the embryo by at least
1mm as viewed with the naked eye
X-ray Radiography
On August 132001 three lOO-seed samples from the Sacramento seed source
were treated for 14 or 8-minute percussion durations The seeds were sent
immediately by overnight delivery to the Faxitron X-Ray Corporation in Wheeling
Illinois where Dr Bob Karrfalt ofthe USDA Forest Service oversaw their imaging
Radiographs were made at SX with a Faxitron Specimen Radiography System Model
MX-20
Scanning Electron Microscope Photography
On February 192002 a Hitachi S-3200N Variable Pressure Scanning
Electron Microscope (Mountain View CA) was used to examine strophiolar cells of
untreated seeds and seeds percussed for 4 minutes Seeds were cut longitudinally
igt their entire length using a razor blade splitting the strophiolar region roughly in
Cross sections of seed samples magnifying the strophiolar region were viewed
600X under the microscope
Analysis
Categorical Analysis of Variance was used to determine treatment source and
(Le time) main effects and interactions The response variable was total
~vlI This procedure is a Chi-square test of homogeneity using the natural log
of germinated to non-germinated seed for each treatment Maximumshy
22
likelihood analysis was used to calculate Chi-square test statistics with their observed
significance levels A single contrast was conducted to cempare treatment means
against the control Observed significance levels less than or equal to 005 were
considered significant Pairwise comparisons were conducted between non-control
treatment mean percents using a conservative alp~a value of 005 divided by the
number of comparisons (Bonferroni approach) and approximate pairwise z statistics
Categorical analyses were performed using Proc Catmod (SAS Institute 1989)
RESULTS
Scarification seed source block and all interactions between factors
influenced germination (Table 3) The main effects are reported in the presence of
these interactions because the overall robustness of treatments and seed sources are of
interest Background levels ofnaturally occurring variation in seed sources are
seen in control germination percentages in Figure 2 In particular time variability
is unlikely to be under the control of the producer Appendix B details experimental
data for source by treatment gennination percents for each of the four blocks
Averaged over all treatments germination by source ranged from 40 to
79 (Figure 2) Averaged over all sources (Figure 3) and for each source (Figure 4
5) percussion scarification durations of4 or 8 minutes yielded greatest germination
Averaged over all sources germination increased with increasing durations of
percussion then leveled off for the 4- and 8-minute treatments (Figure 3) Immersion
and 30-second boiling scarification maximized germination for the hot water
treatments with a decrease in germination when seeds were boiled for 60 seconds
For all but two sources there is a similar ipcreasing trend in germination from
1to 4 minutes ofpercussion (Figure 4) The Holman source increased in germination
1 to 2 minutes then leveled off at 4 minutes The Jemez source changed little
1 to 4 minutes of treatment As treatment duration increased from 4 to 8
mutes five sources decreased in germination (Figure 4a) whereas three sources
24
Table 3 Categorical Analysis of Variance table for germination response to seed treatment seed source block and the interaction of the factors
Source of DF Chi-Square Observed Variability Significance Level
Source 8 98370 lt00001 Trt 7 233876 lt00001
contrast 1 16976 lt00001 SourceTrt 56 97077 lt00001 Block 3 5538 lt00001
SourceBlock 24 10162 lt00001 TrtBlock 21 7564 lt00001
SourceTrtBlock 168 34283 lt00001
S 4)
8 ~
0
sectg ~ 40 ~
N 0 0
20
o f0ltgt ~ ~4 jybshy ((iltgto ~~ K1ltgt 6~b-~ ~~ 0b-~~ ~~ ~b-~ ~b-~b
80
60 -I-- - ~- middotmiddotmiddotmiddotmiddot1
r -] Northern NM Sources
Central NM Sources
Southern NM Sources
sect-o ~
~b-v(i
SOURCE
Figure 2 Effect ofNew Mexico locust seed source on germination for data averaged over all scarification treatments Control germination is shown by solid line within bar
80
~ cd ~ ltl) () 60 ~ ~
N
t 0shyrti tshySltl)
40
0
20
o lmmersion Boiling 30 sec Boiling 60 sec Control 1 min 2min 4 min 8 min
Hot Water Treatments Percussion Treatments
Figure 3 Effect of scarification on germination ofNew Mexico locust for data averaged over all seed sources Means labelled with the same letter are not significantly different
100~----------------------~--~----------------~
80
60
ltU
~
40 IJ
sectgsI
l100
80
60
40
a I middotmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot middotImiddotmiddotmiddotmiddotmiddotmiddot E
_~iii-bull _ _ bull f --~--
t-- -~ ---- -shy ---- shyI ~ ---- - shy
L
fiII
~
b
- shy ---r
- Holman
Sacramento Sandia Gila Jemez
c ~
ltgt ~
Tmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot-I
j~=~=-~~-1
I middotmiddot ~ bull r
t tr
Y -------M-a-gd-a-Ie-n-a--
1 _ _
fCapitan
---- Manzano ~y -_ Raton
2 4 8
Time interval ofpercussion scarification (min)
Figure 4 Effect of seed source by treatment interaction on New Mexico locust germination for percussion scarification pattern a and pattern b
28
tv 0
~ d
~ A s o
1 c3
80
60
40
20
o
TIT
t------=middotfmiddotmiddot-middotmiddot-middotmiddot-middotmiddot 1- --- - - --=- - -shy--cE= - _--shy --shy ----shy ~ ~ ---shy ~ -~- _ --shy-------r 1
I
Raton Holman
-_ Jemez
--- Sandia Manzano Magdalena Capitan Sacramento Gila
Immersion Boiling 30 sec Boiling 60 sec
Hot Water Scarification
Figure 5 Effect of seed source by treatment interaction on New Mexico locust germination for hot water scarification
continued to increase in germination (Figure 4b) There was no difference between
the two treatment levels for the Manzano source (Figure 4b)
The effect ofhot water scarification also varied among sources with few
overall differences between hot water treatments (Figure 5) However boiling for 60
seconds yielded highest germination for the Jemez source whereas increasing
durations ofboiling decreased germination for the Raton source
In addition to consistently high germination 4- and 8-minute percussionshy
treated seeds germinated as fast or faster than all other treatments (Figure 6 Table 4)
050 measured as the number of days needed to complete 50 ofeventual
germination averaged 2 days for both ofthese treatments
X-ray radiographs were taken of seeds collected from the Sacramento source
percussed for 1 4 or 8 minutes (Figure 7) Seeds show signs of damage at 8 minutes
ofpercussion (Figure 8) Compared to an undamaged seed percussed for only 4
minutes (Figure 8a) damage at the 8-minute level includes fractured radicles pitting
of the seed coat and chips in seed coat structure (Figure 8b-d)
Scanning electron microscope (SEM) images at 600X reveal the direct effect
of 4 minutes of percussion on cells of the strophiolar region (Figure 9) Whereas
untreated seeds retain cellular organization in the strophiolar region strophiolar cells
seeds treated for 4 minutes ofpercussion separate and raise resulting in a
~-~e~U appearance
30
Umiddot-~middot--l-~---middotymiddotmiddotmiddot-----middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot--middot--middotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middotmiddotmiddotmiddotmiddotmiddotmiddot- -
050= gt1
-e-- Percussion 8 min -0- Percussion 4 min ---T- Percussion 2 min -----ltil- Percussion 1 min -- Immersion --0- Boiling 30 sec -+- Boiling 60 sec -ltgt- Control
2 4 6 8 10 12
Day Figure 6 Timecourse ofNew Mexico locust gennination following scarification
treatments G50 is the number of days to 50 of eventual germination
31
14
Table 4 Effect of seed source and treatment on G50 (days to 50 germination)
Seed Source Control Immersion B30sec B60sec Plmin P2min P4min P8min mean Raton 3 3 5 7 4 3 2 2 4 Holman 2 2 2 2 2 2 2 2 2
Jemez 4 6 6 5 3 2 2 2 4 Sandia 2 2 2 2 2 2 2 2 2
w tv Manzano 5 2 2 3 3 2 2 2 3
Magdalena 4 2 2 3 4 3 2 1 3
Capitan 5 4 2 2 3 2 2 2 3
Sacramento 3 2 2 2 4 3 2 2 3 Gila 3 2 2 2 2 2 2 2 2 mean 3 3 3 3 3 2 2 2 3 standard 11 14 16 18 09 05 0 03 08 deviation
Note Columns and rows may not agree due to rounding
Figure 7 X-ray radiographs ofSacramento seed source percussed for 1 4 or 8 minutes
33
Figure 8a-d Seed damage close-ups Figure a shows undamaged seed percussed for 4 minutes Figures b-d show seeds percussed for 8 minutes Damage includes fracturing ofradicle in b complete removal ofseed coat in c and pitting ofseed coat in d
34
Untreated Treated
Figure 9 SEM images ofuntreated vs 4-minute percussion-treated strophiolar cells ofNew Mexico locust seeds Treated cells are loosened separated and raised
DISCUSSION
Determining the Optimal Treatment
Averaging results of scarification treatmems across seed sources is a useful
tool to predict which treatment is most likely to be successful for an unknown seed
source Averaged over all sources four- and eight-minute percussion treatments
resulted in the highest germination 815 and 790 respectively and produced
germination that was as fast or faster than all other treatments Although there was no
difference between these two treatments on average an argument for 4 minutes of
percussion as a standard protocol for treating New Mexico locust seeds can be made
Descriptively 4 minutes of percussion improved germination over 8 minutes
ofpercussion for a majority of sources (five out of nine) 8 minutes of percussion
improved germination for three ofnine sources with no difference for a fmal source
For individual sources 4 minutes ofpercussion was always the best or secondmiddot best
treatment and was always equal to or better than all hot water treatments
Germination at 8 minutes ofpercussion was lower than all other percussion durations
for the Jemez and Gila sources and was lower than at least one hot water treatment for
these sources as well
The x-ray radiographs of the Sacramento seed source illustrate the excessive
internal and external seed damage that can occur with 8 minutes of percussion
germination counts 8-minute percussed seeds from Holman and Capitan
ClUll signs of damage that were visible to the naked eye (see below)
r~evertheless some of these seeds still fit the criteria for germination Neither x-ray
36
photography nor visible observations during seed counts revealed seed damage at the
4-minute percussion level In operational trials seeds percussed for 4 minutes
developed into healthy seedlings (Khadduri and Harrington unpublished data)
Strophiole and Seed Coat Behavior
Four minutes ofpercussion successfully breaks seed dormancy in a large
percentage ofNew Mexico locust seeds without excessively damaging the
surrounding seed coat or internal seed coat structures Most papilionoid legume
seeds including seeds ofNew Mexico locust have a specific region of the seed coat
known as the strophiole or lens (Hamly 1932) The strophiole is located on the
cotyledonary lobe of a locust seed (Figure 10 a-c) If seeds are not indiscriminately
damaged the strophiole is the first point of water entry to the embryo (Hopkinson
1997) When a papilionoid seed is percussed for the appropriate amount of time
repeated hits on the integrated seed coat loosen the constrained cells of the strophiolar
region without excessively damaging the rest of the seed coat (Ballard 1976) SEM
photos illuStrate that 4 minutes of percussion loosened and separated strophiolar cells
ofNew Mexico locust seeds when compared with constrained organized strophiolar
cells of untreated seeds (Figure 9) Research-has shown that water passes to the
embryo through the treated strophiole in a controlled manner and regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues
(Kelly and van Staden 1987)
37
10 Strophiole ofunimbibed New Mexico locust seed (a) and following uptake (bc)
38
In contrast hot-water treatments can excessively degrade the seed coat
(Rebman et al 1998 Marunda 1990) Random cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rebman et aI 1998 Marunda 1990) Along with a
proportionately large amount ofun-imbibed (thus under-treated) seeds (Figure l1a)
damage was observed in hot-water treated seeds from six sources during germination
counts Damage included seeds where the radicle failed to elongate (Figure 11 b)
seeds where the radicle elongated but was delayed in freeing itself from the seed coat
(Figure 11c) and seeds where the radicle completely broke off from the embryo
(Figure lId) In addition some seeds germinated in reverse (the cotyledon emerged
fIrst) If the radicle emerged completely from the seed coat they were counted as
germinated
While nearly all seeds treated for 8 minutes ofpercussion imbibed water
damage was evident in x-ray evaluations (Sacramento seed source) and in
germination counts (Holman and Capitan) at this treatment level Seed damage
during germination counts was similar to levels found in hot-water treated seeds
suggesting that over-treatment by both hot water and longer durations of percussion
may result in similar damage to seed structure
The Sacramento and Holman seed sources were two of the fIve sources that
opcrplltlpn in germination from the 4- to 8-minute durations of percussion
the benefIts ofloosening the strophiole as evidenced by imbibition of
all 8-minute treated seeds were outweighed by external and internal seed coat
39
a
b c d
11 Damage to New Mexico locust seeds observed during germination cotmts following hot water scarification 9a Hot water replicate including ~t seedlings and numerous unimbibed seeds 9b Close-up of seed where raallCle failed to elongate 9c Close-up ofseed where radicle elongated but was
-gtIn in freeing itself from the seed coat 9d Close-up ofseed where radicle broke off from embryo
40
damage (which also may be contributing to high imbibition) For the three sources
for which 8 minutes of percussion resulted in maximum germination extended
percussion of seeds necessary to loosen the strophiole may not have excessively
damaged the seed anatomy The Manzano source did not significantly change in
germination from 4 to 8 minutes of percussion suggesting that an increase in
permeable seeds at the 8-minute treatment duration was balanced by the number of
seeds that failed to germinate due to seed damage To summarize the 4-minute
percussion treatment improved germination over 8 minutes ofpercussion for a
majority of sources (five ofnine) the 8-minute percussion treatment improved
germination over 4-minutes ofpercussion for three sources and there was no
difference with the Manzano source Notably for the Raton source 8 minutes of
percussion increased germination by 235 percentage points over 4-minutes of
percussion (7875 vs 5525 respectively) the next best treatment Raton was the
only source to result in germination less than 75 for 4 minutes ofpercussion so a
grower may wish to apply longer percussion durations to lots that do not respond well
to this treatment level
Finally there was an interesting trend in the Jemez and Raton sources seed
that significantly contributed to the source by treatment interaction (Figure 12) A
~u test showed that the Jemez source had a significantly lighter seed weight than
other sources while the Raton source had an average seed weight For the Jemez
the longest duration of boiling increased germination Conversely only short
U4UVll) of percussion were needed to achieve high germination and the longest
41
80 _ Jemez a Raton
iD
~ C iD e iD
Q c 0 ~
c ~ iD 0
20
0------shy
Immersion Boiling 30 sec Boiling 60 sec
Hot Water Scarification
10degTr========~--------~------------------------------
80
60
40
20
_ Jemez Raton
0------shy
1 min 2 min 4 min 8 min
Percussion Scarification
Figure 12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources
42
b
--~----~~~~========~---- duration ofpercussion reduced germination For the Raton source longer durations
ofboiling consistently decreased germination whereas longer percussion treatments
consistently increased germination
A possible explanation is that a thick seed coat is associated with a loose
strophiole structure and that a thin seed coat is associated with a constrained
strophiole This contrast merits further study and may suggest an ecological
adaptation Further investigation ofthis phenomenon may contribute to successful
pre-screening ofunknown seed sources to determine optimal treatment leveL
43
CONCLUSION
Based on results obtained in pilot study (see Appendix) and thesis
experiments 4 minutes ofpercussion is recommended as a standard protocol for
treating an unknown source ofNew Mexico locust seed In addition to laboratory
results the 4-minute percussion treatment has resulted in germination greater than
80 and healthy seedling grow-outs for production purposes (unpublished data) In
laboratory experiments only one hundred seeds per 118 ml (40z) soil tin were
percussed at a time whereas for production we have treated up to five hundred seeds
per tin For large-scale production ofNew Mexico locust seedlings growers may
wish to experiment with larger quantities of seed and larger-size soil tins
Seed storage trials following percussion treatments were not conducted
However germination greater than 80 and healthy seedlings were obtained when
seeds were percussion-treated and subsequently stored at room-temperature (22degC)
for one week (unpublished data) Seed storage trials merit further study as the
standard hot water treatment leaves seeds in a softened condition that is not
conducive to storage
Hydroseeding is an effective method for direct seeding in reclamation
situations Percussion treatment may be conducive to hydro seeding as seeds remain
a dry relatively undamaged yet permeable condition following treatment Sulfuric
seeds also remain dry following treatment but extensive damage to seed coat
poor seedling vigor have been reported with this treatment (Kelly and van Staden
A grow-out study using a hydro seeder on a reclamation site testing percussion
44
against other treatments such as sulfuric acid and mechanical scarification also
merits further study
Papilionoids are the largest subfamily oflegumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust these legumes often play an integral role in the revegetation of
disturbed lands The pilot study (Appendix) indicates that percussion scarification
successfully treats the widely used black locust as well Developing superior
scarification methods to the standard hot water mechanical scarification and acid
treatments may facilitate the use ofpapilionoid legumes in reclamation projects
45
APPENDICES
APPENDIX A
PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR
NEW MEXICO LOCUST AND BLACK LOCUST SEEDS
Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4
Hot water and sulfuric acid soaks are traditional treatments for seeds of many
temperate woody legwnes including locusts However these scarification techniques
often produce inconsistent germination Percussion scarification where seeds are
repeatedly propelled against a hard surface was compared with hot water
scarification to evaluate treatment efficacy for New Mexico locust (Robinia
neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment
seeds were placed in a 98degC water bath which was immediately removed from the
heat source For percussion scarification seeds were placed in a soil sample tin and
agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the
control were followed by 24-hour water soaks Hot water baths resulted in 56 and
41 germination for New Mexico locust and black locust respectively For both
species nearly all durations ofpercussion increased germination over the hot water
treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3
4 and 5 minutes for black locust resulted in at least 90 germination Traditional
scarification treatments randomly degrade the entire seed coat which can lead to
tissue damage during water uptake Percussion scarification specifically weakens the
strophiole the natural source ofwater entry to the seed in papilionoid legwnes
INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom
2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031
48
Following percussion imbibition is controlled through the strophiole and underlying
tissue is protected
Keywords New Mexico locust black locust scarification percussion germination
Introduction
Locust trees (Robinia spp) are aggressive pioneer species that quickly
colonize disturbed land fix atmospheric nitrogen in the soil and stabilize slopes that
are prone to erosion (Klemmedson 1994 Ashby et al 1985) (Figure A1ab)
Recognition of these attributes has contributed to increased demand for locusts in
reclamation projects Locust seeds however exhibit physical dormancy where a
thick seed coat prevents the movement of water and gasses to the embryo (Leadem
1997)
Sulfuric acid or hot water soaks have traditionally been used to break seed
dormancy in macro-propagation of locusts Many growers are moving away from the
use of su1furic acid in treating locust seeds (Dreesen and Harrington 1997) In
addition to being dangerous acid soak durations must be specifically correlated to
seed lot (Olson 1974) Collections of native seed can vary tremendously in seed size
seed weight and hard seededness etc This variability limits the utility of sulfuric
acid soaks as even within a given lot some seeds may be damaged whereas others
are under-treated and fail to imbibe (take up water) A survey of Southwestern
container growers indicates that hot water baths are currently the most common
scarification method for locust seeds (Hine et al 1997) However hot water baths
also produce inconsistent germination (Lin et al 1996)
49
b
Figure AI New Mexico locust a primary invader following disturbance Ala Crown-sprouting at edge ofburn near Los Alamos NM--one year after Cerro Grande fire Alb Colonizing road cut Gila National Forest NM
50
Percussion scarification where seeds are repeatedly propelled against a hard
surface is an alternative dormancy-breaking method that has proven successful in
legumes related to locusts (Hamly 1932 Barton 1947) We compared this alternative
treatment to hot water scarification to evaluate treatment efficacy for New Mexico
locust and black locust seeds
Materials and Methods
Commercial New Mexico locust seeds (Western Native Seed Coaldale CO)
were collected fall 2000 in Huerfano County CO Black locust seeds were collected
September 2000 from Taos County NM by harvesting ripe pods from several trees at
a distance of up to 3 m from ground level Black locust seeds were allowed to air-dry
for 3 weeks and were threshed and separated from large chaffusing a greenhouse
fan A South Dakota Seed Blower (Chicago Illinltois) was used to remove fine chaff
from both seed lots Seeds were storedmiddotat 2-4C until the germination study was
conducted in March 2001
This study compared hot water scarification with percussion scarification
using treatment and species as experimental factors Along with a control
scarification treatments included immersion of seed in boiling water as well as
percussion durations of 1 2 3 45 or 10 minutes Four 100-seed samples were used
to test each species by scarification treatment combination
For the hot water treatment test tubes were filled with 30 mL water and
placed in a water-filled beaker (Figure A2a) The bath was raised to boiling (-98C at
51
b
c
Figure A2 Implementation ofhot water and percussion scarification A2a Hot water bath A2b Paint-shaker set-up A2c Placing soil tin containing seeds at end ofpaint shaker ann
52
Las Cruces NM elevation of 1300 meters) Each 100-seed replicate was placed in a
test tube and each test tube was immediately removed to cool at room temperature
Percussion scarification was implemented using a pneumatic paint shaker
(Central Pneumatic model 00422 Camarillo CA) (Figure A2b) This allowed
standardization of treatment intensity Compressed air pressure was maintained at
530kPa plusmn 30kPa (80psi plusmn 5psi) resulting in approximately 350 oscillations per
minute Each lOO-seed sample was placed in a 118 ml (4 oz) soil tin for shaking To
maximize lateral movement of paint shaker a paint can was used as a spacer to place
soil tins at the end of the shaking arm (Figure A2c) This allowed shaking to occur at
the greatest distance from the pivot point (fulcrum) Following all treatments
including the control seeds were soaked for twenty-four hours in distilled water prior
to plating out
Gennination was evaluated on lab benches at room temperature Each
replicate of seeds was placed in a 100 cm petri dish on moistened filter paper with
humidity maintained by enclosing the petri dishes in ziplock bags Gennination was
monitored daily for 14 days in accordance with International Seed Testing
Association standards for Robinia species (ISTA 1999) Gennination was defined as
protrusion of the radicle from the embryo by at least 1mm as viewed by the naked
eye
Categorical Analysis of Variance using Prpc Catmod (SAS Institute 1989)
was used to detennine treatment differences in germination percentages for each
source This procedure is an extension of a Chi-square test of homogeneity using the
53
natural log of the ratio of germinated to non-germinated seeds for each treatment Pshy
values less than 005 were considered significant A limited set of pairwiseshy
comparisons was conducted to compare treatment mean percents using a
conservative alpha value of 005 divided by the number of comparisons
Results
Percussion maximized germination for both New Mexico locust and black
locust There were three germination response levels for New Mexico locust (Figure
A3) First there was a low response in the control indicating a dormant seed lot
The hot water treatment and I-minute duration of percussion share a second
intermediate response Germination was highest at percussion levels from 2 to 10
minutes
A selected percussion treatment intermediate in time (5 minutes) produced the
steepest germination curve indicating the fastest germination rate (Figure A4) The
hot water treatment produced an intermediate curve or relatively slower germination
rate The control curve was very flat indicating the slowest germination rate for New
Mexico locust
Control seeds germinated poorly for black locust as well (Figure A5) Hot
water scarification improved germination over the control but all levels of percussion
improved germination over hot water The optimal durations of percussion treatment
were bracketed with a significant increase from the 1 to 2 minute percussion level a
high germination response from 2 to 5 minutes of percussion and finally a significant
54
~ d 25 CL)
p t1 0 ~ t1
g Vl ~ Vl
100~---------------------------------------------------------------
80
60
40
20
o Control Hot Water Percussion
I min Percussion
2 min Percussion
3 min
Scarification
Percussion 4 min
Figure A3 Effect of scarification on germination response for New Mexico locust
Percussion 5 min
=significant difference between adjacent treatment bars ns = no significant difference
Percussion 10 min
~ ~ () u agt
Il-t d o ~
~ ~ c3
100middotlr~~~------------------------------------e- Percussion 5min middot 0middot Hot Water -- Control
80-J=middot======
6b-middot-lmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middot--middotmiddot-middot~-middotmiddot -middotmiddot middot middotmiddot-middot 1
40
20
-----~-----------
0 0 0 middotmiddotmiddotmiddotmiddot0middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot0
0 --~-----y-----~------_---~--- T-----~
o shy -TDay 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A4 Effect of selected scarification treatments on germination speed for New Mexico locust First seven days of fourteen-day experiment are shown
Day 7
80
~ cd
5 60 ()
[) Cl d 0 1t1 d
40sect dUl
-J
20
o
ns
Scarification
Figure A5 Effect of scarification on germination response for black locust = significant difference between adjacent treatment bars ns = no significant difference
decrease at the 10-minute level This drop-off suggests that 10 minutes of percussion
over-treats seeds from this lot of-black locust
A selected percussion treatment intermediate in time (5 minutes) also had the
fastest germination rate for black locust (Figure A6)_ The hot water curve is
somewhat flatter indicating a slower germination rate The control curve is very flat
indicating a slow germination rate
A qualitative difference between hot water and percussion-treated seeds was
also observed Along with healthy germinants the hot water treatment produced
over-treated seeds (Figure A 7a) Examples of damaged germinants include seeds
where the radicle did not elongate (Figure A 7b) seeds where the radicle elongated
but was delayed in freeing itself from the seed coat (Figure A7c) and seeds where the
radicle broke off completely from the embryo (Figure A7d) In addition to these
examples of over-treatment there were numerous small dark seeds that failed to
imbibe water For percussion treatment nearly all seeds took up water (Figure A8a)
Germination was rapid and uniform with little damage evident (Figure A8b)
To summarize both New Mexico locust and black locust seed lots were
dormant The standard hot water scarification treatment improved germination but
only to 56 and 41 for New Mexico locust and black locust respectively Nearly
all percussion durations improved total germination as well as germination rate over
the hot water treatment Germination was greater than 90 for percussion durations
of 4 5 and 10 minutes for New Mexico locust and 3 4 and 5 minutes for black locust
58
CI)
sgt t CI) 0
igt 0
sect ttl t E
Vl 0
c3
100r==~==~~=r------------------------------------~ ___ Percussion 5 min
middotmiddotmiddot0middotmiddotmiddot Hot Water -lIf- Control
80r=========~--~~----------------------------~
60
40
20 -~-~---~---------- ---~------------I
omiddot
O
_~_----------lIf-----~bull -----_ shy---shy I
Day 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A6 Effect of selected scarification treatments on germination speed for black locust First seven days of fourteen-day experiment are shown
Day 7
a
b c d
Figure A7 Damage to New Mexico locust seeds observed during germination counts following hot water scarification A7a Hot water replicate including damaged seedlings and numerous unimbibed seeds A7b Close-up ofseed where radicle failed to elongate A7c Close-up ofseed where radicle elongated but was delayed in freeing itself from the seed coat A7d Close-up ofseed where radicle completely broke offfrom embryo
60
a
b
Figure A8 New Mexico locust seeds following percussion treatment at time of germination counts A8a Percussion replicate with uniformly healthy seedlings and near complete imbibition of seeds A8b Close-up ofpercussion-treated seedling
61
Discussion
Papilionoid seeds have a specific anatomical feature known as the strophiole
or lens-the natural site of water entry to the seed (Hamly 1932) The strophiole is
located on the cotyledonary lobe of a locust seed (Figure A9) When a papilionoid
legume seed is percussed for the appropriate amount of time repeated hits on the
integrated seed coat loosen the constrained cells of the strophiolar region without
excessively damaging the rest of the seed coat (Ballard 1976)
When a percussed seed is soaked water enters exclusively through the
strophiole in a controlled manner (Kelly and Staden 1987) This regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues This
contrasts with seeds that have been hot water or acid scarified treatments that can
randomly degrade the seed coat Unlocalized cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rehman et al 1998 Marunda 1990)
Papilionoids are the largest subfamily of legumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust and black locust these legumes often play an integral role in the
revegetation ofdisturbed lands Developing superior scarification methods to the
standard hot water and acid treatments should fadlitate the use ofthese legumes in
restoration projects
62
Figure A9 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (bc)
63
APPENDIXB
GERMINATION PERCENTS AND STANDARD ERRORS FOR SOURCE
BLOCK AND TREATMENT FOR NEW MEXICO LOCUST
Table B 1 Gennination percents and standard errors for source block and treatment for New Mexico locust
Source Block Treatment Germination Percentage Standard Error
Capitan 1 Immersion 74 437 Capitan 2 Immersion 69 462 Capitan 3 Immersion 71 454 Capitan 4 Immersion 67 470 Capitan Boiling 30 sec 79 407 Capitan 2 Boiling 30 sec 76 427 Capitan 3 Boiling 30 sec 72 449 Capitan 4 Boiling 30 sec 76 427 Capitan Boiling 60 sec 71 454 Capitan 2 Boiling 60 sec 54 498 Capitan 3 Boiling 60 sec 67 470 Capitan 4 Boiling 60 sec 76 427 Capitan I Control 5 218 Capitan 2 Control 7 255 Capitan 3 Control 4 196 Capitan 4 Control 5 218 Capitan 1 Percussion I min 47 499 Capitan 2 Percussion I min
470
Capitan Percussion I min 39 488
Capitan 4 Percussion I min 38 485 Capitan I Percussion 2 min 55 497 Capitan Capitan
2
Percussion 2 min Percussion 2 min
71 65
454 477
Capitan 4 Percussion 2 min 56 496 Capitan I Percussion 4 min 86 347 Capitan 2 Percussion 4 min 79 407 Capitan 3 Percussion 4 min 81 392 Capitan 4 Percussion 4 min 82 384 Capitan I Percussion 8 min 90 300 Capitan 2 Percussion 8 min 88 325 Capitan 3 Percussion 8 min 90 300 Capitan 4 Percussion 8 min 90 300 Gila I Immersion 71 454 Gila 2 Immersion 77 421 Gila
Immersion 75 433
Gila 4 Immersion 72 449
Gila I Boiling 30 sec 68 466
Gila 2 Boiling 30 sec 81 392
Gila 3 Boiling 30 sec 54 498
Gila 4 Boiling 30 sec 63 483
Gila I Boiling 60 sec 55 497
Gila 2 Boiling 60 sec 68 466
Gila 3 Boiling 60 sec 57 495
Gila 4 Boiling 60 sec 70 458
65
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Gila 2 Control 13 336 Gila 3 Control 13 336 Gila 4 Control 20 400 Gila 1 Percussion 1 min 66 474 Gila 2 Percussion 1 min 57 495 Gila 3 Percussion 1 min 73 444 Gila 4 Percussion 1 min 82 384 Gila 1 Percussion 2 min 81 392 Gila 2 Percussion 2 min 86 347 Gila 3 Percussion 2 min 85 357 Gila 4 Percussion 2 min 54 498 Gila 1 Percussion 4 min 79 407 Gila 2 Percussion 4 min 83 376 Gila 3 Percussion 4 min 84 367 Gila 4 Percussion 4 min 79 407 Gila 1 Percussion 8 min 51 500 Gila 2 Percussion 8 min 76 427 Gila 3 Percussion 8 min 52 500 Gila 4 Percussion 8 min 74 439 Holman 1 Immersion 83 376 Holman 2 Immersion 86 347 Holman 3 Immersion 81 392 Holman 4 Immersion 90 300 Holman 1 Boiling 30 sec 90 300 Holman 2 Boiling 30 sec 85 357 Holman 3 Boiling 30 sec 87 336 Holman 4 Boiling 30 sec 83 376 Holman 1 Boiling 60 sec 79 407 Holman 2 Boiling 60 sec 80 400 Holman 3 Boiling 60 sec 86 347 Holman 4 Boiling 60 sec 93 255 Holman Control 25 433 Holman 2 Control 19 392 Holman Control 21 407
Holman 4 Control 24 427
Holman Percussion 1 min 77 421
Holman 2 Percussion 1 min 85 357
Holman 3 Percussion 1 min 80 400
Holman 4 Percussion 1 min 89 313
Holman 1 Percussion 2 min 88 325
Holman 2 Percussion 2 min 96 196
Holman 3 Percussion 2 min 93 255
Holman 4 Percussion 2 min 96 196
Holman 1 Percussion 4 min 92 271
Holman 2 Percussion 4 min 97 171
66
I
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Holman 3 Percussion 4 min 91 286 Holman 4 Percussion 4 min 96 196 Holman 1 Percussion 8 min 88 325 Holman 2 Percussion 8 min 85 357 Holman 3 Percussion 8 min 83 376 Holman 4 Percussion 8 min 82 384 Jemez 1 Immersion 53 499 Jemez 2 Immersion 44 496 Jemez 3 Immersion 52 500 Jemez 4 Immersion 70 458 Jemez 1 Boiling 30 sec 58 494 Jemez 2 Boiling 30 sec 55 497 Jemez 3 Boiling 30 sec 48 500 Jemez 4 Boiling 30 sec 65 477 Jemez 1 Boiling 60 sec 68 466 Jemez 2 Boiling 60 sec 63 483 Jemez 3 Boiling 60 sec 73 444 Jemez 4 Boiling 60 sec 89 313 Jemez 1 Control 2 140 Jemez 2 Control 9 286 Jemez 3 Control 6 237 Jemez 4 Control 8 271 Jemez 1 Percussion 1 min 58 494 Jemez 2 Percussion 1 min 63 483 Jemez 3 Percussion 1 min 77 421 Jemez 4 Percussion 1 min 89 313 Jemez 1 Percussion 2 min 68 466 Jemez 2 Percussion 2 min 64 480 Jemez 3 Percussion 2 min 80 400 Jemez 4 Percussion 2 min 83 376 Jemez Percussion 4 min 68 466 Jemez 2 Percussion 4 min 79 407 Jemez 3 Percussion 4 min 70 458 Jemez 4 Percussion 4 min 83 376 Jemez 1 Percussion 8 min 52 500 Jemez 2 Percussion 8 min 59 492 Jemez 3 Percussion 8 min 61 488 Jemez 4 Percussion 8 min 70 458 Magdalena 1 Immersion 69 462 Magdalena 2 Immersion 69 462 Magdalena J Immersion 70 458 Magdalena 4 Immersion 85 357 Magdalena 1 Boiling 30 sec 65 477 Magdalena 2 Boiling 30 sec 71 454 Magdalena 3 Boiling 30 sec 68 466
67
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Magdalena 4 Boiling 30 sec 83 376 Magdalena 1 Boiling 60 sec 63 483 Magdalena 2 Boiling 60 sec 68 466 Magdalena 3 Boiling 60 sec 63 483 Magdalena 4 Boiling 60 sec 82 384 Magdalena 1 Control 11 313 Magdalena 2 Control 6 237 Magdalena 3 Control 5 218 Magdalena 4 Control 4 196 Magdalena Percussion 1 min 32 466 Magdalena 2 Percussion 1 min 45 497 Magdalena 3 Percussion 1 min 46 498 Magdalena 4 Percussion I min 36 480 Magdalena 1 Percussion 2 min 67 470 Magdalena 2 Percussion 2 min 68 466 Magdalena 3 Percussion 2 min 65 477 Magdalena 4 Percussion 2 min 81 392 Magdalena 1 Percussion 4 min 87 336 Magdalena 2 Percussion 4 min 97 171 Magdalena 3 Percussion 4 min 83 376 Magdalena 4 Percussion 4 min 97 171 Magdalena 1 Percussion 8 min 98 lAO Magdalena 2 Percussion 8 min 100 000 Magdalena 3 Percussion 8 min 96 196 Magdalena 4 Percussion 8 min 98 lAO Manzano 1 Immersion 67 470 Manzano 2 Immersion 57 495 Manzano 3 Immersion 72 4049 Manzano 4 Immersion 73 4044 Manzano 1 Boiling 30 sec 70 458 Manzano 2 Boiling 30 sec 64 480 Manzano 3 Boiling 30 sec 71 454 Manzano 4 Boiling 30 sec 80 400 Manzano I Boiling 60 sec 50 500 Manzano 2 Boiling 60 sec 53 499 Manzano 3 Boiling 60 sec 59 492 Manzano 4 Boiling 60 sec 74 439 Manzano I Control 4 196 Manzano 2 Control 8 271 Manzano 3 Control 1 099 Manzano 4 Control 6 237 Manzano 1 Percussion 1 min 52 500 Manzano 2 Percussion 1 min 39 488 Manzano 3 Percussion 1 min 36 480 Manzano 4 Percussion 1 min 51 500
68
Table B 1 Continued
Source Block Treatment Gerrni~ation Percentage Standard Error
Manzano Percussion 2 min 53 499 Manzano 2 Percussion 2 min 58 494 Manzano 3 Percussion 2 min 61 488 Manzano 4 Percussion 2 min 62 485 Manzano 1 Percussion 4 min 76 427 Manzano 2 Percussion 4 min 85 357 Manzano 3 Percussion 4 min 79 407 Manzano 4 Percussion 4 min 83 376 Manzano 1 Percussion 8 min 87 336 Manzano 2 Percussion 8 min 83 376 Manzano 3 Percussion 8 min 83 376 Manzano 4 Percussion 8 min 75 433 Raton 1 Immersion 54 498 Raton 2 Immersion 37 483 Raton 3 Immersion 39 488 Raton 4 Immersion 69 462 Raton 1 Boiling 30 sec 36 480 Raton 2 Boiling 30 sec 20 400 Raton 3 Boiling 30 sec 28 449 Raton 4 Boiling 30 sec 60 490 Raton 1 Boiling 60 sec 15 357 Raton 2 Boiling 60 sec 171 Raton 3 Boiling 60 sec 4 196 Raton 4 Boiling 60 sec 35 477 Raton 1 Control 4 196 Raton 2 Control 12 325 Raton 3 Control 2 140 Raton 4 Control 6 237 Raton 1 Percussion 1 min 52 500 Raton 2 Percussion I min 29 454 Raton 3 Percussion I min 35 477 Raton 4 Percussion 1 min 31 462 Raton 1 Percussion 2 min 55 497 Raton 2 Percussion 2 min 35 477 Raton 3 Percussion 2 min 33 470 Raton 4 Percussion 2 min 42 494 Raton 1 Percussion 4 min 65 477 Raton 2 Percussion 4 min 52 500 Raton Percussion 4 min 51 500 Raton 4 Percussion 4 min 53 499 Raton 1 Percussion 8 min 88 325 Raton 2 Percussion 8 min 80 400 Raton 3 Percussion 8 min 73 444 Raton 4 Percussion 8 min 74 439 Sacramento 1 Immersion 43 495
69
Table B 1 Continued
-Source Block Treatment Germination Percentage Standard Error
Sacramento 2 Immersion 30 458 Sacramento 3 Immersion 38 485 Sacramento 4 Immersion 56 496 Sacramento 1 Boiling 30 sec 47 499 Sacramento 2 Boiling 30 sec 48 500 Sacramento 3 Boiling 30 sec 48 500 Sacramento 4 Boiling 30 sec 56 496 Sacramento 1 Boiling 60 sec 52 500 Sacramento 2 Boiling 60 sec 67 470 Sacramento 3 Boiling 60 sec 43 495 Sacramento 4 Boiling 60 sec 48 500 Sacramento 1 Control 0 000 Sacramento 2 Control 3 171 Sacramento 3 Control 3 171 Sacramento 4 Control 099 Sacramento 1 Percussion 1 min 57 495 Sacramento 2 Percussion 1 min 61 488 Sacramento 3 Percussion 1 min 62 485 Sacramento 4 Percussion 1 min 55 497 Sacramento 1 Percussion 2 min 84 367 Sacramento 2 Percussion 2 min 70 458 Sacramento 3 Percussion 2 min 81 392 Sacramento 4 Percussion 2 min 69 462 Sacramento 1 Percussion 4 min 91 286 Sacramento 2 Percussion 4 min 84 367 Sacramento 3 Percussion 4 min 84 367 Sacramento 4 Percussion 4 min 94 237 Sacramento 1 Percussion 8 min 82 384 Sacramento 2 Percussion 8 min 81 392 Sacramento 3 Percussion 8 min 82 384 Sacramento 4 Percussion 8 min 81 392 Sandia 1 Immersion 76 427 Sandia 2 Immersion 70 458 Sandia 3 Immersion 70 458 Sandia 4 Immersion 81 392 Sandia 1 Boiling 30 sec 78 414 Sandia 2 Boiling 30 sec 71 454 Sandia 3 Boiling 30 sec 63 483 Sandia 4 Boiling 30 sec 78 414 Sandia 1 Boiling 60 sec 67 470 Sandia 2 Boiling 60 sec 65 477 Sandia 3 Boiling 60 sec 74 439 Sandia 4 Boiling 60 sec 66 474 Sandia 1 Control 16 367 Sandia 2 Control 7 255
70
Table B 1 Continued
Source Block Treatment Gennination Percentage Standard Error
Sandia 3 Control 8 271 Sandia 4 Control 16 367 Sandia 1 Percussion 1 min 67 470 Sandia 2 Percussion 1 min 61 488 Sandia 3 Percussion 1 min 66 474 Sandia 4 Percussion 1 min 59 492 Sandia I Percussion 2 min 82 384 Sandia 2 Percussion 2 min 83 376 Sandia 3 Percussion 2 min 81 392 Sandia 4 Percussion 2 min 84 367 Sandia 1 Percussion 4 min 89 313 Sandia 2 Percussion 4 min 86 347 Sandia 3 Percussion 4 min 89 313 Sandia 4 Percussion 4 min 85 357 Sandia 1 Percussion 8 min 80 400 Sandia 2 Percussion 8 min 77 421 Sandia 3 Percussion 8 min 69 462 Sandia 4 Percussion 8 min 66 474
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SerratoValenti G Conara L Ferrando M Modenesi P 1993 Structural and histochemical features ofStylosanthes scabra (Leguminosae Papilionoidae) seed coat as related to water entry Can J Bot 71 834-840
Simpson BJ 1988 A field guide to Texas trees Austin TX Texas Monthly Press 372p
Tran VN Cavanagh AK 1984 Structural aspects ofdormancy In Seed Physiology Vol 2 Murray DR (ed) p1-44 Academic Press Australia
USDA Forest Service 1948 Woody-plant seed manual USDA Misc Pub1654 416p
Vines RA 1960 Trees Shrubs and Woody Vines of the Southwest Austin Univ ofTexas Press 1104 p
Wagner MR Larson RE Craig TP 1992 Ecology and silvics ofNew Mexico locust (Robinia neomexicana) with an emphasis on the arboreal form p53-57 In JW Hanover K Miller and S Plesko (eds) Proc Intemat Confon Black Locust Biology Culture and Utilization Mich State Univ USA
Wilson JK 1944 Immersing seeds of species of Robinia in boiling water hastens germination J For 42 453-454
Zimmerman RW Carpenter SB 1980 First-year coppice production from a 5shyyear old black locust stand on surface mine spoil p309-314 Centr Hardwd For ConfProc University ofMissouri Columbia
A4 Effect of selected scarification treatments on germination speed for New Mexico locust 56
AS Effect of scarification on germination response for black locust 57
A6 Effect of selected scarification treatments on germination speed for black locust 59
A7 Damage to New Mexico locust seeds observed during germination counts following hot water scarification 60
AS New Mexico locust seeds following percussion treatment at time of germination counts 61
A9 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (bc) 63
INTRODUCTION
New Mexico locust (Robinia neomexicana A Gray) a small tree native to the
southwestern United States occurs at elevations from 1200 to 2800 meters This
species fills a successional role in post-disturbance situations As a primary invader
New Mexico locust quickly establishes on burned areas arid flood banks as well as
road cuts (Wagner et aI 1992) Rapid growth crown sprouting and prolific root
suckering favor the successful establishment ofNew Mexico locust on disturbed sites
(Simpson 1988 Gottfried 1980) These attributes also may make New Mexico locust
well suited for steep-slope revegetation where erosion is a problem
New Mexico locust is a nitrogen-fixing legume that tolerates and improves
nutrient-poor soils Stands ofNew Mexico locust increase Nitrogen (N) and Calcium
(Ca) in the forest floor Levels of Carbon (C) Phosphorous (P) Sulfur (8) and
Potassium (K) aIso have been shown to increase in soil beneath New Mexico locust
(Klemmedson 1994)
The ability to colonize rapidly and ameliorate harsh sites contributes to New
Mexico locusts potential as a nurse plant A nurse plant colonizes an inhospitable
site and creates an environment suitable for successional plant establishment New
Mexico locust demonstrates this role in the ponderosa pine (Pinus ponderosa Douglas
ex Lawson amp C Lawson)Gambel oak (Quercus gambelii Nutt) community After a
disturbance such as wildfire New Mexico locust and Gambel oak colonize and
dominate the site until shaded out by ponderosa pine Once shaded these species
become understory shrubs a process taking an average of 15-20 years (Dick-Peddie
1993 Hanks and Dick-Peddie 1974) New Mexico locust gradually declines with
small dense patches averaging about 005 ha beneath pine until the next disturbance
once again offers it a competitive advantage (Klemmedson 1994)
Gottfried (1980) expecting ponderosa pine regeneration to be greater where
New Mexico locust had been eradicated found that survival of planted pine seedlings
was greater where locust had not been removed In a follow-up study Gottfried
(1980) noted that soil moisture in the top 57 cm was highest in 5-year old locust sites
as compared to grass or 20-year old locust sites He concluded that managing an
appropriate cover ofNew Mexico locust could help regenerate pine the later
successional species
The ability ofNew Mexico locust to improve harsh sites also makes it a
candidate for reclaiming disturbances associated with mining Black locust (Robinia
pseudoacacia 1) has been used for decades to reclaim mine spoils and other
disturbed sites in the eastern US and around the world (Keresztesi 1988 Zimmerman
and Carpenter 1980) Ashby et al (1985) described similar positive attributes of
black locust also mentioned previously for New Mexico locust quick cover for
stabilization supply ofN and nutrient-rich litter to the soil and site improvement for
establishment of later successional trees Referring specifically to mine reclamation
the authors note the ability of black locust to grow on a wide range ofmine soil
conditions including extremely acid soils Black locust also shows some tolerance to
soils compacted by grading and topsoiling practices (Ashby et aL 1985) New
2
Mexico locust holds promise as a native southwe~tem counterpart to black locust in
mine reclamation but it has been used infrequently to date
Natural invasion and succession occur slowly on most mine sites (Monsen
1984) While New Mexico locust often colonizes sites naturally there is no
assurance a seed will reach a particular site and establish in a reasonable time frame
The challenge is to introduce New Mexico locust as mother plants to facilitate
colonization Seedling production for outplanting on disturbed sites has been
hampered by poor germination (Lin et al 1996) The goal then is to develop
techniques to overcome poor germination in New Mexico locust thereby facilitating
macropropagation
LITERATURE REVIEW
Seed Dormancy
Low gennination in New Mexico locust is primarily the result of seed
donnancy the failure of an intact viable seed to genninate under favorable
conditions (Bewley 1997) Seed donnancy also can be defined as the inability of the
embryo to genninate because of some inherent constraint (Bewley and Black 1994)
The constraint may originate within the embryo referred to as endogenous or embryo
donnancy or from the tissues surrounding the embryo known as exogenous or coatshy
imposed donnancy (Baskin and Baskin 1998 Leadem 1997 Bewley and Black
1994)
New Mexico locust seed is donnant when the impenneable seed coat does not
allow movement ofwater to the embryo This specific fonn of coat-imposed
donnancy where the restraining tissue involved is an impenneable seed coat is
known as physical donnancy (Baskin and Baskin 1998 Bewley and Black 1994)
Physical donnancy is relatively uncommon in seeds less than 10 of all woody
species exhibit this condition (USDA Forest Service 1948) However it is
characteristic of many genera of the Fabaceae (bean) family For example all
successional trees in the genera Gleditsia and Robinia (of which New Mexico locust
is a member) produce seeds that exhibit physical donnancy (Baskin and Baskin
4
Physical Dormancy
Seeds that exhibit physical dormancy are said to be hard seeded referring to
the thick impermeable seed coat (Leadem 1997) The first line of protection of the
testa in leguminous seeds is the outermost waxy cuticle followed by a subcuticle
consisting ofsuberin Next is a palisade layer consisting of sclereid cells that have
thick lignified secondary walls The most common type of sclereid cell in palisade
layers is the macrosclereid The macrosclereids contain water-repellent substances
including cutin lignin suberin and wax (Rolston 1978) This palisade layer is the
primary barrier to water uptake (Serrato-Valenti et al 1993 Tran and Cavanagh
1984)
All seed coat openings must be blocked to effectively prevent water uptake
The natural opening in a seed where it was attached to the mother plant is the weak
link where physical dormancy is usually broken (Hopkinson 1993 Kuo and Tam
1988) Anatomical structures associated with the seed coat opening vary from
family to family In the Papilionoideae the subfamily of the Fabaceae family
containing New Mexico locust the site ofwater entry is the strophiole The
strophiole is a swelling on the seed coat close to the stalk scar (Hopkinson 1997)
Kelly and van Staden (1987) refer to the strophiole site as the lens where
macrosclereid cells must separate and raise before water can pass through Since
middot6germination ofdormant seeds of the Papilionoideae takes place when the strophiole
permeable (Hopkinson 1993 Kuo and Tam 1988) a logical approach to
5
genninating New Mexico locust artificially would be to loosen the strophiolar cells
and allow water to pass through to the embryo
Evolutionary Role of Physical Dormancy
Why is physical dormancy a mechanism that blocks germination
ecologically advantageous When a plant produc~s seeds with different degrees of
dormancy seeds that are dispersed at one time will germinate over a range of time
(Bewley and Black 1994) Through variability of dormancy within a seed crop seeds
are exposed to changing environmental conditions improving the overall chances of
survival of the seed line (Bewley and Black 1994) Varying expression of dormancy
is under genetic control which indicates that this may be an evolved survival strategy
(Kigel and Galili 1995) A range of dormancy levels may be a particular advantage in
the southwestern US where extreme environmental variations exist
Methods for Breaking Dormancy
There are three common approaches to breaking dormancy in New Mexico
locust seed acid scarification hot water soaks and mechanical treatments In each
case the objective of the treatment is to scarify or abrade the seed coat to make it
permeable to water
Acid Scarification
Acid scarification consists of soaking seeds in concentrated sulfuric acid
rinsing thoroughly with water upon removal followed by subsequent drying before
PlaJlltmg Olson (1974) reports that this scarification method produces the highest
middot~~jllalUll results for all Robinia species Most literature refers to a seminal 1937
6
study conducted by HG Meginnis This stud~ t~ough conducted on black locust
seed is regularly cited in the suggested use of acid to treat New Mexico locust seed
as well (Olson 1974 Vines 1960)
Thee published studies document the specific use of sulfuric acid to treat
New Mexico locust seed Cox and Klett (1984) report that soaking seeds in
concentrated sulfuric acid for thirty minutes improved germination to 34 over an
untreated control of4 Lin et al (1996) found that for two seed lots acid
scarification for twenty-five to eighty minutes produced germination of 80 A third
lot however resulted in less than 20 germination at all durations of 10 to 80
minutes Hine et al (1997) showed that acid treatment levels of 1 2 5 and 10
minutes resulted in less than 20 germination across five sources However there
was an increasing trend in germination with acid soak duration and acid treatments
were not imposed for as long a time as the previous two studies (30 to 80 minutes)
The mixed results obtained by these investigators were probably due to the
widely varying germination response of seed lots to acid treatment For this reason
Olson (1974) recommends correlating duration of acid treatment to each seed lot If
treatment duration is too long the embryo will be permanently damaged If soak
duration is too short the seed will remain impermeable to water and fail to germinate
Compounding the problem of treatment duration is the negative effect acid
have on seedling growth Any type of scarification can injure seeds to some
by disrupting cells essential for growth leading to fungal invasion and
middot-uuu~w injury (Copeland and MacDonald 1995) Some legume seeds subjected
7
to 60 minutes of sulfuric acid scarification (to achieve maximum gennination results)
showed more than 50 seedling mortality after gennination (Kelly and van Staden
1987) Bewley and Black (1994) noted that fungal growth on seeds is an indication
of leakage of sugars organic acids amino acids and proteins
Perhaps the greatest drawback to acid scarification is the dangerous nature of
the treatment itself Difficulties associated with storage and trained use of
concentrated acid may discourage many propagators from using this option (Dreesen
and Harrington 1997) Consequently hot water and to a lesser extent mechanical
scarification are more commonly used and practical treatments for legume seeds
Most southwestern container growers use either of these two treatments to scarify
New Mexico locust seed in particular (Hine et al 1997)
Hot Water Scarification
The widespread use ofhot water scarification to treat locust seed is probably
due to its ease of application and efficacy in comparison to other scarification
treatments Although this practice had been in use for some time Wilson (1944) was
one of the first to report on its systematic application for black locust The treatment
consists of placing seeds in boiling water (4 parts water tomiddot 1 part seed by volume)
removing the vessel from the heat source and allowing the water to cool and seeds to
soak for 24 hours
Hine et al (1997) Lin et al (1996) and Cox and Klett (1984) have examined
the specific application ofhot water scarification on New Mexico locust seed Each
these authors compared hot water to acid scarification Lin et aL (1996) reported
8
60 gennination for two hot water-treated seed lots while a third lot germinated
around 50 (as reported above acid scarification germination ranged from 20 to
80 for these lots) It should be noted that the authors only soaked the seeds in water
from 10 to 80 minutes for this experiment as opposed to the standard 24 hours Cox
and Klett (1984) stated that using the standard 24-hour hot water treatment improved
germination over an acid pre-treatment (which produced less than 20 germination)
but they did not give specific gennination numbers for the hot water treatment Hine
et al (1997) found that across five seed lots germination was 66 for the standard
hot water treatment (as compared to less than 20 germination for acid scarification)
Variations in treatment intensity and duration and their subsequent effects on seed lot
appear to contribute to conflicting conclusions as to whether acid scarification or hot
water treatment is a more successful treatment
Given mixed results ease ofapplication is an important argument for use of
hot water application over acid soaks Safety concerns and material costs for hot
water treatments are low (Dreesen and Harrington 1997) An extension of this is the
practicality ofhot water for treating large batches of seed where a relative increase in
water volume is all that is needed However increases in water and seed even when
inthe same 4 1 proportion can result in dramatically different rates of cooling than in
atest tube experiment Different cooling rates can lead to contrasting results for the
treatment temperature duration and seed lot (USDA Forest Service 1948)
As with acid scarification parameters ofhot water treatment application may
to be optimized to maximize germination Hine et al (1997) found that as
9
temperature was increased from 70degC to 100degC overall germination in New Mexico
locust seed increased The 100degC treatment maximized germination for each of five
sources tested with germination usually above 60
Prolonged exposure of seeds ofmany woody legumes to boiling temperatures
also may improve germination (Briscoe 1996) This approach has not been tested on
New Mexico locust seed Exposing seed to prolonged boiling temperatures may
result in fungal infection and subsequent seedling mortality Marunda (1990) noted
severe fungal infections on both acid and hot water treated Acacia holoserica Cunn
ex G Don seeds attributing the infections to seed damage resulting from the
treatments Identifying an optimal hot water treatment level that does not result in a
high rate of seedling mortality is a logical goal
With hot water treatments as with acid there is also a danger of overtreating
Seeds and reducing germination (Gosling et al 1995) The legume Leucaena
leucocephala (Lam) de Wit germinated equally well after 7 seconds of exposure to
water at 100degC 7 seconds at 90degC 30 seconds at 80degC and 4 minutes at 70degC In
addition acceptably high germination was obtained from treatment at 70degC from 3
minutes to 1 hour whereas no seeds germinated following treatment at 100degC for 2
minutes Perhaps along with a greater margin of error for treatment duration treating
~ew Mexico locust seeds at a lower temperature may result in a comparatively lower
of seedling mortality In practice however nursery growers tend to use the
and quickest treatment available The short treatment length at near-boiling
whether recommended or not may be what is used most frequently
10
Fine-tuning a treatment in this range of treatment temperature and duration may be
the most logical approach
Mechanical Scarification
This treatment involves scratching the seed coat with an abrasive material
(USDA Forest Service 1948) One system employs a rotating drum lined with
sandpaper A paddle spinning at a given number ofrevolutions per minute throws the
seed against the sandpaper (Hine et al 1997) A second method dry tumbling
tumbles seed in a mixer with pea gravel or sharp sand for 5 to 7 days (Dreesen 2001)
These approaches have the advantage that control of temperature is not required as in
water treatments nor is safety an issue as with add scarification
Hine et al (1997) mechanically treated New Mexico locust seed using 100shy
grit sandpaper in a Forsberg (rotating drum) scarifier Gennination was less than
10 They concluded that the seed coats were too thin for their apparatus and
proposed that a finer grit sandpaper would produce more even abrasion Although
germination varied by seed lot Dreesen (2001) reported that using a slow-speed dry
tumbling technique for 5 to 7 days produced gennination rates as high as 90
As with acid and hot water treatments treatment duration must be specifically
shy
correlated to seed lot Treated seeds are also susceptible to fungal infection
iHopkinson 1997) In addition equipment is specialized and may not always be
ltU~CUJl~ to a nursery (USDA Forest Service 1948) Several studies have confinned
degrading ofstrophiolar cells due to acid and hot water scarification (Miklas et al
11
1987 Dell 1980 Hanna 1984) but the effect ofmechanical scarification on the
strophiole has not been studied
Alternative Method for Breaking Dormancy Percussion
Strophiole degradation has been examined in depth using an impaction
treatment more commonly known as percussion Hamly (1932) first proposed that
shaking or impacting seeds could split the macrosc1ereid cells at the strophiole
Hamly successfully released tension ofbent strophiolar cells of impermeable
Melilotus alba Medik (White sweet clover) seeds by delivering physical blows
resulting in cells that had split apart and were only slightly bent Ninety-two percent
oftreated seeds became permeable as compared to 05 of control seeds Barton
(1947) showed that percussion significantly increases permeability in black locust
seed Ballard (1976) reasoned that the integrated nature of the strophiole within the
seed coat means a blow on any part of the seed weakens the strophiole There are no
published studies on the use of percussion on New Mexico locust
Percussion methods vary and often seem SUbjective or ill-defined Hamly
(1932) placed seeds in a glass bottle and shook them three times a second for 20
minutes resulting in 180 oscillations per minute Ballard (1973) describes shaking
seeds in a closed metal container with at least fifty seeds per container at 850-900
JS(~l11alIOIlS per minute for 1 minute Kelly and van Staden (1987) placed seeds into
sealed glass flasks Flasks were shaken by a wrist-shaker at a rate of 250
~UUVJ~ per minute Subsequent germination rates were 45 after 2 hours of
55 after 3 hours 60 after 6 hours and 100 after 12 hours
12
There are several potential advantages of percussion over acid hot water and
mechanical scarification Kelly and van Staden (1987) note that a percussion
treatment only alters macrosclereid cells of the strophiole by loosening separating
and raising them They emphasize that minimal damage is done to the overall
anatomical structure of the seed In acid scarification of the same seed lot radicle
and cotyledon damage occurred Based on photographic evidence the authors
believe that this morphological damage was due to uneven pressure during
imbibition with water entering haphazardly wherever the acid had sufficiently
dissolved the coat This ultimately led to uneven hydration of the embryo In
percussed seeds water only entered through the s~ophiole when the entire seed coat
had been saturated resulting in controlled and even embryo hydration (Kelly and van
Staden 1987)
Although all successful scarification treatments may focus at least in part on
the strophiole for papilionoid legumes (Baskin and Baskin 1998) percussion may be
less damaging to seed coat structures particularly when compared with acid Kelly
and van Staden did not compare hot water or mechanical scarification methods
against percussion However the fungal infection associated with hot water and
mechanical scarification as with acid scarificatio~ have been directly associated with
seed damage (Rehman et al 1998 USDA Forest Service 1948) Embryo damage in
has been observed in hot-water scarified legume seeds (Rehman et al 1998
13
By limiting embryo damage percussion scarification reduces seedling
mortality compared to other treatments Kelly and van Staden (1987) noted that
seedlings from the percussion treatment were morphologically sound and vigorous
when compared to acid treatment seedlings The authors attribute this improved
seedling vigor to the controlled water uptake that limited anatomical damage
Despite a 12-hour percussion treatment morphological damage was not substantially
greater than untreated seeds In addition seedling mass measured 10 days following
germination was not significantly different between percussed and untreated seeds
A final advantage ofpercussion may be the ability to adequately treat the most
dormant seeds while avoiding harm to seeds that have already been made permeable
by the treatment or that were permeable to begin with Instead ofcorroding scalding
or scratching the seed coat percussion appears to be just a loosening of the
strophiole spring (Ballard 1973) Kelly and van Staden (1987) were able to
increase germination with a lengthy treatment (12 hours) Figuring out whether a
substantial cushion oftreatment durations exist for percussion ofNew Mexico locust
seed would be a significant improvement to current scarification methods where seed
damage and seedling health is an issue
The greatest challenge for applying percussion to New Mexico locust seeds is
standardizing a treatment that is both effective and readily reproducible by nurseries
with mechanical scarification this may require the development of specific
~UllDment to properly quantify and execute the treatment
14
f
Objectives
The first objective of the study is to detennine which scarification treatment
results in optimal gennination speed and percentage ofNew Mexico locust The
second objective is to describe treatment interactions with nine seed sources collected
in New Mexico
Hypothesis
A percussion treatment acts exclusively on the main area ofwater uptake in
New Mexico locust seed loosening the strophiole so that imbibition can occur This
direct mechanism ofdonnancy release will result in improved gennination rate and
overall gennination percentage for New Mexico locust seed compared to standard
treatments Standard treatments including acid hot water and mechanical
scarification while eventually weakening the strophiolar cleft cause excessive
morphological damage through generalized seed coat degradation These treatments
are therefore prone to inconsistency due to over and under-treatment
15
MATERIALS AND METHODS
Seed Collection and Cleaning
Seeds were collected from August 25 through September 15 2000 from nine
locations along a latitudinal gradient in New Mexico Seed source locations and
descriptive information are given in Figure 1 and Tables I and 2 Stands were
separated by at least 45 km
Pods were hand picked as they began to ripen and dehisce (open) Collections
were made from ground level to approximately 3 m from several trees at each site
Pods were dried in open-ended brown paper bags for two to four weeks at room
temperature (21-230C) to facilitate cleaning Dried pods were placed in a closed cloth
sack which was pounded against the ground to separate seeds from pods For pods
still containing seeds hand maceration (crunching pods with hands) completed seed
separation Debris was removed by threshing with the aid of a greenhouse fan A
South Dakota Seed Blower (Seedbureau Chicago Illinois) was used to remove
remaining chaff and light seeds by air density separation Seeds were placed in dry
storage in sealed quart-size zip-lock bags within a sealed plastic container Storage
temperatures ranged from 2-4oC Seeds remained in storage for approximately 7
months before the experiment
Experimental Design and Treatment Structure
The experiment compared the impact ofhot water bath and percussion
l11aLl11 on germination properties ofNew Mexico locust seed A single
16
-l
Figure 1 Seed source collection map All sources were collected within New Mexico USA separated by at least 4S lan
IIIRZltItW lIJPIirmiddot rllll1lbullbull_~~~_II
Table 1 Latitude longitude location elevation and collection date ofNew Mexico locust seed sources
Lot title Latitude (N) Longitude (W) Location Elevation (m) Collection Date
Raton 36deg5846 104deg2872 Raton Pass NM 2295 82500
Holman 36deg0306 105deg2429 HolmanNM 2476 911500
Jemez 35deg4500 106deg4100 Jemez Springs NM 1976 9900 00
Sandia 35deg1083 106deg2341 Cedar Crest NM 2432 82600
Manzano 34deg3689 106deg2494 ManzanoNM 2465 9200
Magdalena 33deg5953 107deg0903 Magdalena NM 2544 82900
Capitan 33deg2728 105deg4325 Alto NM 2189 911200
Sacramento 32deg5798 105deg4496 Cloudcroft NM 2630 91400 Gila 32deg5400 107deg4600 Kingston NM 2432 9700
f
Table 2 Mean seed weight and standard error by source
a
Source Mean seed weight (g) for Standard error 100 seeds n = 5
Raton Holman Jemez Sandia Manzano Magdalena Capitan Sacramento Gila
2872 b c 2522 d 1826 e 2753 c 2413 d 2559 d 2443 d 3130 a 3029 ab
00103 00403 00276 00301 00370 00374 00458 00279 00348
a Mean seed weights followed by the same letter are not significantly
different
19
experiment was conducted utilizing a randomized complete block design blocked by
time with a factorial treatment structure Experimental factors were seed source and
scarification treatment Nine seed sources from throughout New Mexico were used
(Figure 1 Table 1) Scarification treatments included a control three levels ofhot
water and four levels of percussion Hot water treatments included immersion at
100degC and boiling for periods of 30 seconds and 60 seconds Percussion treatment
levels were 1 2 4 and 8 minutes One hundred seeds per block were used to test
each treatment combination Four blocks of seed were tested one at a time (all seed
sources included in each run) in close chronological sequence (within 6 weeks) A
total of four 100-seed samples were used to test each source by scarification treatment
combination
Treatments
Hot water treatment of seeds utilized 50 ml test tubes filled with 30 ml
distilled water Each test tube was used to treat one 100-seed sample at a time
resulting in a ratio of4 parts water to 1 part seed by volume Test tubes were placed
in a beaker with the beaker filled with tap water to the same level as the water within
test tubes Nine test tubes (one per seed source) were placed in the beaker
simultaneously so that one replication of each seed sourcescarification treatment
cotnblnatlon was conducted simultaneously Hot water treatments include immersion
boiling For immersion treatments seeds were added to test tubes with the water
(boiling) and the test tubes were immediately removed from the beaker to
For the boiling treatments test tubes remained in the beaker with water at
20
boiling temperature for a set duration after the addition of seeds Following all
treatments test tubes with seeds cooled 24 hours in a test tube rack
Percussion treatment utilized a pneumatic paint shaker (Central Pneumatic
model 00422 Camarillo CA) Compressed air was maintained at 530 kPa plusmn30 kPa
(80 psi plusmn 5 psi) resulting in approximately 350 oscillations per minute Seeds were
placed in 118 ml (4 oz) soil tin cans (US Can Lobard IL) and the lids were secured
with duct tape Each can was used to treat one 100-seed sample To take advantage
ofgreatest lateral movement of the paint shaker a spacer (paint can) was used to
position the soil tin at the end of the shaking arm This allowed shaking to occur at
the greatest distance from the pivot point (fulcrum) Tins were placed perpendicular
to the direction of shaking motion allowing seeds to impact at greatest force by
hitting flat surfaces of the soil tin
Germination Testing
Before gennination testing all seeds were soaked 24 hours in reverse
osmosis-treated water (this was included in the process ofhot water treatment
jU~SCIlOea above) Following soaking seeds were placed in 100 cm Petri dishes lined
90 cm Whatman qualitative 1 filter paper circles (MG Scientific Pleasant
WI) One Petri dish was used for each treatment by source combination
papers were initially moistened with 5 mL reverse osmosis-treated water then
~VU1CUlJl moistened as needed to ensure adequate conditions for gennination
were monitored daily (at 300 PM) for gennination for 14 days in accordance
~UIUlnJlVUa Seed Testing Association (ISTA) standards for Robinia species
21
(1STA 1999) Tests were carried out at room temperature (22degC plusmnOSoC)
Germination was classified as protrusion of the radicle from the embryo by at least
1mm as viewed with the naked eye
X-ray Radiography
On August 132001 three lOO-seed samples from the Sacramento seed source
were treated for 14 or 8-minute percussion durations The seeds were sent
immediately by overnight delivery to the Faxitron X-Ray Corporation in Wheeling
Illinois where Dr Bob Karrfalt ofthe USDA Forest Service oversaw their imaging
Radiographs were made at SX with a Faxitron Specimen Radiography System Model
MX-20
Scanning Electron Microscope Photography
On February 192002 a Hitachi S-3200N Variable Pressure Scanning
Electron Microscope (Mountain View CA) was used to examine strophiolar cells of
untreated seeds and seeds percussed for 4 minutes Seeds were cut longitudinally
igt their entire length using a razor blade splitting the strophiolar region roughly in
Cross sections of seed samples magnifying the strophiolar region were viewed
600X under the microscope
Analysis
Categorical Analysis of Variance was used to determine treatment source and
(Le time) main effects and interactions The response variable was total
~vlI This procedure is a Chi-square test of homogeneity using the natural log
of germinated to non-germinated seed for each treatment Maximumshy
22
likelihood analysis was used to calculate Chi-square test statistics with their observed
significance levels A single contrast was conducted to cempare treatment means
against the control Observed significance levels less than or equal to 005 were
considered significant Pairwise comparisons were conducted between non-control
treatment mean percents using a conservative alp~a value of 005 divided by the
number of comparisons (Bonferroni approach) and approximate pairwise z statistics
Categorical analyses were performed using Proc Catmod (SAS Institute 1989)
RESULTS
Scarification seed source block and all interactions between factors
influenced germination (Table 3) The main effects are reported in the presence of
these interactions because the overall robustness of treatments and seed sources are of
interest Background levels ofnaturally occurring variation in seed sources are
seen in control germination percentages in Figure 2 In particular time variability
is unlikely to be under the control of the producer Appendix B details experimental
data for source by treatment gennination percents for each of the four blocks
Averaged over all treatments germination by source ranged from 40 to
79 (Figure 2) Averaged over all sources (Figure 3) and for each source (Figure 4
5) percussion scarification durations of4 or 8 minutes yielded greatest germination
Averaged over all sources germination increased with increasing durations of
percussion then leveled off for the 4- and 8-minute treatments (Figure 3) Immersion
and 30-second boiling scarification maximized germination for the hot water
treatments with a decrease in germination when seeds were boiled for 60 seconds
For all but two sources there is a similar ipcreasing trend in germination from
1to 4 minutes ofpercussion (Figure 4) The Holman source increased in germination
1 to 2 minutes then leveled off at 4 minutes The Jemez source changed little
1 to 4 minutes of treatment As treatment duration increased from 4 to 8
mutes five sources decreased in germination (Figure 4a) whereas three sources
24
Table 3 Categorical Analysis of Variance table for germination response to seed treatment seed source block and the interaction of the factors
Source of DF Chi-Square Observed Variability Significance Level
Source 8 98370 lt00001 Trt 7 233876 lt00001
contrast 1 16976 lt00001 SourceTrt 56 97077 lt00001 Block 3 5538 lt00001
SourceBlock 24 10162 lt00001 TrtBlock 21 7564 lt00001
SourceTrtBlock 168 34283 lt00001
S 4)
8 ~
0
sectg ~ 40 ~
N 0 0
20
o f0ltgt ~ ~4 jybshy ((iltgto ~~ K1ltgt 6~b-~ ~~ 0b-~~ ~~ ~b-~ ~b-~b
80
60 -I-- - ~- middotmiddotmiddotmiddotmiddot1
r -] Northern NM Sources
Central NM Sources
Southern NM Sources
sect-o ~
~b-v(i
SOURCE
Figure 2 Effect ofNew Mexico locust seed source on germination for data averaged over all scarification treatments Control germination is shown by solid line within bar
80
~ cd ~ ltl) () 60 ~ ~
N
t 0shyrti tshySltl)
40
0
20
o lmmersion Boiling 30 sec Boiling 60 sec Control 1 min 2min 4 min 8 min
Hot Water Treatments Percussion Treatments
Figure 3 Effect of scarification on germination ofNew Mexico locust for data averaged over all seed sources Means labelled with the same letter are not significantly different
100~----------------------~--~----------------~
80
60
ltU
~
40 IJ
sectgsI
l100
80
60
40
a I middotmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot middotImiddotmiddotmiddotmiddotmiddotmiddot E
_~iii-bull _ _ bull f --~--
t-- -~ ---- -shy ---- shyI ~ ---- - shy
L
fiII
~
b
- shy ---r
- Holman
Sacramento Sandia Gila Jemez
c ~
ltgt ~
Tmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot-I
j~=~=-~~-1
I middotmiddot ~ bull r
t tr
Y -------M-a-gd-a-Ie-n-a--
1 _ _
fCapitan
---- Manzano ~y -_ Raton
2 4 8
Time interval ofpercussion scarification (min)
Figure 4 Effect of seed source by treatment interaction on New Mexico locust germination for percussion scarification pattern a and pattern b
28
tv 0
~ d
~ A s o
1 c3
80
60
40
20
o
TIT
t------=middotfmiddotmiddot-middotmiddot-middotmiddot-middotmiddot 1- --- - - --=- - -shy--cE= - _--shy --shy ----shy ~ ~ ---shy ~ -~- _ --shy-------r 1
I
Raton Holman
-_ Jemez
--- Sandia Manzano Magdalena Capitan Sacramento Gila
Immersion Boiling 30 sec Boiling 60 sec
Hot Water Scarification
Figure 5 Effect of seed source by treatment interaction on New Mexico locust germination for hot water scarification
continued to increase in germination (Figure 4b) There was no difference between
the two treatment levels for the Manzano source (Figure 4b)
The effect ofhot water scarification also varied among sources with few
overall differences between hot water treatments (Figure 5) However boiling for 60
seconds yielded highest germination for the Jemez source whereas increasing
durations ofboiling decreased germination for the Raton source
In addition to consistently high germination 4- and 8-minute percussionshy
treated seeds germinated as fast or faster than all other treatments (Figure 6 Table 4)
050 measured as the number of days needed to complete 50 ofeventual
germination averaged 2 days for both ofthese treatments
X-ray radiographs were taken of seeds collected from the Sacramento source
percussed for 1 4 or 8 minutes (Figure 7) Seeds show signs of damage at 8 minutes
ofpercussion (Figure 8) Compared to an undamaged seed percussed for only 4
minutes (Figure 8a) damage at the 8-minute level includes fractured radicles pitting
of the seed coat and chips in seed coat structure (Figure 8b-d)
Scanning electron microscope (SEM) images at 600X reveal the direct effect
of 4 minutes of percussion on cells of the strophiolar region (Figure 9) Whereas
untreated seeds retain cellular organization in the strophiolar region strophiolar cells
seeds treated for 4 minutes ofpercussion separate and raise resulting in a
~-~e~U appearance
30
Umiddot-~middot--l-~---middotymiddotmiddotmiddot-----middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot--middot--middotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middotmiddotmiddotmiddotmiddotmiddotmiddot- -
050= gt1
-e-- Percussion 8 min -0- Percussion 4 min ---T- Percussion 2 min -----ltil- Percussion 1 min -- Immersion --0- Boiling 30 sec -+- Boiling 60 sec -ltgt- Control
2 4 6 8 10 12
Day Figure 6 Timecourse ofNew Mexico locust gennination following scarification
treatments G50 is the number of days to 50 of eventual germination
31
14
Table 4 Effect of seed source and treatment on G50 (days to 50 germination)
Seed Source Control Immersion B30sec B60sec Plmin P2min P4min P8min mean Raton 3 3 5 7 4 3 2 2 4 Holman 2 2 2 2 2 2 2 2 2
Jemez 4 6 6 5 3 2 2 2 4 Sandia 2 2 2 2 2 2 2 2 2
w tv Manzano 5 2 2 3 3 2 2 2 3
Magdalena 4 2 2 3 4 3 2 1 3
Capitan 5 4 2 2 3 2 2 2 3
Sacramento 3 2 2 2 4 3 2 2 3 Gila 3 2 2 2 2 2 2 2 2 mean 3 3 3 3 3 2 2 2 3 standard 11 14 16 18 09 05 0 03 08 deviation
Note Columns and rows may not agree due to rounding
Figure 7 X-ray radiographs ofSacramento seed source percussed for 1 4 or 8 minutes
33
Figure 8a-d Seed damage close-ups Figure a shows undamaged seed percussed for 4 minutes Figures b-d show seeds percussed for 8 minutes Damage includes fracturing ofradicle in b complete removal ofseed coat in c and pitting ofseed coat in d
34
Untreated Treated
Figure 9 SEM images ofuntreated vs 4-minute percussion-treated strophiolar cells ofNew Mexico locust seeds Treated cells are loosened separated and raised
DISCUSSION
Determining the Optimal Treatment
Averaging results of scarification treatmems across seed sources is a useful
tool to predict which treatment is most likely to be successful for an unknown seed
source Averaged over all sources four- and eight-minute percussion treatments
resulted in the highest germination 815 and 790 respectively and produced
germination that was as fast or faster than all other treatments Although there was no
difference between these two treatments on average an argument for 4 minutes of
percussion as a standard protocol for treating New Mexico locust seeds can be made
Descriptively 4 minutes of percussion improved germination over 8 minutes
ofpercussion for a majority of sources (five out of nine) 8 minutes of percussion
improved germination for three ofnine sources with no difference for a fmal source
For individual sources 4 minutes ofpercussion was always the best or secondmiddot best
treatment and was always equal to or better than all hot water treatments
Germination at 8 minutes ofpercussion was lower than all other percussion durations
for the Jemez and Gila sources and was lower than at least one hot water treatment for
these sources as well
The x-ray radiographs of the Sacramento seed source illustrate the excessive
internal and external seed damage that can occur with 8 minutes of percussion
germination counts 8-minute percussed seeds from Holman and Capitan
ClUll signs of damage that were visible to the naked eye (see below)
r~evertheless some of these seeds still fit the criteria for germination Neither x-ray
36
photography nor visible observations during seed counts revealed seed damage at the
4-minute percussion level In operational trials seeds percussed for 4 minutes
developed into healthy seedlings (Khadduri and Harrington unpublished data)
Strophiole and Seed Coat Behavior
Four minutes ofpercussion successfully breaks seed dormancy in a large
percentage ofNew Mexico locust seeds without excessively damaging the
surrounding seed coat or internal seed coat structures Most papilionoid legume
seeds including seeds ofNew Mexico locust have a specific region of the seed coat
known as the strophiole or lens (Hamly 1932) The strophiole is located on the
cotyledonary lobe of a locust seed (Figure 10 a-c) If seeds are not indiscriminately
damaged the strophiole is the first point of water entry to the embryo (Hopkinson
1997) When a papilionoid seed is percussed for the appropriate amount of time
repeated hits on the integrated seed coat loosen the constrained cells of the strophiolar
region without excessively damaging the rest of the seed coat (Ballard 1976) SEM
photos illuStrate that 4 minutes of percussion loosened and separated strophiolar cells
ofNew Mexico locust seeds when compared with constrained organized strophiolar
cells of untreated seeds (Figure 9) Research-has shown that water passes to the
embryo through the treated strophiole in a controlled manner and regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues
(Kelly and van Staden 1987)
37
10 Strophiole ofunimbibed New Mexico locust seed (a) and following uptake (bc)
38
In contrast hot-water treatments can excessively degrade the seed coat
(Rebman et al 1998 Marunda 1990) Random cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rebman et aI 1998 Marunda 1990) Along with a
proportionately large amount ofun-imbibed (thus under-treated) seeds (Figure l1a)
damage was observed in hot-water treated seeds from six sources during germination
counts Damage included seeds where the radicle failed to elongate (Figure 11 b)
seeds where the radicle elongated but was delayed in freeing itself from the seed coat
(Figure 11c) and seeds where the radicle completely broke off from the embryo
(Figure lId) In addition some seeds germinated in reverse (the cotyledon emerged
fIrst) If the radicle emerged completely from the seed coat they were counted as
germinated
While nearly all seeds treated for 8 minutes ofpercussion imbibed water
damage was evident in x-ray evaluations (Sacramento seed source) and in
germination counts (Holman and Capitan) at this treatment level Seed damage
during germination counts was similar to levels found in hot-water treated seeds
suggesting that over-treatment by both hot water and longer durations of percussion
may result in similar damage to seed structure
The Sacramento and Holman seed sources were two of the fIve sources that
opcrplltlpn in germination from the 4- to 8-minute durations of percussion
the benefIts ofloosening the strophiole as evidenced by imbibition of
all 8-minute treated seeds were outweighed by external and internal seed coat
39
a
b c d
11 Damage to New Mexico locust seeds observed during germination cotmts following hot water scarification 9a Hot water replicate including ~t seedlings and numerous unimbibed seeds 9b Close-up of seed where raallCle failed to elongate 9c Close-up ofseed where radicle elongated but was
-gtIn in freeing itself from the seed coat 9d Close-up ofseed where radicle broke off from embryo
40
damage (which also may be contributing to high imbibition) For the three sources
for which 8 minutes of percussion resulted in maximum germination extended
percussion of seeds necessary to loosen the strophiole may not have excessively
damaged the seed anatomy The Manzano source did not significantly change in
germination from 4 to 8 minutes of percussion suggesting that an increase in
permeable seeds at the 8-minute treatment duration was balanced by the number of
seeds that failed to germinate due to seed damage To summarize the 4-minute
percussion treatment improved germination over 8 minutes ofpercussion for a
majority of sources (five ofnine) the 8-minute percussion treatment improved
germination over 4-minutes ofpercussion for three sources and there was no
difference with the Manzano source Notably for the Raton source 8 minutes of
percussion increased germination by 235 percentage points over 4-minutes of
percussion (7875 vs 5525 respectively) the next best treatment Raton was the
only source to result in germination less than 75 for 4 minutes ofpercussion so a
grower may wish to apply longer percussion durations to lots that do not respond well
to this treatment level
Finally there was an interesting trend in the Jemez and Raton sources seed
that significantly contributed to the source by treatment interaction (Figure 12) A
~u test showed that the Jemez source had a significantly lighter seed weight than
other sources while the Raton source had an average seed weight For the Jemez
the longest duration of boiling increased germination Conversely only short
U4UVll) of percussion were needed to achieve high germination and the longest
41
80 _ Jemez a Raton
iD
~ C iD e iD
Q c 0 ~
c ~ iD 0
20
0------shy
Immersion Boiling 30 sec Boiling 60 sec
Hot Water Scarification
10degTr========~--------~------------------------------
80
60
40
20
_ Jemez Raton
0------shy
1 min 2 min 4 min 8 min
Percussion Scarification
Figure 12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources
42
b
--~----~~~~========~---- duration ofpercussion reduced germination For the Raton source longer durations
ofboiling consistently decreased germination whereas longer percussion treatments
consistently increased germination
A possible explanation is that a thick seed coat is associated with a loose
strophiole structure and that a thin seed coat is associated with a constrained
strophiole This contrast merits further study and may suggest an ecological
adaptation Further investigation ofthis phenomenon may contribute to successful
pre-screening ofunknown seed sources to determine optimal treatment leveL
43
CONCLUSION
Based on results obtained in pilot study (see Appendix) and thesis
experiments 4 minutes ofpercussion is recommended as a standard protocol for
treating an unknown source ofNew Mexico locust seed In addition to laboratory
results the 4-minute percussion treatment has resulted in germination greater than
80 and healthy seedling grow-outs for production purposes (unpublished data) In
laboratory experiments only one hundred seeds per 118 ml (40z) soil tin were
percussed at a time whereas for production we have treated up to five hundred seeds
per tin For large-scale production ofNew Mexico locust seedlings growers may
wish to experiment with larger quantities of seed and larger-size soil tins
Seed storage trials following percussion treatments were not conducted
However germination greater than 80 and healthy seedlings were obtained when
seeds were percussion-treated and subsequently stored at room-temperature (22degC)
for one week (unpublished data) Seed storage trials merit further study as the
standard hot water treatment leaves seeds in a softened condition that is not
conducive to storage
Hydroseeding is an effective method for direct seeding in reclamation
situations Percussion treatment may be conducive to hydro seeding as seeds remain
a dry relatively undamaged yet permeable condition following treatment Sulfuric
seeds also remain dry following treatment but extensive damage to seed coat
poor seedling vigor have been reported with this treatment (Kelly and van Staden
A grow-out study using a hydro seeder on a reclamation site testing percussion
44
against other treatments such as sulfuric acid and mechanical scarification also
merits further study
Papilionoids are the largest subfamily oflegumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust these legumes often play an integral role in the revegetation of
disturbed lands The pilot study (Appendix) indicates that percussion scarification
successfully treats the widely used black locust as well Developing superior
scarification methods to the standard hot water mechanical scarification and acid
treatments may facilitate the use ofpapilionoid legumes in reclamation projects
45
APPENDICES
APPENDIX A
PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR
NEW MEXICO LOCUST AND BLACK LOCUST SEEDS
Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4
Hot water and sulfuric acid soaks are traditional treatments for seeds of many
temperate woody legwnes including locusts However these scarification techniques
often produce inconsistent germination Percussion scarification where seeds are
repeatedly propelled against a hard surface was compared with hot water
scarification to evaluate treatment efficacy for New Mexico locust (Robinia
neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment
seeds were placed in a 98degC water bath which was immediately removed from the
heat source For percussion scarification seeds were placed in a soil sample tin and
agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the
control were followed by 24-hour water soaks Hot water baths resulted in 56 and
41 germination for New Mexico locust and black locust respectively For both
species nearly all durations ofpercussion increased germination over the hot water
treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3
4 and 5 minutes for black locust resulted in at least 90 germination Traditional
scarification treatments randomly degrade the entire seed coat which can lead to
tissue damage during water uptake Percussion scarification specifically weakens the
strophiole the natural source ofwater entry to the seed in papilionoid legwnes
INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom
2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031
48
Following percussion imbibition is controlled through the strophiole and underlying
tissue is protected
Keywords New Mexico locust black locust scarification percussion germination
Introduction
Locust trees (Robinia spp) are aggressive pioneer species that quickly
colonize disturbed land fix atmospheric nitrogen in the soil and stabilize slopes that
are prone to erosion (Klemmedson 1994 Ashby et al 1985) (Figure A1ab)
Recognition of these attributes has contributed to increased demand for locusts in
reclamation projects Locust seeds however exhibit physical dormancy where a
thick seed coat prevents the movement of water and gasses to the embryo (Leadem
1997)
Sulfuric acid or hot water soaks have traditionally been used to break seed
dormancy in macro-propagation of locusts Many growers are moving away from the
use of su1furic acid in treating locust seeds (Dreesen and Harrington 1997) In
addition to being dangerous acid soak durations must be specifically correlated to
seed lot (Olson 1974) Collections of native seed can vary tremendously in seed size
seed weight and hard seededness etc This variability limits the utility of sulfuric
acid soaks as even within a given lot some seeds may be damaged whereas others
are under-treated and fail to imbibe (take up water) A survey of Southwestern
container growers indicates that hot water baths are currently the most common
scarification method for locust seeds (Hine et al 1997) However hot water baths
also produce inconsistent germination (Lin et al 1996)
49
b
Figure AI New Mexico locust a primary invader following disturbance Ala Crown-sprouting at edge ofburn near Los Alamos NM--one year after Cerro Grande fire Alb Colonizing road cut Gila National Forest NM
50
Percussion scarification where seeds are repeatedly propelled against a hard
surface is an alternative dormancy-breaking method that has proven successful in
legumes related to locusts (Hamly 1932 Barton 1947) We compared this alternative
treatment to hot water scarification to evaluate treatment efficacy for New Mexico
locust and black locust seeds
Materials and Methods
Commercial New Mexico locust seeds (Western Native Seed Coaldale CO)
were collected fall 2000 in Huerfano County CO Black locust seeds were collected
September 2000 from Taos County NM by harvesting ripe pods from several trees at
a distance of up to 3 m from ground level Black locust seeds were allowed to air-dry
for 3 weeks and were threshed and separated from large chaffusing a greenhouse
fan A South Dakota Seed Blower (Chicago Illinltois) was used to remove fine chaff
from both seed lots Seeds were storedmiddotat 2-4C until the germination study was
conducted in March 2001
This study compared hot water scarification with percussion scarification
using treatment and species as experimental factors Along with a control
scarification treatments included immersion of seed in boiling water as well as
percussion durations of 1 2 3 45 or 10 minutes Four 100-seed samples were used
to test each species by scarification treatment combination
For the hot water treatment test tubes were filled with 30 mL water and
placed in a water-filled beaker (Figure A2a) The bath was raised to boiling (-98C at
51
b
c
Figure A2 Implementation ofhot water and percussion scarification A2a Hot water bath A2b Paint-shaker set-up A2c Placing soil tin containing seeds at end ofpaint shaker ann
52
Las Cruces NM elevation of 1300 meters) Each 100-seed replicate was placed in a
test tube and each test tube was immediately removed to cool at room temperature
Percussion scarification was implemented using a pneumatic paint shaker
(Central Pneumatic model 00422 Camarillo CA) (Figure A2b) This allowed
standardization of treatment intensity Compressed air pressure was maintained at
530kPa plusmn 30kPa (80psi plusmn 5psi) resulting in approximately 350 oscillations per
minute Each lOO-seed sample was placed in a 118 ml (4 oz) soil tin for shaking To
maximize lateral movement of paint shaker a paint can was used as a spacer to place
soil tins at the end of the shaking arm (Figure A2c) This allowed shaking to occur at
the greatest distance from the pivot point (fulcrum) Following all treatments
including the control seeds were soaked for twenty-four hours in distilled water prior
to plating out
Gennination was evaluated on lab benches at room temperature Each
replicate of seeds was placed in a 100 cm petri dish on moistened filter paper with
humidity maintained by enclosing the petri dishes in ziplock bags Gennination was
monitored daily for 14 days in accordance with International Seed Testing
Association standards for Robinia species (ISTA 1999) Gennination was defined as
protrusion of the radicle from the embryo by at least 1mm as viewed by the naked
eye
Categorical Analysis of Variance using Prpc Catmod (SAS Institute 1989)
was used to detennine treatment differences in germination percentages for each
source This procedure is an extension of a Chi-square test of homogeneity using the
53
natural log of the ratio of germinated to non-germinated seeds for each treatment Pshy
values less than 005 were considered significant A limited set of pairwiseshy
comparisons was conducted to compare treatment mean percents using a
conservative alpha value of 005 divided by the number of comparisons
Results
Percussion maximized germination for both New Mexico locust and black
locust There were three germination response levels for New Mexico locust (Figure
A3) First there was a low response in the control indicating a dormant seed lot
The hot water treatment and I-minute duration of percussion share a second
intermediate response Germination was highest at percussion levels from 2 to 10
minutes
A selected percussion treatment intermediate in time (5 minutes) produced the
steepest germination curve indicating the fastest germination rate (Figure A4) The
hot water treatment produced an intermediate curve or relatively slower germination
rate The control curve was very flat indicating the slowest germination rate for New
Mexico locust
Control seeds germinated poorly for black locust as well (Figure A5) Hot
water scarification improved germination over the control but all levels of percussion
improved germination over hot water The optimal durations of percussion treatment
were bracketed with a significant increase from the 1 to 2 minute percussion level a
high germination response from 2 to 5 minutes of percussion and finally a significant
54
~ d 25 CL)
p t1 0 ~ t1
g Vl ~ Vl
100~---------------------------------------------------------------
80
60
40
20
o Control Hot Water Percussion
I min Percussion
2 min Percussion
3 min
Scarification
Percussion 4 min
Figure A3 Effect of scarification on germination response for New Mexico locust
Percussion 5 min
=significant difference between adjacent treatment bars ns = no significant difference
Percussion 10 min
~ ~ () u agt
Il-t d o ~
~ ~ c3
100middotlr~~~------------------------------------e- Percussion 5min middot 0middot Hot Water -- Control
80-J=middot======
6b-middot-lmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middot--middotmiddot-middot~-middotmiddot -middotmiddot middot middotmiddot-middot 1
40
20
-----~-----------
0 0 0 middotmiddotmiddotmiddotmiddot0middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot0
0 --~-----y-----~------_---~--- T-----~
o shy -TDay 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A4 Effect of selected scarification treatments on germination speed for New Mexico locust First seven days of fourteen-day experiment are shown
Day 7
80
~ cd
5 60 ()
[) Cl d 0 1t1 d
40sect dUl
-J
20
o
ns
Scarification
Figure A5 Effect of scarification on germination response for black locust = significant difference between adjacent treatment bars ns = no significant difference
decrease at the 10-minute level This drop-off suggests that 10 minutes of percussion
over-treats seeds from this lot of-black locust
A selected percussion treatment intermediate in time (5 minutes) also had the
fastest germination rate for black locust (Figure A6)_ The hot water curve is
somewhat flatter indicating a slower germination rate The control curve is very flat
indicating a slow germination rate
A qualitative difference between hot water and percussion-treated seeds was
also observed Along with healthy germinants the hot water treatment produced
over-treated seeds (Figure A 7a) Examples of damaged germinants include seeds
where the radicle did not elongate (Figure A 7b) seeds where the radicle elongated
but was delayed in freeing itself from the seed coat (Figure A7c) and seeds where the
radicle broke off completely from the embryo (Figure A7d) In addition to these
examples of over-treatment there were numerous small dark seeds that failed to
imbibe water For percussion treatment nearly all seeds took up water (Figure A8a)
Germination was rapid and uniform with little damage evident (Figure A8b)
To summarize both New Mexico locust and black locust seed lots were
dormant The standard hot water scarification treatment improved germination but
only to 56 and 41 for New Mexico locust and black locust respectively Nearly
all percussion durations improved total germination as well as germination rate over
the hot water treatment Germination was greater than 90 for percussion durations
of 4 5 and 10 minutes for New Mexico locust and 3 4 and 5 minutes for black locust
58
CI)
sgt t CI) 0
igt 0
sect ttl t E
Vl 0
c3
100r==~==~~=r------------------------------------~ ___ Percussion 5 min
middotmiddotmiddot0middotmiddotmiddot Hot Water -lIf- Control
80r=========~--~~----------------------------~
60
40
20 -~-~---~---------- ---~------------I
omiddot
O
_~_----------lIf-----~bull -----_ shy---shy I
Day 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A6 Effect of selected scarification treatments on germination speed for black locust First seven days of fourteen-day experiment are shown
Day 7
a
b c d
Figure A7 Damage to New Mexico locust seeds observed during germination counts following hot water scarification A7a Hot water replicate including damaged seedlings and numerous unimbibed seeds A7b Close-up ofseed where radicle failed to elongate A7c Close-up ofseed where radicle elongated but was delayed in freeing itself from the seed coat A7d Close-up ofseed where radicle completely broke offfrom embryo
60
a
b
Figure A8 New Mexico locust seeds following percussion treatment at time of germination counts A8a Percussion replicate with uniformly healthy seedlings and near complete imbibition of seeds A8b Close-up ofpercussion-treated seedling
61
Discussion
Papilionoid seeds have a specific anatomical feature known as the strophiole
or lens-the natural site of water entry to the seed (Hamly 1932) The strophiole is
located on the cotyledonary lobe of a locust seed (Figure A9) When a papilionoid
legume seed is percussed for the appropriate amount of time repeated hits on the
integrated seed coat loosen the constrained cells of the strophiolar region without
excessively damaging the rest of the seed coat (Ballard 1976)
When a percussed seed is soaked water enters exclusively through the
strophiole in a controlled manner (Kelly and Staden 1987) This regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues This
contrasts with seeds that have been hot water or acid scarified treatments that can
randomly degrade the seed coat Unlocalized cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rehman et al 1998 Marunda 1990)
Papilionoids are the largest subfamily of legumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust and black locust these legumes often play an integral role in the
revegetation ofdisturbed lands Developing superior scarification methods to the
standard hot water and acid treatments should fadlitate the use ofthese legumes in
restoration projects
62
Figure A9 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (bc)
63
APPENDIXB
GERMINATION PERCENTS AND STANDARD ERRORS FOR SOURCE
BLOCK AND TREATMENT FOR NEW MEXICO LOCUST
Table B 1 Gennination percents and standard errors for source block and treatment for New Mexico locust
Source Block Treatment Germination Percentage Standard Error
Capitan 1 Immersion 74 437 Capitan 2 Immersion 69 462 Capitan 3 Immersion 71 454 Capitan 4 Immersion 67 470 Capitan Boiling 30 sec 79 407 Capitan 2 Boiling 30 sec 76 427 Capitan 3 Boiling 30 sec 72 449 Capitan 4 Boiling 30 sec 76 427 Capitan Boiling 60 sec 71 454 Capitan 2 Boiling 60 sec 54 498 Capitan 3 Boiling 60 sec 67 470 Capitan 4 Boiling 60 sec 76 427 Capitan I Control 5 218 Capitan 2 Control 7 255 Capitan 3 Control 4 196 Capitan 4 Control 5 218 Capitan 1 Percussion I min 47 499 Capitan 2 Percussion I min
470
Capitan Percussion I min 39 488
Capitan 4 Percussion I min 38 485 Capitan I Percussion 2 min 55 497 Capitan Capitan
2
Percussion 2 min Percussion 2 min
71 65
454 477
Capitan 4 Percussion 2 min 56 496 Capitan I Percussion 4 min 86 347 Capitan 2 Percussion 4 min 79 407 Capitan 3 Percussion 4 min 81 392 Capitan 4 Percussion 4 min 82 384 Capitan I Percussion 8 min 90 300 Capitan 2 Percussion 8 min 88 325 Capitan 3 Percussion 8 min 90 300 Capitan 4 Percussion 8 min 90 300 Gila I Immersion 71 454 Gila 2 Immersion 77 421 Gila
Immersion 75 433
Gila 4 Immersion 72 449
Gila I Boiling 30 sec 68 466
Gila 2 Boiling 30 sec 81 392
Gila 3 Boiling 30 sec 54 498
Gila 4 Boiling 30 sec 63 483
Gila I Boiling 60 sec 55 497
Gila 2 Boiling 60 sec 68 466
Gila 3 Boiling 60 sec 57 495
Gila 4 Boiling 60 sec 70 458
65
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Gila 2 Control 13 336 Gila 3 Control 13 336 Gila 4 Control 20 400 Gila 1 Percussion 1 min 66 474 Gila 2 Percussion 1 min 57 495 Gila 3 Percussion 1 min 73 444 Gila 4 Percussion 1 min 82 384 Gila 1 Percussion 2 min 81 392 Gila 2 Percussion 2 min 86 347 Gila 3 Percussion 2 min 85 357 Gila 4 Percussion 2 min 54 498 Gila 1 Percussion 4 min 79 407 Gila 2 Percussion 4 min 83 376 Gila 3 Percussion 4 min 84 367 Gila 4 Percussion 4 min 79 407 Gila 1 Percussion 8 min 51 500 Gila 2 Percussion 8 min 76 427 Gila 3 Percussion 8 min 52 500 Gila 4 Percussion 8 min 74 439 Holman 1 Immersion 83 376 Holman 2 Immersion 86 347 Holman 3 Immersion 81 392 Holman 4 Immersion 90 300 Holman 1 Boiling 30 sec 90 300 Holman 2 Boiling 30 sec 85 357 Holman 3 Boiling 30 sec 87 336 Holman 4 Boiling 30 sec 83 376 Holman 1 Boiling 60 sec 79 407 Holman 2 Boiling 60 sec 80 400 Holman 3 Boiling 60 sec 86 347 Holman 4 Boiling 60 sec 93 255 Holman Control 25 433 Holman 2 Control 19 392 Holman Control 21 407
Holman 4 Control 24 427
Holman Percussion 1 min 77 421
Holman 2 Percussion 1 min 85 357
Holman 3 Percussion 1 min 80 400
Holman 4 Percussion 1 min 89 313
Holman 1 Percussion 2 min 88 325
Holman 2 Percussion 2 min 96 196
Holman 3 Percussion 2 min 93 255
Holman 4 Percussion 2 min 96 196
Holman 1 Percussion 4 min 92 271
Holman 2 Percussion 4 min 97 171
66
I
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Holman 3 Percussion 4 min 91 286 Holman 4 Percussion 4 min 96 196 Holman 1 Percussion 8 min 88 325 Holman 2 Percussion 8 min 85 357 Holman 3 Percussion 8 min 83 376 Holman 4 Percussion 8 min 82 384 Jemez 1 Immersion 53 499 Jemez 2 Immersion 44 496 Jemez 3 Immersion 52 500 Jemez 4 Immersion 70 458 Jemez 1 Boiling 30 sec 58 494 Jemez 2 Boiling 30 sec 55 497 Jemez 3 Boiling 30 sec 48 500 Jemez 4 Boiling 30 sec 65 477 Jemez 1 Boiling 60 sec 68 466 Jemez 2 Boiling 60 sec 63 483 Jemez 3 Boiling 60 sec 73 444 Jemez 4 Boiling 60 sec 89 313 Jemez 1 Control 2 140 Jemez 2 Control 9 286 Jemez 3 Control 6 237 Jemez 4 Control 8 271 Jemez 1 Percussion 1 min 58 494 Jemez 2 Percussion 1 min 63 483 Jemez 3 Percussion 1 min 77 421 Jemez 4 Percussion 1 min 89 313 Jemez 1 Percussion 2 min 68 466 Jemez 2 Percussion 2 min 64 480 Jemez 3 Percussion 2 min 80 400 Jemez 4 Percussion 2 min 83 376 Jemez Percussion 4 min 68 466 Jemez 2 Percussion 4 min 79 407 Jemez 3 Percussion 4 min 70 458 Jemez 4 Percussion 4 min 83 376 Jemez 1 Percussion 8 min 52 500 Jemez 2 Percussion 8 min 59 492 Jemez 3 Percussion 8 min 61 488 Jemez 4 Percussion 8 min 70 458 Magdalena 1 Immersion 69 462 Magdalena 2 Immersion 69 462 Magdalena J Immersion 70 458 Magdalena 4 Immersion 85 357 Magdalena 1 Boiling 30 sec 65 477 Magdalena 2 Boiling 30 sec 71 454 Magdalena 3 Boiling 30 sec 68 466
67
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Magdalena 4 Boiling 30 sec 83 376 Magdalena 1 Boiling 60 sec 63 483 Magdalena 2 Boiling 60 sec 68 466 Magdalena 3 Boiling 60 sec 63 483 Magdalena 4 Boiling 60 sec 82 384 Magdalena 1 Control 11 313 Magdalena 2 Control 6 237 Magdalena 3 Control 5 218 Magdalena 4 Control 4 196 Magdalena Percussion 1 min 32 466 Magdalena 2 Percussion 1 min 45 497 Magdalena 3 Percussion 1 min 46 498 Magdalena 4 Percussion I min 36 480 Magdalena 1 Percussion 2 min 67 470 Magdalena 2 Percussion 2 min 68 466 Magdalena 3 Percussion 2 min 65 477 Magdalena 4 Percussion 2 min 81 392 Magdalena 1 Percussion 4 min 87 336 Magdalena 2 Percussion 4 min 97 171 Magdalena 3 Percussion 4 min 83 376 Magdalena 4 Percussion 4 min 97 171 Magdalena 1 Percussion 8 min 98 lAO Magdalena 2 Percussion 8 min 100 000 Magdalena 3 Percussion 8 min 96 196 Magdalena 4 Percussion 8 min 98 lAO Manzano 1 Immersion 67 470 Manzano 2 Immersion 57 495 Manzano 3 Immersion 72 4049 Manzano 4 Immersion 73 4044 Manzano 1 Boiling 30 sec 70 458 Manzano 2 Boiling 30 sec 64 480 Manzano 3 Boiling 30 sec 71 454 Manzano 4 Boiling 30 sec 80 400 Manzano I Boiling 60 sec 50 500 Manzano 2 Boiling 60 sec 53 499 Manzano 3 Boiling 60 sec 59 492 Manzano 4 Boiling 60 sec 74 439 Manzano I Control 4 196 Manzano 2 Control 8 271 Manzano 3 Control 1 099 Manzano 4 Control 6 237 Manzano 1 Percussion 1 min 52 500 Manzano 2 Percussion 1 min 39 488 Manzano 3 Percussion 1 min 36 480 Manzano 4 Percussion 1 min 51 500
68
Table B 1 Continued
Source Block Treatment Gerrni~ation Percentage Standard Error
Manzano Percussion 2 min 53 499 Manzano 2 Percussion 2 min 58 494 Manzano 3 Percussion 2 min 61 488 Manzano 4 Percussion 2 min 62 485 Manzano 1 Percussion 4 min 76 427 Manzano 2 Percussion 4 min 85 357 Manzano 3 Percussion 4 min 79 407 Manzano 4 Percussion 4 min 83 376 Manzano 1 Percussion 8 min 87 336 Manzano 2 Percussion 8 min 83 376 Manzano 3 Percussion 8 min 83 376 Manzano 4 Percussion 8 min 75 433 Raton 1 Immersion 54 498 Raton 2 Immersion 37 483 Raton 3 Immersion 39 488 Raton 4 Immersion 69 462 Raton 1 Boiling 30 sec 36 480 Raton 2 Boiling 30 sec 20 400 Raton 3 Boiling 30 sec 28 449 Raton 4 Boiling 30 sec 60 490 Raton 1 Boiling 60 sec 15 357 Raton 2 Boiling 60 sec 171 Raton 3 Boiling 60 sec 4 196 Raton 4 Boiling 60 sec 35 477 Raton 1 Control 4 196 Raton 2 Control 12 325 Raton 3 Control 2 140 Raton 4 Control 6 237 Raton 1 Percussion 1 min 52 500 Raton 2 Percussion I min 29 454 Raton 3 Percussion I min 35 477 Raton 4 Percussion 1 min 31 462 Raton 1 Percussion 2 min 55 497 Raton 2 Percussion 2 min 35 477 Raton 3 Percussion 2 min 33 470 Raton 4 Percussion 2 min 42 494 Raton 1 Percussion 4 min 65 477 Raton 2 Percussion 4 min 52 500 Raton Percussion 4 min 51 500 Raton 4 Percussion 4 min 53 499 Raton 1 Percussion 8 min 88 325 Raton 2 Percussion 8 min 80 400 Raton 3 Percussion 8 min 73 444 Raton 4 Percussion 8 min 74 439 Sacramento 1 Immersion 43 495
69
Table B 1 Continued
-Source Block Treatment Germination Percentage Standard Error
Sacramento 2 Immersion 30 458 Sacramento 3 Immersion 38 485 Sacramento 4 Immersion 56 496 Sacramento 1 Boiling 30 sec 47 499 Sacramento 2 Boiling 30 sec 48 500 Sacramento 3 Boiling 30 sec 48 500 Sacramento 4 Boiling 30 sec 56 496 Sacramento 1 Boiling 60 sec 52 500 Sacramento 2 Boiling 60 sec 67 470 Sacramento 3 Boiling 60 sec 43 495 Sacramento 4 Boiling 60 sec 48 500 Sacramento 1 Control 0 000 Sacramento 2 Control 3 171 Sacramento 3 Control 3 171 Sacramento 4 Control 099 Sacramento 1 Percussion 1 min 57 495 Sacramento 2 Percussion 1 min 61 488 Sacramento 3 Percussion 1 min 62 485 Sacramento 4 Percussion 1 min 55 497 Sacramento 1 Percussion 2 min 84 367 Sacramento 2 Percussion 2 min 70 458 Sacramento 3 Percussion 2 min 81 392 Sacramento 4 Percussion 2 min 69 462 Sacramento 1 Percussion 4 min 91 286 Sacramento 2 Percussion 4 min 84 367 Sacramento 3 Percussion 4 min 84 367 Sacramento 4 Percussion 4 min 94 237 Sacramento 1 Percussion 8 min 82 384 Sacramento 2 Percussion 8 min 81 392 Sacramento 3 Percussion 8 min 82 384 Sacramento 4 Percussion 8 min 81 392 Sandia 1 Immersion 76 427 Sandia 2 Immersion 70 458 Sandia 3 Immersion 70 458 Sandia 4 Immersion 81 392 Sandia 1 Boiling 30 sec 78 414 Sandia 2 Boiling 30 sec 71 454 Sandia 3 Boiling 30 sec 63 483 Sandia 4 Boiling 30 sec 78 414 Sandia 1 Boiling 60 sec 67 470 Sandia 2 Boiling 60 sec 65 477 Sandia 3 Boiling 60 sec 74 439 Sandia 4 Boiling 60 sec 66 474 Sandia 1 Control 16 367 Sandia 2 Control 7 255
70
Table B 1 Continued
Source Block Treatment Gennination Percentage Standard Error
Sandia 3 Control 8 271 Sandia 4 Control 16 367 Sandia 1 Percussion 1 min 67 470 Sandia 2 Percussion 1 min 61 488 Sandia 3 Percussion 1 min 66 474 Sandia 4 Percussion 1 min 59 492 Sandia I Percussion 2 min 82 384 Sandia 2 Percussion 2 min 83 376 Sandia 3 Percussion 2 min 81 392 Sandia 4 Percussion 2 min 84 367 Sandia 1 Percussion 4 min 89 313 Sandia 2 Percussion 4 min 86 347 Sandia 3 Percussion 4 min 89 313 Sandia 4 Percussion 4 min 85 357 Sandia 1 Percussion 8 min 80 400 Sandia 2 Percussion 8 min 77 421 Sandia 3 Percussion 8 min 69 462 Sandia 4 Percussion 8 min 66 474
LITERATURE CITED
Ashby WC Vogel WG Rogers NF 1985 Black locust in the reclamation equation General Technical Report Northeastern Forest Experiment Station USDA Forest Service NE-I05 12 p
Ballard LAT 1973 Physical barriers to gennination Seed Sci Technoll 285-303
___ 1976 Strophiolar water conduction in the seeds ofTrifolieae induced by action on the testa at nonstrophiolar sites Austr J Plant Physiol3 465-469
Barton LV 1947 Special studies on seed coat impenneability Contrib Boyce Thompson Inst 14 355-362
Baskin CC Baskin JM 1998 Seeds Ecology biogeography and evolution of donnancy and gennination Academic Press London UK 666 p
Bewley JD 1997 Seed germination and donnancy Plant Ce119 1055-1066
Bewley JD Black M 1994 Seeds physiology of development and germination 2nd ed Plenum Press New York NY 445 p
Briscoe CB 1996 Growing Nitrogen Fixing Trees in Acid Soils In MH Powell (ed) Nitrogen Fixing Trees for Acid Soils- A Field ManuaL Winrock International Morrilton AR 22 p
Copeland LO McDonald MB 1995 Principles of Seed Science and Technology 3rded Chapman amp Hall New York NY 409 p
Cox RE Klett JE 1984 Seed gennination requirements of native Colorado plants for use in the landscape Plant Propag 30(2) 6-10
Dell B 1980 Structure and function ofthe strophiolar plug in seeds ofAlbizia Lophantha Am J Bot 67 556-563
Dick-Peddie WA 1993 New Mexico Vegetation Past Present and Future University ofNew Mexico Press Albuquerque NM 244 p
Dreesen DR 2001 Propagation protocol for production ofcontainer Robinia neomexicana plants Los Lunas Plant Materials Center Los Lunas New Mexico In Native Plant Network URL httpwwwnativeplantnetworkorg (accessed 9 January 2002) Moscow (ID) University ofIdaho CoIl Natural Resources For Res Nursery
72
Dreesen DR Harrington JT 1997 Propagation of native plants for restoration projects in the Southwestern US- Preliminary Investigations In Landis TD Thompson JR tech coords National Proceedings Forest and Conservation Nursery Assocs Gen Tech Rep PNW-GTR-419 Portland OR USDA For Ser PNW Res Sta 77-88
Gottfried GJ 1980 Control ofNew Mexican locust and the effect on planted ponderosa pine in central Arizona US For Ser Res Note RM-386 Rocky Mountain For Range ExpStn Fort Collins CO
Gosling PG Samuel YK Jones SK 1995 A systematic examination of germination temperature chipping and water temperaturesoak duration pretreatments on the seeds ofLeucaena leucocephala Seed Sci amp Technol 23521-532
Hamly DH 1932 Softening of the seeds of Melilotus alba Bot Gazette 93 345shy375
Hanna PJ 1984 Anatomical features of the seed coat ofAcacia kempeana (Mueller) which relate to increased germination rate induced by heat treatment New Phytol 96 23-29
Hanks JP Dick-Peddie WA 1974 Vegetation patterns of the White Mountains New Mexico Southwest Naturalist 18(4) 371-382
Hine S Harrington JT Dreesen DR 1997 Seed scarification requirements for Robinia neomexicana In Harrington JTtech coord Proc Third SouthwestemContainer Growers Conf Feb 12-13 1997 Ft Collins CO
Hopkinson JM 1993 The strophiole of leguminous seeds Newsletter- IntI Herbage Seed Production Res Grp 18 7-8
---- 1997 Differences in hardseededness between Cunningham and Tarramba leucaena In Leucnet News The Newsletter of the International LeucaenaRes and Devlt Netwk No4
ISTA (International Seed Testing Association) 1999 International rules for seed testing rules Seed Sci amp Technol27 Supplement
Kelly KM van Staden J 1987 The lens as the site of permeability in the papilionoid seed Aspalathus linearis J Plant Physiol 128(45) 395-404
73
Keresztesi B 1988 Black locust the tree of agriculture Outlook on Agr 17(2) 77~85
Kigel 1 and Galili G 1995 Seed development and germination Marcel Dekker New York 853 p
Klemmedson 10 1994 New Mexico locust and parent material Influence on macronutrients of forest floor and soil Soil Sci Soc Am 1 58 974~980
Kuo WH Tam AW 1988 The pathway of water absorption of mung bean seeds Seed Sci amp Technol16(1) 139~144
Leadem CL 1997 Dormancy--unlocking seed secrets In Landis TD Thompson lR tech coords National Proceedings Forestand Conservation Nursery Assocs Gen Tech Rep PNW-G TR-419 Portland OR USDA For Ser PNW Res Sta 43-52
Lin Y Wagner MR Seweyestewa A Honahni MD DeGomez M McMillin JD 1996 Influence of cultural treatments on seed germination and seedling growth of Robinia neomexicana Forest Farm and Community Tree Res Reports Volume 1
Marunda CT 1990 Effects of seed pretreatment on the development ofAcacia auriculforinis and A holosericea seedlings ACIAR Proceedings Series No 28
Meginnis HG 1937 Sulphuric acid treatment to increase germination of black locust seed USDA Circ 453 34 p
Miklas PN Towensend CE Ladd SL 1987 Seedcoat anatomy and the scarification ofcicer milkvetch seed Crop Sci 27 766-772
Monsen SB 1984 Use of shrubs on mine spoils In The Challenge ofProducing Native Plants for the Intermountain Area Proceedings Intermountain Nurserymans Association 1983 conference Las Vegas NV compo PM Murphy 26-31 Ogden UT USDA For Ser Gen Tech Report INT-l68
Olson DF 1974 Robinia L Locust In Seeds of Woody Plants in the US tech coord CS Schopmeyer 728-731 USDA For Ser Agr Handbk No450
Rehman S Loescher RNl Harris P1C 1998 Dormancy breaking and germination ofAcacia salicina Lindl seeds Seed Sci amp Techno127 553-557
Rolston MP 1978 Water impermeable seed dormancy Bot Rev 44 365-396
74
SAS Institute Inc 1989 SASSTAT Users Guide Version 6 4th Edition Volume 1 Cary NC SAS Institute Inc 943 p
SerratoValenti G Conara L Ferrando M Modenesi P 1993 Structural and histochemical features ofStylosanthes scabra (Leguminosae Papilionoidae) seed coat as related to water entry Can J Bot 71 834-840
Simpson BJ 1988 A field guide to Texas trees Austin TX Texas Monthly Press 372p
Tran VN Cavanagh AK 1984 Structural aspects ofdormancy In Seed Physiology Vol 2 Murray DR (ed) p1-44 Academic Press Australia
USDA Forest Service 1948 Woody-plant seed manual USDA Misc Pub1654 416p
Vines RA 1960 Trees Shrubs and Woody Vines of the Southwest Austin Univ ofTexas Press 1104 p
Wagner MR Larson RE Craig TP 1992 Ecology and silvics ofNew Mexico locust (Robinia neomexicana) with an emphasis on the arboreal form p53-57 In JW Hanover K Miller and S Plesko (eds) Proc Intemat Confon Black Locust Biology Culture and Utilization Mich State Univ USA
Wilson JK 1944 Immersing seeds of species of Robinia in boiling water hastens germination J For 42 453-454
Zimmerman RW Carpenter SB 1980 First-year coppice production from a 5shyyear old black locust stand on surface mine spoil p309-314 Centr Hardwd For ConfProc University ofMissouri Columbia
INTRODUCTION
New Mexico locust (Robinia neomexicana A Gray) a small tree native to the
southwestern United States occurs at elevations from 1200 to 2800 meters This
species fills a successional role in post-disturbance situations As a primary invader
New Mexico locust quickly establishes on burned areas arid flood banks as well as
road cuts (Wagner et aI 1992) Rapid growth crown sprouting and prolific root
suckering favor the successful establishment ofNew Mexico locust on disturbed sites
(Simpson 1988 Gottfried 1980) These attributes also may make New Mexico locust
well suited for steep-slope revegetation where erosion is a problem
New Mexico locust is a nitrogen-fixing legume that tolerates and improves
nutrient-poor soils Stands ofNew Mexico locust increase Nitrogen (N) and Calcium
(Ca) in the forest floor Levels of Carbon (C) Phosphorous (P) Sulfur (8) and
Potassium (K) aIso have been shown to increase in soil beneath New Mexico locust
(Klemmedson 1994)
The ability to colonize rapidly and ameliorate harsh sites contributes to New
Mexico locusts potential as a nurse plant A nurse plant colonizes an inhospitable
site and creates an environment suitable for successional plant establishment New
Mexico locust demonstrates this role in the ponderosa pine (Pinus ponderosa Douglas
ex Lawson amp C Lawson)Gambel oak (Quercus gambelii Nutt) community After a
disturbance such as wildfire New Mexico locust and Gambel oak colonize and
dominate the site until shaded out by ponderosa pine Once shaded these species
become understory shrubs a process taking an average of 15-20 years (Dick-Peddie
1993 Hanks and Dick-Peddie 1974) New Mexico locust gradually declines with
small dense patches averaging about 005 ha beneath pine until the next disturbance
once again offers it a competitive advantage (Klemmedson 1994)
Gottfried (1980) expecting ponderosa pine regeneration to be greater where
New Mexico locust had been eradicated found that survival of planted pine seedlings
was greater where locust had not been removed In a follow-up study Gottfried
(1980) noted that soil moisture in the top 57 cm was highest in 5-year old locust sites
as compared to grass or 20-year old locust sites He concluded that managing an
appropriate cover ofNew Mexico locust could help regenerate pine the later
successional species
The ability ofNew Mexico locust to improve harsh sites also makes it a
candidate for reclaiming disturbances associated with mining Black locust (Robinia
pseudoacacia 1) has been used for decades to reclaim mine spoils and other
disturbed sites in the eastern US and around the world (Keresztesi 1988 Zimmerman
and Carpenter 1980) Ashby et al (1985) described similar positive attributes of
black locust also mentioned previously for New Mexico locust quick cover for
stabilization supply ofN and nutrient-rich litter to the soil and site improvement for
establishment of later successional trees Referring specifically to mine reclamation
the authors note the ability of black locust to grow on a wide range ofmine soil
conditions including extremely acid soils Black locust also shows some tolerance to
soils compacted by grading and topsoiling practices (Ashby et aL 1985) New
2
Mexico locust holds promise as a native southwe~tem counterpart to black locust in
mine reclamation but it has been used infrequently to date
Natural invasion and succession occur slowly on most mine sites (Monsen
1984) While New Mexico locust often colonizes sites naturally there is no
assurance a seed will reach a particular site and establish in a reasonable time frame
The challenge is to introduce New Mexico locust as mother plants to facilitate
colonization Seedling production for outplanting on disturbed sites has been
hampered by poor germination (Lin et al 1996) The goal then is to develop
techniques to overcome poor germination in New Mexico locust thereby facilitating
macropropagation
LITERATURE REVIEW
Seed Dormancy
Low gennination in New Mexico locust is primarily the result of seed
donnancy the failure of an intact viable seed to genninate under favorable
conditions (Bewley 1997) Seed donnancy also can be defined as the inability of the
embryo to genninate because of some inherent constraint (Bewley and Black 1994)
The constraint may originate within the embryo referred to as endogenous or embryo
donnancy or from the tissues surrounding the embryo known as exogenous or coatshy
imposed donnancy (Baskin and Baskin 1998 Leadem 1997 Bewley and Black
1994)
New Mexico locust seed is donnant when the impenneable seed coat does not
allow movement ofwater to the embryo This specific fonn of coat-imposed
donnancy where the restraining tissue involved is an impenneable seed coat is
known as physical donnancy (Baskin and Baskin 1998 Bewley and Black 1994)
Physical donnancy is relatively uncommon in seeds less than 10 of all woody
species exhibit this condition (USDA Forest Service 1948) However it is
characteristic of many genera of the Fabaceae (bean) family For example all
successional trees in the genera Gleditsia and Robinia (of which New Mexico locust
is a member) produce seeds that exhibit physical donnancy (Baskin and Baskin
4
Physical Dormancy
Seeds that exhibit physical dormancy are said to be hard seeded referring to
the thick impermeable seed coat (Leadem 1997) The first line of protection of the
testa in leguminous seeds is the outermost waxy cuticle followed by a subcuticle
consisting ofsuberin Next is a palisade layer consisting of sclereid cells that have
thick lignified secondary walls The most common type of sclereid cell in palisade
layers is the macrosclereid The macrosclereids contain water-repellent substances
including cutin lignin suberin and wax (Rolston 1978) This palisade layer is the
primary barrier to water uptake (Serrato-Valenti et al 1993 Tran and Cavanagh
1984)
All seed coat openings must be blocked to effectively prevent water uptake
The natural opening in a seed where it was attached to the mother plant is the weak
link where physical dormancy is usually broken (Hopkinson 1993 Kuo and Tam
1988) Anatomical structures associated with the seed coat opening vary from
family to family In the Papilionoideae the subfamily of the Fabaceae family
containing New Mexico locust the site ofwater entry is the strophiole The
strophiole is a swelling on the seed coat close to the stalk scar (Hopkinson 1997)
Kelly and van Staden (1987) refer to the strophiole site as the lens where
macrosclereid cells must separate and raise before water can pass through Since
middot6germination ofdormant seeds of the Papilionoideae takes place when the strophiole
permeable (Hopkinson 1993 Kuo and Tam 1988) a logical approach to
5
genninating New Mexico locust artificially would be to loosen the strophiolar cells
and allow water to pass through to the embryo
Evolutionary Role of Physical Dormancy
Why is physical dormancy a mechanism that blocks germination
ecologically advantageous When a plant produc~s seeds with different degrees of
dormancy seeds that are dispersed at one time will germinate over a range of time
(Bewley and Black 1994) Through variability of dormancy within a seed crop seeds
are exposed to changing environmental conditions improving the overall chances of
survival of the seed line (Bewley and Black 1994) Varying expression of dormancy
is under genetic control which indicates that this may be an evolved survival strategy
(Kigel and Galili 1995) A range of dormancy levels may be a particular advantage in
the southwestern US where extreme environmental variations exist
Methods for Breaking Dormancy
There are three common approaches to breaking dormancy in New Mexico
locust seed acid scarification hot water soaks and mechanical treatments In each
case the objective of the treatment is to scarify or abrade the seed coat to make it
permeable to water
Acid Scarification
Acid scarification consists of soaking seeds in concentrated sulfuric acid
rinsing thoroughly with water upon removal followed by subsequent drying before
PlaJlltmg Olson (1974) reports that this scarification method produces the highest
middot~~jllalUll results for all Robinia species Most literature refers to a seminal 1937
6
study conducted by HG Meginnis This stud~ t~ough conducted on black locust
seed is regularly cited in the suggested use of acid to treat New Mexico locust seed
as well (Olson 1974 Vines 1960)
Thee published studies document the specific use of sulfuric acid to treat
New Mexico locust seed Cox and Klett (1984) report that soaking seeds in
concentrated sulfuric acid for thirty minutes improved germination to 34 over an
untreated control of4 Lin et al (1996) found that for two seed lots acid
scarification for twenty-five to eighty minutes produced germination of 80 A third
lot however resulted in less than 20 germination at all durations of 10 to 80
minutes Hine et al (1997) showed that acid treatment levels of 1 2 5 and 10
minutes resulted in less than 20 germination across five sources However there
was an increasing trend in germination with acid soak duration and acid treatments
were not imposed for as long a time as the previous two studies (30 to 80 minutes)
The mixed results obtained by these investigators were probably due to the
widely varying germination response of seed lots to acid treatment For this reason
Olson (1974) recommends correlating duration of acid treatment to each seed lot If
treatment duration is too long the embryo will be permanently damaged If soak
duration is too short the seed will remain impermeable to water and fail to germinate
Compounding the problem of treatment duration is the negative effect acid
have on seedling growth Any type of scarification can injure seeds to some
by disrupting cells essential for growth leading to fungal invasion and
middot-uuu~w injury (Copeland and MacDonald 1995) Some legume seeds subjected
7
to 60 minutes of sulfuric acid scarification (to achieve maximum gennination results)
showed more than 50 seedling mortality after gennination (Kelly and van Staden
1987) Bewley and Black (1994) noted that fungal growth on seeds is an indication
of leakage of sugars organic acids amino acids and proteins
Perhaps the greatest drawback to acid scarification is the dangerous nature of
the treatment itself Difficulties associated with storage and trained use of
concentrated acid may discourage many propagators from using this option (Dreesen
and Harrington 1997) Consequently hot water and to a lesser extent mechanical
scarification are more commonly used and practical treatments for legume seeds
Most southwestern container growers use either of these two treatments to scarify
New Mexico locust seed in particular (Hine et al 1997)
Hot Water Scarification
The widespread use ofhot water scarification to treat locust seed is probably
due to its ease of application and efficacy in comparison to other scarification
treatments Although this practice had been in use for some time Wilson (1944) was
one of the first to report on its systematic application for black locust The treatment
consists of placing seeds in boiling water (4 parts water tomiddot 1 part seed by volume)
removing the vessel from the heat source and allowing the water to cool and seeds to
soak for 24 hours
Hine et al (1997) Lin et al (1996) and Cox and Klett (1984) have examined
the specific application ofhot water scarification on New Mexico locust seed Each
these authors compared hot water to acid scarification Lin et aL (1996) reported
8
60 gennination for two hot water-treated seed lots while a third lot germinated
around 50 (as reported above acid scarification germination ranged from 20 to
80 for these lots) It should be noted that the authors only soaked the seeds in water
from 10 to 80 minutes for this experiment as opposed to the standard 24 hours Cox
and Klett (1984) stated that using the standard 24-hour hot water treatment improved
germination over an acid pre-treatment (which produced less than 20 germination)
but they did not give specific gennination numbers for the hot water treatment Hine
et al (1997) found that across five seed lots germination was 66 for the standard
hot water treatment (as compared to less than 20 germination for acid scarification)
Variations in treatment intensity and duration and their subsequent effects on seed lot
appear to contribute to conflicting conclusions as to whether acid scarification or hot
water treatment is a more successful treatment
Given mixed results ease ofapplication is an important argument for use of
hot water application over acid soaks Safety concerns and material costs for hot
water treatments are low (Dreesen and Harrington 1997) An extension of this is the
practicality ofhot water for treating large batches of seed where a relative increase in
water volume is all that is needed However increases in water and seed even when
inthe same 4 1 proportion can result in dramatically different rates of cooling than in
atest tube experiment Different cooling rates can lead to contrasting results for the
treatment temperature duration and seed lot (USDA Forest Service 1948)
As with acid scarification parameters ofhot water treatment application may
to be optimized to maximize germination Hine et al (1997) found that as
9
temperature was increased from 70degC to 100degC overall germination in New Mexico
locust seed increased The 100degC treatment maximized germination for each of five
sources tested with germination usually above 60
Prolonged exposure of seeds ofmany woody legumes to boiling temperatures
also may improve germination (Briscoe 1996) This approach has not been tested on
New Mexico locust seed Exposing seed to prolonged boiling temperatures may
result in fungal infection and subsequent seedling mortality Marunda (1990) noted
severe fungal infections on both acid and hot water treated Acacia holoserica Cunn
ex G Don seeds attributing the infections to seed damage resulting from the
treatments Identifying an optimal hot water treatment level that does not result in a
high rate of seedling mortality is a logical goal
With hot water treatments as with acid there is also a danger of overtreating
Seeds and reducing germination (Gosling et al 1995) The legume Leucaena
leucocephala (Lam) de Wit germinated equally well after 7 seconds of exposure to
water at 100degC 7 seconds at 90degC 30 seconds at 80degC and 4 minutes at 70degC In
addition acceptably high germination was obtained from treatment at 70degC from 3
minutes to 1 hour whereas no seeds germinated following treatment at 100degC for 2
minutes Perhaps along with a greater margin of error for treatment duration treating
~ew Mexico locust seeds at a lower temperature may result in a comparatively lower
of seedling mortality In practice however nursery growers tend to use the
and quickest treatment available The short treatment length at near-boiling
whether recommended or not may be what is used most frequently
10
Fine-tuning a treatment in this range of treatment temperature and duration may be
the most logical approach
Mechanical Scarification
This treatment involves scratching the seed coat with an abrasive material
(USDA Forest Service 1948) One system employs a rotating drum lined with
sandpaper A paddle spinning at a given number ofrevolutions per minute throws the
seed against the sandpaper (Hine et al 1997) A second method dry tumbling
tumbles seed in a mixer with pea gravel or sharp sand for 5 to 7 days (Dreesen 2001)
These approaches have the advantage that control of temperature is not required as in
water treatments nor is safety an issue as with add scarification
Hine et al (1997) mechanically treated New Mexico locust seed using 100shy
grit sandpaper in a Forsberg (rotating drum) scarifier Gennination was less than
10 They concluded that the seed coats were too thin for their apparatus and
proposed that a finer grit sandpaper would produce more even abrasion Although
germination varied by seed lot Dreesen (2001) reported that using a slow-speed dry
tumbling technique for 5 to 7 days produced gennination rates as high as 90
As with acid and hot water treatments treatment duration must be specifically
shy
correlated to seed lot Treated seeds are also susceptible to fungal infection
iHopkinson 1997) In addition equipment is specialized and may not always be
ltU~CUJl~ to a nursery (USDA Forest Service 1948) Several studies have confinned
degrading ofstrophiolar cells due to acid and hot water scarification (Miklas et al
11
1987 Dell 1980 Hanna 1984) but the effect ofmechanical scarification on the
strophiole has not been studied
Alternative Method for Breaking Dormancy Percussion
Strophiole degradation has been examined in depth using an impaction
treatment more commonly known as percussion Hamly (1932) first proposed that
shaking or impacting seeds could split the macrosc1ereid cells at the strophiole
Hamly successfully released tension ofbent strophiolar cells of impermeable
Melilotus alba Medik (White sweet clover) seeds by delivering physical blows
resulting in cells that had split apart and were only slightly bent Ninety-two percent
oftreated seeds became permeable as compared to 05 of control seeds Barton
(1947) showed that percussion significantly increases permeability in black locust
seed Ballard (1976) reasoned that the integrated nature of the strophiole within the
seed coat means a blow on any part of the seed weakens the strophiole There are no
published studies on the use of percussion on New Mexico locust
Percussion methods vary and often seem SUbjective or ill-defined Hamly
(1932) placed seeds in a glass bottle and shook them three times a second for 20
minutes resulting in 180 oscillations per minute Ballard (1973) describes shaking
seeds in a closed metal container with at least fifty seeds per container at 850-900
JS(~l11alIOIlS per minute for 1 minute Kelly and van Staden (1987) placed seeds into
sealed glass flasks Flasks were shaken by a wrist-shaker at a rate of 250
~UUVJ~ per minute Subsequent germination rates were 45 after 2 hours of
55 after 3 hours 60 after 6 hours and 100 after 12 hours
12
There are several potential advantages of percussion over acid hot water and
mechanical scarification Kelly and van Staden (1987) note that a percussion
treatment only alters macrosclereid cells of the strophiole by loosening separating
and raising them They emphasize that minimal damage is done to the overall
anatomical structure of the seed In acid scarification of the same seed lot radicle
and cotyledon damage occurred Based on photographic evidence the authors
believe that this morphological damage was due to uneven pressure during
imbibition with water entering haphazardly wherever the acid had sufficiently
dissolved the coat This ultimately led to uneven hydration of the embryo In
percussed seeds water only entered through the s~ophiole when the entire seed coat
had been saturated resulting in controlled and even embryo hydration (Kelly and van
Staden 1987)
Although all successful scarification treatments may focus at least in part on
the strophiole for papilionoid legumes (Baskin and Baskin 1998) percussion may be
less damaging to seed coat structures particularly when compared with acid Kelly
and van Staden did not compare hot water or mechanical scarification methods
against percussion However the fungal infection associated with hot water and
mechanical scarification as with acid scarificatio~ have been directly associated with
seed damage (Rehman et al 1998 USDA Forest Service 1948) Embryo damage in
has been observed in hot-water scarified legume seeds (Rehman et al 1998
13
By limiting embryo damage percussion scarification reduces seedling
mortality compared to other treatments Kelly and van Staden (1987) noted that
seedlings from the percussion treatment were morphologically sound and vigorous
when compared to acid treatment seedlings The authors attribute this improved
seedling vigor to the controlled water uptake that limited anatomical damage
Despite a 12-hour percussion treatment morphological damage was not substantially
greater than untreated seeds In addition seedling mass measured 10 days following
germination was not significantly different between percussed and untreated seeds
A final advantage ofpercussion may be the ability to adequately treat the most
dormant seeds while avoiding harm to seeds that have already been made permeable
by the treatment or that were permeable to begin with Instead ofcorroding scalding
or scratching the seed coat percussion appears to be just a loosening of the
strophiole spring (Ballard 1973) Kelly and van Staden (1987) were able to
increase germination with a lengthy treatment (12 hours) Figuring out whether a
substantial cushion oftreatment durations exist for percussion ofNew Mexico locust
seed would be a significant improvement to current scarification methods where seed
damage and seedling health is an issue
The greatest challenge for applying percussion to New Mexico locust seeds is
standardizing a treatment that is both effective and readily reproducible by nurseries
with mechanical scarification this may require the development of specific
~UllDment to properly quantify and execute the treatment
14
f
Objectives
The first objective of the study is to detennine which scarification treatment
results in optimal gennination speed and percentage ofNew Mexico locust The
second objective is to describe treatment interactions with nine seed sources collected
in New Mexico
Hypothesis
A percussion treatment acts exclusively on the main area ofwater uptake in
New Mexico locust seed loosening the strophiole so that imbibition can occur This
direct mechanism ofdonnancy release will result in improved gennination rate and
overall gennination percentage for New Mexico locust seed compared to standard
treatments Standard treatments including acid hot water and mechanical
scarification while eventually weakening the strophiolar cleft cause excessive
morphological damage through generalized seed coat degradation These treatments
are therefore prone to inconsistency due to over and under-treatment
15
MATERIALS AND METHODS
Seed Collection and Cleaning
Seeds were collected from August 25 through September 15 2000 from nine
locations along a latitudinal gradient in New Mexico Seed source locations and
descriptive information are given in Figure 1 and Tables I and 2 Stands were
separated by at least 45 km
Pods were hand picked as they began to ripen and dehisce (open) Collections
were made from ground level to approximately 3 m from several trees at each site
Pods were dried in open-ended brown paper bags for two to four weeks at room
temperature (21-230C) to facilitate cleaning Dried pods were placed in a closed cloth
sack which was pounded against the ground to separate seeds from pods For pods
still containing seeds hand maceration (crunching pods with hands) completed seed
separation Debris was removed by threshing with the aid of a greenhouse fan A
South Dakota Seed Blower (Seedbureau Chicago Illinois) was used to remove
remaining chaff and light seeds by air density separation Seeds were placed in dry
storage in sealed quart-size zip-lock bags within a sealed plastic container Storage
temperatures ranged from 2-4oC Seeds remained in storage for approximately 7
months before the experiment
Experimental Design and Treatment Structure
The experiment compared the impact ofhot water bath and percussion
l11aLl11 on germination properties ofNew Mexico locust seed A single
16
-l
Figure 1 Seed source collection map All sources were collected within New Mexico USA separated by at least 4S lan
IIIRZltItW lIJPIirmiddot rllll1lbullbull_~~~_II
Table 1 Latitude longitude location elevation and collection date ofNew Mexico locust seed sources
Lot title Latitude (N) Longitude (W) Location Elevation (m) Collection Date
Raton 36deg5846 104deg2872 Raton Pass NM 2295 82500
Holman 36deg0306 105deg2429 HolmanNM 2476 911500
Jemez 35deg4500 106deg4100 Jemez Springs NM 1976 9900 00
Sandia 35deg1083 106deg2341 Cedar Crest NM 2432 82600
Manzano 34deg3689 106deg2494 ManzanoNM 2465 9200
Magdalena 33deg5953 107deg0903 Magdalena NM 2544 82900
Capitan 33deg2728 105deg4325 Alto NM 2189 911200
Sacramento 32deg5798 105deg4496 Cloudcroft NM 2630 91400 Gila 32deg5400 107deg4600 Kingston NM 2432 9700
f
Table 2 Mean seed weight and standard error by source
a
Source Mean seed weight (g) for Standard error 100 seeds n = 5
Raton Holman Jemez Sandia Manzano Magdalena Capitan Sacramento Gila
2872 b c 2522 d 1826 e 2753 c 2413 d 2559 d 2443 d 3130 a 3029 ab
00103 00403 00276 00301 00370 00374 00458 00279 00348
a Mean seed weights followed by the same letter are not significantly
different
19
experiment was conducted utilizing a randomized complete block design blocked by
time with a factorial treatment structure Experimental factors were seed source and
scarification treatment Nine seed sources from throughout New Mexico were used
(Figure 1 Table 1) Scarification treatments included a control three levels ofhot
water and four levels of percussion Hot water treatments included immersion at
100degC and boiling for periods of 30 seconds and 60 seconds Percussion treatment
levels were 1 2 4 and 8 minutes One hundred seeds per block were used to test
each treatment combination Four blocks of seed were tested one at a time (all seed
sources included in each run) in close chronological sequence (within 6 weeks) A
total of four 100-seed samples were used to test each source by scarification treatment
combination
Treatments
Hot water treatment of seeds utilized 50 ml test tubes filled with 30 ml
distilled water Each test tube was used to treat one 100-seed sample at a time
resulting in a ratio of4 parts water to 1 part seed by volume Test tubes were placed
in a beaker with the beaker filled with tap water to the same level as the water within
test tubes Nine test tubes (one per seed source) were placed in the beaker
simultaneously so that one replication of each seed sourcescarification treatment
cotnblnatlon was conducted simultaneously Hot water treatments include immersion
boiling For immersion treatments seeds were added to test tubes with the water
(boiling) and the test tubes were immediately removed from the beaker to
For the boiling treatments test tubes remained in the beaker with water at
20
boiling temperature for a set duration after the addition of seeds Following all
treatments test tubes with seeds cooled 24 hours in a test tube rack
Percussion treatment utilized a pneumatic paint shaker (Central Pneumatic
model 00422 Camarillo CA) Compressed air was maintained at 530 kPa plusmn30 kPa
(80 psi plusmn 5 psi) resulting in approximately 350 oscillations per minute Seeds were
placed in 118 ml (4 oz) soil tin cans (US Can Lobard IL) and the lids were secured
with duct tape Each can was used to treat one 100-seed sample To take advantage
ofgreatest lateral movement of the paint shaker a spacer (paint can) was used to
position the soil tin at the end of the shaking arm This allowed shaking to occur at
the greatest distance from the pivot point (fulcrum) Tins were placed perpendicular
to the direction of shaking motion allowing seeds to impact at greatest force by
hitting flat surfaces of the soil tin
Germination Testing
Before gennination testing all seeds were soaked 24 hours in reverse
osmosis-treated water (this was included in the process ofhot water treatment
jU~SCIlOea above) Following soaking seeds were placed in 100 cm Petri dishes lined
90 cm Whatman qualitative 1 filter paper circles (MG Scientific Pleasant
WI) One Petri dish was used for each treatment by source combination
papers were initially moistened with 5 mL reverse osmosis-treated water then
~VU1CUlJl moistened as needed to ensure adequate conditions for gennination
were monitored daily (at 300 PM) for gennination for 14 days in accordance
~UIUlnJlVUa Seed Testing Association (ISTA) standards for Robinia species
21
(1STA 1999) Tests were carried out at room temperature (22degC plusmnOSoC)
Germination was classified as protrusion of the radicle from the embryo by at least
1mm as viewed with the naked eye
X-ray Radiography
On August 132001 three lOO-seed samples from the Sacramento seed source
were treated for 14 or 8-minute percussion durations The seeds were sent
immediately by overnight delivery to the Faxitron X-Ray Corporation in Wheeling
Illinois where Dr Bob Karrfalt ofthe USDA Forest Service oversaw their imaging
Radiographs were made at SX with a Faxitron Specimen Radiography System Model
MX-20
Scanning Electron Microscope Photography
On February 192002 a Hitachi S-3200N Variable Pressure Scanning
Electron Microscope (Mountain View CA) was used to examine strophiolar cells of
untreated seeds and seeds percussed for 4 minutes Seeds were cut longitudinally
igt their entire length using a razor blade splitting the strophiolar region roughly in
Cross sections of seed samples magnifying the strophiolar region were viewed
600X under the microscope
Analysis
Categorical Analysis of Variance was used to determine treatment source and
(Le time) main effects and interactions The response variable was total
~vlI This procedure is a Chi-square test of homogeneity using the natural log
of germinated to non-germinated seed for each treatment Maximumshy
22
likelihood analysis was used to calculate Chi-square test statistics with their observed
significance levels A single contrast was conducted to cempare treatment means
against the control Observed significance levels less than or equal to 005 were
considered significant Pairwise comparisons were conducted between non-control
treatment mean percents using a conservative alp~a value of 005 divided by the
number of comparisons (Bonferroni approach) and approximate pairwise z statistics
Categorical analyses were performed using Proc Catmod (SAS Institute 1989)
RESULTS
Scarification seed source block and all interactions between factors
influenced germination (Table 3) The main effects are reported in the presence of
these interactions because the overall robustness of treatments and seed sources are of
interest Background levels ofnaturally occurring variation in seed sources are
seen in control germination percentages in Figure 2 In particular time variability
is unlikely to be under the control of the producer Appendix B details experimental
data for source by treatment gennination percents for each of the four blocks
Averaged over all treatments germination by source ranged from 40 to
79 (Figure 2) Averaged over all sources (Figure 3) and for each source (Figure 4
5) percussion scarification durations of4 or 8 minutes yielded greatest germination
Averaged over all sources germination increased with increasing durations of
percussion then leveled off for the 4- and 8-minute treatments (Figure 3) Immersion
and 30-second boiling scarification maximized germination for the hot water
treatments with a decrease in germination when seeds were boiled for 60 seconds
For all but two sources there is a similar ipcreasing trend in germination from
1to 4 minutes ofpercussion (Figure 4) The Holman source increased in germination
1 to 2 minutes then leveled off at 4 minutes The Jemez source changed little
1 to 4 minutes of treatment As treatment duration increased from 4 to 8
mutes five sources decreased in germination (Figure 4a) whereas three sources
24
Table 3 Categorical Analysis of Variance table for germination response to seed treatment seed source block and the interaction of the factors
Source of DF Chi-Square Observed Variability Significance Level
Source 8 98370 lt00001 Trt 7 233876 lt00001
contrast 1 16976 lt00001 SourceTrt 56 97077 lt00001 Block 3 5538 lt00001
SourceBlock 24 10162 lt00001 TrtBlock 21 7564 lt00001
SourceTrtBlock 168 34283 lt00001
S 4)
8 ~
0
sectg ~ 40 ~
N 0 0
20
o f0ltgt ~ ~4 jybshy ((iltgto ~~ K1ltgt 6~b-~ ~~ 0b-~~ ~~ ~b-~ ~b-~b
80
60 -I-- - ~- middotmiddotmiddotmiddotmiddot1
r -] Northern NM Sources
Central NM Sources
Southern NM Sources
sect-o ~
~b-v(i
SOURCE
Figure 2 Effect ofNew Mexico locust seed source on germination for data averaged over all scarification treatments Control germination is shown by solid line within bar
80
~ cd ~ ltl) () 60 ~ ~
N
t 0shyrti tshySltl)
40
0
20
o lmmersion Boiling 30 sec Boiling 60 sec Control 1 min 2min 4 min 8 min
Hot Water Treatments Percussion Treatments
Figure 3 Effect of scarification on germination ofNew Mexico locust for data averaged over all seed sources Means labelled with the same letter are not significantly different
100~----------------------~--~----------------~
80
60
ltU
~
40 IJ
sectgsI
l100
80
60
40
a I middotmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot middotImiddotmiddotmiddotmiddotmiddotmiddot E
_~iii-bull _ _ bull f --~--
t-- -~ ---- -shy ---- shyI ~ ---- - shy
L
fiII
~
b
- shy ---r
- Holman
Sacramento Sandia Gila Jemez
c ~
ltgt ~
Tmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot-I
j~=~=-~~-1
I middotmiddot ~ bull r
t tr
Y -------M-a-gd-a-Ie-n-a--
1 _ _
fCapitan
---- Manzano ~y -_ Raton
2 4 8
Time interval ofpercussion scarification (min)
Figure 4 Effect of seed source by treatment interaction on New Mexico locust germination for percussion scarification pattern a and pattern b
28
tv 0
~ d
~ A s o
1 c3
80
60
40
20
o
TIT
t------=middotfmiddotmiddot-middotmiddot-middotmiddot-middotmiddot 1- --- - - --=- - -shy--cE= - _--shy --shy ----shy ~ ~ ---shy ~ -~- _ --shy-------r 1
I
Raton Holman
-_ Jemez
--- Sandia Manzano Magdalena Capitan Sacramento Gila
Immersion Boiling 30 sec Boiling 60 sec
Hot Water Scarification
Figure 5 Effect of seed source by treatment interaction on New Mexico locust germination for hot water scarification
continued to increase in germination (Figure 4b) There was no difference between
the two treatment levels for the Manzano source (Figure 4b)
The effect ofhot water scarification also varied among sources with few
overall differences between hot water treatments (Figure 5) However boiling for 60
seconds yielded highest germination for the Jemez source whereas increasing
durations ofboiling decreased germination for the Raton source
In addition to consistently high germination 4- and 8-minute percussionshy
treated seeds germinated as fast or faster than all other treatments (Figure 6 Table 4)
050 measured as the number of days needed to complete 50 ofeventual
germination averaged 2 days for both ofthese treatments
X-ray radiographs were taken of seeds collected from the Sacramento source
percussed for 1 4 or 8 minutes (Figure 7) Seeds show signs of damage at 8 minutes
ofpercussion (Figure 8) Compared to an undamaged seed percussed for only 4
minutes (Figure 8a) damage at the 8-minute level includes fractured radicles pitting
of the seed coat and chips in seed coat structure (Figure 8b-d)
Scanning electron microscope (SEM) images at 600X reveal the direct effect
of 4 minutes of percussion on cells of the strophiolar region (Figure 9) Whereas
untreated seeds retain cellular organization in the strophiolar region strophiolar cells
seeds treated for 4 minutes ofpercussion separate and raise resulting in a
~-~e~U appearance
30
Umiddot-~middot--l-~---middotymiddotmiddotmiddot-----middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot--middot--middotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middotmiddotmiddotmiddotmiddotmiddotmiddot- -
050= gt1
-e-- Percussion 8 min -0- Percussion 4 min ---T- Percussion 2 min -----ltil- Percussion 1 min -- Immersion --0- Boiling 30 sec -+- Boiling 60 sec -ltgt- Control
2 4 6 8 10 12
Day Figure 6 Timecourse ofNew Mexico locust gennination following scarification
treatments G50 is the number of days to 50 of eventual germination
31
14
Table 4 Effect of seed source and treatment on G50 (days to 50 germination)
Seed Source Control Immersion B30sec B60sec Plmin P2min P4min P8min mean Raton 3 3 5 7 4 3 2 2 4 Holman 2 2 2 2 2 2 2 2 2
Jemez 4 6 6 5 3 2 2 2 4 Sandia 2 2 2 2 2 2 2 2 2
w tv Manzano 5 2 2 3 3 2 2 2 3
Magdalena 4 2 2 3 4 3 2 1 3
Capitan 5 4 2 2 3 2 2 2 3
Sacramento 3 2 2 2 4 3 2 2 3 Gila 3 2 2 2 2 2 2 2 2 mean 3 3 3 3 3 2 2 2 3 standard 11 14 16 18 09 05 0 03 08 deviation
Note Columns and rows may not agree due to rounding
Figure 7 X-ray radiographs ofSacramento seed source percussed for 1 4 or 8 minutes
33
Figure 8a-d Seed damage close-ups Figure a shows undamaged seed percussed for 4 minutes Figures b-d show seeds percussed for 8 minutes Damage includes fracturing ofradicle in b complete removal ofseed coat in c and pitting ofseed coat in d
34
Untreated Treated
Figure 9 SEM images ofuntreated vs 4-minute percussion-treated strophiolar cells ofNew Mexico locust seeds Treated cells are loosened separated and raised
DISCUSSION
Determining the Optimal Treatment
Averaging results of scarification treatmems across seed sources is a useful
tool to predict which treatment is most likely to be successful for an unknown seed
source Averaged over all sources four- and eight-minute percussion treatments
resulted in the highest germination 815 and 790 respectively and produced
germination that was as fast or faster than all other treatments Although there was no
difference between these two treatments on average an argument for 4 minutes of
percussion as a standard protocol for treating New Mexico locust seeds can be made
Descriptively 4 minutes of percussion improved germination over 8 minutes
ofpercussion for a majority of sources (five out of nine) 8 minutes of percussion
improved germination for three ofnine sources with no difference for a fmal source
For individual sources 4 minutes ofpercussion was always the best or secondmiddot best
treatment and was always equal to or better than all hot water treatments
Germination at 8 minutes ofpercussion was lower than all other percussion durations
for the Jemez and Gila sources and was lower than at least one hot water treatment for
these sources as well
The x-ray radiographs of the Sacramento seed source illustrate the excessive
internal and external seed damage that can occur with 8 minutes of percussion
germination counts 8-minute percussed seeds from Holman and Capitan
ClUll signs of damage that were visible to the naked eye (see below)
r~evertheless some of these seeds still fit the criteria for germination Neither x-ray
36
photography nor visible observations during seed counts revealed seed damage at the
4-minute percussion level In operational trials seeds percussed for 4 minutes
developed into healthy seedlings (Khadduri and Harrington unpublished data)
Strophiole and Seed Coat Behavior
Four minutes ofpercussion successfully breaks seed dormancy in a large
percentage ofNew Mexico locust seeds without excessively damaging the
surrounding seed coat or internal seed coat structures Most papilionoid legume
seeds including seeds ofNew Mexico locust have a specific region of the seed coat
known as the strophiole or lens (Hamly 1932) The strophiole is located on the
cotyledonary lobe of a locust seed (Figure 10 a-c) If seeds are not indiscriminately
damaged the strophiole is the first point of water entry to the embryo (Hopkinson
1997) When a papilionoid seed is percussed for the appropriate amount of time
repeated hits on the integrated seed coat loosen the constrained cells of the strophiolar
region without excessively damaging the rest of the seed coat (Ballard 1976) SEM
photos illuStrate that 4 minutes of percussion loosened and separated strophiolar cells
ofNew Mexico locust seeds when compared with constrained organized strophiolar
cells of untreated seeds (Figure 9) Research-has shown that water passes to the
embryo through the treated strophiole in a controlled manner and regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues
(Kelly and van Staden 1987)
37
10 Strophiole ofunimbibed New Mexico locust seed (a) and following uptake (bc)
38
In contrast hot-water treatments can excessively degrade the seed coat
(Rebman et al 1998 Marunda 1990) Random cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rebman et aI 1998 Marunda 1990) Along with a
proportionately large amount ofun-imbibed (thus under-treated) seeds (Figure l1a)
damage was observed in hot-water treated seeds from six sources during germination
counts Damage included seeds where the radicle failed to elongate (Figure 11 b)
seeds where the radicle elongated but was delayed in freeing itself from the seed coat
(Figure 11c) and seeds where the radicle completely broke off from the embryo
(Figure lId) In addition some seeds germinated in reverse (the cotyledon emerged
fIrst) If the radicle emerged completely from the seed coat they were counted as
germinated
While nearly all seeds treated for 8 minutes ofpercussion imbibed water
damage was evident in x-ray evaluations (Sacramento seed source) and in
germination counts (Holman and Capitan) at this treatment level Seed damage
during germination counts was similar to levels found in hot-water treated seeds
suggesting that over-treatment by both hot water and longer durations of percussion
may result in similar damage to seed structure
The Sacramento and Holman seed sources were two of the fIve sources that
opcrplltlpn in germination from the 4- to 8-minute durations of percussion
the benefIts ofloosening the strophiole as evidenced by imbibition of
all 8-minute treated seeds were outweighed by external and internal seed coat
39
a
b c d
11 Damage to New Mexico locust seeds observed during germination cotmts following hot water scarification 9a Hot water replicate including ~t seedlings and numerous unimbibed seeds 9b Close-up of seed where raallCle failed to elongate 9c Close-up ofseed where radicle elongated but was
-gtIn in freeing itself from the seed coat 9d Close-up ofseed where radicle broke off from embryo
40
damage (which also may be contributing to high imbibition) For the three sources
for which 8 minutes of percussion resulted in maximum germination extended
percussion of seeds necessary to loosen the strophiole may not have excessively
damaged the seed anatomy The Manzano source did not significantly change in
germination from 4 to 8 minutes of percussion suggesting that an increase in
permeable seeds at the 8-minute treatment duration was balanced by the number of
seeds that failed to germinate due to seed damage To summarize the 4-minute
percussion treatment improved germination over 8 minutes ofpercussion for a
majority of sources (five ofnine) the 8-minute percussion treatment improved
germination over 4-minutes ofpercussion for three sources and there was no
difference with the Manzano source Notably for the Raton source 8 minutes of
percussion increased germination by 235 percentage points over 4-minutes of
percussion (7875 vs 5525 respectively) the next best treatment Raton was the
only source to result in germination less than 75 for 4 minutes ofpercussion so a
grower may wish to apply longer percussion durations to lots that do not respond well
to this treatment level
Finally there was an interesting trend in the Jemez and Raton sources seed
that significantly contributed to the source by treatment interaction (Figure 12) A
~u test showed that the Jemez source had a significantly lighter seed weight than
other sources while the Raton source had an average seed weight For the Jemez
the longest duration of boiling increased germination Conversely only short
U4UVll) of percussion were needed to achieve high germination and the longest
41
80 _ Jemez a Raton
iD
~ C iD e iD
Q c 0 ~
c ~ iD 0
20
0------shy
Immersion Boiling 30 sec Boiling 60 sec
Hot Water Scarification
10degTr========~--------~------------------------------
80
60
40
20
_ Jemez Raton
0------shy
1 min 2 min 4 min 8 min
Percussion Scarification
Figure 12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources
42
b
--~----~~~~========~---- duration ofpercussion reduced germination For the Raton source longer durations
ofboiling consistently decreased germination whereas longer percussion treatments
consistently increased germination
A possible explanation is that a thick seed coat is associated with a loose
strophiole structure and that a thin seed coat is associated with a constrained
strophiole This contrast merits further study and may suggest an ecological
adaptation Further investigation ofthis phenomenon may contribute to successful
pre-screening ofunknown seed sources to determine optimal treatment leveL
43
CONCLUSION
Based on results obtained in pilot study (see Appendix) and thesis
experiments 4 minutes ofpercussion is recommended as a standard protocol for
treating an unknown source ofNew Mexico locust seed In addition to laboratory
results the 4-minute percussion treatment has resulted in germination greater than
80 and healthy seedling grow-outs for production purposes (unpublished data) In
laboratory experiments only one hundred seeds per 118 ml (40z) soil tin were
percussed at a time whereas for production we have treated up to five hundred seeds
per tin For large-scale production ofNew Mexico locust seedlings growers may
wish to experiment with larger quantities of seed and larger-size soil tins
Seed storage trials following percussion treatments were not conducted
However germination greater than 80 and healthy seedlings were obtained when
seeds were percussion-treated and subsequently stored at room-temperature (22degC)
for one week (unpublished data) Seed storage trials merit further study as the
standard hot water treatment leaves seeds in a softened condition that is not
conducive to storage
Hydroseeding is an effective method for direct seeding in reclamation
situations Percussion treatment may be conducive to hydro seeding as seeds remain
a dry relatively undamaged yet permeable condition following treatment Sulfuric
seeds also remain dry following treatment but extensive damage to seed coat
poor seedling vigor have been reported with this treatment (Kelly and van Staden
A grow-out study using a hydro seeder on a reclamation site testing percussion
44
against other treatments such as sulfuric acid and mechanical scarification also
merits further study
Papilionoids are the largest subfamily oflegumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust these legumes often play an integral role in the revegetation of
disturbed lands The pilot study (Appendix) indicates that percussion scarification
successfully treats the widely used black locust as well Developing superior
scarification methods to the standard hot water mechanical scarification and acid
treatments may facilitate the use ofpapilionoid legumes in reclamation projects
45
APPENDICES
APPENDIX A
PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR
NEW MEXICO LOCUST AND BLACK LOCUST SEEDS
Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4
Hot water and sulfuric acid soaks are traditional treatments for seeds of many
temperate woody legwnes including locusts However these scarification techniques
often produce inconsistent germination Percussion scarification where seeds are
repeatedly propelled against a hard surface was compared with hot water
scarification to evaluate treatment efficacy for New Mexico locust (Robinia
neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment
seeds were placed in a 98degC water bath which was immediately removed from the
heat source For percussion scarification seeds were placed in a soil sample tin and
agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the
control were followed by 24-hour water soaks Hot water baths resulted in 56 and
41 germination for New Mexico locust and black locust respectively For both
species nearly all durations ofpercussion increased germination over the hot water
treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3
4 and 5 minutes for black locust resulted in at least 90 germination Traditional
scarification treatments randomly degrade the entire seed coat which can lead to
tissue damage during water uptake Percussion scarification specifically weakens the
strophiole the natural source ofwater entry to the seed in papilionoid legwnes
INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom
2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031
48
Following percussion imbibition is controlled through the strophiole and underlying
tissue is protected
Keywords New Mexico locust black locust scarification percussion germination
Introduction
Locust trees (Robinia spp) are aggressive pioneer species that quickly
colonize disturbed land fix atmospheric nitrogen in the soil and stabilize slopes that
are prone to erosion (Klemmedson 1994 Ashby et al 1985) (Figure A1ab)
Recognition of these attributes has contributed to increased demand for locusts in
reclamation projects Locust seeds however exhibit physical dormancy where a
thick seed coat prevents the movement of water and gasses to the embryo (Leadem
1997)
Sulfuric acid or hot water soaks have traditionally been used to break seed
dormancy in macro-propagation of locusts Many growers are moving away from the
use of su1furic acid in treating locust seeds (Dreesen and Harrington 1997) In
addition to being dangerous acid soak durations must be specifically correlated to
seed lot (Olson 1974) Collections of native seed can vary tremendously in seed size
seed weight and hard seededness etc This variability limits the utility of sulfuric
acid soaks as even within a given lot some seeds may be damaged whereas others
are under-treated and fail to imbibe (take up water) A survey of Southwestern
container growers indicates that hot water baths are currently the most common
scarification method for locust seeds (Hine et al 1997) However hot water baths
also produce inconsistent germination (Lin et al 1996)
49
b
Figure AI New Mexico locust a primary invader following disturbance Ala Crown-sprouting at edge ofburn near Los Alamos NM--one year after Cerro Grande fire Alb Colonizing road cut Gila National Forest NM
50
Percussion scarification where seeds are repeatedly propelled against a hard
surface is an alternative dormancy-breaking method that has proven successful in
legumes related to locusts (Hamly 1932 Barton 1947) We compared this alternative
treatment to hot water scarification to evaluate treatment efficacy for New Mexico
locust and black locust seeds
Materials and Methods
Commercial New Mexico locust seeds (Western Native Seed Coaldale CO)
were collected fall 2000 in Huerfano County CO Black locust seeds were collected
September 2000 from Taos County NM by harvesting ripe pods from several trees at
a distance of up to 3 m from ground level Black locust seeds were allowed to air-dry
for 3 weeks and were threshed and separated from large chaffusing a greenhouse
fan A South Dakota Seed Blower (Chicago Illinltois) was used to remove fine chaff
from both seed lots Seeds were storedmiddotat 2-4C until the germination study was
conducted in March 2001
This study compared hot water scarification with percussion scarification
using treatment and species as experimental factors Along with a control
scarification treatments included immersion of seed in boiling water as well as
percussion durations of 1 2 3 45 or 10 minutes Four 100-seed samples were used
to test each species by scarification treatment combination
For the hot water treatment test tubes were filled with 30 mL water and
placed in a water-filled beaker (Figure A2a) The bath was raised to boiling (-98C at
51
b
c
Figure A2 Implementation ofhot water and percussion scarification A2a Hot water bath A2b Paint-shaker set-up A2c Placing soil tin containing seeds at end ofpaint shaker ann
52
Las Cruces NM elevation of 1300 meters) Each 100-seed replicate was placed in a
test tube and each test tube was immediately removed to cool at room temperature
Percussion scarification was implemented using a pneumatic paint shaker
(Central Pneumatic model 00422 Camarillo CA) (Figure A2b) This allowed
standardization of treatment intensity Compressed air pressure was maintained at
530kPa plusmn 30kPa (80psi plusmn 5psi) resulting in approximately 350 oscillations per
minute Each lOO-seed sample was placed in a 118 ml (4 oz) soil tin for shaking To
maximize lateral movement of paint shaker a paint can was used as a spacer to place
soil tins at the end of the shaking arm (Figure A2c) This allowed shaking to occur at
the greatest distance from the pivot point (fulcrum) Following all treatments
including the control seeds were soaked for twenty-four hours in distilled water prior
to plating out
Gennination was evaluated on lab benches at room temperature Each
replicate of seeds was placed in a 100 cm petri dish on moistened filter paper with
humidity maintained by enclosing the petri dishes in ziplock bags Gennination was
monitored daily for 14 days in accordance with International Seed Testing
Association standards for Robinia species (ISTA 1999) Gennination was defined as
protrusion of the radicle from the embryo by at least 1mm as viewed by the naked
eye
Categorical Analysis of Variance using Prpc Catmod (SAS Institute 1989)
was used to detennine treatment differences in germination percentages for each
source This procedure is an extension of a Chi-square test of homogeneity using the
53
natural log of the ratio of germinated to non-germinated seeds for each treatment Pshy
values less than 005 were considered significant A limited set of pairwiseshy
comparisons was conducted to compare treatment mean percents using a
conservative alpha value of 005 divided by the number of comparisons
Results
Percussion maximized germination for both New Mexico locust and black
locust There were three germination response levels for New Mexico locust (Figure
A3) First there was a low response in the control indicating a dormant seed lot
The hot water treatment and I-minute duration of percussion share a second
intermediate response Germination was highest at percussion levels from 2 to 10
minutes
A selected percussion treatment intermediate in time (5 minutes) produced the
steepest germination curve indicating the fastest germination rate (Figure A4) The
hot water treatment produced an intermediate curve or relatively slower germination
rate The control curve was very flat indicating the slowest germination rate for New
Mexico locust
Control seeds germinated poorly for black locust as well (Figure A5) Hot
water scarification improved germination over the control but all levels of percussion
improved germination over hot water The optimal durations of percussion treatment
were bracketed with a significant increase from the 1 to 2 minute percussion level a
high germination response from 2 to 5 minutes of percussion and finally a significant
54
~ d 25 CL)
p t1 0 ~ t1
g Vl ~ Vl
100~---------------------------------------------------------------
80
60
40
20
o Control Hot Water Percussion
I min Percussion
2 min Percussion
3 min
Scarification
Percussion 4 min
Figure A3 Effect of scarification on germination response for New Mexico locust
Percussion 5 min
=significant difference between adjacent treatment bars ns = no significant difference
Percussion 10 min
~ ~ () u agt
Il-t d o ~
~ ~ c3
100middotlr~~~------------------------------------e- Percussion 5min middot 0middot Hot Water -- Control
80-J=middot======
6b-middot-lmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middot--middotmiddot-middot~-middotmiddot -middotmiddot middot middotmiddot-middot 1
40
20
-----~-----------
0 0 0 middotmiddotmiddotmiddotmiddot0middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot0
0 --~-----y-----~------_---~--- T-----~
o shy -TDay 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A4 Effect of selected scarification treatments on germination speed for New Mexico locust First seven days of fourteen-day experiment are shown
Day 7
80
~ cd
5 60 ()
[) Cl d 0 1t1 d
40sect dUl
-J
20
o
ns
Scarification
Figure A5 Effect of scarification on germination response for black locust = significant difference between adjacent treatment bars ns = no significant difference
decrease at the 10-minute level This drop-off suggests that 10 minutes of percussion
over-treats seeds from this lot of-black locust
A selected percussion treatment intermediate in time (5 minutes) also had the
fastest germination rate for black locust (Figure A6)_ The hot water curve is
somewhat flatter indicating a slower germination rate The control curve is very flat
indicating a slow germination rate
A qualitative difference between hot water and percussion-treated seeds was
also observed Along with healthy germinants the hot water treatment produced
over-treated seeds (Figure A 7a) Examples of damaged germinants include seeds
where the radicle did not elongate (Figure A 7b) seeds where the radicle elongated
but was delayed in freeing itself from the seed coat (Figure A7c) and seeds where the
radicle broke off completely from the embryo (Figure A7d) In addition to these
examples of over-treatment there were numerous small dark seeds that failed to
imbibe water For percussion treatment nearly all seeds took up water (Figure A8a)
Germination was rapid and uniform with little damage evident (Figure A8b)
To summarize both New Mexico locust and black locust seed lots were
dormant The standard hot water scarification treatment improved germination but
only to 56 and 41 for New Mexico locust and black locust respectively Nearly
all percussion durations improved total germination as well as germination rate over
the hot water treatment Germination was greater than 90 for percussion durations
of 4 5 and 10 minutes for New Mexico locust and 3 4 and 5 minutes for black locust
58
CI)
sgt t CI) 0
igt 0
sect ttl t E
Vl 0
c3
100r==~==~~=r------------------------------------~ ___ Percussion 5 min
middotmiddotmiddot0middotmiddotmiddot Hot Water -lIf- Control
80r=========~--~~----------------------------~
60
40
20 -~-~---~---------- ---~------------I
omiddot
O
_~_----------lIf-----~bull -----_ shy---shy I
Day 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A6 Effect of selected scarification treatments on germination speed for black locust First seven days of fourteen-day experiment are shown
Day 7
a
b c d
Figure A7 Damage to New Mexico locust seeds observed during germination counts following hot water scarification A7a Hot water replicate including damaged seedlings and numerous unimbibed seeds A7b Close-up ofseed where radicle failed to elongate A7c Close-up ofseed where radicle elongated but was delayed in freeing itself from the seed coat A7d Close-up ofseed where radicle completely broke offfrom embryo
60
a
b
Figure A8 New Mexico locust seeds following percussion treatment at time of germination counts A8a Percussion replicate with uniformly healthy seedlings and near complete imbibition of seeds A8b Close-up ofpercussion-treated seedling
61
Discussion
Papilionoid seeds have a specific anatomical feature known as the strophiole
or lens-the natural site of water entry to the seed (Hamly 1932) The strophiole is
located on the cotyledonary lobe of a locust seed (Figure A9) When a papilionoid
legume seed is percussed for the appropriate amount of time repeated hits on the
integrated seed coat loosen the constrained cells of the strophiolar region without
excessively damaging the rest of the seed coat (Ballard 1976)
When a percussed seed is soaked water enters exclusively through the
strophiole in a controlled manner (Kelly and Staden 1987) This regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues This
contrasts with seeds that have been hot water or acid scarified treatments that can
randomly degrade the seed coat Unlocalized cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rehman et al 1998 Marunda 1990)
Papilionoids are the largest subfamily of legumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust and black locust these legumes often play an integral role in the
revegetation ofdisturbed lands Developing superior scarification methods to the
standard hot water and acid treatments should fadlitate the use ofthese legumes in
restoration projects
62
Figure A9 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (bc)
63
APPENDIXB
GERMINATION PERCENTS AND STANDARD ERRORS FOR SOURCE
BLOCK AND TREATMENT FOR NEW MEXICO LOCUST
Table B 1 Gennination percents and standard errors for source block and treatment for New Mexico locust
Source Block Treatment Germination Percentage Standard Error
Capitan 1 Immersion 74 437 Capitan 2 Immersion 69 462 Capitan 3 Immersion 71 454 Capitan 4 Immersion 67 470 Capitan Boiling 30 sec 79 407 Capitan 2 Boiling 30 sec 76 427 Capitan 3 Boiling 30 sec 72 449 Capitan 4 Boiling 30 sec 76 427 Capitan Boiling 60 sec 71 454 Capitan 2 Boiling 60 sec 54 498 Capitan 3 Boiling 60 sec 67 470 Capitan 4 Boiling 60 sec 76 427 Capitan I Control 5 218 Capitan 2 Control 7 255 Capitan 3 Control 4 196 Capitan 4 Control 5 218 Capitan 1 Percussion I min 47 499 Capitan 2 Percussion I min
470
Capitan Percussion I min 39 488
Capitan 4 Percussion I min 38 485 Capitan I Percussion 2 min 55 497 Capitan Capitan
2
Percussion 2 min Percussion 2 min
71 65
454 477
Capitan 4 Percussion 2 min 56 496 Capitan I Percussion 4 min 86 347 Capitan 2 Percussion 4 min 79 407 Capitan 3 Percussion 4 min 81 392 Capitan 4 Percussion 4 min 82 384 Capitan I Percussion 8 min 90 300 Capitan 2 Percussion 8 min 88 325 Capitan 3 Percussion 8 min 90 300 Capitan 4 Percussion 8 min 90 300 Gila I Immersion 71 454 Gila 2 Immersion 77 421 Gila
Immersion 75 433
Gila 4 Immersion 72 449
Gila I Boiling 30 sec 68 466
Gila 2 Boiling 30 sec 81 392
Gila 3 Boiling 30 sec 54 498
Gila 4 Boiling 30 sec 63 483
Gila I Boiling 60 sec 55 497
Gila 2 Boiling 60 sec 68 466
Gila 3 Boiling 60 sec 57 495
Gila 4 Boiling 60 sec 70 458
65
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Gila 2 Control 13 336 Gila 3 Control 13 336 Gila 4 Control 20 400 Gila 1 Percussion 1 min 66 474 Gila 2 Percussion 1 min 57 495 Gila 3 Percussion 1 min 73 444 Gila 4 Percussion 1 min 82 384 Gila 1 Percussion 2 min 81 392 Gila 2 Percussion 2 min 86 347 Gila 3 Percussion 2 min 85 357 Gila 4 Percussion 2 min 54 498 Gila 1 Percussion 4 min 79 407 Gila 2 Percussion 4 min 83 376 Gila 3 Percussion 4 min 84 367 Gila 4 Percussion 4 min 79 407 Gila 1 Percussion 8 min 51 500 Gila 2 Percussion 8 min 76 427 Gila 3 Percussion 8 min 52 500 Gila 4 Percussion 8 min 74 439 Holman 1 Immersion 83 376 Holman 2 Immersion 86 347 Holman 3 Immersion 81 392 Holman 4 Immersion 90 300 Holman 1 Boiling 30 sec 90 300 Holman 2 Boiling 30 sec 85 357 Holman 3 Boiling 30 sec 87 336 Holman 4 Boiling 30 sec 83 376 Holman 1 Boiling 60 sec 79 407 Holman 2 Boiling 60 sec 80 400 Holman 3 Boiling 60 sec 86 347 Holman 4 Boiling 60 sec 93 255 Holman Control 25 433 Holman 2 Control 19 392 Holman Control 21 407
Holman 4 Control 24 427
Holman Percussion 1 min 77 421
Holman 2 Percussion 1 min 85 357
Holman 3 Percussion 1 min 80 400
Holman 4 Percussion 1 min 89 313
Holman 1 Percussion 2 min 88 325
Holman 2 Percussion 2 min 96 196
Holman 3 Percussion 2 min 93 255
Holman 4 Percussion 2 min 96 196
Holman 1 Percussion 4 min 92 271
Holman 2 Percussion 4 min 97 171
66
I
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Holman 3 Percussion 4 min 91 286 Holman 4 Percussion 4 min 96 196 Holman 1 Percussion 8 min 88 325 Holman 2 Percussion 8 min 85 357 Holman 3 Percussion 8 min 83 376 Holman 4 Percussion 8 min 82 384 Jemez 1 Immersion 53 499 Jemez 2 Immersion 44 496 Jemez 3 Immersion 52 500 Jemez 4 Immersion 70 458 Jemez 1 Boiling 30 sec 58 494 Jemez 2 Boiling 30 sec 55 497 Jemez 3 Boiling 30 sec 48 500 Jemez 4 Boiling 30 sec 65 477 Jemez 1 Boiling 60 sec 68 466 Jemez 2 Boiling 60 sec 63 483 Jemez 3 Boiling 60 sec 73 444 Jemez 4 Boiling 60 sec 89 313 Jemez 1 Control 2 140 Jemez 2 Control 9 286 Jemez 3 Control 6 237 Jemez 4 Control 8 271 Jemez 1 Percussion 1 min 58 494 Jemez 2 Percussion 1 min 63 483 Jemez 3 Percussion 1 min 77 421 Jemez 4 Percussion 1 min 89 313 Jemez 1 Percussion 2 min 68 466 Jemez 2 Percussion 2 min 64 480 Jemez 3 Percussion 2 min 80 400 Jemez 4 Percussion 2 min 83 376 Jemez Percussion 4 min 68 466 Jemez 2 Percussion 4 min 79 407 Jemez 3 Percussion 4 min 70 458 Jemez 4 Percussion 4 min 83 376 Jemez 1 Percussion 8 min 52 500 Jemez 2 Percussion 8 min 59 492 Jemez 3 Percussion 8 min 61 488 Jemez 4 Percussion 8 min 70 458 Magdalena 1 Immersion 69 462 Magdalena 2 Immersion 69 462 Magdalena J Immersion 70 458 Magdalena 4 Immersion 85 357 Magdalena 1 Boiling 30 sec 65 477 Magdalena 2 Boiling 30 sec 71 454 Magdalena 3 Boiling 30 sec 68 466
67
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Magdalena 4 Boiling 30 sec 83 376 Magdalena 1 Boiling 60 sec 63 483 Magdalena 2 Boiling 60 sec 68 466 Magdalena 3 Boiling 60 sec 63 483 Magdalena 4 Boiling 60 sec 82 384 Magdalena 1 Control 11 313 Magdalena 2 Control 6 237 Magdalena 3 Control 5 218 Magdalena 4 Control 4 196 Magdalena Percussion 1 min 32 466 Magdalena 2 Percussion 1 min 45 497 Magdalena 3 Percussion 1 min 46 498 Magdalena 4 Percussion I min 36 480 Magdalena 1 Percussion 2 min 67 470 Magdalena 2 Percussion 2 min 68 466 Magdalena 3 Percussion 2 min 65 477 Magdalena 4 Percussion 2 min 81 392 Magdalena 1 Percussion 4 min 87 336 Magdalena 2 Percussion 4 min 97 171 Magdalena 3 Percussion 4 min 83 376 Magdalena 4 Percussion 4 min 97 171 Magdalena 1 Percussion 8 min 98 lAO Magdalena 2 Percussion 8 min 100 000 Magdalena 3 Percussion 8 min 96 196 Magdalena 4 Percussion 8 min 98 lAO Manzano 1 Immersion 67 470 Manzano 2 Immersion 57 495 Manzano 3 Immersion 72 4049 Manzano 4 Immersion 73 4044 Manzano 1 Boiling 30 sec 70 458 Manzano 2 Boiling 30 sec 64 480 Manzano 3 Boiling 30 sec 71 454 Manzano 4 Boiling 30 sec 80 400 Manzano I Boiling 60 sec 50 500 Manzano 2 Boiling 60 sec 53 499 Manzano 3 Boiling 60 sec 59 492 Manzano 4 Boiling 60 sec 74 439 Manzano I Control 4 196 Manzano 2 Control 8 271 Manzano 3 Control 1 099 Manzano 4 Control 6 237 Manzano 1 Percussion 1 min 52 500 Manzano 2 Percussion 1 min 39 488 Manzano 3 Percussion 1 min 36 480 Manzano 4 Percussion 1 min 51 500
68
Table B 1 Continued
Source Block Treatment Gerrni~ation Percentage Standard Error
Manzano Percussion 2 min 53 499 Manzano 2 Percussion 2 min 58 494 Manzano 3 Percussion 2 min 61 488 Manzano 4 Percussion 2 min 62 485 Manzano 1 Percussion 4 min 76 427 Manzano 2 Percussion 4 min 85 357 Manzano 3 Percussion 4 min 79 407 Manzano 4 Percussion 4 min 83 376 Manzano 1 Percussion 8 min 87 336 Manzano 2 Percussion 8 min 83 376 Manzano 3 Percussion 8 min 83 376 Manzano 4 Percussion 8 min 75 433 Raton 1 Immersion 54 498 Raton 2 Immersion 37 483 Raton 3 Immersion 39 488 Raton 4 Immersion 69 462 Raton 1 Boiling 30 sec 36 480 Raton 2 Boiling 30 sec 20 400 Raton 3 Boiling 30 sec 28 449 Raton 4 Boiling 30 sec 60 490 Raton 1 Boiling 60 sec 15 357 Raton 2 Boiling 60 sec 171 Raton 3 Boiling 60 sec 4 196 Raton 4 Boiling 60 sec 35 477 Raton 1 Control 4 196 Raton 2 Control 12 325 Raton 3 Control 2 140 Raton 4 Control 6 237 Raton 1 Percussion 1 min 52 500 Raton 2 Percussion I min 29 454 Raton 3 Percussion I min 35 477 Raton 4 Percussion 1 min 31 462 Raton 1 Percussion 2 min 55 497 Raton 2 Percussion 2 min 35 477 Raton 3 Percussion 2 min 33 470 Raton 4 Percussion 2 min 42 494 Raton 1 Percussion 4 min 65 477 Raton 2 Percussion 4 min 52 500 Raton Percussion 4 min 51 500 Raton 4 Percussion 4 min 53 499 Raton 1 Percussion 8 min 88 325 Raton 2 Percussion 8 min 80 400 Raton 3 Percussion 8 min 73 444 Raton 4 Percussion 8 min 74 439 Sacramento 1 Immersion 43 495
69
Table B 1 Continued
-Source Block Treatment Germination Percentage Standard Error
Sacramento 2 Immersion 30 458 Sacramento 3 Immersion 38 485 Sacramento 4 Immersion 56 496 Sacramento 1 Boiling 30 sec 47 499 Sacramento 2 Boiling 30 sec 48 500 Sacramento 3 Boiling 30 sec 48 500 Sacramento 4 Boiling 30 sec 56 496 Sacramento 1 Boiling 60 sec 52 500 Sacramento 2 Boiling 60 sec 67 470 Sacramento 3 Boiling 60 sec 43 495 Sacramento 4 Boiling 60 sec 48 500 Sacramento 1 Control 0 000 Sacramento 2 Control 3 171 Sacramento 3 Control 3 171 Sacramento 4 Control 099 Sacramento 1 Percussion 1 min 57 495 Sacramento 2 Percussion 1 min 61 488 Sacramento 3 Percussion 1 min 62 485 Sacramento 4 Percussion 1 min 55 497 Sacramento 1 Percussion 2 min 84 367 Sacramento 2 Percussion 2 min 70 458 Sacramento 3 Percussion 2 min 81 392 Sacramento 4 Percussion 2 min 69 462 Sacramento 1 Percussion 4 min 91 286 Sacramento 2 Percussion 4 min 84 367 Sacramento 3 Percussion 4 min 84 367 Sacramento 4 Percussion 4 min 94 237 Sacramento 1 Percussion 8 min 82 384 Sacramento 2 Percussion 8 min 81 392 Sacramento 3 Percussion 8 min 82 384 Sacramento 4 Percussion 8 min 81 392 Sandia 1 Immersion 76 427 Sandia 2 Immersion 70 458 Sandia 3 Immersion 70 458 Sandia 4 Immersion 81 392 Sandia 1 Boiling 30 sec 78 414 Sandia 2 Boiling 30 sec 71 454 Sandia 3 Boiling 30 sec 63 483 Sandia 4 Boiling 30 sec 78 414 Sandia 1 Boiling 60 sec 67 470 Sandia 2 Boiling 60 sec 65 477 Sandia 3 Boiling 60 sec 74 439 Sandia 4 Boiling 60 sec 66 474 Sandia 1 Control 16 367 Sandia 2 Control 7 255
70
Table B 1 Continued
Source Block Treatment Gennination Percentage Standard Error
Sandia 3 Control 8 271 Sandia 4 Control 16 367 Sandia 1 Percussion 1 min 67 470 Sandia 2 Percussion 1 min 61 488 Sandia 3 Percussion 1 min 66 474 Sandia 4 Percussion 1 min 59 492 Sandia I Percussion 2 min 82 384 Sandia 2 Percussion 2 min 83 376 Sandia 3 Percussion 2 min 81 392 Sandia 4 Percussion 2 min 84 367 Sandia 1 Percussion 4 min 89 313 Sandia 2 Percussion 4 min 86 347 Sandia 3 Percussion 4 min 89 313 Sandia 4 Percussion 4 min 85 357 Sandia 1 Percussion 8 min 80 400 Sandia 2 Percussion 8 min 77 421 Sandia 3 Percussion 8 min 69 462 Sandia 4 Percussion 8 min 66 474
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Dell B 1980 Structure and function ofthe strophiolar plug in seeds ofAlbizia Lophantha Am J Bot 67 556-563
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Dreesen DR Harrington JT 1997 Propagation of native plants for restoration projects in the Southwestern US- Preliminary Investigations In Landis TD Thompson JR tech coords National Proceedings Forest and Conservation Nursery Assocs Gen Tech Rep PNW-GTR-419 Portland OR USDA For Ser PNW Res Sta 77-88
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Gosling PG Samuel YK Jones SK 1995 A systematic examination of germination temperature chipping and water temperaturesoak duration pretreatments on the seeds ofLeucaena leucocephala Seed Sci amp Technol 23521-532
Hamly DH 1932 Softening of the seeds of Melilotus alba Bot Gazette 93 345shy375
Hanna PJ 1984 Anatomical features of the seed coat ofAcacia kempeana (Mueller) which relate to increased germination rate induced by heat treatment New Phytol 96 23-29
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74
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Wagner MR Larson RE Craig TP 1992 Ecology and silvics ofNew Mexico locust (Robinia neomexicana) with an emphasis on the arboreal form p53-57 In JW Hanover K Miller and S Plesko (eds) Proc Intemat Confon Black Locust Biology Culture and Utilization Mich State Univ USA
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1993 Hanks and Dick-Peddie 1974) New Mexico locust gradually declines with
small dense patches averaging about 005 ha beneath pine until the next disturbance
once again offers it a competitive advantage (Klemmedson 1994)
Gottfried (1980) expecting ponderosa pine regeneration to be greater where
New Mexico locust had been eradicated found that survival of planted pine seedlings
was greater where locust had not been removed In a follow-up study Gottfried
(1980) noted that soil moisture in the top 57 cm was highest in 5-year old locust sites
as compared to grass or 20-year old locust sites He concluded that managing an
appropriate cover ofNew Mexico locust could help regenerate pine the later
successional species
The ability ofNew Mexico locust to improve harsh sites also makes it a
candidate for reclaiming disturbances associated with mining Black locust (Robinia
pseudoacacia 1) has been used for decades to reclaim mine spoils and other
disturbed sites in the eastern US and around the world (Keresztesi 1988 Zimmerman
and Carpenter 1980) Ashby et al (1985) described similar positive attributes of
black locust also mentioned previously for New Mexico locust quick cover for
stabilization supply ofN and nutrient-rich litter to the soil and site improvement for
establishment of later successional trees Referring specifically to mine reclamation
the authors note the ability of black locust to grow on a wide range ofmine soil
conditions including extremely acid soils Black locust also shows some tolerance to
soils compacted by grading and topsoiling practices (Ashby et aL 1985) New
2
Mexico locust holds promise as a native southwe~tem counterpart to black locust in
mine reclamation but it has been used infrequently to date
Natural invasion and succession occur slowly on most mine sites (Monsen
1984) While New Mexico locust often colonizes sites naturally there is no
assurance a seed will reach a particular site and establish in a reasonable time frame
The challenge is to introduce New Mexico locust as mother plants to facilitate
colonization Seedling production for outplanting on disturbed sites has been
hampered by poor germination (Lin et al 1996) The goal then is to develop
techniques to overcome poor germination in New Mexico locust thereby facilitating
macropropagation
LITERATURE REVIEW
Seed Dormancy
Low gennination in New Mexico locust is primarily the result of seed
donnancy the failure of an intact viable seed to genninate under favorable
conditions (Bewley 1997) Seed donnancy also can be defined as the inability of the
embryo to genninate because of some inherent constraint (Bewley and Black 1994)
The constraint may originate within the embryo referred to as endogenous or embryo
donnancy or from the tissues surrounding the embryo known as exogenous or coatshy
imposed donnancy (Baskin and Baskin 1998 Leadem 1997 Bewley and Black
1994)
New Mexico locust seed is donnant when the impenneable seed coat does not
allow movement ofwater to the embryo This specific fonn of coat-imposed
donnancy where the restraining tissue involved is an impenneable seed coat is
known as physical donnancy (Baskin and Baskin 1998 Bewley and Black 1994)
Physical donnancy is relatively uncommon in seeds less than 10 of all woody
species exhibit this condition (USDA Forest Service 1948) However it is
characteristic of many genera of the Fabaceae (bean) family For example all
successional trees in the genera Gleditsia and Robinia (of which New Mexico locust
is a member) produce seeds that exhibit physical donnancy (Baskin and Baskin
4
Physical Dormancy
Seeds that exhibit physical dormancy are said to be hard seeded referring to
the thick impermeable seed coat (Leadem 1997) The first line of protection of the
testa in leguminous seeds is the outermost waxy cuticle followed by a subcuticle
consisting ofsuberin Next is a palisade layer consisting of sclereid cells that have
thick lignified secondary walls The most common type of sclereid cell in palisade
layers is the macrosclereid The macrosclereids contain water-repellent substances
including cutin lignin suberin and wax (Rolston 1978) This palisade layer is the
primary barrier to water uptake (Serrato-Valenti et al 1993 Tran and Cavanagh
1984)
All seed coat openings must be blocked to effectively prevent water uptake
The natural opening in a seed where it was attached to the mother plant is the weak
link where physical dormancy is usually broken (Hopkinson 1993 Kuo and Tam
1988) Anatomical structures associated with the seed coat opening vary from
family to family In the Papilionoideae the subfamily of the Fabaceae family
containing New Mexico locust the site ofwater entry is the strophiole The
strophiole is a swelling on the seed coat close to the stalk scar (Hopkinson 1997)
Kelly and van Staden (1987) refer to the strophiole site as the lens where
macrosclereid cells must separate and raise before water can pass through Since
middot6germination ofdormant seeds of the Papilionoideae takes place when the strophiole
permeable (Hopkinson 1993 Kuo and Tam 1988) a logical approach to
5
genninating New Mexico locust artificially would be to loosen the strophiolar cells
and allow water to pass through to the embryo
Evolutionary Role of Physical Dormancy
Why is physical dormancy a mechanism that blocks germination
ecologically advantageous When a plant produc~s seeds with different degrees of
dormancy seeds that are dispersed at one time will germinate over a range of time
(Bewley and Black 1994) Through variability of dormancy within a seed crop seeds
are exposed to changing environmental conditions improving the overall chances of
survival of the seed line (Bewley and Black 1994) Varying expression of dormancy
is under genetic control which indicates that this may be an evolved survival strategy
(Kigel and Galili 1995) A range of dormancy levels may be a particular advantage in
the southwestern US where extreme environmental variations exist
Methods for Breaking Dormancy
There are three common approaches to breaking dormancy in New Mexico
locust seed acid scarification hot water soaks and mechanical treatments In each
case the objective of the treatment is to scarify or abrade the seed coat to make it
permeable to water
Acid Scarification
Acid scarification consists of soaking seeds in concentrated sulfuric acid
rinsing thoroughly with water upon removal followed by subsequent drying before
PlaJlltmg Olson (1974) reports that this scarification method produces the highest
middot~~jllalUll results for all Robinia species Most literature refers to a seminal 1937
6
study conducted by HG Meginnis This stud~ t~ough conducted on black locust
seed is regularly cited in the suggested use of acid to treat New Mexico locust seed
as well (Olson 1974 Vines 1960)
Thee published studies document the specific use of sulfuric acid to treat
New Mexico locust seed Cox and Klett (1984) report that soaking seeds in
concentrated sulfuric acid for thirty minutes improved germination to 34 over an
untreated control of4 Lin et al (1996) found that for two seed lots acid
scarification for twenty-five to eighty minutes produced germination of 80 A third
lot however resulted in less than 20 germination at all durations of 10 to 80
minutes Hine et al (1997) showed that acid treatment levels of 1 2 5 and 10
minutes resulted in less than 20 germination across five sources However there
was an increasing trend in germination with acid soak duration and acid treatments
were not imposed for as long a time as the previous two studies (30 to 80 minutes)
The mixed results obtained by these investigators were probably due to the
widely varying germination response of seed lots to acid treatment For this reason
Olson (1974) recommends correlating duration of acid treatment to each seed lot If
treatment duration is too long the embryo will be permanently damaged If soak
duration is too short the seed will remain impermeable to water and fail to germinate
Compounding the problem of treatment duration is the negative effect acid
have on seedling growth Any type of scarification can injure seeds to some
by disrupting cells essential for growth leading to fungal invasion and
middot-uuu~w injury (Copeland and MacDonald 1995) Some legume seeds subjected
7
to 60 minutes of sulfuric acid scarification (to achieve maximum gennination results)
showed more than 50 seedling mortality after gennination (Kelly and van Staden
1987) Bewley and Black (1994) noted that fungal growth on seeds is an indication
of leakage of sugars organic acids amino acids and proteins
Perhaps the greatest drawback to acid scarification is the dangerous nature of
the treatment itself Difficulties associated with storage and trained use of
concentrated acid may discourage many propagators from using this option (Dreesen
and Harrington 1997) Consequently hot water and to a lesser extent mechanical
scarification are more commonly used and practical treatments for legume seeds
Most southwestern container growers use either of these two treatments to scarify
New Mexico locust seed in particular (Hine et al 1997)
Hot Water Scarification
The widespread use ofhot water scarification to treat locust seed is probably
due to its ease of application and efficacy in comparison to other scarification
treatments Although this practice had been in use for some time Wilson (1944) was
one of the first to report on its systematic application for black locust The treatment
consists of placing seeds in boiling water (4 parts water tomiddot 1 part seed by volume)
removing the vessel from the heat source and allowing the water to cool and seeds to
soak for 24 hours
Hine et al (1997) Lin et al (1996) and Cox and Klett (1984) have examined
the specific application ofhot water scarification on New Mexico locust seed Each
these authors compared hot water to acid scarification Lin et aL (1996) reported
8
60 gennination for two hot water-treated seed lots while a third lot germinated
around 50 (as reported above acid scarification germination ranged from 20 to
80 for these lots) It should be noted that the authors only soaked the seeds in water
from 10 to 80 minutes for this experiment as opposed to the standard 24 hours Cox
and Klett (1984) stated that using the standard 24-hour hot water treatment improved
germination over an acid pre-treatment (which produced less than 20 germination)
but they did not give specific gennination numbers for the hot water treatment Hine
et al (1997) found that across five seed lots germination was 66 for the standard
hot water treatment (as compared to less than 20 germination for acid scarification)
Variations in treatment intensity and duration and their subsequent effects on seed lot
appear to contribute to conflicting conclusions as to whether acid scarification or hot
water treatment is a more successful treatment
Given mixed results ease ofapplication is an important argument for use of
hot water application over acid soaks Safety concerns and material costs for hot
water treatments are low (Dreesen and Harrington 1997) An extension of this is the
practicality ofhot water for treating large batches of seed where a relative increase in
water volume is all that is needed However increases in water and seed even when
inthe same 4 1 proportion can result in dramatically different rates of cooling than in
atest tube experiment Different cooling rates can lead to contrasting results for the
treatment temperature duration and seed lot (USDA Forest Service 1948)
As with acid scarification parameters ofhot water treatment application may
to be optimized to maximize germination Hine et al (1997) found that as
9
temperature was increased from 70degC to 100degC overall germination in New Mexico
locust seed increased The 100degC treatment maximized germination for each of five
sources tested with germination usually above 60
Prolonged exposure of seeds ofmany woody legumes to boiling temperatures
also may improve germination (Briscoe 1996) This approach has not been tested on
New Mexico locust seed Exposing seed to prolonged boiling temperatures may
result in fungal infection and subsequent seedling mortality Marunda (1990) noted
severe fungal infections on both acid and hot water treated Acacia holoserica Cunn
ex G Don seeds attributing the infections to seed damage resulting from the
treatments Identifying an optimal hot water treatment level that does not result in a
high rate of seedling mortality is a logical goal
With hot water treatments as with acid there is also a danger of overtreating
Seeds and reducing germination (Gosling et al 1995) The legume Leucaena
leucocephala (Lam) de Wit germinated equally well after 7 seconds of exposure to
water at 100degC 7 seconds at 90degC 30 seconds at 80degC and 4 minutes at 70degC In
addition acceptably high germination was obtained from treatment at 70degC from 3
minutes to 1 hour whereas no seeds germinated following treatment at 100degC for 2
minutes Perhaps along with a greater margin of error for treatment duration treating
~ew Mexico locust seeds at a lower temperature may result in a comparatively lower
of seedling mortality In practice however nursery growers tend to use the
and quickest treatment available The short treatment length at near-boiling
whether recommended or not may be what is used most frequently
10
Fine-tuning a treatment in this range of treatment temperature and duration may be
the most logical approach
Mechanical Scarification
This treatment involves scratching the seed coat with an abrasive material
(USDA Forest Service 1948) One system employs a rotating drum lined with
sandpaper A paddle spinning at a given number ofrevolutions per minute throws the
seed against the sandpaper (Hine et al 1997) A second method dry tumbling
tumbles seed in a mixer with pea gravel or sharp sand for 5 to 7 days (Dreesen 2001)
These approaches have the advantage that control of temperature is not required as in
water treatments nor is safety an issue as with add scarification
Hine et al (1997) mechanically treated New Mexico locust seed using 100shy
grit sandpaper in a Forsberg (rotating drum) scarifier Gennination was less than
10 They concluded that the seed coats were too thin for their apparatus and
proposed that a finer grit sandpaper would produce more even abrasion Although
germination varied by seed lot Dreesen (2001) reported that using a slow-speed dry
tumbling technique for 5 to 7 days produced gennination rates as high as 90
As with acid and hot water treatments treatment duration must be specifically
shy
correlated to seed lot Treated seeds are also susceptible to fungal infection
iHopkinson 1997) In addition equipment is specialized and may not always be
ltU~CUJl~ to a nursery (USDA Forest Service 1948) Several studies have confinned
degrading ofstrophiolar cells due to acid and hot water scarification (Miklas et al
11
1987 Dell 1980 Hanna 1984) but the effect ofmechanical scarification on the
strophiole has not been studied
Alternative Method for Breaking Dormancy Percussion
Strophiole degradation has been examined in depth using an impaction
treatment more commonly known as percussion Hamly (1932) first proposed that
shaking or impacting seeds could split the macrosc1ereid cells at the strophiole
Hamly successfully released tension ofbent strophiolar cells of impermeable
Melilotus alba Medik (White sweet clover) seeds by delivering physical blows
resulting in cells that had split apart and were only slightly bent Ninety-two percent
oftreated seeds became permeable as compared to 05 of control seeds Barton
(1947) showed that percussion significantly increases permeability in black locust
seed Ballard (1976) reasoned that the integrated nature of the strophiole within the
seed coat means a blow on any part of the seed weakens the strophiole There are no
published studies on the use of percussion on New Mexico locust
Percussion methods vary and often seem SUbjective or ill-defined Hamly
(1932) placed seeds in a glass bottle and shook them three times a second for 20
minutes resulting in 180 oscillations per minute Ballard (1973) describes shaking
seeds in a closed metal container with at least fifty seeds per container at 850-900
JS(~l11alIOIlS per minute for 1 minute Kelly and van Staden (1987) placed seeds into
sealed glass flasks Flasks were shaken by a wrist-shaker at a rate of 250
~UUVJ~ per minute Subsequent germination rates were 45 after 2 hours of
55 after 3 hours 60 after 6 hours and 100 after 12 hours
12
There are several potential advantages of percussion over acid hot water and
mechanical scarification Kelly and van Staden (1987) note that a percussion
treatment only alters macrosclereid cells of the strophiole by loosening separating
and raising them They emphasize that minimal damage is done to the overall
anatomical structure of the seed In acid scarification of the same seed lot radicle
and cotyledon damage occurred Based on photographic evidence the authors
believe that this morphological damage was due to uneven pressure during
imbibition with water entering haphazardly wherever the acid had sufficiently
dissolved the coat This ultimately led to uneven hydration of the embryo In
percussed seeds water only entered through the s~ophiole when the entire seed coat
had been saturated resulting in controlled and even embryo hydration (Kelly and van
Staden 1987)
Although all successful scarification treatments may focus at least in part on
the strophiole for papilionoid legumes (Baskin and Baskin 1998) percussion may be
less damaging to seed coat structures particularly when compared with acid Kelly
and van Staden did not compare hot water or mechanical scarification methods
against percussion However the fungal infection associated with hot water and
mechanical scarification as with acid scarificatio~ have been directly associated with
seed damage (Rehman et al 1998 USDA Forest Service 1948) Embryo damage in
has been observed in hot-water scarified legume seeds (Rehman et al 1998
13
By limiting embryo damage percussion scarification reduces seedling
mortality compared to other treatments Kelly and van Staden (1987) noted that
seedlings from the percussion treatment were morphologically sound and vigorous
when compared to acid treatment seedlings The authors attribute this improved
seedling vigor to the controlled water uptake that limited anatomical damage
Despite a 12-hour percussion treatment morphological damage was not substantially
greater than untreated seeds In addition seedling mass measured 10 days following
germination was not significantly different between percussed and untreated seeds
A final advantage ofpercussion may be the ability to adequately treat the most
dormant seeds while avoiding harm to seeds that have already been made permeable
by the treatment or that were permeable to begin with Instead ofcorroding scalding
or scratching the seed coat percussion appears to be just a loosening of the
strophiole spring (Ballard 1973) Kelly and van Staden (1987) were able to
increase germination with a lengthy treatment (12 hours) Figuring out whether a
substantial cushion oftreatment durations exist for percussion ofNew Mexico locust
seed would be a significant improvement to current scarification methods where seed
damage and seedling health is an issue
The greatest challenge for applying percussion to New Mexico locust seeds is
standardizing a treatment that is both effective and readily reproducible by nurseries
with mechanical scarification this may require the development of specific
~UllDment to properly quantify and execute the treatment
14
f
Objectives
The first objective of the study is to detennine which scarification treatment
results in optimal gennination speed and percentage ofNew Mexico locust The
second objective is to describe treatment interactions with nine seed sources collected
in New Mexico
Hypothesis
A percussion treatment acts exclusively on the main area ofwater uptake in
New Mexico locust seed loosening the strophiole so that imbibition can occur This
direct mechanism ofdonnancy release will result in improved gennination rate and
overall gennination percentage for New Mexico locust seed compared to standard
treatments Standard treatments including acid hot water and mechanical
scarification while eventually weakening the strophiolar cleft cause excessive
morphological damage through generalized seed coat degradation These treatments
are therefore prone to inconsistency due to over and under-treatment
15
MATERIALS AND METHODS
Seed Collection and Cleaning
Seeds were collected from August 25 through September 15 2000 from nine
locations along a latitudinal gradient in New Mexico Seed source locations and
descriptive information are given in Figure 1 and Tables I and 2 Stands were
separated by at least 45 km
Pods were hand picked as they began to ripen and dehisce (open) Collections
were made from ground level to approximately 3 m from several trees at each site
Pods were dried in open-ended brown paper bags for two to four weeks at room
temperature (21-230C) to facilitate cleaning Dried pods were placed in a closed cloth
sack which was pounded against the ground to separate seeds from pods For pods
still containing seeds hand maceration (crunching pods with hands) completed seed
separation Debris was removed by threshing with the aid of a greenhouse fan A
South Dakota Seed Blower (Seedbureau Chicago Illinois) was used to remove
remaining chaff and light seeds by air density separation Seeds were placed in dry
storage in sealed quart-size zip-lock bags within a sealed plastic container Storage
temperatures ranged from 2-4oC Seeds remained in storage for approximately 7
months before the experiment
Experimental Design and Treatment Structure
The experiment compared the impact ofhot water bath and percussion
l11aLl11 on germination properties ofNew Mexico locust seed A single
16
-l
Figure 1 Seed source collection map All sources were collected within New Mexico USA separated by at least 4S lan
IIIRZltItW lIJPIirmiddot rllll1lbullbull_~~~_II
Table 1 Latitude longitude location elevation and collection date ofNew Mexico locust seed sources
Lot title Latitude (N) Longitude (W) Location Elevation (m) Collection Date
Raton 36deg5846 104deg2872 Raton Pass NM 2295 82500
Holman 36deg0306 105deg2429 HolmanNM 2476 911500
Jemez 35deg4500 106deg4100 Jemez Springs NM 1976 9900 00
Sandia 35deg1083 106deg2341 Cedar Crest NM 2432 82600
Manzano 34deg3689 106deg2494 ManzanoNM 2465 9200
Magdalena 33deg5953 107deg0903 Magdalena NM 2544 82900
Capitan 33deg2728 105deg4325 Alto NM 2189 911200
Sacramento 32deg5798 105deg4496 Cloudcroft NM 2630 91400 Gila 32deg5400 107deg4600 Kingston NM 2432 9700
f
Table 2 Mean seed weight and standard error by source
a
Source Mean seed weight (g) for Standard error 100 seeds n = 5
Raton Holman Jemez Sandia Manzano Magdalena Capitan Sacramento Gila
2872 b c 2522 d 1826 e 2753 c 2413 d 2559 d 2443 d 3130 a 3029 ab
00103 00403 00276 00301 00370 00374 00458 00279 00348
a Mean seed weights followed by the same letter are not significantly
different
19
experiment was conducted utilizing a randomized complete block design blocked by
time with a factorial treatment structure Experimental factors were seed source and
scarification treatment Nine seed sources from throughout New Mexico were used
(Figure 1 Table 1) Scarification treatments included a control three levels ofhot
water and four levels of percussion Hot water treatments included immersion at
100degC and boiling for periods of 30 seconds and 60 seconds Percussion treatment
levels were 1 2 4 and 8 minutes One hundred seeds per block were used to test
each treatment combination Four blocks of seed were tested one at a time (all seed
sources included in each run) in close chronological sequence (within 6 weeks) A
total of four 100-seed samples were used to test each source by scarification treatment
combination
Treatments
Hot water treatment of seeds utilized 50 ml test tubes filled with 30 ml
distilled water Each test tube was used to treat one 100-seed sample at a time
resulting in a ratio of4 parts water to 1 part seed by volume Test tubes were placed
in a beaker with the beaker filled with tap water to the same level as the water within
test tubes Nine test tubes (one per seed source) were placed in the beaker
simultaneously so that one replication of each seed sourcescarification treatment
cotnblnatlon was conducted simultaneously Hot water treatments include immersion
boiling For immersion treatments seeds were added to test tubes with the water
(boiling) and the test tubes were immediately removed from the beaker to
For the boiling treatments test tubes remained in the beaker with water at
20
boiling temperature for a set duration after the addition of seeds Following all
treatments test tubes with seeds cooled 24 hours in a test tube rack
Percussion treatment utilized a pneumatic paint shaker (Central Pneumatic
model 00422 Camarillo CA) Compressed air was maintained at 530 kPa plusmn30 kPa
(80 psi plusmn 5 psi) resulting in approximately 350 oscillations per minute Seeds were
placed in 118 ml (4 oz) soil tin cans (US Can Lobard IL) and the lids were secured
with duct tape Each can was used to treat one 100-seed sample To take advantage
ofgreatest lateral movement of the paint shaker a spacer (paint can) was used to
position the soil tin at the end of the shaking arm This allowed shaking to occur at
the greatest distance from the pivot point (fulcrum) Tins were placed perpendicular
to the direction of shaking motion allowing seeds to impact at greatest force by
hitting flat surfaces of the soil tin
Germination Testing
Before gennination testing all seeds were soaked 24 hours in reverse
osmosis-treated water (this was included in the process ofhot water treatment
jU~SCIlOea above) Following soaking seeds were placed in 100 cm Petri dishes lined
90 cm Whatman qualitative 1 filter paper circles (MG Scientific Pleasant
WI) One Petri dish was used for each treatment by source combination
papers were initially moistened with 5 mL reverse osmosis-treated water then
~VU1CUlJl moistened as needed to ensure adequate conditions for gennination
were monitored daily (at 300 PM) for gennination for 14 days in accordance
~UIUlnJlVUa Seed Testing Association (ISTA) standards for Robinia species
21
(1STA 1999) Tests were carried out at room temperature (22degC plusmnOSoC)
Germination was classified as protrusion of the radicle from the embryo by at least
1mm as viewed with the naked eye
X-ray Radiography
On August 132001 three lOO-seed samples from the Sacramento seed source
were treated for 14 or 8-minute percussion durations The seeds were sent
immediately by overnight delivery to the Faxitron X-Ray Corporation in Wheeling
Illinois where Dr Bob Karrfalt ofthe USDA Forest Service oversaw their imaging
Radiographs were made at SX with a Faxitron Specimen Radiography System Model
MX-20
Scanning Electron Microscope Photography
On February 192002 a Hitachi S-3200N Variable Pressure Scanning
Electron Microscope (Mountain View CA) was used to examine strophiolar cells of
untreated seeds and seeds percussed for 4 minutes Seeds were cut longitudinally
igt their entire length using a razor blade splitting the strophiolar region roughly in
Cross sections of seed samples magnifying the strophiolar region were viewed
600X under the microscope
Analysis
Categorical Analysis of Variance was used to determine treatment source and
(Le time) main effects and interactions The response variable was total
~vlI This procedure is a Chi-square test of homogeneity using the natural log
of germinated to non-germinated seed for each treatment Maximumshy
22
likelihood analysis was used to calculate Chi-square test statistics with their observed
significance levels A single contrast was conducted to cempare treatment means
against the control Observed significance levels less than or equal to 005 were
considered significant Pairwise comparisons were conducted between non-control
treatment mean percents using a conservative alp~a value of 005 divided by the
number of comparisons (Bonferroni approach) and approximate pairwise z statistics
Categorical analyses were performed using Proc Catmod (SAS Institute 1989)
RESULTS
Scarification seed source block and all interactions between factors
influenced germination (Table 3) The main effects are reported in the presence of
these interactions because the overall robustness of treatments and seed sources are of
interest Background levels ofnaturally occurring variation in seed sources are
seen in control germination percentages in Figure 2 In particular time variability
is unlikely to be under the control of the producer Appendix B details experimental
data for source by treatment gennination percents for each of the four blocks
Averaged over all treatments germination by source ranged from 40 to
79 (Figure 2) Averaged over all sources (Figure 3) and for each source (Figure 4
5) percussion scarification durations of4 or 8 minutes yielded greatest germination
Averaged over all sources germination increased with increasing durations of
percussion then leveled off for the 4- and 8-minute treatments (Figure 3) Immersion
and 30-second boiling scarification maximized germination for the hot water
treatments with a decrease in germination when seeds were boiled for 60 seconds
For all but two sources there is a similar ipcreasing trend in germination from
1to 4 minutes ofpercussion (Figure 4) The Holman source increased in germination
1 to 2 minutes then leveled off at 4 minutes The Jemez source changed little
1 to 4 minutes of treatment As treatment duration increased from 4 to 8
mutes five sources decreased in germination (Figure 4a) whereas three sources
24
Table 3 Categorical Analysis of Variance table for germination response to seed treatment seed source block and the interaction of the factors
Source of DF Chi-Square Observed Variability Significance Level
Source 8 98370 lt00001 Trt 7 233876 lt00001
contrast 1 16976 lt00001 SourceTrt 56 97077 lt00001 Block 3 5538 lt00001
SourceBlock 24 10162 lt00001 TrtBlock 21 7564 lt00001
SourceTrtBlock 168 34283 lt00001
S 4)
8 ~
0
sectg ~ 40 ~
N 0 0
20
o f0ltgt ~ ~4 jybshy ((iltgto ~~ K1ltgt 6~b-~ ~~ 0b-~~ ~~ ~b-~ ~b-~b
80
60 -I-- - ~- middotmiddotmiddotmiddotmiddot1
r -] Northern NM Sources
Central NM Sources
Southern NM Sources
sect-o ~
~b-v(i
SOURCE
Figure 2 Effect ofNew Mexico locust seed source on germination for data averaged over all scarification treatments Control germination is shown by solid line within bar
80
~ cd ~ ltl) () 60 ~ ~
N
t 0shyrti tshySltl)
40
0
20
o lmmersion Boiling 30 sec Boiling 60 sec Control 1 min 2min 4 min 8 min
Hot Water Treatments Percussion Treatments
Figure 3 Effect of scarification on germination ofNew Mexico locust for data averaged over all seed sources Means labelled with the same letter are not significantly different
100~----------------------~--~----------------~
80
60
ltU
~
40 IJ
sectgsI
l100
80
60
40
a I middotmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot middotImiddotmiddotmiddotmiddotmiddotmiddot E
_~iii-bull _ _ bull f --~--
t-- -~ ---- -shy ---- shyI ~ ---- - shy
L
fiII
~
b
- shy ---r
- Holman
Sacramento Sandia Gila Jemez
c ~
ltgt ~
Tmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot-I
j~=~=-~~-1
I middotmiddot ~ bull r
t tr
Y -------M-a-gd-a-Ie-n-a--
1 _ _
fCapitan
---- Manzano ~y -_ Raton
2 4 8
Time interval ofpercussion scarification (min)
Figure 4 Effect of seed source by treatment interaction on New Mexico locust germination for percussion scarification pattern a and pattern b
28
tv 0
~ d
~ A s o
1 c3
80
60
40
20
o
TIT
t------=middotfmiddotmiddot-middotmiddot-middotmiddot-middotmiddot 1- --- - - --=- - -shy--cE= - _--shy --shy ----shy ~ ~ ---shy ~ -~- _ --shy-------r 1
I
Raton Holman
-_ Jemez
--- Sandia Manzano Magdalena Capitan Sacramento Gila
Immersion Boiling 30 sec Boiling 60 sec
Hot Water Scarification
Figure 5 Effect of seed source by treatment interaction on New Mexico locust germination for hot water scarification
continued to increase in germination (Figure 4b) There was no difference between
the two treatment levels for the Manzano source (Figure 4b)
The effect ofhot water scarification also varied among sources with few
overall differences between hot water treatments (Figure 5) However boiling for 60
seconds yielded highest germination for the Jemez source whereas increasing
durations ofboiling decreased germination for the Raton source
In addition to consistently high germination 4- and 8-minute percussionshy
treated seeds germinated as fast or faster than all other treatments (Figure 6 Table 4)
050 measured as the number of days needed to complete 50 ofeventual
germination averaged 2 days for both ofthese treatments
X-ray radiographs were taken of seeds collected from the Sacramento source
percussed for 1 4 or 8 minutes (Figure 7) Seeds show signs of damage at 8 minutes
ofpercussion (Figure 8) Compared to an undamaged seed percussed for only 4
minutes (Figure 8a) damage at the 8-minute level includes fractured radicles pitting
of the seed coat and chips in seed coat structure (Figure 8b-d)
Scanning electron microscope (SEM) images at 600X reveal the direct effect
of 4 minutes of percussion on cells of the strophiolar region (Figure 9) Whereas
untreated seeds retain cellular organization in the strophiolar region strophiolar cells
seeds treated for 4 minutes ofpercussion separate and raise resulting in a
~-~e~U appearance
30
Umiddot-~middot--l-~---middotymiddotmiddotmiddot-----middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot--middot--middotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middotmiddotmiddotmiddotmiddotmiddotmiddot- -
050= gt1
-e-- Percussion 8 min -0- Percussion 4 min ---T- Percussion 2 min -----ltil- Percussion 1 min -- Immersion --0- Boiling 30 sec -+- Boiling 60 sec -ltgt- Control
2 4 6 8 10 12
Day Figure 6 Timecourse ofNew Mexico locust gennination following scarification
treatments G50 is the number of days to 50 of eventual germination
31
14
Table 4 Effect of seed source and treatment on G50 (days to 50 germination)
Seed Source Control Immersion B30sec B60sec Plmin P2min P4min P8min mean Raton 3 3 5 7 4 3 2 2 4 Holman 2 2 2 2 2 2 2 2 2
Jemez 4 6 6 5 3 2 2 2 4 Sandia 2 2 2 2 2 2 2 2 2
w tv Manzano 5 2 2 3 3 2 2 2 3
Magdalena 4 2 2 3 4 3 2 1 3
Capitan 5 4 2 2 3 2 2 2 3
Sacramento 3 2 2 2 4 3 2 2 3 Gila 3 2 2 2 2 2 2 2 2 mean 3 3 3 3 3 2 2 2 3 standard 11 14 16 18 09 05 0 03 08 deviation
Note Columns and rows may not agree due to rounding
Figure 7 X-ray radiographs ofSacramento seed source percussed for 1 4 or 8 minutes
33
Figure 8a-d Seed damage close-ups Figure a shows undamaged seed percussed for 4 minutes Figures b-d show seeds percussed for 8 minutes Damage includes fracturing ofradicle in b complete removal ofseed coat in c and pitting ofseed coat in d
34
Untreated Treated
Figure 9 SEM images ofuntreated vs 4-minute percussion-treated strophiolar cells ofNew Mexico locust seeds Treated cells are loosened separated and raised
DISCUSSION
Determining the Optimal Treatment
Averaging results of scarification treatmems across seed sources is a useful
tool to predict which treatment is most likely to be successful for an unknown seed
source Averaged over all sources four- and eight-minute percussion treatments
resulted in the highest germination 815 and 790 respectively and produced
germination that was as fast or faster than all other treatments Although there was no
difference between these two treatments on average an argument for 4 minutes of
percussion as a standard protocol for treating New Mexico locust seeds can be made
Descriptively 4 minutes of percussion improved germination over 8 minutes
ofpercussion for a majority of sources (five out of nine) 8 minutes of percussion
improved germination for three ofnine sources with no difference for a fmal source
For individual sources 4 minutes ofpercussion was always the best or secondmiddot best
treatment and was always equal to or better than all hot water treatments
Germination at 8 minutes ofpercussion was lower than all other percussion durations
for the Jemez and Gila sources and was lower than at least one hot water treatment for
these sources as well
The x-ray radiographs of the Sacramento seed source illustrate the excessive
internal and external seed damage that can occur with 8 minutes of percussion
germination counts 8-minute percussed seeds from Holman and Capitan
ClUll signs of damage that were visible to the naked eye (see below)
r~evertheless some of these seeds still fit the criteria for germination Neither x-ray
36
photography nor visible observations during seed counts revealed seed damage at the
4-minute percussion level In operational trials seeds percussed for 4 minutes
developed into healthy seedlings (Khadduri and Harrington unpublished data)
Strophiole and Seed Coat Behavior
Four minutes ofpercussion successfully breaks seed dormancy in a large
percentage ofNew Mexico locust seeds without excessively damaging the
surrounding seed coat or internal seed coat structures Most papilionoid legume
seeds including seeds ofNew Mexico locust have a specific region of the seed coat
known as the strophiole or lens (Hamly 1932) The strophiole is located on the
cotyledonary lobe of a locust seed (Figure 10 a-c) If seeds are not indiscriminately
damaged the strophiole is the first point of water entry to the embryo (Hopkinson
1997) When a papilionoid seed is percussed for the appropriate amount of time
repeated hits on the integrated seed coat loosen the constrained cells of the strophiolar
region without excessively damaging the rest of the seed coat (Ballard 1976) SEM
photos illuStrate that 4 minutes of percussion loosened and separated strophiolar cells
ofNew Mexico locust seeds when compared with constrained organized strophiolar
cells of untreated seeds (Figure 9) Research-has shown that water passes to the
embryo through the treated strophiole in a controlled manner and regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues
(Kelly and van Staden 1987)
37
10 Strophiole ofunimbibed New Mexico locust seed (a) and following uptake (bc)
38
In contrast hot-water treatments can excessively degrade the seed coat
(Rebman et al 1998 Marunda 1990) Random cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rebman et aI 1998 Marunda 1990) Along with a
proportionately large amount ofun-imbibed (thus under-treated) seeds (Figure l1a)
damage was observed in hot-water treated seeds from six sources during germination
counts Damage included seeds where the radicle failed to elongate (Figure 11 b)
seeds where the radicle elongated but was delayed in freeing itself from the seed coat
(Figure 11c) and seeds where the radicle completely broke off from the embryo
(Figure lId) In addition some seeds germinated in reverse (the cotyledon emerged
fIrst) If the radicle emerged completely from the seed coat they were counted as
germinated
While nearly all seeds treated for 8 minutes ofpercussion imbibed water
damage was evident in x-ray evaluations (Sacramento seed source) and in
germination counts (Holman and Capitan) at this treatment level Seed damage
during germination counts was similar to levels found in hot-water treated seeds
suggesting that over-treatment by both hot water and longer durations of percussion
may result in similar damage to seed structure
The Sacramento and Holman seed sources were two of the fIve sources that
opcrplltlpn in germination from the 4- to 8-minute durations of percussion
the benefIts ofloosening the strophiole as evidenced by imbibition of
all 8-minute treated seeds were outweighed by external and internal seed coat
39
a
b c d
11 Damage to New Mexico locust seeds observed during germination cotmts following hot water scarification 9a Hot water replicate including ~t seedlings and numerous unimbibed seeds 9b Close-up of seed where raallCle failed to elongate 9c Close-up ofseed where radicle elongated but was
-gtIn in freeing itself from the seed coat 9d Close-up ofseed where radicle broke off from embryo
40
damage (which also may be contributing to high imbibition) For the three sources
for which 8 minutes of percussion resulted in maximum germination extended
percussion of seeds necessary to loosen the strophiole may not have excessively
damaged the seed anatomy The Manzano source did not significantly change in
germination from 4 to 8 minutes of percussion suggesting that an increase in
permeable seeds at the 8-minute treatment duration was balanced by the number of
seeds that failed to germinate due to seed damage To summarize the 4-minute
percussion treatment improved germination over 8 minutes ofpercussion for a
majority of sources (five ofnine) the 8-minute percussion treatment improved
germination over 4-minutes ofpercussion for three sources and there was no
difference with the Manzano source Notably for the Raton source 8 minutes of
percussion increased germination by 235 percentage points over 4-minutes of
percussion (7875 vs 5525 respectively) the next best treatment Raton was the
only source to result in germination less than 75 for 4 minutes ofpercussion so a
grower may wish to apply longer percussion durations to lots that do not respond well
to this treatment level
Finally there was an interesting trend in the Jemez and Raton sources seed
that significantly contributed to the source by treatment interaction (Figure 12) A
~u test showed that the Jemez source had a significantly lighter seed weight than
other sources while the Raton source had an average seed weight For the Jemez
the longest duration of boiling increased germination Conversely only short
U4UVll) of percussion were needed to achieve high germination and the longest
41
80 _ Jemez a Raton
iD
~ C iD e iD
Q c 0 ~
c ~ iD 0
20
0------shy
Immersion Boiling 30 sec Boiling 60 sec
Hot Water Scarification
10degTr========~--------~------------------------------
80
60
40
20
_ Jemez Raton
0------shy
1 min 2 min 4 min 8 min
Percussion Scarification
Figure 12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources
42
b
--~----~~~~========~---- duration ofpercussion reduced germination For the Raton source longer durations
ofboiling consistently decreased germination whereas longer percussion treatments
consistently increased germination
A possible explanation is that a thick seed coat is associated with a loose
strophiole structure and that a thin seed coat is associated with a constrained
strophiole This contrast merits further study and may suggest an ecological
adaptation Further investigation ofthis phenomenon may contribute to successful
pre-screening ofunknown seed sources to determine optimal treatment leveL
43
CONCLUSION
Based on results obtained in pilot study (see Appendix) and thesis
experiments 4 minutes ofpercussion is recommended as a standard protocol for
treating an unknown source ofNew Mexico locust seed In addition to laboratory
results the 4-minute percussion treatment has resulted in germination greater than
80 and healthy seedling grow-outs for production purposes (unpublished data) In
laboratory experiments only one hundred seeds per 118 ml (40z) soil tin were
percussed at a time whereas for production we have treated up to five hundred seeds
per tin For large-scale production ofNew Mexico locust seedlings growers may
wish to experiment with larger quantities of seed and larger-size soil tins
Seed storage trials following percussion treatments were not conducted
However germination greater than 80 and healthy seedlings were obtained when
seeds were percussion-treated and subsequently stored at room-temperature (22degC)
for one week (unpublished data) Seed storage trials merit further study as the
standard hot water treatment leaves seeds in a softened condition that is not
conducive to storage
Hydroseeding is an effective method for direct seeding in reclamation
situations Percussion treatment may be conducive to hydro seeding as seeds remain
a dry relatively undamaged yet permeable condition following treatment Sulfuric
seeds also remain dry following treatment but extensive damage to seed coat
poor seedling vigor have been reported with this treatment (Kelly and van Staden
A grow-out study using a hydro seeder on a reclamation site testing percussion
44
against other treatments such as sulfuric acid and mechanical scarification also
merits further study
Papilionoids are the largest subfamily oflegumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust these legumes often play an integral role in the revegetation of
disturbed lands The pilot study (Appendix) indicates that percussion scarification
successfully treats the widely used black locust as well Developing superior
scarification methods to the standard hot water mechanical scarification and acid
treatments may facilitate the use ofpapilionoid legumes in reclamation projects
45
APPENDICES
APPENDIX A
PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR
NEW MEXICO LOCUST AND BLACK LOCUST SEEDS
Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4
Hot water and sulfuric acid soaks are traditional treatments for seeds of many
temperate woody legwnes including locusts However these scarification techniques
often produce inconsistent germination Percussion scarification where seeds are
repeatedly propelled against a hard surface was compared with hot water
scarification to evaluate treatment efficacy for New Mexico locust (Robinia
neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment
seeds were placed in a 98degC water bath which was immediately removed from the
heat source For percussion scarification seeds were placed in a soil sample tin and
agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the
control were followed by 24-hour water soaks Hot water baths resulted in 56 and
41 germination for New Mexico locust and black locust respectively For both
species nearly all durations ofpercussion increased germination over the hot water
treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3
4 and 5 minutes for black locust resulted in at least 90 germination Traditional
scarification treatments randomly degrade the entire seed coat which can lead to
tissue damage during water uptake Percussion scarification specifically weakens the
strophiole the natural source ofwater entry to the seed in papilionoid legwnes
INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom
2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031
48
Following percussion imbibition is controlled through the strophiole and underlying
tissue is protected
Keywords New Mexico locust black locust scarification percussion germination
Introduction
Locust trees (Robinia spp) are aggressive pioneer species that quickly
colonize disturbed land fix atmospheric nitrogen in the soil and stabilize slopes that
are prone to erosion (Klemmedson 1994 Ashby et al 1985) (Figure A1ab)
Recognition of these attributes has contributed to increased demand for locusts in
reclamation projects Locust seeds however exhibit physical dormancy where a
thick seed coat prevents the movement of water and gasses to the embryo (Leadem
1997)
Sulfuric acid or hot water soaks have traditionally been used to break seed
dormancy in macro-propagation of locusts Many growers are moving away from the
use of su1furic acid in treating locust seeds (Dreesen and Harrington 1997) In
addition to being dangerous acid soak durations must be specifically correlated to
seed lot (Olson 1974) Collections of native seed can vary tremendously in seed size
seed weight and hard seededness etc This variability limits the utility of sulfuric
acid soaks as even within a given lot some seeds may be damaged whereas others
are under-treated and fail to imbibe (take up water) A survey of Southwestern
container growers indicates that hot water baths are currently the most common
scarification method for locust seeds (Hine et al 1997) However hot water baths
also produce inconsistent germination (Lin et al 1996)
49
b
Figure AI New Mexico locust a primary invader following disturbance Ala Crown-sprouting at edge ofburn near Los Alamos NM--one year after Cerro Grande fire Alb Colonizing road cut Gila National Forest NM
50
Percussion scarification where seeds are repeatedly propelled against a hard
surface is an alternative dormancy-breaking method that has proven successful in
legumes related to locusts (Hamly 1932 Barton 1947) We compared this alternative
treatment to hot water scarification to evaluate treatment efficacy for New Mexico
locust and black locust seeds
Materials and Methods
Commercial New Mexico locust seeds (Western Native Seed Coaldale CO)
were collected fall 2000 in Huerfano County CO Black locust seeds were collected
September 2000 from Taos County NM by harvesting ripe pods from several trees at
a distance of up to 3 m from ground level Black locust seeds were allowed to air-dry
for 3 weeks and were threshed and separated from large chaffusing a greenhouse
fan A South Dakota Seed Blower (Chicago Illinltois) was used to remove fine chaff
from both seed lots Seeds were storedmiddotat 2-4C until the germination study was
conducted in March 2001
This study compared hot water scarification with percussion scarification
using treatment and species as experimental factors Along with a control
scarification treatments included immersion of seed in boiling water as well as
percussion durations of 1 2 3 45 or 10 minutes Four 100-seed samples were used
to test each species by scarification treatment combination
For the hot water treatment test tubes were filled with 30 mL water and
placed in a water-filled beaker (Figure A2a) The bath was raised to boiling (-98C at
51
b
c
Figure A2 Implementation ofhot water and percussion scarification A2a Hot water bath A2b Paint-shaker set-up A2c Placing soil tin containing seeds at end ofpaint shaker ann
52
Las Cruces NM elevation of 1300 meters) Each 100-seed replicate was placed in a
test tube and each test tube was immediately removed to cool at room temperature
Percussion scarification was implemented using a pneumatic paint shaker
(Central Pneumatic model 00422 Camarillo CA) (Figure A2b) This allowed
standardization of treatment intensity Compressed air pressure was maintained at
530kPa plusmn 30kPa (80psi plusmn 5psi) resulting in approximately 350 oscillations per
minute Each lOO-seed sample was placed in a 118 ml (4 oz) soil tin for shaking To
maximize lateral movement of paint shaker a paint can was used as a spacer to place
soil tins at the end of the shaking arm (Figure A2c) This allowed shaking to occur at
the greatest distance from the pivot point (fulcrum) Following all treatments
including the control seeds were soaked for twenty-four hours in distilled water prior
to plating out
Gennination was evaluated on lab benches at room temperature Each
replicate of seeds was placed in a 100 cm petri dish on moistened filter paper with
humidity maintained by enclosing the petri dishes in ziplock bags Gennination was
monitored daily for 14 days in accordance with International Seed Testing
Association standards for Robinia species (ISTA 1999) Gennination was defined as
protrusion of the radicle from the embryo by at least 1mm as viewed by the naked
eye
Categorical Analysis of Variance using Prpc Catmod (SAS Institute 1989)
was used to detennine treatment differences in germination percentages for each
source This procedure is an extension of a Chi-square test of homogeneity using the
53
natural log of the ratio of germinated to non-germinated seeds for each treatment Pshy
values less than 005 were considered significant A limited set of pairwiseshy
comparisons was conducted to compare treatment mean percents using a
conservative alpha value of 005 divided by the number of comparisons
Results
Percussion maximized germination for both New Mexico locust and black
locust There were three germination response levels for New Mexico locust (Figure
A3) First there was a low response in the control indicating a dormant seed lot
The hot water treatment and I-minute duration of percussion share a second
intermediate response Germination was highest at percussion levels from 2 to 10
minutes
A selected percussion treatment intermediate in time (5 minutes) produced the
steepest germination curve indicating the fastest germination rate (Figure A4) The
hot water treatment produced an intermediate curve or relatively slower germination
rate The control curve was very flat indicating the slowest germination rate for New
Mexico locust
Control seeds germinated poorly for black locust as well (Figure A5) Hot
water scarification improved germination over the control but all levels of percussion
improved germination over hot water The optimal durations of percussion treatment
were bracketed with a significant increase from the 1 to 2 minute percussion level a
high germination response from 2 to 5 minutes of percussion and finally a significant
54
~ d 25 CL)
p t1 0 ~ t1
g Vl ~ Vl
100~---------------------------------------------------------------
80
60
40
20
o Control Hot Water Percussion
I min Percussion
2 min Percussion
3 min
Scarification
Percussion 4 min
Figure A3 Effect of scarification on germination response for New Mexico locust
Percussion 5 min
=significant difference between adjacent treatment bars ns = no significant difference
Percussion 10 min
~ ~ () u agt
Il-t d o ~
~ ~ c3
100middotlr~~~------------------------------------e- Percussion 5min middot 0middot Hot Water -- Control
80-J=middot======
6b-middot-lmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middot--middotmiddot-middot~-middotmiddot -middotmiddot middot middotmiddot-middot 1
40
20
-----~-----------
0 0 0 middotmiddotmiddotmiddotmiddot0middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot0
0 --~-----y-----~------_---~--- T-----~
o shy -TDay 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A4 Effect of selected scarification treatments on germination speed for New Mexico locust First seven days of fourteen-day experiment are shown
Day 7
80
~ cd
5 60 ()
[) Cl d 0 1t1 d
40sect dUl
-J
20
o
ns
Scarification
Figure A5 Effect of scarification on germination response for black locust = significant difference between adjacent treatment bars ns = no significant difference
decrease at the 10-minute level This drop-off suggests that 10 minutes of percussion
over-treats seeds from this lot of-black locust
A selected percussion treatment intermediate in time (5 minutes) also had the
fastest germination rate for black locust (Figure A6)_ The hot water curve is
somewhat flatter indicating a slower germination rate The control curve is very flat
indicating a slow germination rate
A qualitative difference between hot water and percussion-treated seeds was
also observed Along with healthy germinants the hot water treatment produced
over-treated seeds (Figure A 7a) Examples of damaged germinants include seeds
where the radicle did not elongate (Figure A 7b) seeds where the radicle elongated
but was delayed in freeing itself from the seed coat (Figure A7c) and seeds where the
radicle broke off completely from the embryo (Figure A7d) In addition to these
examples of over-treatment there were numerous small dark seeds that failed to
imbibe water For percussion treatment nearly all seeds took up water (Figure A8a)
Germination was rapid and uniform with little damage evident (Figure A8b)
To summarize both New Mexico locust and black locust seed lots were
dormant The standard hot water scarification treatment improved germination but
only to 56 and 41 for New Mexico locust and black locust respectively Nearly
all percussion durations improved total germination as well as germination rate over
the hot water treatment Germination was greater than 90 for percussion durations
of 4 5 and 10 minutes for New Mexico locust and 3 4 and 5 minutes for black locust
58
CI)
sgt t CI) 0
igt 0
sect ttl t E
Vl 0
c3
100r==~==~~=r------------------------------------~ ___ Percussion 5 min
middotmiddotmiddot0middotmiddotmiddot Hot Water -lIf- Control
80r=========~--~~----------------------------~
60
40
20 -~-~---~---------- ---~------------I
omiddot
O
_~_----------lIf-----~bull -----_ shy---shy I
Day 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A6 Effect of selected scarification treatments on germination speed for black locust First seven days of fourteen-day experiment are shown
Day 7
a
b c d
Figure A7 Damage to New Mexico locust seeds observed during germination counts following hot water scarification A7a Hot water replicate including damaged seedlings and numerous unimbibed seeds A7b Close-up ofseed where radicle failed to elongate A7c Close-up ofseed where radicle elongated but was delayed in freeing itself from the seed coat A7d Close-up ofseed where radicle completely broke offfrom embryo
60
a
b
Figure A8 New Mexico locust seeds following percussion treatment at time of germination counts A8a Percussion replicate with uniformly healthy seedlings and near complete imbibition of seeds A8b Close-up ofpercussion-treated seedling
61
Discussion
Papilionoid seeds have a specific anatomical feature known as the strophiole
or lens-the natural site of water entry to the seed (Hamly 1932) The strophiole is
located on the cotyledonary lobe of a locust seed (Figure A9) When a papilionoid
legume seed is percussed for the appropriate amount of time repeated hits on the
integrated seed coat loosen the constrained cells of the strophiolar region without
excessively damaging the rest of the seed coat (Ballard 1976)
When a percussed seed is soaked water enters exclusively through the
strophiole in a controlled manner (Kelly and Staden 1987) This regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues This
contrasts with seeds that have been hot water or acid scarified treatments that can
randomly degrade the seed coat Unlocalized cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rehman et al 1998 Marunda 1990)
Papilionoids are the largest subfamily of legumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust and black locust these legumes often play an integral role in the
revegetation ofdisturbed lands Developing superior scarification methods to the
standard hot water and acid treatments should fadlitate the use ofthese legumes in
restoration projects
62
Figure A9 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (bc)
63
APPENDIXB
GERMINATION PERCENTS AND STANDARD ERRORS FOR SOURCE
BLOCK AND TREATMENT FOR NEW MEXICO LOCUST
Table B 1 Gennination percents and standard errors for source block and treatment for New Mexico locust
Source Block Treatment Germination Percentage Standard Error
Capitan 1 Immersion 74 437 Capitan 2 Immersion 69 462 Capitan 3 Immersion 71 454 Capitan 4 Immersion 67 470 Capitan Boiling 30 sec 79 407 Capitan 2 Boiling 30 sec 76 427 Capitan 3 Boiling 30 sec 72 449 Capitan 4 Boiling 30 sec 76 427 Capitan Boiling 60 sec 71 454 Capitan 2 Boiling 60 sec 54 498 Capitan 3 Boiling 60 sec 67 470 Capitan 4 Boiling 60 sec 76 427 Capitan I Control 5 218 Capitan 2 Control 7 255 Capitan 3 Control 4 196 Capitan 4 Control 5 218 Capitan 1 Percussion I min 47 499 Capitan 2 Percussion I min
470
Capitan Percussion I min 39 488
Capitan 4 Percussion I min 38 485 Capitan I Percussion 2 min 55 497 Capitan Capitan
2
Percussion 2 min Percussion 2 min
71 65
454 477
Capitan 4 Percussion 2 min 56 496 Capitan I Percussion 4 min 86 347 Capitan 2 Percussion 4 min 79 407 Capitan 3 Percussion 4 min 81 392 Capitan 4 Percussion 4 min 82 384 Capitan I Percussion 8 min 90 300 Capitan 2 Percussion 8 min 88 325 Capitan 3 Percussion 8 min 90 300 Capitan 4 Percussion 8 min 90 300 Gila I Immersion 71 454 Gila 2 Immersion 77 421 Gila
Immersion 75 433
Gila 4 Immersion 72 449
Gila I Boiling 30 sec 68 466
Gila 2 Boiling 30 sec 81 392
Gila 3 Boiling 30 sec 54 498
Gila 4 Boiling 30 sec 63 483
Gila I Boiling 60 sec 55 497
Gila 2 Boiling 60 sec 68 466
Gila 3 Boiling 60 sec 57 495
Gila 4 Boiling 60 sec 70 458
65
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Gila 2 Control 13 336 Gila 3 Control 13 336 Gila 4 Control 20 400 Gila 1 Percussion 1 min 66 474 Gila 2 Percussion 1 min 57 495 Gila 3 Percussion 1 min 73 444 Gila 4 Percussion 1 min 82 384 Gila 1 Percussion 2 min 81 392 Gila 2 Percussion 2 min 86 347 Gila 3 Percussion 2 min 85 357 Gila 4 Percussion 2 min 54 498 Gila 1 Percussion 4 min 79 407 Gila 2 Percussion 4 min 83 376 Gila 3 Percussion 4 min 84 367 Gila 4 Percussion 4 min 79 407 Gila 1 Percussion 8 min 51 500 Gila 2 Percussion 8 min 76 427 Gila 3 Percussion 8 min 52 500 Gila 4 Percussion 8 min 74 439 Holman 1 Immersion 83 376 Holman 2 Immersion 86 347 Holman 3 Immersion 81 392 Holman 4 Immersion 90 300 Holman 1 Boiling 30 sec 90 300 Holman 2 Boiling 30 sec 85 357 Holman 3 Boiling 30 sec 87 336 Holman 4 Boiling 30 sec 83 376 Holman 1 Boiling 60 sec 79 407 Holman 2 Boiling 60 sec 80 400 Holman 3 Boiling 60 sec 86 347 Holman 4 Boiling 60 sec 93 255 Holman Control 25 433 Holman 2 Control 19 392 Holman Control 21 407
Holman 4 Control 24 427
Holman Percussion 1 min 77 421
Holman 2 Percussion 1 min 85 357
Holman 3 Percussion 1 min 80 400
Holman 4 Percussion 1 min 89 313
Holman 1 Percussion 2 min 88 325
Holman 2 Percussion 2 min 96 196
Holman 3 Percussion 2 min 93 255
Holman 4 Percussion 2 min 96 196
Holman 1 Percussion 4 min 92 271
Holman 2 Percussion 4 min 97 171
66
I
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Holman 3 Percussion 4 min 91 286 Holman 4 Percussion 4 min 96 196 Holman 1 Percussion 8 min 88 325 Holman 2 Percussion 8 min 85 357 Holman 3 Percussion 8 min 83 376 Holman 4 Percussion 8 min 82 384 Jemez 1 Immersion 53 499 Jemez 2 Immersion 44 496 Jemez 3 Immersion 52 500 Jemez 4 Immersion 70 458 Jemez 1 Boiling 30 sec 58 494 Jemez 2 Boiling 30 sec 55 497 Jemez 3 Boiling 30 sec 48 500 Jemez 4 Boiling 30 sec 65 477 Jemez 1 Boiling 60 sec 68 466 Jemez 2 Boiling 60 sec 63 483 Jemez 3 Boiling 60 sec 73 444 Jemez 4 Boiling 60 sec 89 313 Jemez 1 Control 2 140 Jemez 2 Control 9 286 Jemez 3 Control 6 237 Jemez 4 Control 8 271 Jemez 1 Percussion 1 min 58 494 Jemez 2 Percussion 1 min 63 483 Jemez 3 Percussion 1 min 77 421 Jemez 4 Percussion 1 min 89 313 Jemez 1 Percussion 2 min 68 466 Jemez 2 Percussion 2 min 64 480 Jemez 3 Percussion 2 min 80 400 Jemez 4 Percussion 2 min 83 376 Jemez Percussion 4 min 68 466 Jemez 2 Percussion 4 min 79 407 Jemez 3 Percussion 4 min 70 458 Jemez 4 Percussion 4 min 83 376 Jemez 1 Percussion 8 min 52 500 Jemez 2 Percussion 8 min 59 492 Jemez 3 Percussion 8 min 61 488 Jemez 4 Percussion 8 min 70 458 Magdalena 1 Immersion 69 462 Magdalena 2 Immersion 69 462 Magdalena J Immersion 70 458 Magdalena 4 Immersion 85 357 Magdalena 1 Boiling 30 sec 65 477 Magdalena 2 Boiling 30 sec 71 454 Magdalena 3 Boiling 30 sec 68 466
67
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Magdalena 4 Boiling 30 sec 83 376 Magdalena 1 Boiling 60 sec 63 483 Magdalena 2 Boiling 60 sec 68 466 Magdalena 3 Boiling 60 sec 63 483 Magdalena 4 Boiling 60 sec 82 384 Magdalena 1 Control 11 313 Magdalena 2 Control 6 237 Magdalena 3 Control 5 218 Magdalena 4 Control 4 196 Magdalena Percussion 1 min 32 466 Magdalena 2 Percussion 1 min 45 497 Magdalena 3 Percussion 1 min 46 498 Magdalena 4 Percussion I min 36 480 Magdalena 1 Percussion 2 min 67 470 Magdalena 2 Percussion 2 min 68 466 Magdalena 3 Percussion 2 min 65 477 Magdalena 4 Percussion 2 min 81 392 Magdalena 1 Percussion 4 min 87 336 Magdalena 2 Percussion 4 min 97 171 Magdalena 3 Percussion 4 min 83 376 Magdalena 4 Percussion 4 min 97 171 Magdalena 1 Percussion 8 min 98 lAO Magdalena 2 Percussion 8 min 100 000 Magdalena 3 Percussion 8 min 96 196 Magdalena 4 Percussion 8 min 98 lAO Manzano 1 Immersion 67 470 Manzano 2 Immersion 57 495 Manzano 3 Immersion 72 4049 Manzano 4 Immersion 73 4044 Manzano 1 Boiling 30 sec 70 458 Manzano 2 Boiling 30 sec 64 480 Manzano 3 Boiling 30 sec 71 454 Manzano 4 Boiling 30 sec 80 400 Manzano I Boiling 60 sec 50 500 Manzano 2 Boiling 60 sec 53 499 Manzano 3 Boiling 60 sec 59 492 Manzano 4 Boiling 60 sec 74 439 Manzano I Control 4 196 Manzano 2 Control 8 271 Manzano 3 Control 1 099 Manzano 4 Control 6 237 Manzano 1 Percussion 1 min 52 500 Manzano 2 Percussion 1 min 39 488 Manzano 3 Percussion 1 min 36 480 Manzano 4 Percussion 1 min 51 500
68
Table B 1 Continued
Source Block Treatment Gerrni~ation Percentage Standard Error
Manzano Percussion 2 min 53 499 Manzano 2 Percussion 2 min 58 494 Manzano 3 Percussion 2 min 61 488 Manzano 4 Percussion 2 min 62 485 Manzano 1 Percussion 4 min 76 427 Manzano 2 Percussion 4 min 85 357 Manzano 3 Percussion 4 min 79 407 Manzano 4 Percussion 4 min 83 376 Manzano 1 Percussion 8 min 87 336 Manzano 2 Percussion 8 min 83 376 Manzano 3 Percussion 8 min 83 376 Manzano 4 Percussion 8 min 75 433 Raton 1 Immersion 54 498 Raton 2 Immersion 37 483 Raton 3 Immersion 39 488 Raton 4 Immersion 69 462 Raton 1 Boiling 30 sec 36 480 Raton 2 Boiling 30 sec 20 400 Raton 3 Boiling 30 sec 28 449 Raton 4 Boiling 30 sec 60 490 Raton 1 Boiling 60 sec 15 357 Raton 2 Boiling 60 sec 171 Raton 3 Boiling 60 sec 4 196 Raton 4 Boiling 60 sec 35 477 Raton 1 Control 4 196 Raton 2 Control 12 325 Raton 3 Control 2 140 Raton 4 Control 6 237 Raton 1 Percussion 1 min 52 500 Raton 2 Percussion I min 29 454 Raton 3 Percussion I min 35 477 Raton 4 Percussion 1 min 31 462 Raton 1 Percussion 2 min 55 497 Raton 2 Percussion 2 min 35 477 Raton 3 Percussion 2 min 33 470 Raton 4 Percussion 2 min 42 494 Raton 1 Percussion 4 min 65 477 Raton 2 Percussion 4 min 52 500 Raton Percussion 4 min 51 500 Raton 4 Percussion 4 min 53 499 Raton 1 Percussion 8 min 88 325 Raton 2 Percussion 8 min 80 400 Raton 3 Percussion 8 min 73 444 Raton 4 Percussion 8 min 74 439 Sacramento 1 Immersion 43 495
69
Table B 1 Continued
-Source Block Treatment Germination Percentage Standard Error
Sacramento 2 Immersion 30 458 Sacramento 3 Immersion 38 485 Sacramento 4 Immersion 56 496 Sacramento 1 Boiling 30 sec 47 499 Sacramento 2 Boiling 30 sec 48 500 Sacramento 3 Boiling 30 sec 48 500 Sacramento 4 Boiling 30 sec 56 496 Sacramento 1 Boiling 60 sec 52 500 Sacramento 2 Boiling 60 sec 67 470 Sacramento 3 Boiling 60 sec 43 495 Sacramento 4 Boiling 60 sec 48 500 Sacramento 1 Control 0 000 Sacramento 2 Control 3 171 Sacramento 3 Control 3 171 Sacramento 4 Control 099 Sacramento 1 Percussion 1 min 57 495 Sacramento 2 Percussion 1 min 61 488 Sacramento 3 Percussion 1 min 62 485 Sacramento 4 Percussion 1 min 55 497 Sacramento 1 Percussion 2 min 84 367 Sacramento 2 Percussion 2 min 70 458 Sacramento 3 Percussion 2 min 81 392 Sacramento 4 Percussion 2 min 69 462 Sacramento 1 Percussion 4 min 91 286 Sacramento 2 Percussion 4 min 84 367 Sacramento 3 Percussion 4 min 84 367 Sacramento 4 Percussion 4 min 94 237 Sacramento 1 Percussion 8 min 82 384 Sacramento 2 Percussion 8 min 81 392 Sacramento 3 Percussion 8 min 82 384 Sacramento 4 Percussion 8 min 81 392 Sandia 1 Immersion 76 427 Sandia 2 Immersion 70 458 Sandia 3 Immersion 70 458 Sandia 4 Immersion 81 392 Sandia 1 Boiling 30 sec 78 414 Sandia 2 Boiling 30 sec 71 454 Sandia 3 Boiling 30 sec 63 483 Sandia 4 Boiling 30 sec 78 414 Sandia 1 Boiling 60 sec 67 470 Sandia 2 Boiling 60 sec 65 477 Sandia 3 Boiling 60 sec 74 439 Sandia 4 Boiling 60 sec 66 474 Sandia 1 Control 16 367 Sandia 2 Control 7 255
70
Table B 1 Continued
Source Block Treatment Gennination Percentage Standard Error
Sandia 3 Control 8 271 Sandia 4 Control 16 367 Sandia 1 Percussion 1 min 67 470 Sandia 2 Percussion 1 min 61 488 Sandia 3 Percussion 1 min 66 474 Sandia 4 Percussion 1 min 59 492 Sandia I Percussion 2 min 82 384 Sandia 2 Percussion 2 min 83 376 Sandia 3 Percussion 2 min 81 392 Sandia 4 Percussion 2 min 84 367 Sandia 1 Percussion 4 min 89 313 Sandia 2 Percussion 4 min 86 347 Sandia 3 Percussion 4 min 89 313 Sandia 4 Percussion 4 min 85 357 Sandia 1 Percussion 8 min 80 400 Sandia 2 Percussion 8 min 77 421 Sandia 3 Percussion 8 min 69 462 Sandia 4 Percussion 8 min 66 474
LITERATURE CITED
Ashby WC Vogel WG Rogers NF 1985 Black locust in the reclamation equation General Technical Report Northeastern Forest Experiment Station USDA Forest Service NE-I05 12 p
Ballard LAT 1973 Physical barriers to gennination Seed Sci Technoll 285-303
___ 1976 Strophiolar water conduction in the seeds ofTrifolieae induced by action on the testa at nonstrophiolar sites Austr J Plant Physiol3 465-469
Barton LV 1947 Special studies on seed coat impenneability Contrib Boyce Thompson Inst 14 355-362
Baskin CC Baskin JM 1998 Seeds Ecology biogeography and evolution of donnancy and gennination Academic Press London UK 666 p
Bewley JD 1997 Seed germination and donnancy Plant Ce119 1055-1066
Bewley JD Black M 1994 Seeds physiology of development and germination 2nd ed Plenum Press New York NY 445 p
Briscoe CB 1996 Growing Nitrogen Fixing Trees in Acid Soils In MH Powell (ed) Nitrogen Fixing Trees for Acid Soils- A Field ManuaL Winrock International Morrilton AR 22 p
Copeland LO McDonald MB 1995 Principles of Seed Science and Technology 3rded Chapman amp Hall New York NY 409 p
Cox RE Klett JE 1984 Seed gennination requirements of native Colorado plants for use in the landscape Plant Propag 30(2) 6-10
Dell B 1980 Structure and function ofthe strophiolar plug in seeds ofAlbizia Lophantha Am J Bot 67 556-563
Dick-Peddie WA 1993 New Mexico Vegetation Past Present and Future University ofNew Mexico Press Albuquerque NM 244 p
Dreesen DR 2001 Propagation protocol for production ofcontainer Robinia neomexicana plants Los Lunas Plant Materials Center Los Lunas New Mexico In Native Plant Network URL httpwwwnativeplantnetworkorg (accessed 9 January 2002) Moscow (ID) University ofIdaho CoIl Natural Resources For Res Nursery
72
Dreesen DR Harrington JT 1997 Propagation of native plants for restoration projects in the Southwestern US- Preliminary Investigations In Landis TD Thompson JR tech coords National Proceedings Forest and Conservation Nursery Assocs Gen Tech Rep PNW-GTR-419 Portland OR USDA For Ser PNW Res Sta 77-88
Gottfried GJ 1980 Control ofNew Mexican locust and the effect on planted ponderosa pine in central Arizona US For Ser Res Note RM-386 Rocky Mountain For Range ExpStn Fort Collins CO
Gosling PG Samuel YK Jones SK 1995 A systematic examination of germination temperature chipping and water temperaturesoak duration pretreatments on the seeds ofLeucaena leucocephala Seed Sci amp Technol 23521-532
Hamly DH 1932 Softening of the seeds of Melilotus alba Bot Gazette 93 345shy375
Hanna PJ 1984 Anatomical features of the seed coat ofAcacia kempeana (Mueller) which relate to increased germination rate induced by heat treatment New Phytol 96 23-29
Hanks JP Dick-Peddie WA 1974 Vegetation patterns of the White Mountains New Mexico Southwest Naturalist 18(4) 371-382
Hine S Harrington JT Dreesen DR 1997 Seed scarification requirements for Robinia neomexicana In Harrington JTtech coord Proc Third SouthwestemContainer Growers Conf Feb 12-13 1997 Ft Collins CO
Hopkinson JM 1993 The strophiole of leguminous seeds Newsletter- IntI Herbage Seed Production Res Grp 18 7-8
---- 1997 Differences in hardseededness between Cunningham and Tarramba leucaena In Leucnet News The Newsletter of the International LeucaenaRes and Devlt Netwk No4
ISTA (International Seed Testing Association) 1999 International rules for seed testing rules Seed Sci amp Technol27 Supplement
Kelly KM van Staden J 1987 The lens as the site of permeability in the papilionoid seed Aspalathus linearis J Plant Physiol 128(45) 395-404
73
Keresztesi B 1988 Black locust the tree of agriculture Outlook on Agr 17(2) 77~85
Kigel 1 and Galili G 1995 Seed development and germination Marcel Dekker New York 853 p
Klemmedson 10 1994 New Mexico locust and parent material Influence on macronutrients of forest floor and soil Soil Sci Soc Am 1 58 974~980
Kuo WH Tam AW 1988 The pathway of water absorption of mung bean seeds Seed Sci amp Technol16(1) 139~144
Leadem CL 1997 Dormancy--unlocking seed secrets In Landis TD Thompson lR tech coords National Proceedings Forestand Conservation Nursery Assocs Gen Tech Rep PNW-G TR-419 Portland OR USDA For Ser PNW Res Sta 43-52
Lin Y Wagner MR Seweyestewa A Honahni MD DeGomez M McMillin JD 1996 Influence of cultural treatments on seed germination and seedling growth of Robinia neomexicana Forest Farm and Community Tree Res Reports Volume 1
Marunda CT 1990 Effects of seed pretreatment on the development ofAcacia auriculforinis and A holosericea seedlings ACIAR Proceedings Series No 28
Meginnis HG 1937 Sulphuric acid treatment to increase germination of black locust seed USDA Circ 453 34 p
Miklas PN Towensend CE Ladd SL 1987 Seedcoat anatomy and the scarification ofcicer milkvetch seed Crop Sci 27 766-772
Monsen SB 1984 Use of shrubs on mine spoils In The Challenge ofProducing Native Plants for the Intermountain Area Proceedings Intermountain Nurserymans Association 1983 conference Las Vegas NV compo PM Murphy 26-31 Ogden UT USDA For Ser Gen Tech Report INT-l68
Olson DF 1974 Robinia L Locust In Seeds of Woody Plants in the US tech coord CS Schopmeyer 728-731 USDA For Ser Agr Handbk No450
Rehman S Loescher RNl Harris P1C 1998 Dormancy breaking and germination ofAcacia salicina Lindl seeds Seed Sci amp Techno127 553-557
Rolston MP 1978 Water impermeable seed dormancy Bot Rev 44 365-396
74
SAS Institute Inc 1989 SASSTAT Users Guide Version 6 4th Edition Volume 1 Cary NC SAS Institute Inc 943 p
SerratoValenti G Conara L Ferrando M Modenesi P 1993 Structural and histochemical features ofStylosanthes scabra (Leguminosae Papilionoidae) seed coat as related to water entry Can J Bot 71 834-840
Simpson BJ 1988 A field guide to Texas trees Austin TX Texas Monthly Press 372p
Tran VN Cavanagh AK 1984 Structural aspects ofdormancy In Seed Physiology Vol 2 Murray DR (ed) p1-44 Academic Press Australia
USDA Forest Service 1948 Woody-plant seed manual USDA Misc Pub1654 416p
Vines RA 1960 Trees Shrubs and Woody Vines of the Southwest Austin Univ ofTexas Press 1104 p
Wagner MR Larson RE Craig TP 1992 Ecology and silvics ofNew Mexico locust (Robinia neomexicana) with an emphasis on the arboreal form p53-57 In JW Hanover K Miller and S Plesko (eds) Proc Intemat Confon Black Locust Biology Culture and Utilization Mich State Univ USA
Wilson JK 1944 Immersing seeds of species of Robinia in boiling water hastens germination J For 42 453-454
Zimmerman RW Carpenter SB 1980 First-year coppice production from a 5shyyear old black locust stand on surface mine spoil p309-314 Centr Hardwd For ConfProc University ofMissouri Columbia
Mexico locust holds promise as a native southwe~tem counterpart to black locust in
mine reclamation but it has been used infrequently to date
Natural invasion and succession occur slowly on most mine sites (Monsen
1984) While New Mexico locust often colonizes sites naturally there is no
assurance a seed will reach a particular site and establish in a reasonable time frame
The challenge is to introduce New Mexico locust as mother plants to facilitate
colonization Seedling production for outplanting on disturbed sites has been
hampered by poor germination (Lin et al 1996) The goal then is to develop
techniques to overcome poor germination in New Mexico locust thereby facilitating
macropropagation
LITERATURE REVIEW
Seed Dormancy
Low gennination in New Mexico locust is primarily the result of seed
donnancy the failure of an intact viable seed to genninate under favorable
conditions (Bewley 1997) Seed donnancy also can be defined as the inability of the
embryo to genninate because of some inherent constraint (Bewley and Black 1994)
The constraint may originate within the embryo referred to as endogenous or embryo
donnancy or from the tissues surrounding the embryo known as exogenous or coatshy
imposed donnancy (Baskin and Baskin 1998 Leadem 1997 Bewley and Black
1994)
New Mexico locust seed is donnant when the impenneable seed coat does not
allow movement ofwater to the embryo This specific fonn of coat-imposed
donnancy where the restraining tissue involved is an impenneable seed coat is
known as physical donnancy (Baskin and Baskin 1998 Bewley and Black 1994)
Physical donnancy is relatively uncommon in seeds less than 10 of all woody
species exhibit this condition (USDA Forest Service 1948) However it is
characteristic of many genera of the Fabaceae (bean) family For example all
successional trees in the genera Gleditsia and Robinia (of which New Mexico locust
is a member) produce seeds that exhibit physical donnancy (Baskin and Baskin
4
Physical Dormancy
Seeds that exhibit physical dormancy are said to be hard seeded referring to
the thick impermeable seed coat (Leadem 1997) The first line of protection of the
testa in leguminous seeds is the outermost waxy cuticle followed by a subcuticle
consisting ofsuberin Next is a palisade layer consisting of sclereid cells that have
thick lignified secondary walls The most common type of sclereid cell in palisade
layers is the macrosclereid The macrosclereids contain water-repellent substances
including cutin lignin suberin and wax (Rolston 1978) This palisade layer is the
primary barrier to water uptake (Serrato-Valenti et al 1993 Tran and Cavanagh
1984)
All seed coat openings must be blocked to effectively prevent water uptake
The natural opening in a seed where it was attached to the mother plant is the weak
link where physical dormancy is usually broken (Hopkinson 1993 Kuo and Tam
1988) Anatomical structures associated with the seed coat opening vary from
family to family In the Papilionoideae the subfamily of the Fabaceae family
containing New Mexico locust the site ofwater entry is the strophiole The
strophiole is a swelling on the seed coat close to the stalk scar (Hopkinson 1997)
Kelly and van Staden (1987) refer to the strophiole site as the lens where
macrosclereid cells must separate and raise before water can pass through Since
middot6germination ofdormant seeds of the Papilionoideae takes place when the strophiole
permeable (Hopkinson 1993 Kuo and Tam 1988) a logical approach to
5
genninating New Mexico locust artificially would be to loosen the strophiolar cells
and allow water to pass through to the embryo
Evolutionary Role of Physical Dormancy
Why is physical dormancy a mechanism that blocks germination
ecologically advantageous When a plant produc~s seeds with different degrees of
dormancy seeds that are dispersed at one time will germinate over a range of time
(Bewley and Black 1994) Through variability of dormancy within a seed crop seeds
are exposed to changing environmental conditions improving the overall chances of
survival of the seed line (Bewley and Black 1994) Varying expression of dormancy
is under genetic control which indicates that this may be an evolved survival strategy
(Kigel and Galili 1995) A range of dormancy levels may be a particular advantage in
the southwestern US where extreme environmental variations exist
Methods for Breaking Dormancy
There are three common approaches to breaking dormancy in New Mexico
locust seed acid scarification hot water soaks and mechanical treatments In each
case the objective of the treatment is to scarify or abrade the seed coat to make it
permeable to water
Acid Scarification
Acid scarification consists of soaking seeds in concentrated sulfuric acid
rinsing thoroughly with water upon removal followed by subsequent drying before
PlaJlltmg Olson (1974) reports that this scarification method produces the highest
middot~~jllalUll results for all Robinia species Most literature refers to a seminal 1937
6
study conducted by HG Meginnis This stud~ t~ough conducted on black locust
seed is regularly cited in the suggested use of acid to treat New Mexico locust seed
as well (Olson 1974 Vines 1960)
Thee published studies document the specific use of sulfuric acid to treat
New Mexico locust seed Cox and Klett (1984) report that soaking seeds in
concentrated sulfuric acid for thirty minutes improved germination to 34 over an
untreated control of4 Lin et al (1996) found that for two seed lots acid
scarification for twenty-five to eighty minutes produced germination of 80 A third
lot however resulted in less than 20 germination at all durations of 10 to 80
minutes Hine et al (1997) showed that acid treatment levels of 1 2 5 and 10
minutes resulted in less than 20 germination across five sources However there
was an increasing trend in germination with acid soak duration and acid treatments
were not imposed for as long a time as the previous two studies (30 to 80 minutes)
The mixed results obtained by these investigators were probably due to the
widely varying germination response of seed lots to acid treatment For this reason
Olson (1974) recommends correlating duration of acid treatment to each seed lot If
treatment duration is too long the embryo will be permanently damaged If soak
duration is too short the seed will remain impermeable to water and fail to germinate
Compounding the problem of treatment duration is the negative effect acid
have on seedling growth Any type of scarification can injure seeds to some
by disrupting cells essential for growth leading to fungal invasion and
middot-uuu~w injury (Copeland and MacDonald 1995) Some legume seeds subjected
7
to 60 minutes of sulfuric acid scarification (to achieve maximum gennination results)
showed more than 50 seedling mortality after gennination (Kelly and van Staden
1987) Bewley and Black (1994) noted that fungal growth on seeds is an indication
of leakage of sugars organic acids amino acids and proteins
Perhaps the greatest drawback to acid scarification is the dangerous nature of
the treatment itself Difficulties associated with storage and trained use of
concentrated acid may discourage many propagators from using this option (Dreesen
and Harrington 1997) Consequently hot water and to a lesser extent mechanical
scarification are more commonly used and practical treatments for legume seeds
Most southwestern container growers use either of these two treatments to scarify
New Mexico locust seed in particular (Hine et al 1997)
Hot Water Scarification
The widespread use ofhot water scarification to treat locust seed is probably
due to its ease of application and efficacy in comparison to other scarification
treatments Although this practice had been in use for some time Wilson (1944) was
one of the first to report on its systematic application for black locust The treatment
consists of placing seeds in boiling water (4 parts water tomiddot 1 part seed by volume)
removing the vessel from the heat source and allowing the water to cool and seeds to
soak for 24 hours
Hine et al (1997) Lin et al (1996) and Cox and Klett (1984) have examined
the specific application ofhot water scarification on New Mexico locust seed Each
these authors compared hot water to acid scarification Lin et aL (1996) reported
8
60 gennination for two hot water-treated seed lots while a third lot germinated
around 50 (as reported above acid scarification germination ranged from 20 to
80 for these lots) It should be noted that the authors only soaked the seeds in water
from 10 to 80 minutes for this experiment as opposed to the standard 24 hours Cox
and Klett (1984) stated that using the standard 24-hour hot water treatment improved
germination over an acid pre-treatment (which produced less than 20 germination)
but they did not give specific gennination numbers for the hot water treatment Hine
et al (1997) found that across five seed lots germination was 66 for the standard
hot water treatment (as compared to less than 20 germination for acid scarification)
Variations in treatment intensity and duration and their subsequent effects on seed lot
appear to contribute to conflicting conclusions as to whether acid scarification or hot
water treatment is a more successful treatment
Given mixed results ease ofapplication is an important argument for use of
hot water application over acid soaks Safety concerns and material costs for hot
water treatments are low (Dreesen and Harrington 1997) An extension of this is the
practicality ofhot water for treating large batches of seed where a relative increase in
water volume is all that is needed However increases in water and seed even when
inthe same 4 1 proportion can result in dramatically different rates of cooling than in
atest tube experiment Different cooling rates can lead to contrasting results for the
treatment temperature duration and seed lot (USDA Forest Service 1948)
As with acid scarification parameters ofhot water treatment application may
to be optimized to maximize germination Hine et al (1997) found that as
9
temperature was increased from 70degC to 100degC overall germination in New Mexico
locust seed increased The 100degC treatment maximized germination for each of five
sources tested with germination usually above 60
Prolonged exposure of seeds ofmany woody legumes to boiling temperatures
also may improve germination (Briscoe 1996) This approach has not been tested on
New Mexico locust seed Exposing seed to prolonged boiling temperatures may
result in fungal infection and subsequent seedling mortality Marunda (1990) noted
severe fungal infections on both acid and hot water treated Acacia holoserica Cunn
ex G Don seeds attributing the infections to seed damage resulting from the
treatments Identifying an optimal hot water treatment level that does not result in a
high rate of seedling mortality is a logical goal
With hot water treatments as with acid there is also a danger of overtreating
Seeds and reducing germination (Gosling et al 1995) The legume Leucaena
leucocephala (Lam) de Wit germinated equally well after 7 seconds of exposure to
water at 100degC 7 seconds at 90degC 30 seconds at 80degC and 4 minutes at 70degC In
addition acceptably high germination was obtained from treatment at 70degC from 3
minutes to 1 hour whereas no seeds germinated following treatment at 100degC for 2
minutes Perhaps along with a greater margin of error for treatment duration treating
~ew Mexico locust seeds at a lower temperature may result in a comparatively lower
of seedling mortality In practice however nursery growers tend to use the
and quickest treatment available The short treatment length at near-boiling
whether recommended or not may be what is used most frequently
10
Fine-tuning a treatment in this range of treatment temperature and duration may be
the most logical approach
Mechanical Scarification
This treatment involves scratching the seed coat with an abrasive material
(USDA Forest Service 1948) One system employs a rotating drum lined with
sandpaper A paddle spinning at a given number ofrevolutions per minute throws the
seed against the sandpaper (Hine et al 1997) A second method dry tumbling
tumbles seed in a mixer with pea gravel or sharp sand for 5 to 7 days (Dreesen 2001)
These approaches have the advantage that control of temperature is not required as in
water treatments nor is safety an issue as with add scarification
Hine et al (1997) mechanically treated New Mexico locust seed using 100shy
grit sandpaper in a Forsberg (rotating drum) scarifier Gennination was less than
10 They concluded that the seed coats were too thin for their apparatus and
proposed that a finer grit sandpaper would produce more even abrasion Although
germination varied by seed lot Dreesen (2001) reported that using a slow-speed dry
tumbling technique for 5 to 7 days produced gennination rates as high as 90
As with acid and hot water treatments treatment duration must be specifically
shy
correlated to seed lot Treated seeds are also susceptible to fungal infection
iHopkinson 1997) In addition equipment is specialized and may not always be
ltU~CUJl~ to a nursery (USDA Forest Service 1948) Several studies have confinned
degrading ofstrophiolar cells due to acid and hot water scarification (Miklas et al
11
1987 Dell 1980 Hanna 1984) but the effect ofmechanical scarification on the
strophiole has not been studied
Alternative Method for Breaking Dormancy Percussion
Strophiole degradation has been examined in depth using an impaction
treatment more commonly known as percussion Hamly (1932) first proposed that
shaking or impacting seeds could split the macrosc1ereid cells at the strophiole
Hamly successfully released tension ofbent strophiolar cells of impermeable
Melilotus alba Medik (White sweet clover) seeds by delivering physical blows
resulting in cells that had split apart and were only slightly bent Ninety-two percent
oftreated seeds became permeable as compared to 05 of control seeds Barton
(1947) showed that percussion significantly increases permeability in black locust
seed Ballard (1976) reasoned that the integrated nature of the strophiole within the
seed coat means a blow on any part of the seed weakens the strophiole There are no
published studies on the use of percussion on New Mexico locust
Percussion methods vary and often seem SUbjective or ill-defined Hamly
(1932) placed seeds in a glass bottle and shook them three times a second for 20
minutes resulting in 180 oscillations per minute Ballard (1973) describes shaking
seeds in a closed metal container with at least fifty seeds per container at 850-900
JS(~l11alIOIlS per minute for 1 minute Kelly and van Staden (1987) placed seeds into
sealed glass flasks Flasks were shaken by a wrist-shaker at a rate of 250
~UUVJ~ per minute Subsequent germination rates were 45 after 2 hours of
55 after 3 hours 60 after 6 hours and 100 after 12 hours
12
There are several potential advantages of percussion over acid hot water and
mechanical scarification Kelly and van Staden (1987) note that a percussion
treatment only alters macrosclereid cells of the strophiole by loosening separating
and raising them They emphasize that minimal damage is done to the overall
anatomical structure of the seed In acid scarification of the same seed lot radicle
and cotyledon damage occurred Based on photographic evidence the authors
believe that this morphological damage was due to uneven pressure during
imbibition with water entering haphazardly wherever the acid had sufficiently
dissolved the coat This ultimately led to uneven hydration of the embryo In
percussed seeds water only entered through the s~ophiole when the entire seed coat
had been saturated resulting in controlled and even embryo hydration (Kelly and van
Staden 1987)
Although all successful scarification treatments may focus at least in part on
the strophiole for papilionoid legumes (Baskin and Baskin 1998) percussion may be
less damaging to seed coat structures particularly when compared with acid Kelly
and van Staden did not compare hot water or mechanical scarification methods
against percussion However the fungal infection associated with hot water and
mechanical scarification as with acid scarificatio~ have been directly associated with
seed damage (Rehman et al 1998 USDA Forest Service 1948) Embryo damage in
has been observed in hot-water scarified legume seeds (Rehman et al 1998
13
By limiting embryo damage percussion scarification reduces seedling
mortality compared to other treatments Kelly and van Staden (1987) noted that
seedlings from the percussion treatment were morphologically sound and vigorous
when compared to acid treatment seedlings The authors attribute this improved
seedling vigor to the controlled water uptake that limited anatomical damage
Despite a 12-hour percussion treatment morphological damage was not substantially
greater than untreated seeds In addition seedling mass measured 10 days following
germination was not significantly different between percussed and untreated seeds
A final advantage ofpercussion may be the ability to adequately treat the most
dormant seeds while avoiding harm to seeds that have already been made permeable
by the treatment or that were permeable to begin with Instead ofcorroding scalding
or scratching the seed coat percussion appears to be just a loosening of the
strophiole spring (Ballard 1973) Kelly and van Staden (1987) were able to
increase germination with a lengthy treatment (12 hours) Figuring out whether a
substantial cushion oftreatment durations exist for percussion ofNew Mexico locust
seed would be a significant improvement to current scarification methods where seed
damage and seedling health is an issue
The greatest challenge for applying percussion to New Mexico locust seeds is
standardizing a treatment that is both effective and readily reproducible by nurseries
with mechanical scarification this may require the development of specific
~UllDment to properly quantify and execute the treatment
14
f
Objectives
The first objective of the study is to detennine which scarification treatment
results in optimal gennination speed and percentage ofNew Mexico locust The
second objective is to describe treatment interactions with nine seed sources collected
in New Mexico
Hypothesis
A percussion treatment acts exclusively on the main area ofwater uptake in
New Mexico locust seed loosening the strophiole so that imbibition can occur This
direct mechanism ofdonnancy release will result in improved gennination rate and
overall gennination percentage for New Mexico locust seed compared to standard
treatments Standard treatments including acid hot water and mechanical
scarification while eventually weakening the strophiolar cleft cause excessive
morphological damage through generalized seed coat degradation These treatments
are therefore prone to inconsistency due to over and under-treatment
15
MATERIALS AND METHODS
Seed Collection and Cleaning
Seeds were collected from August 25 through September 15 2000 from nine
locations along a latitudinal gradient in New Mexico Seed source locations and
descriptive information are given in Figure 1 and Tables I and 2 Stands were
separated by at least 45 km
Pods were hand picked as they began to ripen and dehisce (open) Collections
were made from ground level to approximately 3 m from several trees at each site
Pods were dried in open-ended brown paper bags for two to four weeks at room
temperature (21-230C) to facilitate cleaning Dried pods were placed in a closed cloth
sack which was pounded against the ground to separate seeds from pods For pods
still containing seeds hand maceration (crunching pods with hands) completed seed
separation Debris was removed by threshing with the aid of a greenhouse fan A
South Dakota Seed Blower (Seedbureau Chicago Illinois) was used to remove
remaining chaff and light seeds by air density separation Seeds were placed in dry
storage in sealed quart-size zip-lock bags within a sealed plastic container Storage
temperatures ranged from 2-4oC Seeds remained in storage for approximately 7
months before the experiment
Experimental Design and Treatment Structure
The experiment compared the impact ofhot water bath and percussion
l11aLl11 on germination properties ofNew Mexico locust seed A single
16
-l
Figure 1 Seed source collection map All sources were collected within New Mexico USA separated by at least 4S lan
IIIRZltItW lIJPIirmiddot rllll1lbullbull_~~~_II
Table 1 Latitude longitude location elevation and collection date ofNew Mexico locust seed sources
Lot title Latitude (N) Longitude (W) Location Elevation (m) Collection Date
Raton 36deg5846 104deg2872 Raton Pass NM 2295 82500
Holman 36deg0306 105deg2429 HolmanNM 2476 911500
Jemez 35deg4500 106deg4100 Jemez Springs NM 1976 9900 00
Sandia 35deg1083 106deg2341 Cedar Crest NM 2432 82600
Manzano 34deg3689 106deg2494 ManzanoNM 2465 9200
Magdalena 33deg5953 107deg0903 Magdalena NM 2544 82900
Capitan 33deg2728 105deg4325 Alto NM 2189 911200
Sacramento 32deg5798 105deg4496 Cloudcroft NM 2630 91400 Gila 32deg5400 107deg4600 Kingston NM 2432 9700
f
Table 2 Mean seed weight and standard error by source
a
Source Mean seed weight (g) for Standard error 100 seeds n = 5
Raton Holman Jemez Sandia Manzano Magdalena Capitan Sacramento Gila
2872 b c 2522 d 1826 e 2753 c 2413 d 2559 d 2443 d 3130 a 3029 ab
00103 00403 00276 00301 00370 00374 00458 00279 00348
a Mean seed weights followed by the same letter are not significantly
different
19
experiment was conducted utilizing a randomized complete block design blocked by
time with a factorial treatment structure Experimental factors were seed source and
scarification treatment Nine seed sources from throughout New Mexico were used
(Figure 1 Table 1) Scarification treatments included a control three levels ofhot
water and four levels of percussion Hot water treatments included immersion at
100degC and boiling for periods of 30 seconds and 60 seconds Percussion treatment
levels were 1 2 4 and 8 minutes One hundred seeds per block were used to test
each treatment combination Four blocks of seed were tested one at a time (all seed
sources included in each run) in close chronological sequence (within 6 weeks) A
total of four 100-seed samples were used to test each source by scarification treatment
combination
Treatments
Hot water treatment of seeds utilized 50 ml test tubes filled with 30 ml
distilled water Each test tube was used to treat one 100-seed sample at a time
resulting in a ratio of4 parts water to 1 part seed by volume Test tubes were placed
in a beaker with the beaker filled with tap water to the same level as the water within
test tubes Nine test tubes (one per seed source) were placed in the beaker
simultaneously so that one replication of each seed sourcescarification treatment
cotnblnatlon was conducted simultaneously Hot water treatments include immersion
boiling For immersion treatments seeds were added to test tubes with the water
(boiling) and the test tubes were immediately removed from the beaker to
For the boiling treatments test tubes remained in the beaker with water at
20
boiling temperature for a set duration after the addition of seeds Following all
treatments test tubes with seeds cooled 24 hours in a test tube rack
Percussion treatment utilized a pneumatic paint shaker (Central Pneumatic
model 00422 Camarillo CA) Compressed air was maintained at 530 kPa plusmn30 kPa
(80 psi plusmn 5 psi) resulting in approximately 350 oscillations per minute Seeds were
placed in 118 ml (4 oz) soil tin cans (US Can Lobard IL) and the lids were secured
with duct tape Each can was used to treat one 100-seed sample To take advantage
ofgreatest lateral movement of the paint shaker a spacer (paint can) was used to
position the soil tin at the end of the shaking arm This allowed shaking to occur at
the greatest distance from the pivot point (fulcrum) Tins were placed perpendicular
to the direction of shaking motion allowing seeds to impact at greatest force by
hitting flat surfaces of the soil tin
Germination Testing
Before gennination testing all seeds were soaked 24 hours in reverse
osmosis-treated water (this was included in the process ofhot water treatment
jU~SCIlOea above) Following soaking seeds were placed in 100 cm Petri dishes lined
90 cm Whatman qualitative 1 filter paper circles (MG Scientific Pleasant
WI) One Petri dish was used for each treatment by source combination
papers were initially moistened with 5 mL reverse osmosis-treated water then
~VU1CUlJl moistened as needed to ensure adequate conditions for gennination
were monitored daily (at 300 PM) for gennination for 14 days in accordance
~UIUlnJlVUa Seed Testing Association (ISTA) standards for Robinia species
21
(1STA 1999) Tests were carried out at room temperature (22degC plusmnOSoC)
Germination was classified as protrusion of the radicle from the embryo by at least
1mm as viewed with the naked eye
X-ray Radiography
On August 132001 three lOO-seed samples from the Sacramento seed source
were treated for 14 or 8-minute percussion durations The seeds were sent
immediately by overnight delivery to the Faxitron X-Ray Corporation in Wheeling
Illinois where Dr Bob Karrfalt ofthe USDA Forest Service oversaw their imaging
Radiographs were made at SX with a Faxitron Specimen Radiography System Model
MX-20
Scanning Electron Microscope Photography
On February 192002 a Hitachi S-3200N Variable Pressure Scanning
Electron Microscope (Mountain View CA) was used to examine strophiolar cells of
untreated seeds and seeds percussed for 4 minutes Seeds were cut longitudinally
igt their entire length using a razor blade splitting the strophiolar region roughly in
Cross sections of seed samples magnifying the strophiolar region were viewed
600X under the microscope
Analysis
Categorical Analysis of Variance was used to determine treatment source and
(Le time) main effects and interactions The response variable was total
~vlI This procedure is a Chi-square test of homogeneity using the natural log
of germinated to non-germinated seed for each treatment Maximumshy
22
likelihood analysis was used to calculate Chi-square test statistics with their observed
significance levels A single contrast was conducted to cempare treatment means
against the control Observed significance levels less than or equal to 005 were
considered significant Pairwise comparisons were conducted between non-control
treatment mean percents using a conservative alp~a value of 005 divided by the
number of comparisons (Bonferroni approach) and approximate pairwise z statistics
Categorical analyses were performed using Proc Catmod (SAS Institute 1989)
RESULTS
Scarification seed source block and all interactions between factors
influenced germination (Table 3) The main effects are reported in the presence of
these interactions because the overall robustness of treatments and seed sources are of
interest Background levels ofnaturally occurring variation in seed sources are
seen in control germination percentages in Figure 2 In particular time variability
is unlikely to be under the control of the producer Appendix B details experimental
data for source by treatment gennination percents for each of the four blocks
Averaged over all treatments germination by source ranged from 40 to
79 (Figure 2) Averaged over all sources (Figure 3) and for each source (Figure 4
5) percussion scarification durations of4 or 8 minutes yielded greatest germination
Averaged over all sources germination increased with increasing durations of
percussion then leveled off for the 4- and 8-minute treatments (Figure 3) Immersion
and 30-second boiling scarification maximized germination for the hot water
treatments with a decrease in germination when seeds were boiled for 60 seconds
For all but two sources there is a similar ipcreasing trend in germination from
1to 4 minutes ofpercussion (Figure 4) The Holman source increased in germination
1 to 2 minutes then leveled off at 4 minutes The Jemez source changed little
1 to 4 minutes of treatment As treatment duration increased from 4 to 8
mutes five sources decreased in germination (Figure 4a) whereas three sources
24
Table 3 Categorical Analysis of Variance table for germination response to seed treatment seed source block and the interaction of the factors
Source of DF Chi-Square Observed Variability Significance Level
Source 8 98370 lt00001 Trt 7 233876 lt00001
contrast 1 16976 lt00001 SourceTrt 56 97077 lt00001 Block 3 5538 lt00001
SourceBlock 24 10162 lt00001 TrtBlock 21 7564 lt00001
SourceTrtBlock 168 34283 lt00001
S 4)
8 ~
0
sectg ~ 40 ~
N 0 0
20
o f0ltgt ~ ~4 jybshy ((iltgto ~~ K1ltgt 6~b-~ ~~ 0b-~~ ~~ ~b-~ ~b-~b
80
60 -I-- - ~- middotmiddotmiddotmiddotmiddot1
r -] Northern NM Sources
Central NM Sources
Southern NM Sources
sect-o ~
~b-v(i
SOURCE
Figure 2 Effect ofNew Mexico locust seed source on germination for data averaged over all scarification treatments Control germination is shown by solid line within bar
80
~ cd ~ ltl) () 60 ~ ~
N
t 0shyrti tshySltl)
40
0
20
o lmmersion Boiling 30 sec Boiling 60 sec Control 1 min 2min 4 min 8 min
Hot Water Treatments Percussion Treatments
Figure 3 Effect of scarification on germination ofNew Mexico locust for data averaged over all seed sources Means labelled with the same letter are not significantly different
100~----------------------~--~----------------~
80
60
ltU
~
40 IJ
sectgsI
l100
80
60
40
a I middotmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot middotImiddotmiddotmiddotmiddotmiddotmiddot E
_~iii-bull _ _ bull f --~--
t-- -~ ---- -shy ---- shyI ~ ---- - shy
L
fiII
~
b
- shy ---r
- Holman
Sacramento Sandia Gila Jemez
c ~
ltgt ~
Tmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot-I
j~=~=-~~-1
I middotmiddot ~ bull r
t tr
Y -------M-a-gd-a-Ie-n-a--
1 _ _
fCapitan
---- Manzano ~y -_ Raton
2 4 8
Time interval ofpercussion scarification (min)
Figure 4 Effect of seed source by treatment interaction on New Mexico locust germination for percussion scarification pattern a and pattern b
28
tv 0
~ d
~ A s o
1 c3
80
60
40
20
o
TIT
t------=middotfmiddotmiddot-middotmiddot-middotmiddot-middotmiddot 1- --- - - --=- - -shy--cE= - _--shy --shy ----shy ~ ~ ---shy ~ -~- _ --shy-------r 1
I
Raton Holman
-_ Jemez
--- Sandia Manzano Magdalena Capitan Sacramento Gila
Immersion Boiling 30 sec Boiling 60 sec
Hot Water Scarification
Figure 5 Effect of seed source by treatment interaction on New Mexico locust germination for hot water scarification
continued to increase in germination (Figure 4b) There was no difference between
the two treatment levels for the Manzano source (Figure 4b)
The effect ofhot water scarification also varied among sources with few
overall differences between hot water treatments (Figure 5) However boiling for 60
seconds yielded highest germination for the Jemez source whereas increasing
durations ofboiling decreased germination for the Raton source
In addition to consistently high germination 4- and 8-minute percussionshy
treated seeds germinated as fast or faster than all other treatments (Figure 6 Table 4)
050 measured as the number of days needed to complete 50 ofeventual
germination averaged 2 days for both ofthese treatments
X-ray radiographs were taken of seeds collected from the Sacramento source
percussed for 1 4 or 8 minutes (Figure 7) Seeds show signs of damage at 8 minutes
ofpercussion (Figure 8) Compared to an undamaged seed percussed for only 4
minutes (Figure 8a) damage at the 8-minute level includes fractured radicles pitting
of the seed coat and chips in seed coat structure (Figure 8b-d)
Scanning electron microscope (SEM) images at 600X reveal the direct effect
of 4 minutes of percussion on cells of the strophiolar region (Figure 9) Whereas
untreated seeds retain cellular organization in the strophiolar region strophiolar cells
seeds treated for 4 minutes ofpercussion separate and raise resulting in a
~-~e~U appearance
30
Umiddot-~middot--l-~---middotymiddotmiddotmiddot-----middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot--middot--middotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middotmiddotmiddotmiddotmiddotmiddotmiddot- -
050= gt1
-e-- Percussion 8 min -0- Percussion 4 min ---T- Percussion 2 min -----ltil- Percussion 1 min -- Immersion --0- Boiling 30 sec -+- Boiling 60 sec -ltgt- Control
2 4 6 8 10 12
Day Figure 6 Timecourse ofNew Mexico locust gennination following scarification
treatments G50 is the number of days to 50 of eventual germination
31
14
Table 4 Effect of seed source and treatment on G50 (days to 50 germination)
Seed Source Control Immersion B30sec B60sec Plmin P2min P4min P8min mean Raton 3 3 5 7 4 3 2 2 4 Holman 2 2 2 2 2 2 2 2 2
Jemez 4 6 6 5 3 2 2 2 4 Sandia 2 2 2 2 2 2 2 2 2
w tv Manzano 5 2 2 3 3 2 2 2 3
Magdalena 4 2 2 3 4 3 2 1 3
Capitan 5 4 2 2 3 2 2 2 3
Sacramento 3 2 2 2 4 3 2 2 3 Gila 3 2 2 2 2 2 2 2 2 mean 3 3 3 3 3 2 2 2 3 standard 11 14 16 18 09 05 0 03 08 deviation
Note Columns and rows may not agree due to rounding
Figure 7 X-ray radiographs ofSacramento seed source percussed for 1 4 or 8 minutes
33
Figure 8a-d Seed damage close-ups Figure a shows undamaged seed percussed for 4 minutes Figures b-d show seeds percussed for 8 minutes Damage includes fracturing ofradicle in b complete removal ofseed coat in c and pitting ofseed coat in d
34
Untreated Treated
Figure 9 SEM images ofuntreated vs 4-minute percussion-treated strophiolar cells ofNew Mexico locust seeds Treated cells are loosened separated and raised
DISCUSSION
Determining the Optimal Treatment
Averaging results of scarification treatmems across seed sources is a useful
tool to predict which treatment is most likely to be successful for an unknown seed
source Averaged over all sources four- and eight-minute percussion treatments
resulted in the highest germination 815 and 790 respectively and produced
germination that was as fast or faster than all other treatments Although there was no
difference between these two treatments on average an argument for 4 minutes of
percussion as a standard protocol for treating New Mexico locust seeds can be made
Descriptively 4 minutes of percussion improved germination over 8 minutes
ofpercussion for a majority of sources (five out of nine) 8 minutes of percussion
improved germination for three ofnine sources with no difference for a fmal source
For individual sources 4 minutes ofpercussion was always the best or secondmiddot best
treatment and was always equal to or better than all hot water treatments
Germination at 8 minutes ofpercussion was lower than all other percussion durations
for the Jemez and Gila sources and was lower than at least one hot water treatment for
these sources as well
The x-ray radiographs of the Sacramento seed source illustrate the excessive
internal and external seed damage that can occur with 8 minutes of percussion
germination counts 8-minute percussed seeds from Holman and Capitan
ClUll signs of damage that were visible to the naked eye (see below)
r~evertheless some of these seeds still fit the criteria for germination Neither x-ray
36
photography nor visible observations during seed counts revealed seed damage at the
4-minute percussion level In operational trials seeds percussed for 4 minutes
developed into healthy seedlings (Khadduri and Harrington unpublished data)
Strophiole and Seed Coat Behavior
Four minutes ofpercussion successfully breaks seed dormancy in a large
percentage ofNew Mexico locust seeds without excessively damaging the
surrounding seed coat or internal seed coat structures Most papilionoid legume
seeds including seeds ofNew Mexico locust have a specific region of the seed coat
known as the strophiole or lens (Hamly 1932) The strophiole is located on the
cotyledonary lobe of a locust seed (Figure 10 a-c) If seeds are not indiscriminately
damaged the strophiole is the first point of water entry to the embryo (Hopkinson
1997) When a papilionoid seed is percussed for the appropriate amount of time
repeated hits on the integrated seed coat loosen the constrained cells of the strophiolar
region without excessively damaging the rest of the seed coat (Ballard 1976) SEM
photos illuStrate that 4 minutes of percussion loosened and separated strophiolar cells
ofNew Mexico locust seeds when compared with constrained organized strophiolar
cells of untreated seeds (Figure 9) Research-has shown that water passes to the
embryo through the treated strophiole in a controlled manner and regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues
(Kelly and van Staden 1987)
37
10 Strophiole ofunimbibed New Mexico locust seed (a) and following uptake (bc)
38
In contrast hot-water treatments can excessively degrade the seed coat
(Rebman et al 1998 Marunda 1990) Random cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rebman et aI 1998 Marunda 1990) Along with a
proportionately large amount ofun-imbibed (thus under-treated) seeds (Figure l1a)
damage was observed in hot-water treated seeds from six sources during germination
counts Damage included seeds where the radicle failed to elongate (Figure 11 b)
seeds where the radicle elongated but was delayed in freeing itself from the seed coat
(Figure 11c) and seeds where the radicle completely broke off from the embryo
(Figure lId) In addition some seeds germinated in reverse (the cotyledon emerged
fIrst) If the radicle emerged completely from the seed coat they were counted as
germinated
While nearly all seeds treated for 8 minutes ofpercussion imbibed water
damage was evident in x-ray evaluations (Sacramento seed source) and in
germination counts (Holman and Capitan) at this treatment level Seed damage
during germination counts was similar to levels found in hot-water treated seeds
suggesting that over-treatment by both hot water and longer durations of percussion
may result in similar damage to seed structure
The Sacramento and Holman seed sources were two of the fIve sources that
opcrplltlpn in germination from the 4- to 8-minute durations of percussion
the benefIts ofloosening the strophiole as evidenced by imbibition of
all 8-minute treated seeds were outweighed by external and internal seed coat
39
a
b c d
11 Damage to New Mexico locust seeds observed during germination cotmts following hot water scarification 9a Hot water replicate including ~t seedlings and numerous unimbibed seeds 9b Close-up of seed where raallCle failed to elongate 9c Close-up ofseed where radicle elongated but was
-gtIn in freeing itself from the seed coat 9d Close-up ofseed where radicle broke off from embryo
40
damage (which also may be contributing to high imbibition) For the three sources
for which 8 minutes of percussion resulted in maximum germination extended
percussion of seeds necessary to loosen the strophiole may not have excessively
damaged the seed anatomy The Manzano source did not significantly change in
germination from 4 to 8 minutes of percussion suggesting that an increase in
permeable seeds at the 8-minute treatment duration was balanced by the number of
seeds that failed to germinate due to seed damage To summarize the 4-minute
percussion treatment improved germination over 8 minutes ofpercussion for a
majority of sources (five ofnine) the 8-minute percussion treatment improved
germination over 4-minutes ofpercussion for three sources and there was no
difference with the Manzano source Notably for the Raton source 8 minutes of
percussion increased germination by 235 percentage points over 4-minutes of
percussion (7875 vs 5525 respectively) the next best treatment Raton was the
only source to result in germination less than 75 for 4 minutes ofpercussion so a
grower may wish to apply longer percussion durations to lots that do not respond well
to this treatment level
Finally there was an interesting trend in the Jemez and Raton sources seed
that significantly contributed to the source by treatment interaction (Figure 12) A
~u test showed that the Jemez source had a significantly lighter seed weight than
other sources while the Raton source had an average seed weight For the Jemez
the longest duration of boiling increased germination Conversely only short
U4UVll) of percussion were needed to achieve high germination and the longest
41
80 _ Jemez a Raton
iD
~ C iD e iD
Q c 0 ~
c ~ iD 0
20
0------shy
Immersion Boiling 30 sec Boiling 60 sec
Hot Water Scarification
10degTr========~--------~------------------------------
80
60
40
20
_ Jemez Raton
0------shy
1 min 2 min 4 min 8 min
Percussion Scarification
Figure 12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources
42
b
--~----~~~~========~---- duration ofpercussion reduced germination For the Raton source longer durations
ofboiling consistently decreased germination whereas longer percussion treatments
consistently increased germination
A possible explanation is that a thick seed coat is associated with a loose
strophiole structure and that a thin seed coat is associated with a constrained
strophiole This contrast merits further study and may suggest an ecological
adaptation Further investigation ofthis phenomenon may contribute to successful
pre-screening ofunknown seed sources to determine optimal treatment leveL
43
CONCLUSION
Based on results obtained in pilot study (see Appendix) and thesis
experiments 4 minutes ofpercussion is recommended as a standard protocol for
treating an unknown source ofNew Mexico locust seed In addition to laboratory
results the 4-minute percussion treatment has resulted in germination greater than
80 and healthy seedling grow-outs for production purposes (unpublished data) In
laboratory experiments only one hundred seeds per 118 ml (40z) soil tin were
percussed at a time whereas for production we have treated up to five hundred seeds
per tin For large-scale production ofNew Mexico locust seedlings growers may
wish to experiment with larger quantities of seed and larger-size soil tins
Seed storage trials following percussion treatments were not conducted
However germination greater than 80 and healthy seedlings were obtained when
seeds were percussion-treated and subsequently stored at room-temperature (22degC)
for one week (unpublished data) Seed storage trials merit further study as the
standard hot water treatment leaves seeds in a softened condition that is not
conducive to storage
Hydroseeding is an effective method for direct seeding in reclamation
situations Percussion treatment may be conducive to hydro seeding as seeds remain
a dry relatively undamaged yet permeable condition following treatment Sulfuric
seeds also remain dry following treatment but extensive damage to seed coat
poor seedling vigor have been reported with this treatment (Kelly and van Staden
A grow-out study using a hydro seeder on a reclamation site testing percussion
44
against other treatments such as sulfuric acid and mechanical scarification also
merits further study
Papilionoids are the largest subfamily oflegumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust these legumes often play an integral role in the revegetation of
disturbed lands The pilot study (Appendix) indicates that percussion scarification
successfully treats the widely used black locust as well Developing superior
scarification methods to the standard hot water mechanical scarification and acid
treatments may facilitate the use ofpapilionoid legumes in reclamation projects
45
APPENDICES
APPENDIX A
PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR
NEW MEXICO LOCUST AND BLACK LOCUST SEEDS
Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4
Hot water and sulfuric acid soaks are traditional treatments for seeds of many
temperate woody legwnes including locusts However these scarification techniques
often produce inconsistent germination Percussion scarification where seeds are
repeatedly propelled against a hard surface was compared with hot water
scarification to evaluate treatment efficacy for New Mexico locust (Robinia
neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment
seeds were placed in a 98degC water bath which was immediately removed from the
heat source For percussion scarification seeds were placed in a soil sample tin and
agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the
control were followed by 24-hour water soaks Hot water baths resulted in 56 and
41 germination for New Mexico locust and black locust respectively For both
species nearly all durations ofpercussion increased germination over the hot water
treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3
4 and 5 minutes for black locust resulted in at least 90 germination Traditional
scarification treatments randomly degrade the entire seed coat which can lead to
tissue damage during water uptake Percussion scarification specifically weakens the
strophiole the natural source ofwater entry to the seed in papilionoid legwnes
INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom
2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031
48
Following percussion imbibition is controlled through the strophiole and underlying
tissue is protected
Keywords New Mexico locust black locust scarification percussion germination
Introduction
Locust trees (Robinia spp) are aggressive pioneer species that quickly
colonize disturbed land fix atmospheric nitrogen in the soil and stabilize slopes that
are prone to erosion (Klemmedson 1994 Ashby et al 1985) (Figure A1ab)
Recognition of these attributes has contributed to increased demand for locusts in
reclamation projects Locust seeds however exhibit physical dormancy where a
thick seed coat prevents the movement of water and gasses to the embryo (Leadem
1997)
Sulfuric acid or hot water soaks have traditionally been used to break seed
dormancy in macro-propagation of locusts Many growers are moving away from the
use of su1furic acid in treating locust seeds (Dreesen and Harrington 1997) In
addition to being dangerous acid soak durations must be specifically correlated to
seed lot (Olson 1974) Collections of native seed can vary tremendously in seed size
seed weight and hard seededness etc This variability limits the utility of sulfuric
acid soaks as even within a given lot some seeds may be damaged whereas others
are under-treated and fail to imbibe (take up water) A survey of Southwestern
container growers indicates that hot water baths are currently the most common
scarification method for locust seeds (Hine et al 1997) However hot water baths
also produce inconsistent germination (Lin et al 1996)
49
b
Figure AI New Mexico locust a primary invader following disturbance Ala Crown-sprouting at edge ofburn near Los Alamos NM--one year after Cerro Grande fire Alb Colonizing road cut Gila National Forest NM
50
Percussion scarification where seeds are repeatedly propelled against a hard
surface is an alternative dormancy-breaking method that has proven successful in
legumes related to locusts (Hamly 1932 Barton 1947) We compared this alternative
treatment to hot water scarification to evaluate treatment efficacy for New Mexico
locust and black locust seeds
Materials and Methods
Commercial New Mexico locust seeds (Western Native Seed Coaldale CO)
were collected fall 2000 in Huerfano County CO Black locust seeds were collected
September 2000 from Taos County NM by harvesting ripe pods from several trees at
a distance of up to 3 m from ground level Black locust seeds were allowed to air-dry
for 3 weeks and were threshed and separated from large chaffusing a greenhouse
fan A South Dakota Seed Blower (Chicago Illinltois) was used to remove fine chaff
from both seed lots Seeds were storedmiddotat 2-4C until the germination study was
conducted in March 2001
This study compared hot water scarification with percussion scarification
using treatment and species as experimental factors Along with a control
scarification treatments included immersion of seed in boiling water as well as
percussion durations of 1 2 3 45 or 10 minutes Four 100-seed samples were used
to test each species by scarification treatment combination
For the hot water treatment test tubes were filled with 30 mL water and
placed in a water-filled beaker (Figure A2a) The bath was raised to boiling (-98C at
51
b
c
Figure A2 Implementation ofhot water and percussion scarification A2a Hot water bath A2b Paint-shaker set-up A2c Placing soil tin containing seeds at end ofpaint shaker ann
52
Las Cruces NM elevation of 1300 meters) Each 100-seed replicate was placed in a
test tube and each test tube was immediately removed to cool at room temperature
Percussion scarification was implemented using a pneumatic paint shaker
(Central Pneumatic model 00422 Camarillo CA) (Figure A2b) This allowed
standardization of treatment intensity Compressed air pressure was maintained at
530kPa plusmn 30kPa (80psi plusmn 5psi) resulting in approximately 350 oscillations per
minute Each lOO-seed sample was placed in a 118 ml (4 oz) soil tin for shaking To
maximize lateral movement of paint shaker a paint can was used as a spacer to place
soil tins at the end of the shaking arm (Figure A2c) This allowed shaking to occur at
the greatest distance from the pivot point (fulcrum) Following all treatments
including the control seeds were soaked for twenty-four hours in distilled water prior
to plating out
Gennination was evaluated on lab benches at room temperature Each
replicate of seeds was placed in a 100 cm petri dish on moistened filter paper with
humidity maintained by enclosing the petri dishes in ziplock bags Gennination was
monitored daily for 14 days in accordance with International Seed Testing
Association standards for Robinia species (ISTA 1999) Gennination was defined as
protrusion of the radicle from the embryo by at least 1mm as viewed by the naked
eye
Categorical Analysis of Variance using Prpc Catmod (SAS Institute 1989)
was used to detennine treatment differences in germination percentages for each
source This procedure is an extension of a Chi-square test of homogeneity using the
53
natural log of the ratio of germinated to non-germinated seeds for each treatment Pshy
values less than 005 were considered significant A limited set of pairwiseshy
comparisons was conducted to compare treatment mean percents using a
conservative alpha value of 005 divided by the number of comparisons
Results
Percussion maximized germination for both New Mexico locust and black
locust There were three germination response levels for New Mexico locust (Figure
A3) First there was a low response in the control indicating a dormant seed lot
The hot water treatment and I-minute duration of percussion share a second
intermediate response Germination was highest at percussion levels from 2 to 10
minutes
A selected percussion treatment intermediate in time (5 minutes) produced the
steepest germination curve indicating the fastest germination rate (Figure A4) The
hot water treatment produced an intermediate curve or relatively slower germination
rate The control curve was very flat indicating the slowest germination rate for New
Mexico locust
Control seeds germinated poorly for black locust as well (Figure A5) Hot
water scarification improved germination over the control but all levels of percussion
improved germination over hot water The optimal durations of percussion treatment
were bracketed with a significant increase from the 1 to 2 minute percussion level a
high germination response from 2 to 5 minutes of percussion and finally a significant
54
~ d 25 CL)
p t1 0 ~ t1
g Vl ~ Vl
100~---------------------------------------------------------------
80
60
40
20
o Control Hot Water Percussion
I min Percussion
2 min Percussion
3 min
Scarification
Percussion 4 min
Figure A3 Effect of scarification on germination response for New Mexico locust
Percussion 5 min
=significant difference between adjacent treatment bars ns = no significant difference
Percussion 10 min
~ ~ () u agt
Il-t d o ~
~ ~ c3
100middotlr~~~------------------------------------e- Percussion 5min middot 0middot Hot Water -- Control
80-J=middot======
6b-middot-lmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middot--middotmiddot-middot~-middotmiddot -middotmiddot middot middotmiddot-middot 1
40
20
-----~-----------
0 0 0 middotmiddotmiddotmiddotmiddot0middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot0
0 --~-----y-----~------_---~--- T-----~
o shy -TDay 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A4 Effect of selected scarification treatments on germination speed for New Mexico locust First seven days of fourteen-day experiment are shown
Day 7
80
~ cd
5 60 ()
[) Cl d 0 1t1 d
40sect dUl
-J
20
o
ns
Scarification
Figure A5 Effect of scarification on germination response for black locust = significant difference between adjacent treatment bars ns = no significant difference
decrease at the 10-minute level This drop-off suggests that 10 minutes of percussion
over-treats seeds from this lot of-black locust
A selected percussion treatment intermediate in time (5 minutes) also had the
fastest germination rate for black locust (Figure A6)_ The hot water curve is
somewhat flatter indicating a slower germination rate The control curve is very flat
indicating a slow germination rate
A qualitative difference between hot water and percussion-treated seeds was
also observed Along with healthy germinants the hot water treatment produced
over-treated seeds (Figure A 7a) Examples of damaged germinants include seeds
where the radicle did not elongate (Figure A 7b) seeds where the radicle elongated
but was delayed in freeing itself from the seed coat (Figure A7c) and seeds where the
radicle broke off completely from the embryo (Figure A7d) In addition to these
examples of over-treatment there were numerous small dark seeds that failed to
imbibe water For percussion treatment nearly all seeds took up water (Figure A8a)
Germination was rapid and uniform with little damage evident (Figure A8b)
To summarize both New Mexico locust and black locust seed lots were
dormant The standard hot water scarification treatment improved germination but
only to 56 and 41 for New Mexico locust and black locust respectively Nearly
all percussion durations improved total germination as well as germination rate over
the hot water treatment Germination was greater than 90 for percussion durations
of 4 5 and 10 minutes for New Mexico locust and 3 4 and 5 minutes for black locust
58
CI)
sgt t CI) 0
igt 0
sect ttl t E
Vl 0
c3
100r==~==~~=r------------------------------------~ ___ Percussion 5 min
middotmiddotmiddot0middotmiddotmiddot Hot Water -lIf- Control
80r=========~--~~----------------------------~
60
40
20 -~-~---~---------- ---~------------I
omiddot
O
_~_----------lIf-----~bull -----_ shy---shy I
Day 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A6 Effect of selected scarification treatments on germination speed for black locust First seven days of fourteen-day experiment are shown
Day 7
a
b c d
Figure A7 Damage to New Mexico locust seeds observed during germination counts following hot water scarification A7a Hot water replicate including damaged seedlings and numerous unimbibed seeds A7b Close-up ofseed where radicle failed to elongate A7c Close-up ofseed where radicle elongated but was delayed in freeing itself from the seed coat A7d Close-up ofseed where radicle completely broke offfrom embryo
60
a
b
Figure A8 New Mexico locust seeds following percussion treatment at time of germination counts A8a Percussion replicate with uniformly healthy seedlings and near complete imbibition of seeds A8b Close-up ofpercussion-treated seedling
61
Discussion
Papilionoid seeds have a specific anatomical feature known as the strophiole
or lens-the natural site of water entry to the seed (Hamly 1932) The strophiole is
located on the cotyledonary lobe of a locust seed (Figure A9) When a papilionoid
legume seed is percussed for the appropriate amount of time repeated hits on the
integrated seed coat loosen the constrained cells of the strophiolar region without
excessively damaging the rest of the seed coat (Ballard 1976)
When a percussed seed is soaked water enters exclusively through the
strophiole in a controlled manner (Kelly and Staden 1987) This regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues This
contrasts with seeds that have been hot water or acid scarified treatments that can
randomly degrade the seed coat Unlocalized cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rehman et al 1998 Marunda 1990)
Papilionoids are the largest subfamily of legumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust and black locust these legumes often play an integral role in the
revegetation ofdisturbed lands Developing superior scarification methods to the
standard hot water and acid treatments should fadlitate the use ofthese legumes in
restoration projects
62
Figure A9 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (bc)
63
APPENDIXB
GERMINATION PERCENTS AND STANDARD ERRORS FOR SOURCE
BLOCK AND TREATMENT FOR NEW MEXICO LOCUST
Table B 1 Gennination percents and standard errors for source block and treatment for New Mexico locust
Source Block Treatment Germination Percentage Standard Error
Capitan 1 Immersion 74 437 Capitan 2 Immersion 69 462 Capitan 3 Immersion 71 454 Capitan 4 Immersion 67 470 Capitan Boiling 30 sec 79 407 Capitan 2 Boiling 30 sec 76 427 Capitan 3 Boiling 30 sec 72 449 Capitan 4 Boiling 30 sec 76 427 Capitan Boiling 60 sec 71 454 Capitan 2 Boiling 60 sec 54 498 Capitan 3 Boiling 60 sec 67 470 Capitan 4 Boiling 60 sec 76 427 Capitan I Control 5 218 Capitan 2 Control 7 255 Capitan 3 Control 4 196 Capitan 4 Control 5 218 Capitan 1 Percussion I min 47 499 Capitan 2 Percussion I min
470
Capitan Percussion I min 39 488
Capitan 4 Percussion I min 38 485 Capitan I Percussion 2 min 55 497 Capitan Capitan
2
Percussion 2 min Percussion 2 min
71 65
454 477
Capitan 4 Percussion 2 min 56 496 Capitan I Percussion 4 min 86 347 Capitan 2 Percussion 4 min 79 407 Capitan 3 Percussion 4 min 81 392 Capitan 4 Percussion 4 min 82 384 Capitan I Percussion 8 min 90 300 Capitan 2 Percussion 8 min 88 325 Capitan 3 Percussion 8 min 90 300 Capitan 4 Percussion 8 min 90 300 Gila I Immersion 71 454 Gila 2 Immersion 77 421 Gila
Immersion 75 433
Gila 4 Immersion 72 449
Gila I Boiling 30 sec 68 466
Gila 2 Boiling 30 sec 81 392
Gila 3 Boiling 30 sec 54 498
Gila 4 Boiling 30 sec 63 483
Gila I Boiling 60 sec 55 497
Gila 2 Boiling 60 sec 68 466
Gila 3 Boiling 60 sec 57 495
Gila 4 Boiling 60 sec 70 458
65
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Gila 2 Control 13 336 Gila 3 Control 13 336 Gila 4 Control 20 400 Gila 1 Percussion 1 min 66 474 Gila 2 Percussion 1 min 57 495 Gila 3 Percussion 1 min 73 444 Gila 4 Percussion 1 min 82 384 Gila 1 Percussion 2 min 81 392 Gila 2 Percussion 2 min 86 347 Gila 3 Percussion 2 min 85 357 Gila 4 Percussion 2 min 54 498 Gila 1 Percussion 4 min 79 407 Gila 2 Percussion 4 min 83 376 Gila 3 Percussion 4 min 84 367 Gila 4 Percussion 4 min 79 407 Gila 1 Percussion 8 min 51 500 Gila 2 Percussion 8 min 76 427 Gila 3 Percussion 8 min 52 500 Gila 4 Percussion 8 min 74 439 Holman 1 Immersion 83 376 Holman 2 Immersion 86 347 Holman 3 Immersion 81 392 Holman 4 Immersion 90 300 Holman 1 Boiling 30 sec 90 300 Holman 2 Boiling 30 sec 85 357 Holman 3 Boiling 30 sec 87 336 Holman 4 Boiling 30 sec 83 376 Holman 1 Boiling 60 sec 79 407 Holman 2 Boiling 60 sec 80 400 Holman 3 Boiling 60 sec 86 347 Holman 4 Boiling 60 sec 93 255 Holman Control 25 433 Holman 2 Control 19 392 Holman Control 21 407
Holman 4 Control 24 427
Holman Percussion 1 min 77 421
Holman 2 Percussion 1 min 85 357
Holman 3 Percussion 1 min 80 400
Holman 4 Percussion 1 min 89 313
Holman 1 Percussion 2 min 88 325
Holman 2 Percussion 2 min 96 196
Holman 3 Percussion 2 min 93 255
Holman 4 Percussion 2 min 96 196
Holman 1 Percussion 4 min 92 271
Holman 2 Percussion 4 min 97 171
66
I
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Holman 3 Percussion 4 min 91 286 Holman 4 Percussion 4 min 96 196 Holman 1 Percussion 8 min 88 325 Holman 2 Percussion 8 min 85 357 Holman 3 Percussion 8 min 83 376 Holman 4 Percussion 8 min 82 384 Jemez 1 Immersion 53 499 Jemez 2 Immersion 44 496 Jemez 3 Immersion 52 500 Jemez 4 Immersion 70 458 Jemez 1 Boiling 30 sec 58 494 Jemez 2 Boiling 30 sec 55 497 Jemez 3 Boiling 30 sec 48 500 Jemez 4 Boiling 30 sec 65 477 Jemez 1 Boiling 60 sec 68 466 Jemez 2 Boiling 60 sec 63 483 Jemez 3 Boiling 60 sec 73 444 Jemez 4 Boiling 60 sec 89 313 Jemez 1 Control 2 140 Jemez 2 Control 9 286 Jemez 3 Control 6 237 Jemez 4 Control 8 271 Jemez 1 Percussion 1 min 58 494 Jemez 2 Percussion 1 min 63 483 Jemez 3 Percussion 1 min 77 421 Jemez 4 Percussion 1 min 89 313 Jemez 1 Percussion 2 min 68 466 Jemez 2 Percussion 2 min 64 480 Jemez 3 Percussion 2 min 80 400 Jemez 4 Percussion 2 min 83 376 Jemez Percussion 4 min 68 466 Jemez 2 Percussion 4 min 79 407 Jemez 3 Percussion 4 min 70 458 Jemez 4 Percussion 4 min 83 376 Jemez 1 Percussion 8 min 52 500 Jemez 2 Percussion 8 min 59 492 Jemez 3 Percussion 8 min 61 488 Jemez 4 Percussion 8 min 70 458 Magdalena 1 Immersion 69 462 Magdalena 2 Immersion 69 462 Magdalena J Immersion 70 458 Magdalena 4 Immersion 85 357 Magdalena 1 Boiling 30 sec 65 477 Magdalena 2 Boiling 30 sec 71 454 Magdalena 3 Boiling 30 sec 68 466
67
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Magdalena 4 Boiling 30 sec 83 376 Magdalena 1 Boiling 60 sec 63 483 Magdalena 2 Boiling 60 sec 68 466 Magdalena 3 Boiling 60 sec 63 483 Magdalena 4 Boiling 60 sec 82 384 Magdalena 1 Control 11 313 Magdalena 2 Control 6 237 Magdalena 3 Control 5 218 Magdalena 4 Control 4 196 Magdalena Percussion 1 min 32 466 Magdalena 2 Percussion 1 min 45 497 Magdalena 3 Percussion 1 min 46 498 Magdalena 4 Percussion I min 36 480 Magdalena 1 Percussion 2 min 67 470 Magdalena 2 Percussion 2 min 68 466 Magdalena 3 Percussion 2 min 65 477 Magdalena 4 Percussion 2 min 81 392 Magdalena 1 Percussion 4 min 87 336 Magdalena 2 Percussion 4 min 97 171 Magdalena 3 Percussion 4 min 83 376 Magdalena 4 Percussion 4 min 97 171 Magdalena 1 Percussion 8 min 98 lAO Magdalena 2 Percussion 8 min 100 000 Magdalena 3 Percussion 8 min 96 196 Magdalena 4 Percussion 8 min 98 lAO Manzano 1 Immersion 67 470 Manzano 2 Immersion 57 495 Manzano 3 Immersion 72 4049 Manzano 4 Immersion 73 4044 Manzano 1 Boiling 30 sec 70 458 Manzano 2 Boiling 30 sec 64 480 Manzano 3 Boiling 30 sec 71 454 Manzano 4 Boiling 30 sec 80 400 Manzano I Boiling 60 sec 50 500 Manzano 2 Boiling 60 sec 53 499 Manzano 3 Boiling 60 sec 59 492 Manzano 4 Boiling 60 sec 74 439 Manzano I Control 4 196 Manzano 2 Control 8 271 Manzano 3 Control 1 099 Manzano 4 Control 6 237 Manzano 1 Percussion 1 min 52 500 Manzano 2 Percussion 1 min 39 488 Manzano 3 Percussion 1 min 36 480 Manzano 4 Percussion 1 min 51 500
68
Table B 1 Continued
Source Block Treatment Gerrni~ation Percentage Standard Error
Manzano Percussion 2 min 53 499 Manzano 2 Percussion 2 min 58 494 Manzano 3 Percussion 2 min 61 488 Manzano 4 Percussion 2 min 62 485 Manzano 1 Percussion 4 min 76 427 Manzano 2 Percussion 4 min 85 357 Manzano 3 Percussion 4 min 79 407 Manzano 4 Percussion 4 min 83 376 Manzano 1 Percussion 8 min 87 336 Manzano 2 Percussion 8 min 83 376 Manzano 3 Percussion 8 min 83 376 Manzano 4 Percussion 8 min 75 433 Raton 1 Immersion 54 498 Raton 2 Immersion 37 483 Raton 3 Immersion 39 488 Raton 4 Immersion 69 462 Raton 1 Boiling 30 sec 36 480 Raton 2 Boiling 30 sec 20 400 Raton 3 Boiling 30 sec 28 449 Raton 4 Boiling 30 sec 60 490 Raton 1 Boiling 60 sec 15 357 Raton 2 Boiling 60 sec 171 Raton 3 Boiling 60 sec 4 196 Raton 4 Boiling 60 sec 35 477 Raton 1 Control 4 196 Raton 2 Control 12 325 Raton 3 Control 2 140 Raton 4 Control 6 237 Raton 1 Percussion 1 min 52 500 Raton 2 Percussion I min 29 454 Raton 3 Percussion I min 35 477 Raton 4 Percussion 1 min 31 462 Raton 1 Percussion 2 min 55 497 Raton 2 Percussion 2 min 35 477 Raton 3 Percussion 2 min 33 470 Raton 4 Percussion 2 min 42 494 Raton 1 Percussion 4 min 65 477 Raton 2 Percussion 4 min 52 500 Raton Percussion 4 min 51 500 Raton 4 Percussion 4 min 53 499 Raton 1 Percussion 8 min 88 325 Raton 2 Percussion 8 min 80 400 Raton 3 Percussion 8 min 73 444 Raton 4 Percussion 8 min 74 439 Sacramento 1 Immersion 43 495
69
Table B 1 Continued
-Source Block Treatment Germination Percentage Standard Error
Sacramento 2 Immersion 30 458 Sacramento 3 Immersion 38 485 Sacramento 4 Immersion 56 496 Sacramento 1 Boiling 30 sec 47 499 Sacramento 2 Boiling 30 sec 48 500 Sacramento 3 Boiling 30 sec 48 500 Sacramento 4 Boiling 30 sec 56 496 Sacramento 1 Boiling 60 sec 52 500 Sacramento 2 Boiling 60 sec 67 470 Sacramento 3 Boiling 60 sec 43 495 Sacramento 4 Boiling 60 sec 48 500 Sacramento 1 Control 0 000 Sacramento 2 Control 3 171 Sacramento 3 Control 3 171 Sacramento 4 Control 099 Sacramento 1 Percussion 1 min 57 495 Sacramento 2 Percussion 1 min 61 488 Sacramento 3 Percussion 1 min 62 485 Sacramento 4 Percussion 1 min 55 497 Sacramento 1 Percussion 2 min 84 367 Sacramento 2 Percussion 2 min 70 458 Sacramento 3 Percussion 2 min 81 392 Sacramento 4 Percussion 2 min 69 462 Sacramento 1 Percussion 4 min 91 286 Sacramento 2 Percussion 4 min 84 367 Sacramento 3 Percussion 4 min 84 367 Sacramento 4 Percussion 4 min 94 237 Sacramento 1 Percussion 8 min 82 384 Sacramento 2 Percussion 8 min 81 392 Sacramento 3 Percussion 8 min 82 384 Sacramento 4 Percussion 8 min 81 392 Sandia 1 Immersion 76 427 Sandia 2 Immersion 70 458 Sandia 3 Immersion 70 458 Sandia 4 Immersion 81 392 Sandia 1 Boiling 30 sec 78 414 Sandia 2 Boiling 30 sec 71 454 Sandia 3 Boiling 30 sec 63 483 Sandia 4 Boiling 30 sec 78 414 Sandia 1 Boiling 60 sec 67 470 Sandia 2 Boiling 60 sec 65 477 Sandia 3 Boiling 60 sec 74 439 Sandia 4 Boiling 60 sec 66 474 Sandia 1 Control 16 367 Sandia 2 Control 7 255
70
Table B 1 Continued
Source Block Treatment Gennination Percentage Standard Error
Sandia 3 Control 8 271 Sandia 4 Control 16 367 Sandia 1 Percussion 1 min 67 470 Sandia 2 Percussion 1 min 61 488 Sandia 3 Percussion 1 min 66 474 Sandia 4 Percussion 1 min 59 492 Sandia I Percussion 2 min 82 384 Sandia 2 Percussion 2 min 83 376 Sandia 3 Percussion 2 min 81 392 Sandia 4 Percussion 2 min 84 367 Sandia 1 Percussion 4 min 89 313 Sandia 2 Percussion 4 min 86 347 Sandia 3 Percussion 4 min 89 313 Sandia 4 Percussion 4 min 85 357 Sandia 1 Percussion 8 min 80 400 Sandia 2 Percussion 8 min 77 421 Sandia 3 Percussion 8 min 69 462 Sandia 4 Percussion 8 min 66 474
LITERATURE CITED
Ashby WC Vogel WG Rogers NF 1985 Black locust in the reclamation equation General Technical Report Northeastern Forest Experiment Station USDA Forest Service NE-I05 12 p
Ballard LAT 1973 Physical barriers to gennination Seed Sci Technoll 285-303
___ 1976 Strophiolar water conduction in the seeds ofTrifolieae induced by action on the testa at nonstrophiolar sites Austr J Plant Physiol3 465-469
Barton LV 1947 Special studies on seed coat impenneability Contrib Boyce Thompson Inst 14 355-362
Baskin CC Baskin JM 1998 Seeds Ecology biogeography and evolution of donnancy and gennination Academic Press London UK 666 p
Bewley JD 1997 Seed germination and donnancy Plant Ce119 1055-1066
Bewley JD Black M 1994 Seeds physiology of development and germination 2nd ed Plenum Press New York NY 445 p
Briscoe CB 1996 Growing Nitrogen Fixing Trees in Acid Soils In MH Powell (ed) Nitrogen Fixing Trees for Acid Soils- A Field ManuaL Winrock International Morrilton AR 22 p
Copeland LO McDonald MB 1995 Principles of Seed Science and Technology 3rded Chapman amp Hall New York NY 409 p
Cox RE Klett JE 1984 Seed gennination requirements of native Colorado plants for use in the landscape Plant Propag 30(2) 6-10
Dell B 1980 Structure and function ofthe strophiolar plug in seeds ofAlbizia Lophantha Am J Bot 67 556-563
Dick-Peddie WA 1993 New Mexico Vegetation Past Present and Future University ofNew Mexico Press Albuquerque NM 244 p
Dreesen DR 2001 Propagation protocol for production ofcontainer Robinia neomexicana plants Los Lunas Plant Materials Center Los Lunas New Mexico In Native Plant Network URL httpwwwnativeplantnetworkorg (accessed 9 January 2002) Moscow (ID) University ofIdaho CoIl Natural Resources For Res Nursery
72
Dreesen DR Harrington JT 1997 Propagation of native plants for restoration projects in the Southwestern US- Preliminary Investigations In Landis TD Thompson JR tech coords National Proceedings Forest and Conservation Nursery Assocs Gen Tech Rep PNW-GTR-419 Portland OR USDA For Ser PNW Res Sta 77-88
Gottfried GJ 1980 Control ofNew Mexican locust and the effect on planted ponderosa pine in central Arizona US For Ser Res Note RM-386 Rocky Mountain For Range ExpStn Fort Collins CO
Gosling PG Samuel YK Jones SK 1995 A systematic examination of germination temperature chipping and water temperaturesoak duration pretreatments on the seeds ofLeucaena leucocephala Seed Sci amp Technol 23521-532
Hamly DH 1932 Softening of the seeds of Melilotus alba Bot Gazette 93 345shy375
Hanna PJ 1984 Anatomical features of the seed coat ofAcacia kempeana (Mueller) which relate to increased germination rate induced by heat treatment New Phytol 96 23-29
Hanks JP Dick-Peddie WA 1974 Vegetation patterns of the White Mountains New Mexico Southwest Naturalist 18(4) 371-382
Hine S Harrington JT Dreesen DR 1997 Seed scarification requirements for Robinia neomexicana In Harrington JTtech coord Proc Third SouthwestemContainer Growers Conf Feb 12-13 1997 Ft Collins CO
Hopkinson JM 1993 The strophiole of leguminous seeds Newsletter- IntI Herbage Seed Production Res Grp 18 7-8
---- 1997 Differences in hardseededness between Cunningham and Tarramba leucaena In Leucnet News The Newsletter of the International LeucaenaRes and Devlt Netwk No4
ISTA (International Seed Testing Association) 1999 International rules for seed testing rules Seed Sci amp Technol27 Supplement
Kelly KM van Staden J 1987 The lens as the site of permeability in the papilionoid seed Aspalathus linearis J Plant Physiol 128(45) 395-404
73
Keresztesi B 1988 Black locust the tree of agriculture Outlook on Agr 17(2) 77~85
Kigel 1 and Galili G 1995 Seed development and germination Marcel Dekker New York 853 p
Klemmedson 10 1994 New Mexico locust and parent material Influence on macronutrients of forest floor and soil Soil Sci Soc Am 1 58 974~980
Kuo WH Tam AW 1988 The pathway of water absorption of mung bean seeds Seed Sci amp Technol16(1) 139~144
Leadem CL 1997 Dormancy--unlocking seed secrets In Landis TD Thompson lR tech coords National Proceedings Forestand Conservation Nursery Assocs Gen Tech Rep PNW-G TR-419 Portland OR USDA For Ser PNW Res Sta 43-52
Lin Y Wagner MR Seweyestewa A Honahni MD DeGomez M McMillin JD 1996 Influence of cultural treatments on seed germination and seedling growth of Robinia neomexicana Forest Farm and Community Tree Res Reports Volume 1
Marunda CT 1990 Effects of seed pretreatment on the development ofAcacia auriculforinis and A holosericea seedlings ACIAR Proceedings Series No 28
Meginnis HG 1937 Sulphuric acid treatment to increase germination of black locust seed USDA Circ 453 34 p
Miklas PN Towensend CE Ladd SL 1987 Seedcoat anatomy and the scarification ofcicer milkvetch seed Crop Sci 27 766-772
Monsen SB 1984 Use of shrubs on mine spoils In The Challenge ofProducing Native Plants for the Intermountain Area Proceedings Intermountain Nurserymans Association 1983 conference Las Vegas NV compo PM Murphy 26-31 Ogden UT USDA For Ser Gen Tech Report INT-l68
Olson DF 1974 Robinia L Locust In Seeds of Woody Plants in the US tech coord CS Schopmeyer 728-731 USDA For Ser Agr Handbk No450
Rehman S Loescher RNl Harris P1C 1998 Dormancy breaking and germination ofAcacia salicina Lindl seeds Seed Sci amp Techno127 553-557
Rolston MP 1978 Water impermeable seed dormancy Bot Rev 44 365-396
74
SAS Institute Inc 1989 SASSTAT Users Guide Version 6 4th Edition Volume 1 Cary NC SAS Institute Inc 943 p
SerratoValenti G Conara L Ferrando M Modenesi P 1993 Structural and histochemical features ofStylosanthes scabra (Leguminosae Papilionoidae) seed coat as related to water entry Can J Bot 71 834-840
Simpson BJ 1988 A field guide to Texas trees Austin TX Texas Monthly Press 372p
Tran VN Cavanagh AK 1984 Structural aspects ofdormancy In Seed Physiology Vol 2 Murray DR (ed) p1-44 Academic Press Australia
USDA Forest Service 1948 Woody-plant seed manual USDA Misc Pub1654 416p
Vines RA 1960 Trees Shrubs and Woody Vines of the Southwest Austin Univ ofTexas Press 1104 p
Wagner MR Larson RE Craig TP 1992 Ecology and silvics ofNew Mexico locust (Robinia neomexicana) with an emphasis on the arboreal form p53-57 In JW Hanover K Miller and S Plesko (eds) Proc Intemat Confon Black Locust Biology Culture and Utilization Mich State Univ USA
Wilson JK 1944 Immersing seeds of species of Robinia in boiling water hastens germination J For 42 453-454
Zimmerman RW Carpenter SB 1980 First-year coppice production from a 5shyyear old black locust stand on surface mine spoil p309-314 Centr Hardwd For ConfProc University ofMissouri Columbia
LITERATURE REVIEW
Seed Dormancy
Low gennination in New Mexico locust is primarily the result of seed
donnancy the failure of an intact viable seed to genninate under favorable
conditions (Bewley 1997) Seed donnancy also can be defined as the inability of the
embryo to genninate because of some inherent constraint (Bewley and Black 1994)
The constraint may originate within the embryo referred to as endogenous or embryo
donnancy or from the tissues surrounding the embryo known as exogenous or coatshy
imposed donnancy (Baskin and Baskin 1998 Leadem 1997 Bewley and Black
1994)
New Mexico locust seed is donnant when the impenneable seed coat does not
allow movement ofwater to the embryo This specific fonn of coat-imposed
donnancy where the restraining tissue involved is an impenneable seed coat is
known as physical donnancy (Baskin and Baskin 1998 Bewley and Black 1994)
Physical donnancy is relatively uncommon in seeds less than 10 of all woody
species exhibit this condition (USDA Forest Service 1948) However it is
characteristic of many genera of the Fabaceae (bean) family For example all
successional trees in the genera Gleditsia and Robinia (of which New Mexico locust
is a member) produce seeds that exhibit physical donnancy (Baskin and Baskin
4
Physical Dormancy
Seeds that exhibit physical dormancy are said to be hard seeded referring to
the thick impermeable seed coat (Leadem 1997) The first line of protection of the
testa in leguminous seeds is the outermost waxy cuticle followed by a subcuticle
consisting ofsuberin Next is a palisade layer consisting of sclereid cells that have
thick lignified secondary walls The most common type of sclereid cell in palisade
layers is the macrosclereid The macrosclereids contain water-repellent substances
including cutin lignin suberin and wax (Rolston 1978) This palisade layer is the
primary barrier to water uptake (Serrato-Valenti et al 1993 Tran and Cavanagh
1984)
All seed coat openings must be blocked to effectively prevent water uptake
The natural opening in a seed where it was attached to the mother plant is the weak
link where physical dormancy is usually broken (Hopkinson 1993 Kuo and Tam
1988) Anatomical structures associated with the seed coat opening vary from
family to family In the Papilionoideae the subfamily of the Fabaceae family
containing New Mexico locust the site ofwater entry is the strophiole The
strophiole is a swelling on the seed coat close to the stalk scar (Hopkinson 1997)
Kelly and van Staden (1987) refer to the strophiole site as the lens where
macrosclereid cells must separate and raise before water can pass through Since
middot6germination ofdormant seeds of the Papilionoideae takes place when the strophiole
permeable (Hopkinson 1993 Kuo and Tam 1988) a logical approach to
5
genninating New Mexico locust artificially would be to loosen the strophiolar cells
and allow water to pass through to the embryo
Evolutionary Role of Physical Dormancy
Why is physical dormancy a mechanism that blocks germination
ecologically advantageous When a plant produc~s seeds with different degrees of
dormancy seeds that are dispersed at one time will germinate over a range of time
(Bewley and Black 1994) Through variability of dormancy within a seed crop seeds
are exposed to changing environmental conditions improving the overall chances of
survival of the seed line (Bewley and Black 1994) Varying expression of dormancy
is under genetic control which indicates that this may be an evolved survival strategy
(Kigel and Galili 1995) A range of dormancy levels may be a particular advantage in
the southwestern US where extreme environmental variations exist
Methods for Breaking Dormancy
There are three common approaches to breaking dormancy in New Mexico
locust seed acid scarification hot water soaks and mechanical treatments In each
case the objective of the treatment is to scarify or abrade the seed coat to make it
permeable to water
Acid Scarification
Acid scarification consists of soaking seeds in concentrated sulfuric acid
rinsing thoroughly with water upon removal followed by subsequent drying before
PlaJlltmg Olson (1974) reports that this scarification method produces the highest
middot~~jllalUll results for all Robinia species Most literature refers to a seminal 1937
6
study conducted by HG Meginnis This stud~ t~ough conducted on black locust
seed is regularly cited in the suggested use of acid to treat New Mexico locust seed
as well (Olson 1974 Vines 1960)
Thee published studies document the specific use of sulfuric acid to treat
New Mexico locust seed Cox and Klett (1984) report that soaking seeds in
concentrated sulfuric acid for thirty minutes improved germination to 34 over an
untreated control of4 Lin et al (1996) found that for two seed lots acid
scarification for twenty-five to eighty minutes produced germination of 80 A third
lot however resulted in less than 20 germination at all durations of 10 to 80
minutes Hine et al (1997) showed that acid treatment levels of 1 2 5 and 10
minutes resulted in less than 20 germination across five sources However there
was an increasing trend in germination with acid soak duration and acid treatments
were not imposed for as long a time as the previous two studies (30 to 80 minutes)
The mixed results obtained by these investigators were probably due to the
widely varying germination response of seed lots to acid treatment For this reason
Olson (1974) recommends correlating duration of acid treatment to each seed lot If
treatment duration is too long the embryo will be permanently damaged If soak
duration is too short the seed will remain impermeable to water and fail to germinate
Compounding the problem of treatment duration is the negative effect acid
have on seedling growth Any type of scarification can injure seeds to some
by disrupting cells essential for growth leading to fungal invasion and
middot-uuu~w injury (Copeland and MacDonald 1995) Some legume seeds subjected
7
to 60 minutes of sulfuric acid scarification (to achieve maximum gennination results)
showed more than 50 seedling mortality after gennination (Kelly and van Staden
1987) Bewley and Black (1994) noted that fungal growth on seeds is an indication
of leakage of sugars organic acids amino acids and proteins
Perhaps the greatest drawback to acid scarification is the dangerous nature of
the treatment itself Difficulties associated with storage and trained use of
concentrated acid may discourage many propagators from using this option (Dreesen
and Harrington 1997) Consequently hot water and to a lesser extent mechanical
scarification are more commonly used and practical treatments for legume seeds
Most southwestern container growers use either of these two treatments to scarify
New Mexico locust seed in particular (Hine et al 1997)
Hot Water Scarification
The widespread use ofhot water scarification to treat locust seed is probably
due to its ease of application and efficacy in comparison to other scarification
treatments Although this practice had been in use for some time Wilson (1944) was
one of the first to report on its systematic application for black locust The treatment
consists of placing seeds in boiling water (4 parts water tomiddot 1 part seed by volume)
removing the vessel from the heat source and allowing the water to cool and seeds to
soak for 24 hours
Hine et al (1997) Lin et al (1996) and Cox and Klett (1984) have examined
the specific application ofhot water scarification on New Mexico locust seed Each
these authors compared hot water to acid scarification Lin et aL (1996) reported
8
60 gennination for two hot water-treated seed lots while a third lot germinated
around 50 (as reported above acid scarification germination ranged from 20 to
80 for these lots) It should be noted that the authors only soaked the seeds in water
from 10 to 80 minutes for this experiment as opposed to the standard 24 hours Cox
and Klett (1984) stated that using the standard 24-hour hot water treatment improved
germination over an acid pre-treatment (which produced less than 20 germination)
but they did not give specific gennination numbers for the hot water treatment Hine
et al (1997) found that across five seed lots germination was 66 for the standard
hot water treatment (as compared to less than 20 germination for acid scarification)
Variations in treatment intensity and duration and their subsequent effects on seed lot
appear to contribute to conflicting conclusions as to whether acid scarification or hot
water treatment is a more successful treatment
Given mixed results ease ofapplication is an important argument for use of
hot water application over acid soaks Safety concerns and material costs for hot
water treatments are low (Dreesen and Harrington 1997) An extension of this is the
practicality ofhot water for treating large batches of seed where a relative increase in
water volume is all that is needed However increases in water and seed even when
inthe same 4 1 proportion can result in dramatically different rates of cooling than in
atest tube experiment Different cooling rates can lead to contrasting results for the
treatment temperature duration and seed lot (USDA Forest Service 1948)
As with acid scarification parameters ofhot water treatment application may
to be optimized to maximize germination Hine et al (1997) found that as
9
temperature was increased from 70degC to 100degC overall germination in New Mexico
locust seed increased The 100degC treatment maximized germination for each of five
sources tested with germination usually above 60
Prolonged exposure of seeds ofmany woody legumes to boiling temperatures
also may improve germination (Briscoe 1996) This approach has not been tested on
New Mexico locust seed Exposing seed to prolonged boiling temperatures may
result in fungal infection and subsequent seedling mortality Marunda (1990) noted
severe fungal infections on both acid and hot water treated Acacia holoserica Cunn
ex G Don seeds attributing the infections to seed damage resulting from the
treatments Identifying an optimal hot water treatment level that does not result in a
high rate of seedling mortality is a logical goal
With hot water treatments as with acid there is also a danger of overtreating
Seeds and reducing germination (Gosling et al 1995) The legume Leucaena
leucocephala (Lam) de Wit germinated equally well after 7 seconds of exposure to
water at 100degC 7 seconds at 90degC 30 seconds at 80degC and 4 minutes at 70degC In
addition acceptably high germination was obtained from treatment at 70degC from 3
minutes to 1 hour whereas no seeds germinated following treatment at 100degC for 2
minutes Perhaps along with a greater margin of error for treatment duration treating
~ew Mexico locust seeds at a lower temperature may result in a comparatively lower
of seedling mortality In practice however nursery growers tend to use the
and quickest treatment available The short treatment length at near-boiling
whether recommended or not may be what is used most frequently
10
Fine-tuning a treatment in this range of treatment temperature and duration may be
the most logical approach
Mechanical Scarification
This treatment involves scratching the seed coat with an abrasive material
(USDA Forest Service 1948) One system employs a rotating drum lined with
sandpaper A paddle spinning at a given number ofrevolutions per minute throws the
seed against the sandpaper (Hine et al 1997) A second method dry tumbling
tumbles seed in a mixer with pea gravel or sharp sand for 5 to 7 days (Dreesen 2001)
These approaches have the advantage that control of temperature is not required as in
water treatments nor is safety an issue as with add scarification
Hine et al (1997) mechanically treated New Mexico locust seed using 100shy
grit sandpaper in a Forsberg (rotating drum) scarifier Gennination was less than
10 They concluded that the seed coats were too thin for their apparatus and
proposed that a finer grit sandpaper would produce more even abrasion Although
germination varied by seed lot Dreesen (2001) reported that using a slow-speed dry
tumbling technique for 5 to 7 days produced gennination rates as high as 90
As with acid and hot water treatments treatment duration must be specifically
shy
correlated to seed lot Treated seeds are also susceptible to fungal infection
iHopkinson 1997) In addition equipment is specialized and may not always be
ltU~CUJl~ to a nursery (USDA Forest Service 1948) Several studies have confinned
degrading ofstrophiolar cells due to acid and hot water scarification (Miklas et al
11
1987 Dell 1980 Hanna 1984) but the effect ofmechanical scarification on the
strophiole has not been studied
Alternative Method for Breaking Dormancy Percussion
Strophiole degradation has been examined in depth using an impaction
treatment more commonly known as percussion Hamly (1932) first proposed that
shaking or impacting seeds could split the macrosc1ereid cells at the strophiole
Hamly successfully released tension ofbent strophiolar cells of impermeable
Melilotus alba Medik (White sweet clover) seeds by delivering physical blows
resulting in cells that had split apart and were only slightly bent Ninety-two percent
oftreated seeds became permeable as compared to 05 of control seeds Barton
(1947) showed that percussion significantly increases permeability in black locust
seed Ballard (1976) reasoned that the integrated nature of the strophiole within the
seed coat means a blow on any part of the seed weakens the strophiole There are no
published studies on the use of percussion on New Mexico locust
Percussion methods vary and often seem SUbjective or ill-defined Hamly
(1932) placed seeds in a glass bottle and shook them three times a second for 20
minutes resulting in 180 oscillations per minute Ballard (1973) describes shaking
seeds in a closed metal container with at least fifty seeds per container at 850-900
JS(~l11alIOIlS per minute for 1 minute Kelly and van Staden (1987) placed seeds into
sealed glass flasks Flasks were shaken by a wrist-shaker at a rate of 250
~UUVJ~ per minute Subsequent germination rates were 45 after 2 hours of
55 after 3 hours 60 after 6 hours and 100 after 12 hours
12
There are several potential advantages of percussion over acid hot water and
mechanical scarification Kelly and van Staden (1987) note that a percussion
treatment only alters macrosclereid cells of the strophiole by loosening separating
and raising them They emphasize that minimal damage is done to the overall
anatomical structure of the seed In acid scarification of the same seed lot radicle
and cotyledon damage occurred Based on photographic evidence the authors
believe that this morphological damage was due to uneven pressure during
imbibition with water entering haphazardly wherever the acid had sufficiently
dissolved the coat This ultimately led to uneven hydration of the embryo In
percussed seeds water only entered through the s~ophiole when the entire seed coat
had been saturated resulting in controlled and even embryo hydration (Kelly and van
Staden 1987)
Although all successful scarification treatments may focus at least in part on
the strophiole for papilionoid legumes (Baskin and Baskin 1998) percussion may be
less damaging to seed coat structures particularly when compared with acid Kelly
and van Staden did not compare hot water or mechanical scarification methods
against percussion However the fungal infection associated with hot water and
mechanical scarification as with acid scarificatio~ have been directly associated with
seed damage (Rehman et al 1998 USDA Forest Service 1948) Embryo damage in
has been observed in hot-water scarified legume seeds (Rehman et al 1998
13
By limiting embryo damage percussion scarification reduces seedling
mortality compared to other treatments Kelly and van Staden (1987) noted that
seedlings from the percussion treatment were morphologically sound and vigorous
when compared to acid treatment seedlings The authors attribute this improved
seedling vigor to the controlled water uptake that limited anatomical damage
Despite a 12-hour percussion treatment morphological damage was not substantially
greater than untreated seeds In addition seedling mass measured 10 days following
germination was not significantly different between percussed and untreated seeds
A final advantage ofpercussion may be the ability to adequately treat the most
dormant seeds while avoiding harm to seeds that have already been made permeable
by the treatment or that were permeable to begin with Instead ofcorroding scalding
or scratching the seed coat percussion appears to be just a loosening of the
strophiole spring (Ballard 1973) Kelly and van Staden (1987) were able to
increase germination with a lengthy treatment (12 hours) Figuring out whether a
substantial cushion oftreatment durations exist for percussion ofNew Mexico locust
seed would be a significant improvement to current scarification methods where seed
damage and seedling health is an issue
The greatest challenge for applying percussion to New Mexico locust seeds is
standardizing a treatment that is both effective and readily reproducible by nurseries
with mechanical scarification this may require the development of specific
~UllDment to properly quantify and execute the treatment
14
f
Objectives
The first objective of the study is to detennine which scarification treatment
results in optimal gennination speed and percentage ofNew Mexico locust The
second objective is to describe treatment interactions with nine seed sources collected
in New Mexico
Hypothesis
A percussion treatment acts exclusively on the main area ofwater uptake in
New Mexico locust seed loosening the strophiole so that imbibition can occur This
direct mechanism ofdonnancy release will result in improved gennination rate and
overall gennination percentage for New Mexico locust seed compared to standard
treatments Standard treatments including acid hot water and mechanical
scarification while eventually weakening the strophiolar cleft cause excessive
morphological damage through generalized seed coat degradation These treatments
are therefore prone to inconsistency due to over and under-treatment
15
MATERIALS AND METHODS
Seed Collection and Cleaning
Seeds were collected from August 25 through September 15 2000 from nine
locations along a latitudinal gradient in New Mexico Seed source locations and
descriptive information are given in Figure 1 and Tables I and 2 Stands were
separated by at least 45 km
Pods were hand picked as they began to ripen and dehisce (open) Collections
were made from ground level to approximately 3 m from several trees at each site
Pods were dried in open-ended brown paper bags for two to four weeks at room
temperature (21-230C) to facilitate cleaning Dried pods were placed in a closed cloth
sack which was pounded against the ground to separate seeds from pods For pods
still containing seeds hand maceration (crunching pods with hands) completed seed
separation Debris was removed by threshing with the aid of a greenhouse fan A
South Dakota Seed Blower (Seedbureau Chicago Illinois) was used to remove
remaining chaff and light seeds by air density separation Seeds were placed in dry
storage in sealed quart-size zip-lock bags within a sealed plastic container Storage
temperatures ranged from 2-4oC Seeds remained in storage for approximately 7
months before the experiment
Experimental Design and Treatment Structure
The experiment compared the impact ofhot water bath and percussion
l11aLl11 on germination properties ofNew Mexico locust seed A single
16
-l
Figure 1 Seed source collection map All sources were collected within New Mexico USA separated by at least 4S lan
IIIRZltItW lIJPIirmiddot rllll1lbullbull_~~~_II
Table 1 Latitude longitude location elevation and collection date ofNew Mexico locust seed sources
Lot title Latitude (N) Longitude (W) Location Elevation (m) Collection Date
Raton 36deg5846 104deg2872 Raton Pass NM 2295 82500
Holman 36deg0306 105deg2429 HolmanNM 2476 911500
Jemez 35deg4500 106deg4100 Jemez Springs NM 1976 9900 00
Sandia 35deg1083 106deg2341 Cedar Crest NM 2432 82600
Manzano 34deg3689 106deg2494 ManzanoNM 2465 9200
Magdalena 33deg5953 107deg0903 Magdalena NM 2544 82900
Capitan 33deg2728 105deg4325 Alto NM 2189 911200
Sacramento 32deg5798 105deg4496 Cloudcroft NM 2630 91400 Gila 32deg5400 107deg4600 Kingston NM 2432 9700
f
Table 2 Mean seed weight and standard error by source
a
Source Mean seed weight (g) for Standard error 100 seeds n = 5
Raton Holman Jemez Sandia Manzano Magdalena Capitan Sacramento Gila
2872 b c 2522 d 1826 e 2753 c 2413 d 2559 d 2443 d 3130 a 3029 ab
00103 00403 00276 00301 00370 00374 00458 00279 00348
a Mean seed weights followed by the same letter are not significantly
different
19
experiment was conducted utilizing a randomized complete block design blocked by
time with a factorial treatment structure Experimental factors were seed source and
scarification treatment Nine seed sources from throughout New Mexico were used
(Figure 1 Table 1) Scarification treatments included a control three levels ofhot
water and four levels of percussion Hot water treatments included immersion at
100degC and boiling for periods of 30 seconds and 60 seconds Percussion treatment
levels were 1 2 4 and 8 minutes One hundred seeds per block were used to test
each treatment combination Four blocks of seed were tested one at a time (all seed
sources included in each run) in close chronological sequence (within 6 weeks) A
total of four 100-seed samples were used to test each source by scarification treatment
combination
Treatments
Hot water treatment of seeds utilized 50 ml test tubes filled with 30 ml
distilled water Each test tube was used to treat one 100-seed sample at a time
resulting in a ratio of4 parts water to 1 part seed by volume Test tubes were placed
in a beaker with the beaker filled with tap water to the same level as the water within
test tubes Nine test tubes (one per seed source) were placed in the beaker
simultaneously so that one replication of each seed sourcescarification treatment
cotnblnatlon was conducted simultaneously Hot water treatments include immersion
boiling For immersion treatments seeds were added to test tubes with the water
(boiling) and the test tubes were immediately removed from the beaker to
For the boiling treatments test tubes remained in the beaker with water at
20
boiling temperature for a set duration after the addition of seeds Following all
treatments test tubes with seeds cooled 24 hours in a test tube rack
Percussion treatment utilized a pneumatic paint shaker (Central Pneumatic
model 00422 Camarillo CA) Compressed air was maintained at 530 kPa plusmn30 kPa
(80 psi plusmn 5 psi) resulting in approximately 350 oscillations per minute Seeds were
placed in 118 ml (4 oz) soil tin cans (US Can Lobard IL) and the lids were secured
with duct tape Each can was used to treat one 100-seed sample To take advantage
ofgreatest lateral movement of the paint shaker a spacer (paint can) was used to
position the soil tin at the end of the shaking arm This allowed shaking to occur at
the greatest distance from the pivot point (fulcrum) Tins were placed perpendicular
to the direction of shaking motion allowing seeds to impact at greatest force by
hitting flat surfaces of the soil tin
Germination Testing
Before gennination testing all seeds were soaked 24 hours in reverse
osmosis-treated water (this was included in the process ofhot water treatment
jU~SCIlOea above) Following soaking seeds were placed in 100 cm Petri dishes lined
90 cm Whatman qualitative 1 filter paper circles (MG Scientific Pleasant
WI) One Petri dish was used for each treatment by source combination
papers were initially moistened with 5 mL reverse osmosis-treated water then
~VU1CUlJl moistened as needed to ensure adequate conditions for gennination
were monitored daily (at 300 PM) for gennination for 14 days in accordance
~UIUlnJlVUa Seed Testing Association (ISTA) standards for Robinia species
21
(1STA 1999) Tests were carried out at room temperature (22degC plusmnOSoC)
Germination was classified as protrusion of the radicle from the embryo by at least
1mm as viewed with the naked eye
X-ray Radiography
On August 132001 three lOO-seed samples from the Sacramento seed source
were treated for 14 or 8-minute percussion durations The seeds were sent
immediately by overnight delivery to the Faxitron X-Ray Corporation in Wheeling
Illinois where Dr Bob Karrfalt ofthe USDA Forest Service oversaw their imaging
Radiographs were made at SX with a Faxitron Specimen Radiography System Model
MX-20
Scanning Electron Microscope Photography
On February 192002 a Hitachi S-3200N Variable Pressure Scanning
Electron Microscope (Mountain View CA) was used to examine strophiolar cells of
untreated seeds and seeds percussed for 4 minutes Seeds were cut longitudinally
igt their entire length using a razor blade splitting the strophiolar region roughly in
Cross sections of seed samples magnifying the strophiolar region were viewed
600X under the microscope
Analysis
Categorical Analysis of Variance was used to determine treatment source and
(Le time) main effects and interactions The response variable was total
~vlI This procedure is a Chi-square test of homogeneity using the natural log
of germinated to non-germinated seed for each treatment Maximumshy
22
likelihood analysis was used to calculate Chi-square test statistics with their observed
significance levels A single contrast was conducted to cempare treatment means
against the control Observed significance levels less than or equal to 005 were
considered significant Pairwise comparisons were conducted between non-control
treatment mean percents using a conservative alp~a value of 005 divided by the
number of comparisons (Bonferroni approach) and approximate pairwise z statistics
Categorical analyses were performed using Proc Catmod (SAS Institute 1989)
RESULTS
Scarification seed source block and all interactions between factors
influenced germination (Table 3) The main effects are reported in the presence of
these interactions because the overall robustness of treatments and seed sources are of
interest Background levels ofnaturally occurring variation in seed sources are
seen in control germination percentages in Figure 2 In particular time variability
is unlikely to be under the control of the producer Appendix B details experimental
data for source by treatment gennination percents for each of the four blocks
Averaged over all treatments germination by source ranged from 40 to
79 (Figure 2) Averaged over all sources (Figure 3) and for each source (Figure 4
5) percussion scarification durations of4 or 8 minutes yielded greatest germination
Averaged over all sources germination increased with increasing durations of
percussion then leveled off for the 4- and 8-minute treatments (Figure 3) Immersion
and 30-second boiling scarification maximized germination for the hot water
treatments with a decrease in germination when seeds were boiled for 60 seconds
For all but two sources there is a similar ipcreasing trend in germination from
1to 4 minutes ofpercussion (Figure 4) The Holman source increased in germination
1 to 2 minutes then leveled off at 4 minutes The Jemez source changed little
1 to 4 minutes of treatment As treatment duration increased from 4 to 8
mutes five sources decreased in germination (Figure 4a) whereas three sources
24
Table 3 Categorical Analysis of Variance table for germination response to seed treatment seed source block and the interaction of the factors
Source of DF Chi-Square Observed Variability Significance Level
Source 8 98370 lt00001 Trt 7 233876 lt00001
contrast 1 16976 lt00001 SourceTrt 56 97077 lt00001 Block 3 5538 lt00001
SourceBlock 24 10162 lt00001 TrtBlock 21 7564 lt00001
SourceTrtBlock 168 34283 lt00001
S 4)
8 ~
0
sectg ~ 40 ~
N 0 0
20
o f0ltgt ~ ~4 jybshy ((iltgto ~~ K1ltgt 6~b-~ ~~ 0b-~~ ~~ ~b-~ ~b-~b
80
60 -I-- - ~- middotmiddotmiddotmiddotmiddot1
r -] Northern NM Sources
Central NM Sources
Southern NM Sources
sect-o ~
~b-v(i
SOURCE
Figure 2 Effect ofNew Mexico locust seed source on germination for data averaged over all scarification treatments Control germination is shown by solid line within bar
80
~ cd ~ ltl) () 60 ~ ~
N
t 0shyrti tshySltl)
40
0
20
o lmmersion Boiling 30 sec Boiling 60 sec Control 1 min 2min 4 min 8 min
Hot Water Treatments Percussion Treatments
Figure 3 Effect of scarification on germination ofNew Mexico locust for data averaged over all seed sources Means labelled with the same letter are not significantly different
100~----------------------~--~----------------~
80
60
ltU
~
40 IJ
sectgsI
l100
80
60
40
a I middotmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot middotImiddotmiddotmiddotmiddotmiddotmiddot E
_~iii-bull _ _ bull f --~--
t-- -~ ---- -shy ---- shyI ~ ---- - shy
L
fiII
~
b
- shy ---r
- Holman
Sacramento Sandia Gila Jemez
c ~
ltgt ~
Tmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot-I
j~=~=-~~-1
I middotmiddot ~ bull r
t tr
Y -------M-a-gd-a-Ie-n-a--
1 _ _
fCapitan
---- Manzano ~y -_ Raton
2 4 8
Time interval ofpercussion scarification (min)
Figure 4 Effect of seed source by treatment interaction on New Mexico locust germination for percussion scarification pattern a and pattern b
28
tv 0
~ d
~ A s o
1 c3
80
60
40
20
o
TIT
t------=middotfmiddotmiddot-middotmiddot-middotmiddot-middotmiddot 1- --- - - --=- - -shy--cE= - _--shy --shy ----shy ~ ~ ---shy ~ -~- _ --shy-------r 1
I
Raton Holman
-_ Jemez
--- Sandia Manzano Magdalena Capitan Sacramento Gila
Immersion Boiling 30 sec Boiling 60 sec
Hot Water Scarification
Figure 5 Effect of seed source by treatment interaction on New Mexico locust germination for hot water scarification
continued to increase in germination (Figure 4b) There was no difference between
the two treatment levels for the Manzano source (Figure 4b)
The effect ofhot water scarification also varied among sources with few
overall differences between hot water treatments (Figure 5) However boiling for 60
seconds yielded highest germination for the Jemez source whereas increasing
durations ofboiling decreased germination for the Raton source
In addition to consistently high germination 4- and 8-minute percussionshy
treated seeds germinated as fast or faster than all other treatments (Figure 6 Table 4)
050 measured as the number of days needed to complete 50 ofeventual
germination averaged 2 days for both ofthese treatments
X-ray radiographs were taken of seeds collected from the Sacramento source
percussed for 1 4 or 8 minutes (Figure 7) Seeds show signs of damage at 8 minutes
ofpercussion (Figure 8) Compared to an undamaged seed percussed for only 4
minutes (Figure 8a) damage at the 8-minute level includes fractured radicles pitting
of the seed coat and chips in seed coat structure (Figure 8b-d)
Scanning electron microscope (SEM) images at 600X reveal the direct effect
of 4 minutes of percussion on cells of the strophiolar region (Figure 9) Whereas
untreated seeds retain cellular organization in the strophiolar region strophiolar cells
seeds treated for 4 minutes ofpercussion separate and raise resulting in a
~-~e~U appearance
30
Umiddot-~middot--l-~---middotymiddotmiddotmiddot-----middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot--middot--middotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middotmiddotmiddotmiddotmiddotmiddotmiddot- -
050= gt1
-e-- Percussion 8 min -0- Percussion 4 min ---T- Percussion 2 min -----ltil- Percussion 1 min -- Immersion --0- Boiling 30 sec -+- Boiling 60 sec -ltgt- Control
2 4 6 8 10 12
Day Figure 6 Timecourse ofNew Mexico locust gennination following scarification
treatments G50 is the number of days to 50 of eventual germination
31
14
Table 4 Effect of seed source and treatment on G50 (days to 50 germination)
Seed Source Control Immersion B30sec B60sec Plmin P2min P4min P8min mean Raton 3 3 5 7 4 3 2 2 4 Holman 2 2 2 2 2 2 2 2 2
Jemez 4 6 6 5 3 2 2 2 4 Sandia 2 2 2 2 2 2 2 2 2
w tv Manzano 5 2 2 3 3 2 2 2 3
Magdalena 4 2 2 3 4 3 2 1 3
Capitan 5 4 2 2 3 2 2 2 3
Sacramento 3 2 2 2 4 3 2 2 3 Gila 3 2 2 2 2 2 2 2 2 mean 3 3 3 3 3 2 2 2 3 standard 11 14 16 18 09 05 0 03 08 deviation
Note Columns and rows may not agree due to rounding
Figure 7 X-ray radiographs ofSacramento seed source percussed for 1 4 or 8 minutes
33
Figure 8a-d Seed damage close-ups Figure a shows undamaged seed percussed for 4 minutes Figures b-d show seeds percussed for 8 minutes Damage includes fracturing ofradicle in b complete removal ofseed coat in c and pitting ofseed coat in d
34
Untreated Treated
Figure 9 SEM images ofuntreated vs 4-minute percussion-treated strophiolar cells ofNew Mexico locust seeds Treated cells are loosened separated and raised
DISCUSSION
Determining the Optimal Treatment
Averaging results of scarification treatmems across seed sources is a useful
tool to predict which treatment is most likely to be successful for an unknown seed
source Averaged over all sources four- and eight-minute percussion treatments
resulted in the highest germination 815 and 790 respectively and produced
germination that was as fast or faster than all other treatments Although there was no
difference between these two treatments on average an argument for 4 minutes of
percussion as a standard protocol for treating New Mexico locust seeds can be made
Descriptively 4 minutes of percussion improved germination over 8 minutes
ofpercussion for a majority of sources (five out of nine) 8 minutes of percussion
improved germination for three ofnine sources with no difference for a fmal source
For individual sources 4 minutes ofpercussion was always the best or secondmiddot best
treatment and was always equal to or better than all hot water treatments
Germination at 8 minutes ofpercussion was lower than all other percussion durations
for the Jemez and Gila sources and was lower than at least one hot water treatment for
these sources as well
The x-ray radiographs of the Sacramento seed source illustrate the excessive
internal and external seed damage that can occur with 8 minutes of percussion
germination counts 8-minute percussed seeds from Holman and Capitan
ClUll signs of damage that were visible to the naked eye (see below)
r~evertheless some of these seeds still fit the criteria for germination Neither x-ray
36
photography nor visible observations during seed counts revealed seed damage at the
4-minute percussion level In operational trials seeds percussed for 4 minutes
developed into healthy seedlings (Khadduri and Harrington unpublished data)
Strophiole and Seed Coat Behavior
Four minutes ofpercussion successfully breaks seed dormancy in a large
percentage ofNew Mexico locust seeds without excessively damaging the
surrounding seed coat or internal seed coat structures Most papilionoid legume
seeds including seeds ofNew Mexico locust have a specific region of the seed coat
known as the strophiole or lens (Hamly 1932) The strophiole is located on the
cotyledonary lobe of a locust seed (Figure 10 a-c) If seeds are not indiscriminately
damaged the strophiole is the first point of water entry to the embryo (Hopkinson
1997) When a papilionoid seed is percussed for the appropriate amount of time
repeated hits on the integrated seed coat loosen the constrained cells of the strophiolar
region without excessively damaging the rest of the seed coat (Ballard 1976) SEM
photos illuStrate that 4 minutes of percussion loosened and separated strophiolar cells
ofNew Mexico locust seeds when compared with constrained organized strophiolar
cells of untreated seeds (Figure 9) Research-has shown that water passes to the
embryo through the treated strophiole in a controlled manner and regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues
(Kelly and van Staden 1987)
37
10 Strophiole ofunimbibed New Mexico locust seed (a) and following uptake (bc)
38
In contrast hot-water treatments can excessively degrade the seed coat
(Rebman et al 1998 Marunda 1990) Random cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rebman et aI 1998 Marunda 1990) Along with a
proportionately large amount ofun-imbibed (thus under-treated) seeds (Figure l1a)
damage was observed in hot-water treated seeds from six sources during germination
counts Damage included seeds where the radicle failed to elongate (Figure 11 b)
seeds where the radicle elongated but was delayed in freeing itself from the seed coat
(Figure 11c) and seeds where the radicle completely broke off from the embryo
(Figure lId) In addition some seeds germinated in reverse (the cotyledon emerged
fIrst) If the radicle emerged completely from the seed coat they were counted as
germinated
While nearly all seeds treated for 8 minutes ofpercussion imbibed water
damage was evident in x-ray evaluations (Sacramento seed source) and in
germination counts (Holman and Capitan) at this treatment level Seed damage
during germination counts was similar to levels found in hot-water treated seeds
suggesting that over-treatment by both hot water and longer durations of percussion
may result in similar damage to seed structure
The Sacramento and Holman seed sources were two of the fIve sources that
opcrplltlpn in germination from the 4- to 8-minute durations of percussion
the benefIts ofloosening the strophiole as evidenced by imbibition of
all 8-minute treated seeds were outweighed by external and internal seed coat
39
a
b c d
11 Damage to New Mexico locust seeds observed during germination cotmts following hot water scarification 9a Hot water replicate including ~t seedlings and numerous unimbibed seeds 9b Close-up of seed where raallCle failed to elongate 9c Close-up ofseed where radicle elongated but was
-gtIn in freeing itself from the seed coat 9d Close-up ofseed where radicle broke off from embryo
40
damage (which also may be contributing to high imbibition) For the three sources
for which 8 minutes of percussion resulted in maximum germination extended
percussion of seeds necessary to loosen the strophiole may not have excessively
damaged the seed anatomy The Manzano source did not significantly change in
germination from 4 to 8 minutes of percussion suggesting that an increase in
permeable seeds at the 8-minute treatment duration was balanced by the number of
seeds that failed to germinate due to seed damage To summarize the 4-minute
percussion treatment improved germination over 8 minutes ofpercussion for a
majority of sources (five ofnine) the 8-minute percussion treatment improved
germination over 4-minutes ofpercussion for three sources and there was no
difference with the Manzano source Notably for the Raton source 8 minutes of
percussion increased germination by 235 percentage points over 4-minutes of
percussion (7875 vs 5525 respectively) the next best treatment Raton was the
only source to result in germination less than 75 for 4 minutes ofpercussion so a
grower may wish to apply longer percussion durations to lots that do not respond well
to this treatment level
Finally there was an interesting trend in the Jemez and Raton sources seed
that significantly contributed to the source by treatment interaction (Figure 12) A
~u test showed that the Jemez source had a significantly lighter seed weight than
other sources while the Raton source had an average seed weight For the Jemez
the longest duration of boiling increased germination Conversely only short
U4UVll) of percussion were needed to achieve high germination and the longest
41
80 _ Jemez a Raton
iD
~ C iD e iD
Q c 0 ~
c ~ iD 0
20
0------shy
Immersion Boiling 30 sec Boiling 60 sec
Hot Water Scarification
10degTr========~--------~------------------------------
80
60
40
20
_ Jemez Raton
0------shy
1 min 2 min 4 min 8 min
Percussion Scarification
Figure 12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources
42
b
--~----~~~~========~---- duration ofpercussion reduced germination For the Raton source longer durations
ofboiling consistently decreased germination whereas longer percussion treatments
consistently increased germination
A possible explanation is that a thick seed coat is associated with a loose
strophiole structure and that a thin seed coat is associated with a constrained
strophiole This contrast merits further study and may suggest an ecological
adaptation Further investigation ofthis phenomenon may contribute to successful
pre-screening ofunknown seed sources to determine optimal treatment leveL
43
CONCLUSION
Based on results obtained in pilot study (see Appendix) and thesis
experiments 4 minutes ofpercussion is recommended as a standard protocol for
treating an unknown source ofNew Mexico locust seed In addition to laboratory
results the 4-minute percussion treatment has resulted in germination greater than
80 and healthy seedling grow-outs for production purposes (unpublished data) In
laboratory experiments only one hundred seeds per 118 ml (40z) soil tin were
percussed at a time whereas for production we have treated up to five hundred seeds
per tin For large-scale production ofNew Mexico locust seedlings growers may
wish to experiment with larger quantities of seed and larger-size soil tins
Seed storage trials following percussion treatments were not conducted
However germination greater than 80 and healthy seedlings were obtained when
seeds were percussion-treated and subsequently stored at room-temperature (22degC)
for one week (unpublished data) Seed storage trials merit further study as the
standard hot water treatment leaves seeds in a softened condition that is not
conducive to storage
Hydroseeding is an effective method for direct seeding in reclamation
situations Percussion treatment may be conducive to hydro seeding as seeds remain
a dry relatively undamaged yet permeable condition following treatment Sulfuric
seeds also remain dry following treatment but extensive damage to seed coat
poor seedling vigor have been reported with this treatment (Kelly and van Staden
A grow-out study using a hydro seeder on a reclamation site testing percussion
44
against other treatments such as sulfuric acid and mechanical scarification also
merits further study
Papilionoids are the largest subfamily oflegumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust these legumes often play an integral role in the revegetation of
disturbed lands The pilot study (Appendix) indicates that percussion scarification
successfully treats the widely used black locust as well Developing superior
scarification methods to the standard hot water mechanical scarification and acid
treatments may facilitate the use ofpapilionoid legumes in reclamation projects
45
APPENDICES
APPENDIX A
PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR
NEW MEXICO LOCUST AND BLACK LOCUST SEEDS
Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4
Hot water and sulfuric acid soaks are traditional treatments for seeds of many
temperate woody legwnes including locusts However these scarification techniques
often produce inconsistent germination Percussion scarification where seeds are
repeatedly propelled against a hard surface was compared with hot water
scarification to evaluate treatment efficacy for New Mexico locust (Robinia
neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment
seeds were placed in a 98degC water bath which was immediately removed from the
heat source For percussion scarification seeds were placed in a soil sample tin and
agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the
control were followed by 24-hour water soaks Hot water baths resulted in 56 and
41 germination for New Mexico locust and black locust respectively For both
species nearly all durations ofpercussion increased germination over the hot water
treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3
4 and 5 minutes for black locust resulted in at least 90 germination Traditional
scarification treatments randomly degrade the entire seed coat which can lead to
tissue damage during water uptake Percussion scarification specifically weakens the
strophiole the natural source ofwater entry to the seed in papilionoid legwnes
INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom
2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031
48
Following percussion imbibition is controlled through the strophiole and underlying
tissue is protected
Keywords New Mexico locust black locust scarification percussion germination
Introduction
Locust trees (Robinia spp) are aggressive pioneer species that quickly
colonize disturbed land fix atmospheric nitrogen in the soil and stabilize slopes that
are prone to erosion (Klemmedson 1994 Ashby et al 1985) (Figure A1ab)
Recognition of these attributes has contributed to increased demand for locusts in
reclamation projects Locust seeds however exhibit physical dormancy where a
thick seed coat prevents the movement of water and gasses to the embryo (Leadem
1997)
Sulfuric acid or hot water soaks have traditionally been used to break seed
dormancy in macro-propagation of locusts Many growers are moving away from the
use of su1furic acid in treating locust seeds (Dreesen and Harrington 1997) In
addition to being dangerous acid soak durations must be specifically correlated to
seed lot (Olson 1974) Collections of native seed can vary tremendously in seed size
seed weight and hard seededness etc This variability limits the utility of sulfuric
acid soaks as even within a given lot some seeds may be damaged whereas others
are under-treated and fail to imbibe (take up water) A survey of Southwestern
container growers indicates that hot water baths are currently the most common
scarification method for locust seeds (Hine et al 1997) However hot water baths
also produce inconsistent germination (Lin et al 1996)
49
b
Figure AI New Mexico locust a primary invader following disturbance Ala Crown-sprouting at edge ofburn near Los Alamos NM--one year after Cerro Grande fire Alb Colonizing road cut Gila National Forest NM
50
Percussion scarification where seeds are repeatedly propelled against a hard
surface is an alternative dormancy-breaking method that has proven successful in
legumes related to locusts (Hamly 1932 Barton 1947) We compared this alternative
treatment to hot water scarification to evaluate treatment efficacy for New Mexico
locust and black locust seeds
Materials and Methods
Commercial New Mexico locust seeds (Western Native Seed Coaldale CO)
were collected fall 2000 in Huerfano County CO Black locust seeds were collected
September 2000 from Taos County NM by harvesting ripe pods from several trees at
a distance of up to 3 m from ground level Black locust seeds were allowed to air-dry
for 3 weeks and were threshed and separated from large chaffusing a greenhouse
fan A South Dakota Seed Blower (Chicago Illinltois) was used to remove fine chaff
from both seed lots Seeds were storedmiddotat 2-4C until the germination study was
conducted in March 2001
This study compared hot water scarification with percussion scarification
using treatment and species as experimental factors Along with a control
scarification treatments included immersion of seed in boiling water as well as
percussion durations of 1 2 3 45 or 10 minutes Four 100-seed samples were used
to test each species by scarification treatment combination
For the hot water treatment test tubes were filled with 30 mL water and
placed in a water-filled beaker (Figure A2a) The bath was raised to boiling (-98C at
51
b
c
Figure A2 Implementation ofhot water and percussion scarification A2a Hot water bath A2b Paint-shaker set-up A2c Placing soil tin containing seeds at end ofpaint shaker ann
52
Las Cruces NM elevation of 1300 meters) Each 100-seed replicate was placed in a
test tube and each test tube was immediately removed to cool at room temperature
Percussion scarification was implemented using a pneumatic paint shaker
(Central Pneumatic model 00422 Camarillo CA) (Figure A2b) This allowed
standardization of treatment intensity Compressed air pressure was maintained at
530kPa plusmn 30kPa (80psi plusmn 5psi) resulting in approximately 350 oscillations per
minute Each lOO-seed sample was placed in a 118 ml (4 oz) soil tin for shaking To
maximize lateral movement of paint shaker a paint can was used as a spacer to place
soil tins at the end of the shaking arm (Figure A2c) This allowed shaking to occur at
the greatest distance from the pivot point (fulcrum) Following all treatments
including the control seeds were soaked for twenty-four hours in distilled water prior
to plating out
Gennination was evaluated on lab benches at room temperature Each
replicate of seeds was placed in a 100 cm petri dish on moistened filter paper with
humidity maintained by enclosing the petri dishes in ziplock bags Gennination was
monitored daily for 14 days in accordance with International Seed Testing
Association standards for Robinia species (ISTA 1999) Gennination was defined as
protrusion of the radicle from the embryo by at least 1mm as viewed by the naked
eye
Categorical Analysis of Variance using Prpc Catmod (SAS Institute 1989)
was used to detennine treatment differences in germination percentages for each
source This procedure is an extension of a Chi-square test of homogeneity using the
53
natural log of the ratio of germinated to non-germinated seeds for each treatment Pshy
values less than 005 were considered significant A limited set of pairwiseshy
comparisons was conducted to compare treatment mean percents using a
conservative alpha value of 005 divided by the number of comparisons
Results
Percussion maximized germination for both New Mexico locust and black
locust There were three germination response levels for New Mexico locust (Figure
A3) First there was a low response in the control indicating a dormant seed lot
The hot water treatment and I-minute duration of percussion share a second
intermediate response Germination was highest at percussion levels from 2 to 10
minutes
A selected percussion treatment intermediate in time (5 minutes) produced the
steepest germination curve indicating the fastest germination rate (Figure A4) The
hot water treatment produced an intermediate curve or relatively slower germination
rate The control curve was very flat indicating the slowest germination rate for New
Mexico locust
Control seeds germinated poorly for black locust as well (Figure A5) Hot
water scarification improved germination over the control but all levels of percussion
improved germination over hot water The optimal durations of percussion treatment
were bracketed with a significant increase from the 1 to 2 minute percussion level a
high germination response from 2 to 5 minutes of percussion and finally a significant
54
~ d 25 CL)
p t1 0 ~ t1
g Vl ~ Vl
100~---------------------------------------------------------------
80
60
40
20
o Control Hot Water Percussion
I min Percussion
2 min Percussion
3 min
Scarification
Percussion 4 min
Figure A3 Effect of scarification on germination response for New Mexico locust
Percussion 5 min
=significant difference between adjacent treatment bars ns = no significant difference
Percussion 10 min
~ ~ () u agt
Il-t d o ~
~ ~ c3
100middotlr~~~------------------------------------e- Percussion 5min middot 0middot Hot Water -- Control
80-J=middot======
6b-middot-lmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middot--middotmiddot-middot~-middotmiddot -middotmiddot middot middotmiddot-middot 1
40
20
-----~-----------
0 0 0 middotmiddotmiddotmiddotmiddot0middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot0
0 --~-----y-----~------_---~--- T-----~
o shy -TDay 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A4 Effect of selected scarification treatments on germination speed for New Mexico locust First seven days of fourteen-day experiment are shown
Day 7
80
~ cd
5 60 ()
[) Cl d 0 1t1 d
40sect dUl
-J
20
o
ns
Scarification
Figure A5 Effect of scarification on germination response for black locust = significant difference between adjacent treatment bars ns = no significant difference
decrease at the 10-minute level This drop-off suggests that 10 minutes of percussion
over-treats seeds from this lot of-black locust
A selected percussion treatment intermediate in time (5 minutes) also had the
fastest germination rate for black locust (Figure A6)_ The hot water curve is
somewhat flatter indicating a slower germination rate The control curve is very flat
indicating a slow germination rate
A qualitative difference between hot water and percussion-treated seeds was
also observed Along with healthy germinants the hot water treatment produced
over-treated seeds (Figure A 7a) Examples of damaged germinants include seeds
where the radicle did not elongate (Figure A 7b) seeds where the radicle elongated
but was delayed in freeing itself from the seed coat (Figure A7c) and seeds where the
radicle broke off completely from the embryo (Figure A7d) In addition to these
examples of over-treatment there were numerous small dark seeds that failed to
imbibe water For percussion treatment nearly all seeds took up water (Figure A8a)
Germination was rapid and uniform with little damage evident (Figure A8b)
To summarize both New Mexico locust and black locust seed lots were
dormant The standard hot water scarification treatment improved germination but
only to 56 and 41 for New Mexico locust and black locust respectively Nearly
all percussion durations improved total germination as well as germination rate over
the hot water treatment Germination was greater than 90 for percussion durations
of 4 5 and 10 minutes for New Mexico locust and 3 4 and 5 minutes for black locust
58
CI)
sgt t CI) 0
igt 0
sect ttl t E
Vl 0
c3
100r==~==~~=r------------------------------------~ ___ Percussion 5 min
middotmiddotmiddot0middotmiddotmiddot Hot Water -lIf- Control
80r=========~--~~----------------------------~
60
40
20 -~-~---~---------- ---~------------I
omiddot
O
_~_----------lIf-----~bull -----_ shy---shy I
Day 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A6 Effect of selected scarification treatments on germination speed for black locust First seven days of fourteen-day experiment are shown
Day 7
a
b c d
Figure A7 Damage to New Mexico locust seeds observed during germination counts following hot water scarification A7a Hot water replicate including damaged seedlings and numerous unimbibed seeds A7b Close-up ofseed where radicle failed to elongate A7c Close-up ofseed where radicle elongated but was delayed in freeing itself from the seed coat A7d Close-up ofseed where radicle completely broke offfrom embryo
60
a
b
Figure A8 New Mexico locust seeds following percussion treatment at time of germination counts A8a Percussion replicate with uniformly healthy seedlings and near complete imbibition of seeds A8b Close-up ofpercussion-treated seedling
61
Discussion
Papilionoid seeds have a specific anatomical feature known as the strophiole
or lens-the natural site of water entry to the seed (Hamly 1932) The strophiole is
located on the cotyledonary lobe of a locust seed (Figure A9) When a papilionoid
legume seed is percussed for the appropriate amount of time repeated hits on the
integrated seed coat loosen the constrained cells of the strophiolar region without
excessively damaging the rest of the seed coat (Ballard 1976)
When a percussed seed is soaked water enters exclusively through the
strophiole in a controlled manner (Kelly and Staden 1987) This regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues This
contrasts with seeds that have been hot water or acid scarified treatments that can
randomly degrade the seed coat Unlocalized cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rehman et al 1998 Marunda 1990)
Papilionoids are the largest subfamily of legumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust and black locust these legumes often play an integral role in the
revegetation ofdisturbed lands Developing superior scarification methods to the
standard hot water and acid treatments should fadlitate the use ofthese legumes in
restoration projects
62
Figure A9 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (bc)
63
APPENDIXB
GERMINATION PERCENTS AND STANDARD ERRORS FOR SOURCE
BLOCK AND TREATMENT FOR NEW MEXICO LOCUST
Table B 1 Gennination percents and standard errors for source block and treatment for New Mexico locust
Source Block Treatment Germination Percentage Standard Error
Capitan 1 Immersion 74 437 Capitan 2 Immersion 69 462 Capitan 3 Immersion 71 454 Capitan 4 Immersion 67 470 Capitan Boiling 30 sec 79 407 Capitan 2 Boiling 30 sec 76 427 Capitan 3 Boiling 30 sec 72 449 Capitan 4 Boiling 30 sec 76 427 Capitan Boiling 60 sec 71 454 Capitan 2 Boiling 60 sec 54 498 Capitan 3 Boiling 60 sec 67 470 Capitan 4 Boiling 60 sec 76 427 Capitan I Control 5 218 Capitan 2 Control 7 255 Capitan 3 Control 4 196 Capitan 4 Control 5 218 Capitan 1 Percussion I min 47 499 Capitan 2 Percussion I min
470
Capitan Percussion I min 39 488
Capitan 4 Percussion I min 38 485 Capitan I Percussion 2 min 55 497 Capitan Capitan
2
Percussion 2 min Percussion 2 min
71 65
454 477
Capitan 4 Percussion 2 min 56 496 Capitan I Percussion 4 min 86 347 Capitan 2 Percussion 4 min 79 407 Capitan 3 Percussion 4 min 81 392 Capitan 4 Percussion 4 min 82 384 Capitan I Percussion 8 min 90 300 Capitan 2 Percussion 8 min 88 325 Capitan 3 Percussion 8 min 90 300 Capitan 4 Percussion 8 min 90 300 Gila I Immersion 71 454 Gila 2 Immersion 77 421 Gila
Immersion 75 433
Gila 4 Immersion 72 449
Gila I Boiling 30 sec 68 466
Gila 2 Boiling 30 sec 81 392
Gila 3 Boiling 30 sec 54 498
Gila 4 Boiling 30 sec 63 483
Gila I Boiling 60 sec 55 497
Gila 2 Boiling 60 sec 68 466
Gila 3 Boiling 60 sec 57 495
Gila 4 Boiling 60 sec 70 458
65
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Gila 2 Control 13 336 Gila 3 Control 13 336 Gila 4 Control 20 400 Gila 1 Percussion 1 min 66 474 Gila 2 Percussion 1 min 57 495 Gila 3 Percussion 1 min 73 444 Gila 4 Percussion 1 min 82 384 Gila 1 Percussion 2 min 81 392 Gila 2 Percussion 2 min 86 347 Gila 3 Percussion 2 min 85 357 Gila 4 Percussion 2 min 54 498 Gila 1 Percussion 4 min 79 407 Gila 2 Percussion 4 min 83 376 Gila 3 Percussion 4 min 84 367 Gila 4 Percussion 4 min 79 407 Gila 1 Percussion 8 min 51 500 Gila 2 Percussion 8 min 76 427 Gila 3 Percussion 8 min 52 500 Gila 4 Percussion 8 min 74 439 Holman 1 Immersion 83 376 Holman 2 Immersion 86 347 Holman 3 Immersion 81 392 Holman 4 Immersion 90 300 Holman 1 Boiling 30 sec 90 300 Holman 2 Boiling 30 sec 85 357 Holman 3 Boiling 30 sec 87 336 Holman 4 Boiling 30 sec 83 376 Holman 1 Boiling 60 sec 79 407 Holman 2 Boiling 60 sec 80 400 Holman 3 Boiling 60 sec 86 347 Holman 4 Boiling 60 sec 93 255 Holman Control 25 433 Holman 2 Control 19 392 Holman Control 21 407
Holman 4 Control 24 427
Holman Percussion 1 min 77 421
Holman 2 Percussion 1 min 85 357
Holman 3 Percussion 1 min 80 400
Holman 4 Percussion 1 min 89 313
Holman 1 Percussion 2 min 88 325
Holman 2 Percussion 2 min 96 196
Holman 3 Percussion 2 min 93 255
Holman 4 Percussion 2 min 96 196
Holman 1 Percussion 4 min 92 271
Holman 2 Percussion 4 min 97 171
66
I
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Holman 3 Percussion 4 min 91 286 Holman 4 Percussion 4 min 96 196 Holman 1 Percussion 8 min 88 325 Holman 2 Percussion 8 min 85 357 Holman 3 Percussion 8 min 83 376 Holman 4 Percussion 8 min 82 384 Jemez 1 Immersion 53 499 Jemez 2 Immersion 44 496 Jemez 3 Immersion 52 500 Jemez 4 Immersion 70 458 Jemez 1 Boiling 30 sec 58 494 Jemez 2 Boiling 30 sec 55 497 Jemez 3 Boiling 30 sec 48 500 Jemez 4 Boiling 30 sec 65 477 Jemez 1 Boiling 60 sec 68 466 Jemez 2 Boiling 60 sec 63 483 Jemez 3 Boiling 60 sec 73 444 Jemez 4 Boiling 60 sec 89 313 Jemez 1 Control 2 140 Jemez 2 Control 9 286 Jemez 3 Control 6 237 Jemez 4 Control 8 271 Jemez 1 Percussion 1 min 58 494 Jemez 2 Percussion 1 min 63 483 Jemez 3 Percussion 1 min 77 421 Jemez 4 Percussion 1 min 89 313 Jemez 1 Percussion 2 min 68 466 Jemez 2 Percussion 2 min 64 480 Jemez 3 Percussion 2 min 80 400 Jemez 4 Percussion 2 min 83 376 Jemez Percussion 4 min 68 466 Jemez 2 Percussion 4 min 79 407 Jemez 3 Percussion 4 min 70 458 Jemez 4 Percussion 4 min 83 376 Jemez 1 Percussion 8 min 52 500 Jemez 2 Percussion 8 min 59 492 Jemez 3 Percussion 8 min 61 488 Jemez 4 Percussion 8 min 70 458 Magdalena 1 Immersion 69 462 Magdalena 2 Immersion 69 462 Magdalena J Immersion 70 458 Magdalena 4 Immersion 85 357 Magdalena 1 Boiling 30 sec 65 477 Magdalena 2 Boiling 30 sec 71 454 Magdalena 3 Boiling 30 sec 68 466
67
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Magdalena 4 Boiling 30 sec 83 376 Magdalena 1 Boiling 60 sec 63 483 Magdalena 2 Boiling 60 sec 68 466 Magdalena 3 Boiling 60 sec 63 483 Magdalena 4 Boiling 60 sec 82 384 Magdalena 1 Control 11 313 Magdalena 2 Control 6 237 Magdalena 3 Control 5 218 Magdalena 4 Control 4 196 Magdalena Percussion 1 min 32 466 Magdalena 2 Percussion 1 min 45 497 Magdalena 3 Percussion 1 min 46 498 Magdalena 4 Percussion I min 36 480 Magdalena 1 Percussion 2 min 67 470 Magdalena 2 Percussion 2 min 68 466 Magdalena 3 Percussion 2 min 65 477 Magdalena 4 Percussion 2 min 81 392 Magdalena 1 Percussion 4 min 87 336 Magdalena 2 Percussion 4 min 97 171 Magdalena 3 Percussion 4 min 83 376 Magdalena 4 Percussion 4 min 97 171 Magdalena 1 Percussion 8 min 98 lAO Magdalena 2 Percussion 8 min 100 000 Magdalena 3 Percussion 8 min 96 196 Magdalena 4 Percussion 8 min 98 lAO Manzano 1 Immersion 67 470 Manzano 2 Immersion 57 495 Manzano 3 Immersion 72 4049 Manzano 4 Immersion 73 4044 Manzano 1 Boiling 30 sec 70 458 Manzano 2 Boiling 30 sec 64 480 Manzano 3 Boiling 30 sec 71 454 Manzano 4 Boiling 30 sec 80 400 Manzano I Boiling 60 sec 50 500 Manzano 2 Boiling 60 sec 53 499 Manzano 3 Boiling 60 sec 59 492 Manzano 4 Boiling 60 sec 74 439 Manzano I Control 4 196 Manzano 2 Control 8 271 Manzano 3 Control 1 099 Manzano 4 Control 6 237 Manzano 1 Percussion 1 min 52 500 Manzano 2 Percussion 1 min 39 488 Manzano 3 Percussion 1 min 36 480 Manzano 4 Percussion 1 min 51 500
68
Table B 1 Continued
Source Block Treatment Gerrni~ation Percentage Standard Error
Manzano Percussion 2 min 53 499 Manzano 2 Percussion 2 min 58 494 Manzano 3 Percussion 2 min 61 488 Manzano 4 Percussion 2 min 62 485 Manzano 1 Percussion 4 min 76 427 Manzano 2 Percussion 4 min 85 357 Manzano 3 Percussion 4 min 79 407 Manzano 4 Percussion 4 min 83 376 Manzano 1 Percussion 8 min 87 336 Manzano 2 Percussion 8 min 83 376 Manzano 3 Percussion 8 min 83 376 Manzano 4 Percussion 8 min 75 433 Raton 1 Immersion 54 498 Raton 2 Immersion 37 483 Raton 3 Immersion 39 488 Raton 4 Immersion 69 462 Raton 1 Boiling 30 sec 36 480 Raton 2 Boiling 30 sec 20 400 Raton 3 Boiling 30 sec 28 449 Raton 4 Boiling 30 sec 60 490 Raton 1 Boiling 60 sec 15 357 Raton 2 Boiling 60 sec 171 Raton 3 Boiling 60 sec 4 196 Raton 4 Boiling 60 sec 35 477 Raton 1 Control 4 196 Raton 2 Control 12 325 Raton 3 Control 2 140 Raton 4 Control 6 237 Raton 1 Percussion 1 min 52 500 Raton 2 Percussion I min 29 454 Raton 3 Percussion I min 35 477 Raton 4 Percussion 1 min 31 462 Raton 1 Percussion 2 min 55 497 Raton 2 Percussion 2 min 35 477 Raton 3 Percussion 2 min 33 470 Raton 4 Percussion 2 min 42 494 Raton 1 Percussion 4 min 65 477 Raton 2 Percussion 4 min 52 500 Raton Percussion 4 min 51 500 Raton 4 Percussion 4 min 53 499 Raton 1 Percussion 8 min 88 325 Raton 2 Percussion 8 min 80 400 Raton 3 Percussion 8 min 73 444 Raton 4 Percussion 8 min 74 439 Sacramento 1 Immersion 43 495
69
Table B 1 Continued
-Source Block Treatment Germination Percentage Standard Error
Sacramento 2 Immersion 30 458 Sacramento 3 Immersion 38 485 Sacramento 4 Immersion 56 496 Sacramento 1 Boiling 30 sec 47 499 Sacramento 2 Boiling 30 sec 48 500 Sacramento 3 Boiling 30 sec 48 500 Sacramento 4 Boiling 30 sec 56 496 Sacramento 1 Boiling 60 sec 52 500 Sacramento 2 Boiling 60 sec 67 470 Sacramento 3 Boiling 60 sec 43 495 Sacramento 4 Boiling 60 sec 48 500 Sacramento 1 Control 0 000 Sacramento 2 Control 3 171 Sacramento 3 Control 3 171 Sacramento 4 Control 099 Sacramento 1 Percussion 1 min 57 495 Sacramento 2 Percussion 1 min 61 488 Sacramento 3 Percussion 1 min 62 485 Sacramento 4 Percussion 1 min 55 497 Sacramento 1 Percussion 2 min 84 367 Sacramento 2 Percussion 2 min 70 458 Sacramento 3 Percussion 2 min 81 392 Sacramento 4 Percussion 2 min 69 462 Sacramento 1 Percussion 4 min 91 286 Sacramento 2 Percussion 4 min 84 367 Sacramento 3 Percussion 4 min 84 367 Sacramento 4 Percussion 4 min 94 237 Sacramento 1 Percussion 8 min 82 384 Sacramento 2 Percussion 8 min 81 392 Sacramento 3 Percussion 8 min 82 384 Sacramento 4 Percussion 8 min 81 392 Sandia 1 Immersion 76 427 Sandia 2 Immersion 70 458 Sandia 3 Immersion 70 458 Sandia 4 Immersion 81 392 Sandia 1 Boiling 30 sec 78 414 Sandia 2 Boiling 30 sec 71 454 Sandia 3 Boiling 30 sec 63 483 Sandia 4 Boiling 30 sec 78 414 Sandia 1 Boiling 60 sec 67 470 Sandia 2 Boiling 60 sec 65 477 Sandia 3 Boiling 60 sec 74 439 Sandia 4 Boiling 60 sec 66 474 Sandia 1 Control 16 367 Sandia 2 Control 7 255
70
Table B 1 Continued
Source Block Treatment Gennination Percentage Standard Error
Sandia 3 Control 8 271 Sandia 4 Control 16 367 Sandia 1 Percussion 1 min 67 470 Sandia 2 Percussion 1 min 61 488 Sandia 3 Percussion 1 min 66 474 Sandia 4 Percussion 1 min 59 492 Sandia I Percussion 2 min 82 384 Sandia 2 Percussion 2 min 83 376 Sandia 3 Percussion 2 min 81 392 Sandia 4 Percussion 2 min 84 367 Sandia 1 Percussion 4 min 89 313 Sandia 2 Percussion 4 min 86 347 Sandia 3 Percussion 4 min 89 313 Sandia 4 Percussion 4 min 85 357 Sandia 1 Percussion 8 min 80 400 Sandia 2 Percussion 8 min 77 421 Sandia 3 Percussion 8 min 69 462 Sandia 4 Percussion 8 min 66 474
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Marunda CT 1990 Effects of seed pretreatment on the development ofAcacia auriculforinis and A holosericea seedlings ACIAR Proceedings Series No 28
Meginnis HG 1937 Sulphuric acid treatment to increase germination of black locust seed USDA Circ 453 34 p
Miklas PN Towensend CE Ladd SL 1987 Seedcoat anatomy and the scarification ofcicer milkvetch seed Crop Sci 27 766-772
Monsen SB 1984 Use of shrubs on mine spoils In The Challenge ofProducing Native Plants for the Intermountain Area Proceedings Intermountain Nurserymans Association 1983 conference Las Vegas NV compo PM Murphy 26-31 Ogden UT USDA For Ser Gen Tech Report INT-l68
Olson DF 1974 Robinia L Locust In Seeds of Woody Plants in the US tech coord CS Schopmeyer 728-731 USDA For Ser Agr Handbk No450
Rehman S Loescher RNl Harris P1C 1998 Dormancy breaking and germination ofAcacia salicina Lindl seeds Seed Sci amp Techno127 553-557
Rolston MP 1978 Water impermeable seed dormancy Bot Rev 44 365-396
74
SAS Institute Inc 1989 SASSTAT Users Guide Version 6 4th Edition Volume 1 Cary NC SAS Institute Inc 943 p
SerratoValenti G Conara L Ferrando M Modenesi P 1993 Structural and histochemical features ofStylosanthes scabra (Leguminosae Papilionoidae) seed coat as related to water entry Can J Bot 71 834-840
Simpson BJ 1988 A field guide to Texas trees Austin TX Texas Monthly Press 372p
Tran VN Cavanagh AK 1984 Structural aspects ofdormancy In Seed Physiology Vol 2 Murray DR (ed) p1-44 Academic Press Australia
USDA Forest Service 1948 Woody-plant seed manual USDA Misc Pub1654 416p
Vines RA 1960 Trees Shrubs and Woody Vines of the Southwest Austin Univ ofTexas Press 1104 p
Wagner MR Larson RE Craig TP 1992 Ecology and silvics ofNew Mexico locust (Robinia neomexicana) with an emphasis on the arboreal form p53-57 In JW Hanover K Miller and S Plesko (eds) Proc Intemat Confon Black Locust Biology Culture and Utilization Mich State Univ USA
Wilson JK 1944 Immersing seeds of species of Robinia in boiling water hastens germination J For 42 453-454
Zimmerman RW Carpenter SB 1980 First-year coppice production from a 5shyyear old black locust stand on surface mine spoil p309-314 Centr Hardwd For ConfProc University ofMissouri Columbia
Physical Dormancy
Seeds that exhibit physical dormancy are said to be hard seeded referring to
the thick impermeable seed coat (Leadem 1997) The first line of protection of the
testa in leguminous seeds is the outermost waxy cuticle followed by a subcuticle
consisting ofsuberin Next is a palisade layer consisting of sclereid cells that have
thick lignified secondary walls The most common type of sclereid cell in palisade
layers is the macrosclereid The macrosclereids contain water-repellent substances
including cutin lignin suberin and wax (Rolston 1978) This palisade layer is the
primary barrier to water uptake (Serrato-Valenti et al 1993 Tran and Cavanagh
1984)
All seed coat openings must be blocked to effectively prevent water uptake
The natural opening in a seed where it was attached to the mother plant is the weak
link where physical dormancy is usually broken (Hopkinson 1993 Kuo and Tam
1988) Anatomical structures associated with the seed coat opening vary from
family to family In the Papilionoideae the subfamily of the Fabaceae family
containing New Mexico locust the site ofwater entry is the strophiole The
strophiole is a swelling on the seed coat close to the stalk scar (Hopkinson 1997)
Kelly and van Staden (1987) refer to the strophiole site as the lens where
macrosclereid cells must separate and raise before water can pass through Since
middot6germination ofdormant seeds of the Papilionoideae takes place when the strophiole
permeable (Hopkinson 1993 Kuo and Tam 1988) a logical approach to
5
genninating New Mexico locust artificially would be to loosen the strophiolar cells
and allow water to pass through to the embryo
Evolutionary Role of Physical Dormancy
Why is physical dormancy a mechanism that blocks germination
ecologically advantageous When a plant produc~s seeds with different degrees of
dormancy seeds that are dispersed at one time will germinate over a range of time
(Bewley and Black 1994) Through variability of dormancy within a seed crop seeds
are exposed to changing environmental conditions improving the overall chances of
survival of the seed line (Bewley and Black 1994) Varying expression of dormancy
is under genetic control which indicates that this may be an evolved survival strategy
(Kigel and Galili 1995) A range of dormancy levels may be a particular advantage in
the southwestern US where extreme environmental variations exist
Methods for Breaking Dormancy
There are three common approaches to breaking dormancy in New Mexico
locust seed acid scarification hot water soaks and mechanical treatments In each
case the objective of the treatment is to scarify or abrade the seed coat to make it
permeable to water
Acid Scarification
Acid scarification consists of soaking seeds in concentrated sulfuric acid
rinsing thoroughly with water upon removal followed by subsequent drying before
PlaJlltmg Olson (1974) reports that this scarification method produces the highest
middot~~jllalUll results for all Robinia species Most literature refers to a seminal 1937
6
study conducted by HG Meginnis This stud~ t~ough conducted on black locust
seed is regularly cited in the suggested use of acid to treat New Mexico locust seed
as well (Olson 1974 Vines 1960)
Thee published studies document the specific use of sulfuric acid to treat
New Mexico locust seed Cox and Klett (1984) report that soaking seeds in
concentrated sulfuric acid for thirty minutes improved germination to 34 over an
untreated control of4 Lin et al (1996) found that for two seed lots acid
scarification for twenty-five to eighty minutes produced germination of 80 A third
lot however resulted in less than 20 germination at all durations of 10 to 80
minutes Hine et al (1997) showed that acid treatment levels of 1 2 5 and 10
minutes resulted in less than 20 germination across five sources However there
was an increasing trend in germination with acid soak duration and acid treatments
were not imposed for as long a time as the previous two studies (30 to 80 minutes)
The mixed results obtained by these investigators were probably due to the
widely varying germination response of seed lots to acid treatment For this reason
Olson (1974) recommends correlating duration of acid treatment to each seed lot If
treatment duration is too long the embryo will be permanently damaged If soak
duration is too short the seed will remain impermeable to water and fail to germinate
Compounding the problem of treatment duration is the negative effect acid
have on seedling growth Any type of scarification can injure seeds to some
by disrupting cells essential for growth leading to fungal invasion and
middot-uuu~w injury (Copeland and MacDonald 1995) Some legume seeds subjected
7
to 60 minutes of sulfuric acid scarification (to achieve maximum gennination results)
showed more than 50 seedling mortality after gennination (Kelly and van Staden
1987) Bewley and Black (1994) noted that fungal growth on seeds is an indication
of leakage of sugars organic acids amino acids and proteins
Perhaps the greatest drawback to acid scarification is the dangerous nature of
the treatment itself Difficulties associated with storage and trained use of
concentrated acid may discourage many propagators from using this option (Dreesen
and Harrington 1997) Consequently hot water and to a lesser extent mechanical
scarification are more commonly used and practical treatments for legume seeds
Most southwestern container growers use either of these two treatments to scarify
New Mexico locust seed in particular (Hine et al 1997)
Hot Water Scarification
The widespread use ofhot water scarification to treat locust seed is probably
due to its ease of application and efficacy in comparison to other scarification
treatments Although this practice had been in use for some time Wilson (1944) was
one of the first to report on its systematic application for black locust The treatment
consists of placing seeds in boiling water (4 parts water tomiddot 1 part seed by volume)
removing the vessel from the heat source and allowing the water to cool and seeds to
soak for 24 hours
Hine et al (1997) Lin et al (1996) and Cox and Klett (1984) have examined
the specific application ofhot water scarification on New Mexico locust seed Each
these authors compared hot water to acid scarification Lin et aL (1996) reported
8
60 gennination for two hot water-treated seed lots while a third lot germinated
around 50 (as reported above acid scarification germination ranged from 20 to
80 for these lots) It should be noted that the authors only soaked the seeds in water
from 10 to 80 minutes for this experiment as opposed to the standard 24 hours Cox
and Klett (1984) stated that using the standard 24-hour hot water treatment improved
germination over an acid pre-treatment (which produced less than 20 germination)
but they did not give specific gennination numbers for the hot water treatment Hine
et al (1997) found that across five seed lots germination was 66 for the standard
hot water treatment (as compared to less than 20 germination for acid scarification)
Variations in treatment intensity and duration and their subsequent effects on seed lot
appear to contribute to conflicting conclusions as to whether acid scarification or hot
water treatment is a more successful treatment
Given mixed results ease ofapplication is an important argument for use of
hot water application over acid soaks Safety concerns and material costs for hot
water treatments are low (Dreesen and Harrington 1997) An extension of this is the
practicality ofhot water for treating large batches of seed where a relative increase in
water volume is all that is needed However increases in water and seed even when
inthe same 4 1 proportion can result in dramatically different rates of cooling than in
atest tube experiment Different cooling rates can lead to contrasting results for the
treatment temperature duration and seed lot (USDA Forest Service 1948)
As with acid scarification parameters ofhot water treatment application may
to be optimized to maximize germination Hine et al (1997) found that as
9
temperature was increased from 70degC to 100degC overall germination in New Mexico
locust seed increased The 100degC treatment maximized germination for each of five
sources tested with germination usually above 60
Prolonged exposure of seeds ofmany woody legumes to boiling temperatures
also may improve germination (Briscoe 1996) This approach has not been tested on
New Mexico locust seed Exposing seed to prolonged boiling temperatures may
result in fungal infection and subsequent seedling mortality Marunda (1990) noted
severe fungal infections on both acid and hot water treated Acacia holoserica Cunn
ex G Don seeds attributing the infections to seed damage resulting from the
treatments Identifying an optimal hot water treatment level that does not result in a
high rate of seedling mortality is a logical goal
With hot water treatments as with acid there is also a danger of overtreating
Seeds and reducing germination (Gosling et al 1995) The legume Leucaena
leucocephala (Lam) de Wit germinated equally well after 7 seconds of exposure to
water at 100degC 7 seconds at 90degC 30 seconds at 80degC and 4 minutes at 70degC In
addition acceptably high germination was obtained from treatment at 70degC from 3
minutes to 1 hour whereas no seeds germinated following treatment at 100degC for 2
minutes Perhaps along with a greater margin of error for treatment duration treating
~ew Mexico locust seeds at a lower temperature may result in a comparatively lower
of seedling mortality In practice however nursery growers tend to use the
and quickest treatment available The short treatment length at near-boiling
whether recommended or not may be what is used most frequently
10
Fine-tuning a treatment in this range of treatment temperature and duration may be
the most logical approach
Mechanical Scarification
This treatment involves scratching the seed coat with an abrasive material
(USDA Forest Service 1948) One system employs a rotating drum lined with
sandpaper A paddle spinning at a given number ofrevolutions per minute throws the
seed against the sandpaper (Hine et al 1997) A second method dry tumbling
tumbles seed in a mixer with pea gravel or sharp sand for 5 to 7 days (Dreesen 2001)
These approaches have the advantage that control of temperature is not required as in
water treatments nor is safety an issue as with add scarification
Hine et al (1997) mechanically treated New Mexico locust seed using 100shy
grit sandpaper in a Forsberg (rotating drum) scarifier Gennination was less than
10 They concluded that the seed coats were too thin for their apparatus and
proposed that a finer grit sandpaper would produce more even abrasion Although
germination varied by seed lot Dreesen (2001) reported that using a slow-speed dry
tumbling technique for 5 to 7 days produced gennination rates as high as 90
As with acid and hot water treatments treatment duration must be specifically
shy
correlated to seed lot Treated seeds are also susceptible to fungal infection
iHopkinson 1997) In addition equipment is specialized and may not always be
ltU~CUJl~ to a nursery (USDA Forest Service 1948) Several studies have confinned
degrading ofstrophiolar cells due to acid and hot water scarification (Miklas et al
11
1987 Dell 1980 Hanna 1984) but the effect ofmechanical scarification on the
strophiole has not been studied
Alternative Method for Breaking Dormancy Percussion
Strophiole degradation has been examined in depth using an impaction
treatment more commonly known as percussion Hamly (1932) first proposed that
shaking or impacting seeds could split the macrosc1ereid cells at the strophiole
Hamly successfully released tension ofbent strophiolar cells of impermeable
Melilotus alba Medik (White sweet clover) seeds by delivering physical blows
resulting in cells that had split apart and were only slightly bent Ninety-two percent
oftreated seeds became permeable as compared to 05 of control seeds Barton
(1947) showed that percussion significantly increases permeability in black locust
seed Ballard (1976) reasoned that the integrated nature of the strophiole within the
seed coat means a blow on any part of the seed weakens the strophiole There are no
published studies on the use of percussion on New Mexico locust
Percussion methods vary and often seem SUbjective or ill-defined Hamly
(1932) placed seeds in a glass bottle and shook them three times a second for 20
minutes resulting in 180 oscillations per minute Ballard (1973) describes shaking
seeds in a closed metal container with at least fifty seeds per container at 850-900
JS(~l11alIOIlS per minute for 1 minute Kelly and van Staden (1987) placed seeds into
sealed glass flasks Flasks were shaken by a wrist-shaker at a rate of 250
~UUVJ~ per minute Subsequent germination rates were 45 after 2 hours of
55 after 3 hours 60 after 6 hours and 100 after 12 hours
12
There are several potential advantages of percussion over acid hot water and
mechanical scarification Kelly and van Staden (1987) note that a percussion
treatment only alters macrosclereid cells of the strophiole by loosening separating
and raising them They emphasize that minimal damage is done to the overall
anatomical structure of the seed In acid scarification of the same seed lot radicle
and cotyledon damage occurred Based on photographic evidence the authors
believe that this morphological damage was due to uneven pressure during
imbibition with water entering haphazardly wherever the acid had sufficiently
dissolved the coat This ultimately led to uneven hydration of the embryo In
percussed seeds water only entered through the s~ophiole when the entire seed coat
had been saturated resulting in controlled and even embryo hydration (Kelly and van
Staden 1987)
Although all successful scarification treatments may focus at least in part on
the strophiole for papilionoid legumes (Baskin and Baskin 1998) percussion may be
less damaging to seed coat structures particularly when compared with acid Kelly
and van Staden did not compare hot water or mechanical scarification methods
against percussion However the fungal infection associated with hot water and
mechanical scarification as with acid scarificatio~ have been directly associated with
seed damage (Rehman et al 1998 USDA Forest Service 1948) Embryo damage in
has been observed in hot-water scarified legume seeds (Rehman et al 1998
13
By limiting embryo damage percussion scarification reduces seedling
mortality compared to other treatments Kelly and van Staden (1987) noted that
seedlings from the percussion treatment were morphologically sound and vigorous
when compared to acid treatment seedlings The authors attribute this improved
seedling vigor to the controlled water uptake that limited anatomical damage
Despite a 12-hour percussion treatment morphological damage was not substantially
greater than untreated seeds In addition seedling mass measured 10 days following
germination was not significantly different between percussed and untreated seeds
A final advantage ofpercussion may be the ability to adequately treat the most
dormant seeds while avoiding harm to seeds that have already been made permeable
by the treatment or that were permeable to begin with Instead ofcorroding scalding
or scratching the seed coat percussion appears to be just a loosening of the
strophiole spring (Ballard 1973) Kelly and van Staden (1987) were able to
increase germination with a lengthy treatment (12 hours) Figuring out whether a
substantial cushion oftreatment durations exist for percussion ofNew Mexico locust
seed would be a significant improvement to current scarification methods where seed
damage and seedling health is an issue
The greatest challenge for applying percussion to New Mexico locust seeds is
standardizing a treatment that is both effective and readily reproducible by nurseries
with mechanical scarification this may require the development of specific
~UllDment to properly quantify and execute the treatment
14
f
Objectives
The first objective of the study is to detennine which scarification treatment
results in optimal gennination speed and percentage ofNew Mexico locust The
second objective is to describe treatment interactions with nine seed sources collected
in New Mexico
Hypothesis
A percussion treatment acts exclusively on the main area ofwater uptake in
New Mexico locust seed loosening the strophiole so that imbibition can occur This
direct mechanism ofdonnancy release will result in improved gennination rate and
overall gennination percentage for New Mexico locust seed compared to standard
treatments Standard treatments including acid hot water and mechanical
scarification while eventually weakening the strophiolar cleft cause excessive
morphological damage through generalized seed coat degradation These treatments
are therefore prone to inconsistency due to over and under-treatment
15
MATERIALS AND METHODS
Seed Collection and Cleaning
Seeds were collected from August 25 through September 15 2000 from nine
locations along a latitudinal gradient in New Mexico Seed source locations and
descriptive information are given in Figure 1 and Tables I and 2 Stands were
separated by at least 45 km
Pods were hand picked as they began to ripen and dehisce (open) Collections
were made from ground level to approximately 3 m from several trees at each site
Pods were dried in open-ended brown paper bags for two to four weeks at room
temperature (21-230C) to facilitate cleaning Dried pods were placed in a closed cloth
sack which was pounded against the ground to separate seeds from pods For pods
still containing seeds hand maceration (crunching pods with hands) completed seed
separation Debris was removed by threshing with the aid of a greenhouse fan A
South Dakota Seed Blower (Seedbureau Chicago Illinois) was used to remove
remaining chaff and light seeds by air density separation Seeds were placed in dry
storage in sealed quart-size zip-lock bags within a sealed plastic container Storage
temperatures ranged from 2-4oC Seeds remained in storage for approximately 7
months before the experiment
Experimental Design and Treatment Structure
The experiment compared the impact ofhot water bath and percussion
l11aLl11 on germination properties ofNew Mexico locust seed A single
16
-l
Figure 1 Seed source collection map All sources were collected within New Mexico USA separated by at least 4S lan
IIIRZltItW lIJPIirmiddot rllll1lbullbull_~~~_II
Table 1 Latitude longitude location elevation and collection date ofNew Mexico locust seed sources
Lot title Latitude (N) Longitude (W) Location Elevation (m) Collection Date
Raton 36deg5846 104deg2872 Raton Pass NM 2295 82500
Holman 36deg0306 105deg2429 HolmanNM 2476 911500
Jemez 35deg4500 106deg4100 Jemez Springs NM 1976 9900 00
Sandia 35deg1083 106deg2341 Cedar Crest NM 2432 82600
Manzano 34deg3689 106deg2494 ManzanoNM 2465 9200
Magdalena 33deg5953 107deg0903 Magdalena NM 2544 82900
Capitan 33deg2728 105deg4325 Alto NM 2189 911200
Sacramento 32deg5798 105deg4496 Cloudcroft NM 2630 91400 Gila 32deg5400 107deg4600 Kingston NM 2432 9700
f
Table 2 Mean seed weight and standard error by source
a
Source Mean seed weight (g) for Standard error 100 seeds n = 5
Raton Holman Jemez Sandia Manzano Magdalena Capitan Sacramento Gila
2872 b c 2522 d 1826 e 2753 c 2413 d 2559 d 2443 d 3130 a 3029 ab
00103 00403 00276 00301 00370 00374 00458 00279 00348
a Mean seed weights followed by the same letter are not significantly
different
19
experiment was conducted utilizing a randomized complete block design blocked by
time with a factorial treatment structure Experimental factors were seed source and
scarification treatment Nine seed sources from throughout New Mexico were used
(Figure 1 Table 1) Scarification treatments included a control three levels ofhot
water and four levels of percussion Hot water treatments included immersion at
100degC and boiling for periods of 30 seconds and 60 seconds Percussion treatment
levels were 1 2 4 and 8 minutes One hundred seeds per block were used to test
each treatment combination Four blocks of seed were tested one at a time (all seed
sources included in each run) in close chronological sequence (within 6 weeks) A
total of four 100-seed samples were used to test each source by scarification treatment
combination
Treatments
Hot water treatment of seeds utilized 50 ml test tubes filled with 30 ml
distilled water Each test tube was used to treat one 100-seed sample at a time
resulting in a ratio of4 parts water to 1 part seed by volume Test tubes were placed
in a beaker with the beaker filled with tap water to the same level as the water within
test tubes Nine test tubes (one per seed source) were placed in the beaker
simultaneously so that one replication of each seed sourcescarification treatment
cotnblnatlon was conducted simultaneously Hot water treatments include immersion
boiling For immersion treatments seeds were added to test tubes with the water
(boiling) and the test tubes were immediately removed from the beaker to
For the boiling treatments test tubes remained in the beaker with water at
20
boiling temperature for a set duration after the addition of seeds Following all
treatments test tubes with seeds cooled 24 hours in a test tube rack
Percussion treatment utilized a pneumatic paint shaker (Central Pneumatic
model 00422 Camarillo CA) Compressed air was maintained at 530 kPa plusmn30 kPa
(80 psi plusmn 5 psi) resulting in approximately 350 oscillations per minute Seeds were
placed in 118 ml (4 oz) soil tin cans (US Can Lobard IL) and the lids were secured
with duct tape Each can was used to treat one 100-seed sample To take advantage
ofgreatest lateral movement of the paint shaker a spacer (paint can) was used to
position the soil tin at the end of the shaking arm This allowed shaking to occur at
the greatest distance from the pivot point (fulcrum) Tins were placed perpendicular
to the direction of shaking motion allowing seeds to impact at greatest force by
hitting flat surfaces of the soil tin
Germination Testing
Before gennination testing all seeds were soaked 24 hours in reverse
osmosis-treated water (this was included in the process ofhot water treatment
jU~SCIlOea above) Following soaking seeds were placed in 100 cm Petri dishes lined
90 cm Whatman qualitative 1 filter paper circles (MG Scientific Pleasant
WI) One Petri dish was used for each treatment by source combination
papers were initially moistened with 5 mL reverse osmosis-treated water then
~VU1CUlJl moistened as needed to ensure adequate conditions for gennination
were monitored daily (at 300 PM) for gennination for 14 days in accordance
~UIUlnJlVUa Seed Testing Association (ISTA) standards for Robinia species
21
(1STA 1999) Tests were carried out at room temperature (22degC plusmnOSoC)
Germination was classified as protrusion of the radicle from the embryo by at least
1mm as viewed with the naked eye
X-ray Radiography
On August 132001 three lOO-seed samples from the Sacramento seed source
were treated for 14 or 8-minute percussion durations The seeds were sent
immediately by overnight delivery to the Faxitron X-Ray Corporation in Wheeling
Illinois where Dr Bob Karrfalt ofthe USDA Forest Service oversaw their imaging
Radiographs were made at SX with a Faxitron Specimen Radiography System Model
MX-20
Scanning Electron Microscope Photography
On February 192002 a Hitachi S-3200N Variable Pressure Scanning
Electron Microscope (Mountain View CA) was used to examine strophiolar cells of
untreated seeds and seeds percussed for 4 minutes Seeds were cut longitudinally
igt their entire length using a razor blade splitting the strophiolar region roughly in
Cross sections of seed samples magnifying the strophiolar region were viewed
600X under the microscope
Analysis
Categorical Analysis of Variance was used to determine treatment source and
(Le time) main effects and interactions The response variable was total
~vlI This procedure is a Chi-square test of homogeneity using the natural log
of germinated to non-germinated seed for each treatment Maximumshy
22
likelihood analysis was used to calculate Chi-square test statistics with their observed
significance levels A single contrast was conducted to cempare treatment means
against the control Observed significance levels less than or equal to 005 were
considered significant Pairwise comparisons were conducted between non-control
treatment mean percents using a conservative alp~a value of 005 divided by the
number of comparisons (Bonferroni approach) and approximate pairwise z statistics
Categorical analyses were performed using Proc Catmod (SAS Institute 1989)
RESULTS
Scarification seed source block and all interactions between factors
influenced germination (Table 3) The main effects are reported in the presence of
these interactions because the overall robustness of treatments and seed sources are of
interest Background levels ofnaturally occurring variation in seed sources are
seen in control germination percentages in Figure 2 In particular time variability
is unlikely to be under the control of the producer Appendix B details experimental
data for source by treatment gennination percents for each of the four blocks
Averaged over all treatments germination by source ranged from 40 to
79 (Figure 2) Averaged over all sources (Figure 3) and for each source (Figure 4
5) percussion scarification durations of4 or 8 minutes yielded greatest germination
Averaged over all sources germination increased with increasing durations of
percussion then leveled off for the 4- and 8-minute treatments (Figure 3) Immersion
and 30-second boiling scarification maximized germination for the hot water
treatments with a decrease in germination when seeds were boiled for 60 seconds
For all but two sources there is a similar ipcreasing trend in germination from
1to 4 minutes ofpercussion (Figure 4) The Holman source increased in germination
1 to 2 minutes then leveled off at 4 minutes The Jemez source changed little
1 to 4 minutes of treatment As treatment duration increased from 4 to 8
mutes five sources decreased in germination (Figure 4a) whereas three sources
24
Table 3 Categorical Analysis of Variance table for germination response to seed treatment seed source block and the interaction of the factors
Source of DF Chi-Square Observed Variability Significance Level
Source 8 98370 lt00001 Trt 7 233876 lt00001
contrast 1 16976 lt00001 SourceTrt 56 97077 lt00001 Block 3 5538 lt00001
SourceBlock 24 10162 lt00001 TrtBlock 21 7564 lt00001
SourceTrtBlock 168 34283 lt00001
S 4)
8 ~
0
sectg ~ 40 ~
N 0 0
20
o f0ltgt ~ ~4 jybshy ((iltgto ~~ K1ltgt 6~b-~ ~~ 0b-~~ ~~ ~b-~ ~b-~b
80
60 -I-- - ~- middotmiddotmiddotmiddotmiddot1
r -] Northern NM Sources
Central NM Sources
Southern NM Sources
sect-o ~
~b-v(i
SOURCE
Figure 2 Effect ofNew Mexico locust seed source on germination for data averaged over all scarification treatments Control germination is shown by solid line within bar
80
~ cd ~ ltl) () 60 ~ ~
N
t 0shyrti tshySltl)
40
0
20
o lmmersion Boiling 30 sec Boiling 60 sec Control 1 min 2min 4 min 8 min
Hot Water Treatments Percussion Treatments
Figure 3 Effect of scarification on germination ofNew Mexico locust for data averaged over all seed sources Means labelled with the same letter are not significantly different
100~----------------------~--~----------------~
80
60
ltU
~
40 IJ
sectgsI
l100
80
60
40
a I middotmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot middotImiddotmiddotmiddotmiddotmiddotmiddot E
_~iii-bull _ _ bull f --~--
t-- -~ ---- -shy ---- shyI ~ ---- - shy
L
fiII
~
b
- shy ---r
- Holman
Sacramento Sandia Gila Jemez
c ~
ltgt ~
Tmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot-I
j~=~=-~~-1
I middotmiddot ~ bull r
t tr
Y -------M-a-gd-a-Ie-n-a--
1 _ _
fCapitan
---- Manzano ~y -_ Raton
2 4 8
Time interval ofpercussion scarification (min)
Figure 4 Effect of seed source by treatment interaction on New Mexico locust germination for percussion scarification pattern a and pattern b
28
tv 0
~ d
~ A s o
1 c3
80
60
40
20
o
TIT
t------=middotfmiddotmiddot-middotmiddot-middotmiddot-middotmiddot 1- --- - - --=- - -shy--cE= - _--shy --shy ----shy ~ ~ ---shy ~ -~- _ --shy-------r 1
I
Raton Holman
-_ Jemez
--- Sandia Manzano Magdalena Capitan Sacramento Gila
Immersion Boiling 30 sec Boiling 60 sec
Hot Water Scarification
Figure 5 Effect of seed source by treatment interaction on New Mexico locust germination for hot water scarification
continued to increase in germination (Figure 4b) There was no difference between
the two treatment levels for the Manzano source (Figure 4b)
The effect ofhot water scarification also varied among sources with few
overall differences between hot water treatments (Figure 5) However boiling for 60
seconds yielded highest germination for the Jemez source whereas increasing
durations ofboiling decreased germination for the Raton source
In addition to consistently high germination 4- and 8-minute percussionshy
treated seeds germinated as fast or faster than all other treatments (Figure 6 Table 4)
050 measured as the number of days needed to complete 50 ofeventual
germination averaged 2 days for both ofthese treatments
X-ray radiographs were taken of seeds collected from the Sacramento source
percussed for 1 4 or 8 minutes (Figure 7) Seeds show signs of damage at 8 minutes
ofpercussion (Figure 8) Compared to an undamaged seed percussed for only 4
minutes (Figure 8a) damage at the 8-minute level includes fractured radicles pitting
of the seed coat and chips in seed coat structure (Figure 8b-d)
Scanning electron microscope (SEM) images at 600X reveal the direct effect
of 4 minutes of percussion on cells of the strophiolar region (Figure 9) Whereas
untreated seeds retain cellular organization in the strophiolar region strophiolar cells
seeds treated for 4 minutes ofpercussion separate and raise resulting in a
~-~e~U appearance
30
Umiddot-~middot--l-~---middotymiddotmiddotmiddot-----middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot--middot--middotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middotmiddotmiddotmiddotmiddotmiddotmiddot- -
050= gt1
-e-- Percussion 8 min -0- Percussion 4 min ---T- Percussion 2 min -----ltil- Percussion 1 min -- Immersion --0- Boiling 30 sec -+- Boiling 60 sec -ltgt- Control
2 4 6 8 10 12
Day Figure 6 Timecourse ofNew Mexico locust gennination following scarification
treatments G50 is the number of days to 50 of eventual germination
31
14
Table 4 Effect of seed source and treatment on G50 (days to 50 germination)
Seed Source Control Immersion B30sec B60sec Plmin P2min P4min P8min mean Raton 3 3 5 7 4 3 2 2 4 Holman 2 2 2 2 2 2 2 2 2
Jemez 4 6 6 5 3 2 2 2 4 Sandia 2 2 2 2 2 2 2 2 2
w tv Manzano 5 2 2 3 3 2 2 2 3
Magdalena 4 2 2 3 4 3 2 1 3
Capitan 5 4 2 2 3 2 2 2 3
Sacramento 3 2 2 2 4 3 2 2 3 Gila 3 2 2 2 2 2 2 2 2 mean 3 3 3 3 3 2 2 2 3 standard 11 14 16 18 09 05 0 03 08 deviation
Note Columns and rows may not agree due to rounding
Figure 7 X-ray radiographs ofSacramento seed source percussed for 1 4 or 8 minutes
33
Figure 8a-d Seed damage close-ups Figure a shows undamaged seed percussed for 4 minutes Figures b-d show seeds percussed for 8 minutes Damage includes fracturing ofradicle in b complete removal ofseed coat in c and pitting ofseed coat in d
34
Untreated Treated
Figure 9 SEM images ofuntreated vs 4-minute percussion-treated strophiolar cells ofNew Mexico locust seeds Treated cells are loosened separated and raised
DISCUSSION
Determining the Optimal Treatment
Averaging results of scarification treatmems across seed sources is a useful
tool to predict which treatment is most likely to be successful for an unknown seed
source Averaged over all sources four- and eight-minute percussion treatments
resulted in the highest germination 815 and 790 respectively and produced
germination that was as fast or faster than all other treatments Although there was no
difference between these two treatments on average an argument for 4 minutes of
percussion as a standard protocol for treating New Mexico locust seeds can be made
Descriptively 4 minutes of percussion improved germination over 8 minutes
ofpercussion for a majority of sources (five out of nine) 8 minutes of percussion
improved germination for three ofnine sources with no difference for a fmal source
For individual sources 4 minutes ofpercussion was always the best or secondmiddot best
treatment and was always equal to or better than all hot water treatments
Germination at 8 minutes ofpercussion was lower than all other percussion durations
for the Jemez and Gila sources and was lower than at least one hot water treatment for
these sources as well
The x-ray radiographs of the Sacramento seed source illustrate the excessive
internal and external seed damage that can occur with 8 minutes of percussion
germination counts 8-minute percussed seeds from Holman and Capitan
ClUll signs of damage that were visible to the naked eye (see below)
r~evertheless some of these seeds still fit the criteria for germination Neither x-ray
36
photography nor visible observations during seed counts revealed seed damage at the
4-minute percussion level In operational trials seeds percussed for 4 minutes
developed into healthy seedlings (Khadduri and Harrington unpublished data)
Strophiole and Seed Coat Behavior
Four minutes ofpercussion successfully breaks seed dormancy in a large
percentage ofNew Mexico locust seeds without excessively damaging the
surrounding seed coat or internal seed coat structures Most papilionoid legume
seeds including seeds ofNew Mexico locust have a specific region of the seed coat
known as the strophiole or lens (Hamly 1932) The strophiole is located on the
cotyledonary lobe of a locust seed (Figure 10 a-c) If seeds are not indiscriminately
damaged the strophiole is the first point of water entry to the embryo (Hopkinson
1997) When a papilionoid seed is percussed for the appropriate amount of time
repeated hits on the integrated seed coat loosen the constrained cells of the strophiolar
region without excessively damaging the rest of the seed coat (Ballard 1976) SEM
photos illuStrate that 4 minutes of percussion loosened and separated strophiolar cells
ofNew Mexico locust seeds when compared with constrained organized strophiolar
cells of untreated seeds (Figure 9) Research-has shown that water passes to the
embryo through the treated strophiole in a controlled manner and regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues
(Kelly and van Staden 1987)
37
10 Strophiole ofunimbibed New Mexico locust seed (a) and following uptake (bc)
38
In contrast hot-water treatments can excessively degrade the seed coat
(Rebman et al 1998 Marunda 1990) Random cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rebman et aI 1998 Marunda 1990) Along with a
proportionately large amount ofun-imbibed (thus under-treated) seeds (Figure l1a)
damage was observed in hot-water treated seeds from six sources during germination
counts Damage included seeds where the radicle failed to elongate (Figure 11 b)
seeds where the radicle elongated but was delayed in freeing itself from the seed coat
(Figure 11c) and seeds where the radicle completely broke off from the embryo
(Figure lId) In addition some seeds germinated in reverse (the cotyledon emerged
fIrst) If the radicle emerged completely from the seed coat they were counted as
germinated
While nearly all seeds treated for 8 minutes ofpercussion imbibed water
damage was evident in x-ray evaluations (Sacramento seed source) and in
germination counts (Holman and Capitan) at this treatment level Seed damage
during germination counts was similar to levels found in hot-water treated seeds
suggesting that over-treatment by both hot water and longer durations of percussion
may result in similar damage to seed structure
The Sacramento and Holman seed sources were two of the fIve sources that
opcrplltlpn in germination from the 4- to 8-minute durations of percussion
the benefIts ofloosening the strophiole as evidenced by imbibition of
all 8-minute treated seeds were outweighed by external and internal seed coat
39
a
b c d
11 Damage to New Mexico locust seeds observed during germination cotmts following hot water scarification 9a Hot water replicate including ~t seedlings and numerous unimbibed seeds 9b Close-up of seed where raallCle failed to elongate 9c Close-up ofseed where radicle elongated but was
-gtIn in freeing itself from the seed coat 9d Close-up ofseed where radicle broke off from embryo
40
damage (which also may be contributing to high imbibition) For the three sources
for which 8 minutes of percussion resulted in maximum germination extended
percussion of seeds necessary to loosen the strophiole may not have excessively
damaged the seed anatomy The Manzano source did not significantly change in
germination from 4 to 8 minutes of percussion suggesting that an increase in
permeable seeds at the 8-minute treatment duration was balanced by the number of
seeds that failed to germinate due to seed damage To summarize the 4-minute
percussion treatment improved germination over 8 minutes ofpercussion for a
majority of sources (five ofnine) the 8-minute percussion treatment improved
germination over 4-minutes ofpercussion for three sources and there was no
difference with the Manzano source Notably for the Raton source 8 minutes of
percussion increased germination by 235 percentage points over 4-minutes of
percussion (7875 vs 5525 respectively) the next best treatment Raton was the
only source to result in germination less than 75 for 4 minutes ofpercussion so a
grower may wish to apply longer percussion durations to lots that do not respond well
to this treatment level
Finally there was an interesting trend in the Jemez and Raton sources seed
that significantly contributed to the source by treatment interaction (Figure 12) A
~u test showed that the Jemez source had a significantly lighter seed weight than
other sources while the Raton source had an average seed weight For the Jemez
the longest duration of boiling increased germination Conversely only short
U4UVll) of percussion were needed to achieve high germination and the longest
41
80 _ Jemez a Raton
iD
~ C iD e iD
Q c 0 ~
c ~ iD 0
20
0------shy
Immersion Boiling 30 sec Boiling 60 sec
Hot Water Scarification
10degTr========~--------~------------------------------
80
60
40
20
_ Jemez Raton
0------shy
1 min 2 min 4 min 8 min
Percussion Scarification
Figure 12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources
42
b
--~----~~~~========~---- duration ofpercussion reduced germination For the Raton source longer durations
ofboiling consistently decreased germination whereas longer percussion treatments
consistently increased germination
A possible explanation is that a thick seed coat is associated with a loose
strophiole structure and that a thin seed coat is associated with a constrained
strophiole This contrast merits further study and may suggest an ecological
adaptation Further investigation ofthis phenomenon may contribute to successful
pre-screening ofunknown seed sources to determine optimal treatment leveL
43
CONCLUSION
Based on results obtained in pilot study (see Appendix) and thesis
experiments 4 minutes ofpercussion is recommended as a standard protocol for
treating an unknown source ofNew Mexico locust seed In addition to laboratory
results the 4-minute percussion treatment has resulted in germination greater than
80 and healthy seedling grow-outs for production purposes (unpublished data) In
laboratory experiments only one hundred seeds per 118 ml (40z) soil tin were
percussed at a time whereas for production we have treated up to five hundred seeds
per tin For large-scale production ofNew Mexico locust seedlings growers may
wish to experiment with larger quantities of seed and larger-size soil tins
Seed storage trials following percussion treatments were not conducted
However germination greater than 80 and healthy seedlings were obtained when
seeds were percussion-treated and subsequently stored at room-temperature (22degC)
for one week (unpublished data) Seed storage trials merit further study as the
standard hot water treatment leaves seeds in a softened condition that is not
conducive to storage
Hydroseeding is an effective method for direct seeding in reclamation
situations Percussion treatment may be conducive to hydro seeding as seeds remain
a dry relatively undamaged yet permeable condition following treatment Sulfuric
seeds also remain dry following treatment but extensive damage to seed coat
poor seedling vigor have been reported with this treatment (Kelly and van Staden
A grow-out study using a hydro seeder on a reclamation site testing percussion
44
against other treatments such as sulfuric acid and mechanical scarification also
merits further study
Papilionoids are the largest subfamily oflegumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust these legumes often play an integral role in the revegetation of
disturbed lands The pilot study (Appendix) indicates that percussion scarification
successfully treats the widely used black locust as well Developing superior
scarification methods to the standard hot water mechanical scarification and acid
treatments may facilitate the use ofpapilionoid legumes in reclamation projects
45
APPENDICES
APPENDIX A
PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR
NEW MEXICO LOCUST AND BLACK LOCUST SEEDS
Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4
Hot water and sulfuric acid soaks are traditional treatments for seeds of many
temperate woody legwnes including locusts However these scarification techniques
often produce inconsistent germination Percussion scarification where seeds are
repeatedly propelled against a hard surface was compared with hot water
scarification to evaluate treatment efficacy for New Mexico locust (Robinia
neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment
seeds were placed in a 98degC water bath which was immediately removed from the
heat source For percussion scarification seeds were placed in a soil sample tin and
agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the
control were followed by 24-hour water soaks Hot water baths resulted in 56 and
41 germination for New Mexico locust and black locust respectively For both
species nearly all durations ofpercussion increased germination over the hot water
treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3
4 and 5 minutes for black locust resulted in at least 90 germination Traditional
scarification treatments randomly degrade the entire seed coat which can lead to
tissue damage during water uptake Percussion scarification specifically weakens the
strophiole the natural source ofwater entry to the seed in papilionoid legwnes
INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom
2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031
48
Following percussion imbibition is controlled through the strophiole and underlying
tissue is protected
Keywords New Mexico locust black locust scarification percussion germination
Introduction
Locust trees (Robinia spp) are aggressive pioneer species that quickly
colonize disturbed land fix atmospheric nitrogen in the soil and stabilize slopes that
are prone to erosion (Klemmedson 1994 Ashby et al 1985) (Figure A1ab)
Recognition of these attributes has contributed to increased demand for locusts in
reclamation projects Locust seeds however exhibit physical dormancy where a
thick seed coat prevents the movement of water and gasses to the embryo (Leadem
1997)
Sulfuric acid or hot water soaks have traditionally been used to break seed
dormancy in macro-propagation of locusts Many growers are moving away from the
use of su1furic acid in treating locust seeds (Dreesen and Harrington 1997) In
addition to being dangerous acid soak durations must be specifically correlated to
seed lot (Olson 1974) Collections of native seed can vary tremendously in seed size
seed weight and hard seededness etc This variability limits the utility of sulfuric
acid soaks as even within a given lot some seeds may be damaged whereas others
are under-treated and fail to imbibe (take up water) A survey of Southwestern
container growers indicates that hot water baths are currently the most common
scarification method for locust seeds (Hine et al 1997) However hot water baths
also produce inconsistent germination (Lin et al 1996)
49
b
Figure AI New Mexico locust a primary invader following disturbance Ala Crown-sprouting at edge ofburn near Los Alamos NM--one year after Cerro Grande fire Alb Colonizing road cut Gila National Forest NM
50
Percussion scarification where seeds are repeatedly propelled against a hard
surface is an alternative dormancy-breaking method that has proven successful in
legumes related to locusts (Hamly 1932 Barton 1947) We compared this alternative
treatment to hot water scarification to evaluate treatment efficacy for New Mexico
locust and black locust seeds
Materials and Methods
Commercial New Mexico locust seeds (Western Native Seed Coaldale CO)
were collected fall 2000 in Huerfano County CO Black locust seeds were collected
September 2000 from Taos County NM by harvesting ripe pods from several trees at
a distance of up to 3 m from ground level Black locust seeds were allowed to air-dry
for 3 weeks and were threshed and separated from large chaffusing a greenhouse
fan A South Dakota Seed Blower (Chicago Illinltois) was used to remove fine chaff
from both seed lots Seeds were storedmiddotat 2-4C until the germination study was
conducted in March 2001
This study compared hot water scarification with percussion scarification
using treatment and species as experimental factors Along with a control
scarification treatments included immersion of seed in boiling water as well as
percussion durations of 1 2 3 45 or 10 minutes Four 100-seed samples were used
to test each species by scarification treatment combination
For the hot water treatment test tubes were filled with 30 mL water and
placed in a water-filled beaker (Figure A2a) The bath was raised to boiling (-98C at
51
b
c
Figure A2 Implementation ofhot water and percussion scarification A2a Hot water bath A2b Paint-shaker set-up A2c Placing soil tin containing seeds at end ofpaint shaker ann
52
Las Cruces NM elevation of 1300 meters) Each 100-seed replicate was placed in a
test tube and each test tube was immediately removed to cool at room temperature
Percussion scarification was implemented using a pneumatic paint shaker
(Central Pneumatic model 00422 Camarillo CA) (Figure A2b) This allowed
standardization of treatment intensity Compressed air pressure was maintained at
530kPa plusmn 30kPa (80psi plusmn 5psi) resulting in approximately 350 oscillations per
minute Each lOO-seed sample was placed in a 118 ml (4 oz) soil tin for shaking To
maximize lateral movement of paint shaker a paint can was used as a spacer to place
soil tins at the end of the shaking arm (Figure A2c) This allowed shaking to occur at
the greatest distance from the pivot point (fulcrum) Following all treatments
including the control seeds were soaked for twenty-four hours in distilled water prior
to plating out
Gennination was evaluated on lab benches at room temperature Each
replicate of seeds was placed in a 100 cm petri dish on moistened filter paper with
humidity maintained by enclosing the petri dishes in ziplock bags Gennination was
monitored daily for 14 days in accordance with International Seed Testing
Association standards for Robinia species (ISTA 1999) Gennination was defined as
protrusion of the radicle from the embryo by at least 1mm as viewed by the naked
eye
Categorical Analysis of Variance using Prpc Catmod (SAS Institute 1989)
was used to detennine treatment differences in germination percentages for each
source This procedure is an extension of a Chi-square test of homogeneity using the
53
natural log of the ratio of germinated to non-germinated seeds for each treatment Pshy
values less than 005 were considered significant A limited set of pairwiseshy
comparisons was conducted to compare treatment mean percents using a
conservative alpha value of 005 divided by the number of comparisons
Results
Percussion maximized germination for both New Mexico locust and black
locust There were three germination response levels for New Mexico locust (Figure
A3) First there was a low response in the control indicating a dormant seed lot
The hot water treatment and I-minute duration of percussion share a second
intermediate response Germination was highest at percussion levels from 2 to 10
minutes
A selected percussion treatment intermediate in time (5 minutes) produced the
steepest germination curve indicating the fastest germination rate (Figure A4) The
hot water treatment produced an intermediate curve or relatively slower germination
rate The control curve was very flat indicating the slowest germination rate for New
Mexico locust
Control seeds germinated poorly for black locust as well (Figure A5) Hot
water scarification improved germination over the control but all levels of percussion
improved germination over hot water The optimal durations of percussion treatment
were bracketed with a significant increase from the 1 to 2 minute percussion level a
high germination response from 2 to 5 minutes of percussion and finally a significant
54
~ d 25 CL)
p t1 0 ~ t1
g Vl ~ Vl
100~---------------------------------------------------------------
80
60
40
20
o Control Hot Water Percussion
I min Percussion
2 min Percussion
3 min
Scarification
Percussion 4 min
Figure A3 Effect of scarification on germination response for New Mexico locust
Percussion 5 min
=significant difference between adjacent treatment bars ns = no significant difference
Percussion 10 min
~ ~ () u agt
Il-t d o ~
~ ~ c3
100middotlr~~~------------------------------------e- Percussion 5min middot 0middot Hot Water -- Control
80-J=middot======
6b-middot-lmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middot--middotmiddot-middot~-middotmiddot -middotmiddot middot middotmiddot-middot 1
40
20
-----~-----------
0 0 0 middotmiddotmiddotmiddotmiddot0middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot0
0 --~-----y-----~------_---~--- T-----~
o shy -TDay 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A4 Effect of selected scarification treatments on germination speed for New Mexico locust First seven days of fourteen-day experiment are shown
Day 7
80
~ cd
5 60 ()
[) Cl d 0 1t1 d
40sect dUl
-J
20
o
ns
Scarification
Figure A5 Effect of scarification on germination response for black locust = significant difference between adjacent treatment bars ns = no significant difference
decrease at the 10-minute level This drop-off suggests that 10 minutes of percussion
over-treats seeds from this lot of-black locust
A selected percussion treatment intermediate in time (5 minutes) also had the
fastest germination rate for black locust (Figure A6)_ The hot water curve is
somewhat flatter indicating a slower germination rate The control curve is very flat
indicating a slow germination rate
A qualitative difference between hot water and percussion-treated seeds was
also observed Along with healthy germinants the hot water treatment produced
over-treated seeds (Figure A 7a) Examples of damaged germinants include seeds
where the radicle did not elongate (Figure A 7b) seeds where the radicle elongated
but was delayed in freeing itself from the seed coat (Figure A7c) and seeds where the
radicle broke off completely from the embryo (Figure A7d) In addition to these
examples of over-treatment there were numerous small dark seeds that failed to
imbibe water For percussion treatment nearly all seeds took up water (Figure A8a)
Germination was rapid and uniform with little damage evident (Figure A8b)
To summarize both New Mexico locust and black locust seed lots were
dormant The standard hot water scarification treatment improved germination but
only to 56 and 41 for New Mexico locust and black locust respectively Nearly
all percussion durations improved total germination as well as germination rate over
the hot water treatment Germination was greater than 90 for percussion durations
of 4 5 and 10 minutes for New Mexico locust and 3 4 and 5 minutes for black locust
58
CI)
sgt t CI) 0
igt 0
sect ttl t E
Vl 0
c3
100r==~==~~=r------------------------------------~ ___ Percussion 5 min
middotmiddotmiddot0middotmiddotmiddot Hot Water -lIf- Control
80r=========~--~~----------------------------~
60
40
20 -~-~---~---------- ---~------------I
omiddot
O
_~_----------lIf-----~bull -----_ shy---shy I
Day 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A6 Effect of selected scarification treatments on germination speed for black locust First seven days of fourteen-day experiment are shown
Day 7
a
b c d
Figure A7 Damage to New Mexico locust seeds observed during germination counts following hot water scarification A7a Hot water replicate including damaged seedlings and numerous unimbibed seeds A7b Close-up ofseed where radicle failed to elongate A7c Close-up ofseed where radicle elongated but was delayed in freeing itself from the seed coat A7d Close-up ofseed where radicle completely broke offfrom embryo
60
a
b
Figure A8 New Mexico locust seeds following percussion treatment at time of germination counts A8a Percussion replicate with uniformly healthy seedlings and near complete imbibition of seeds A8b Close-up ofpercussion-treated seedling
61
Discussion
Papilionoid seeds have a specific anatomical feature known as the strophiole
or lens-the natural site of water entry to the seed (Hamly 1932) The strophiole is
located on the cotyledonary lobe of a locust seed (Figure A9) When a papilionoid
legume seed is percussed for the appropriate amount of time repeated hits on the
integrated seed coat loosen the constrained cells of the strophiolar region without
excessively damaging the rest of the seed coat (Ballard 1976)
When a percussed seed is soaked water enters exclusively through the
strophiole in a controlled manner (Kelly and Staden 1987) This regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues This
contrasts with seeds that have been hot water or acid scarified treatments that can
randomly degrade the seed coat Unlocalized cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rehman et al 1998 Marunda 1990)
Papilionoids are the largest subfamily of legumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust and black locust these legumes often play an integral role in the
revegetation ofdisturbed lands Developing superior scarification methods to the
standard hot water and acid treatments should fadlitate the use ofthese legumes in
restoration projects
62
Figure A9 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (bc)
63
APPENDIXB
GERMINATION PERCENTS AND STANDARD ERRORS FOR SOURCE
BLOCK AND TREATMENT FOR NEW MEXICO LOCUST
Table B 1 Gennination percents and standard errors for source block and treatment for New Mexico locust
Source Block Treatment Germination Percentage Standard Error
Capitan 1 Immersion 74 437 Capitan 2 Immersion 69 462 Capitan 3 Immersion 71 454 Capitan 4 Immersion 67 470 Capitan Boiling 30 sec 79 407 Capitan 2 Boiling 30 sec 76 427 Capitan 3 Boiling 30 sec 72 449 Capitan 4 Boiling 30 sec 76 427 Capitan Boiling 60 sec 71 454 Capitan 2 Boiling 60 sec 54 498 Capitan 3 Boiling 60 sec 67 470 Capitan 4 Boiling 60 sec 76 427 Capitan I Control 5 218 Capitan 2 Control 7 255 Capitan 3 Control 4 196 Capitan 4 Control 5 218 Capitan 1 Percussion I min 47 499 Capitan 2 Percussion I min
470
Capitan Percussion I min 39 488
Capitan 4 Percussion I min 38 485 Capitan I Percussion 2 min 55 497 Capitan Capitan
2
Percussion 2 min Percussion 2 min
71 65
454 477
Capitan 4 Percussion 2 min 56 496 Capitan I Percussion 4 min 86 347 Capitan 2 Percussion 4 min 79 407 Capitan 3 Percussion 4 min 81 392 Capitan 4 Percussion 4 min 82 384 Capitan I Percussion 8 min 90 300 Capitan 2 Percussion 8 min 88 325 Capitan 3 Percussion 8 min 90 300 Capitan 4 Percussion 8 min 90 300 Gila I Immersion 71 454 Gila 2 Immersion 77 421 Gila
Immersion 75 433
Gila 4 Immersion 72 449
Gila I Boiling 30 sec 68 466
Gila 2 Boiling 30 sec 81 392
Gila 3 Boiling 30 sec 54 498
Gila 4 Boiling 30 sec 63 483
Gila I Boiling 60 sec 55 497
Gila 2 Boiling 60 sec 68 466
Gila 3 Boiling 60 sec 57 495
Gila 4 Boiling 60 sec 70 458
65
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Gila 2 Control 13 336 Gila 3 Control 13 336 Gila 4 Control 20 400 Gila 1 Percussion 1 min 66 474 Gila 2 Percussion 1 min 57 495 Gila 3 Percussion 1 min 73 444 Gila 4 Percussion 1 min 82 384 Gila 1 Percussion 2 min 81 392 Gila 2 Percussion 2 min 86 347 Gila 3 Percussion 2 min 85 357 Gila 4 Percussion 2 min 54 498 Gila 1 Percussion 4 min 79 407 Gila 2 Percussion 4 min 83 376 Gila 3 Percussion 4 min 84 367 Gila 4 Percussion 4 min 79 407 Gila 1 Percussion 8 min 51 500 Gila 2 Percussion 8 min 76 427 Gila 3 Percussion 8 min 52 500 Gila 4 Percussion 8 min 74 439 Holman 1 Immersion 83 376 Holman 2 Immersion 86 347 Holman 3 Immersion 81 392 Holman 4 Immersion 90 300 Holman 1 Boiling 30 sec 90 300 Holman 2 Boiling 30 sec 85 357 Holman 3 Boiling 30 sec 87 336 Holman 4 Boiling 30 sec 83 376 Holman 1 Boiling 60 sec 79 407 Holman 2 Boiling 60 sec 80 400 Holman 3 Boiling 60 sec 86 347 Holman 4 Boiling 60 sec 93 255 Holman Control 25 433 Holman 2 Control 19 392 Holman Control 21 407
Holman 4 Control 24 427
Holman Percussion 1 min 77 421
Holman 2 Percussion 1 min 85 357
Holman 3 Percussion 1 min 80 400
Holman 4 Percussion 1 min 89 313
Holman 1 Percussion 2 min 88 325
Holman 2 Percussion 2 min 96 196
Holman 3 Percussion 2 min 93 255
Holman 4 Percussion 2 min 96 196
Holman 1 Percussion 4 min 92 271
Holman 2 Percussion 4 min 97 171
66
I
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Holman 3 Percussion 4 min 91 286 Holman 4 Percussion 4 min 96 196 Holman 1 Percussion 8 min 88 325 Holman 2 Percussion 8 min 85 357 Holman 3 Percussion 8 min 83 376 Holman 4 Percussion 8 min 82 384 Jemez 1 Immersion 53 499 Jemez 2 Immersion 44 496 Jemez 3 Immersion 52 500 Jemez 4 Immersion 70 458 Jemez 1 Boiling 30 sec 58 494 Jemez 2 Boiling 30 sec 55 497 Jemez 3 Boiling 30 sec 48 500 Jemez 4 Boiling 30 sec 65 477 Jemez 1 Boiling 60 sec 68 466 Jemez 2 Boiling 60 sec 63 483 Jemez 3 Boiling 60 sec 73 444 Jemez 4 Boiling 60 sec 89 313 Jemez 1 Control 2 140 Jemez 2 Control 9 286 Jemez 3 Control 6 237 Jemez 4 Control 8 271 Jemez 1 Percussion 1 min 58 494 Jemez 2 Percussion 1 min 63 483 Jemez 3 Percussion 1 min 77 421 Jemez 4 Percussion 1 min 89 313 Jemez 1 Percussion 2 min 68 466 Jemez 2 Percussion 2 min 64 480 Jemez 3 Percussion 2 min 80 400 Jemez 4 Percussion 2 min 83 376 Jemez Percussion 4 min 68 466 Jemez 2 Percussion 4 min 79 407 Jemez 3 Percussion 4 min 70 458 Jemez 4 Percussion 4 min 83 376 Jemez 1 Percussion 8 min 52 500 Jemez 2 Percussion 8 min 59 492 Jemez 3 Percussion 8 min 61 488 Jemez 4 Percussion 8 min 70 458 Magdalena 1 Immersion 69 462 Magdalena 2 Immersion 69 462 Magdalena J Immersion 70 458 Magdalena 4 Immersion 85 357 Magdalena 1 Boiling 30 sec 65 477 Magdalena 2 Boiling 30 sec 71 454 Magdalena 3 Boiling 30 sec 68 466
67
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Magdalena 4 Boiling 30 sec 83 376 Magdalena 1 Boiling 60 sec 63 483 Magdalena 2 Boiling 60 sec 68 466 Magdalena 3 Boiling 60 sec 63 483 Magdalena 4 Boiling 60 sec 82 384 Magdalena 1 Control 11 313 Magdalena 2 Control 6 237 Magdalena 3 Control 5 218 Magdalena 4 Control 4 196 Magdalena Percussion 1 min 32 466 Magdalena 2 Percussion 1 min 45 497 Magdalena 3 Percussion 1 min 46 498 Magdalena 4 Percussion I min 36 480 Magdalena 1 Percussion 2 min 67 470 Magdalena 2 Percussion 2 min 68 466 Magdalena 3 Percussion 2 min 65 477 Magdalena 4 Percussion 2 min 81 392 Magdalena 1 Percussion 4 min 87 336 Magdalena 2 Percussion 4 min 97 171 Magdalena 3 Percussion 4 min 83 376 Magdalena 4 Percussion 4 min 97 171 Magdalena 1 Percussion 8 min 98 lAO Magdalena 2 Percussion 8 min 100 000 Magdalena 3 Percussion 8 min 96 196 Magdalena 4 Percussion 8 min 98 lAO Manzano 1 Immersion 67 470 Manzano 2 Immersion 57 495 Manzano 3 Immersion 72 4049 Manzano 4 Immersion 73 4044 Manzano 1 Boiling 30 sec 70 458 Manzano 2 Boiling 30 sec 64 480 Manzano 3 Boiling 30 sec 71 454 Manzano 4 Boiling 30 sec 80 400 Manzano I Boiling 60 sec 50 500 Manzano 2 Boiling 60 sec 53 499 Manzano 3 Boiling 60 sec 59 492 Manzano 4 Boiling 60 sec 74 439 Manzano I Control 4 196 Manzano 2 Control 8 271 Manzano 3 Control 1 099 Manzano 4 Control 6 237 Manzano 1 Percussion 1 min 52 500 Manzano 2 Percussion 1 min 39 488 Manzano 3 Percussion 1 min 36 480 Manzano 4 Percussion 1 min 51 500
68
Table B 1 Continued
Source Block Treatment Gerrni~ation Percentage Standard Error
Manzano Percussion 2 min 53 499 Manzano 2 Percussion 2 min 58 494 Manzano 3 Percussion 2 min 61 488 Manzano 4 Percussion 2 min 62 485 Manzano 1 Percussion 4 min 76 427 Manzano 2 Percussion 4 min 85 357 Manzano 3 Percussion 4 min 79 407 Manzano 4 Percussion 4 min 83 376 Manzano 1 Percussion 8 min 87 336 Manzano 2 Percussion 8 min 83 376 Manzano 3 Percussion 8 min 83 376 Manzano 4 Percussion 8 min 75 433 Raton 1 Immersion 54 498 Raton 2 Immersion 37 483 Raton 3 Immersion 39 488 Raton 4 Immersion 69 462 Raton 1 Boiling 30 sec 36 480 Raton 2 Boiling 30 sec 20 400 Raton 3 Boiling 30 sec 28 449 Raton 4 Boiling 30 sec 60 490 Raton 1 Boiling 60 sec 15 357 Raton 2 Boiling 60 sec 171 Raton 3 Boiling 60 sec 4 196 Raton 4 Boiling 60 sec 35 477 Raton 1 Control 4 196 Raton 2 Control 12 325 Raton 3 Control 2 140 Raton 4 Control 6 237 Raton 1 Percussion 1 min 52 500 Raton 2 Percussion I min 29 454 Raton 3 Percussion I min 35 477 Raton 4 Percussion 1 min 31 462 Raton 1 Percussion 2 min 55 497 Raton 2 Percussion 2 min 35 477 Raton 3 Percussion 2 min 33 470 Raton 4 Percussion 2 min 42 494 Raton 1 Percussion 4 min 65 477 Raton 2 Percussion 4 min 52 500 Raton Percussion 4 min 51 500 Raton 4 Percussion 4 min 53 499 Raton 1 Percussion 8 min 88 325 Raton 2 Percussion 8 min 80 400 Raton 3 Percussion 8 min 73 444 Raton 4 Percussion 8 min 74 439 Sacramento 1 Immersion 43 495
69
Table B 1 Continued
-Source Block Treatment Germination Percentage Standard Error
Sacramento 2 Immersion 30 458 Sacramento 3 Immersion 38 485 Sacramento 4 Immersion 56 496 Sacramento 1 Boiling 30 sec 47 499 Sacramento 2 Boiling 30 sec 48 500 Sacramento 3 Boiling 30 sec 48 500 Sacramento 4 Boiling 30 sec 56 496 Sacramento 1 Boiling 60 sec 52 500 Sacramento 2 Boiling 60 sec 67 470 Sacramento 3 Boiling 60 sec 43 495 Sacramento 4 Boiling 60 sec 48 500 Sacramento 1 Control 0 000 Sacramento 2 Control 3 171 Sacramento 3 Control 3 171 Sacramento 4 Control 099 Sacramento 1 Percussion 1 min 57 495 Sacramento 2 Percussion 1 min 61 488 Sacramento 3 Percussion 1 min 62 485 Sacramento 4 Percussion 1 min 55 497 Sacramento 1 Percussion 2 min 84 367 Sacramento 2 Percussion 2 min 70 458 Sacramento 3 Percussion 2 min 81 392 Sacramento 4 Percussion 2 min 69 462 Sacramento 1 Percussion 4 min 91 286 Sacramento 2 Percussion 4 min 84 367 Sacramento 3 Percussion 4 min 84 367 Sacramento 4 Percussion 4 min 94 237 Sacramento 1 Percussion 8 min 82 384 Sacramento 2 Percussion 8 min 81 392 Sacramento 3 Percussion 8 min 82 384 Sacramento 4 Percussion 8 min 81 392 Sandia 1 Immersion 76 427 Sandia 2 Immersion 70 458 Sandia 3 Immersion 70 458 Sandia 4 Immersion 81 392 Sandia 1 Boiling 30 sec 78 414 Sandia 2 Boiling 30 sec 71 454 Sandia 3 Boiling 30 sec 63 483 Sandia 4 Boiling 30 sec 78 414 Sandia 1 Boiling 60 sec 67 470 Sandia 2 Boiling 60 sec 65 477 Sandia 3 Boiling 60 sec 74 439 Sandia 4 Boiling 60 sec 66 474 Sandia 1 Control 16 367 Sandia 2 Control 7 255
70
Table B 1 Continued
Source Block Treatment Gennination Percentage Standard Error
Sandia 3 Control 8 271 Sandia 4 Control 16 367 Sandia 1 Percussion 1 min 67 470 Sandia 2 Percussion 1 min 61 488 Sandia 3 Percussion 1 min 66 474 Sandia 4 Percussion 1 min 59 492 Sandia I Percussion 2 min 82 384 Sandia 2 Percussion 2 min 83 376 Sandia 3 Percussion 2 min 81 392 Sandia 4 Percussion 2 min 84 367 Sandia 1 Percussion 4 min 89 313 Sandia 2 Percussion 4 min 86 347 Sandia 3 Percussion 4 min 89 313 Sandia 4 Percussion 4 min 85 357 Sandia 1 Percussion 8 min 80 400 Sandia 2 Percussion 8 min 77 421 Sandia 3 Percussion 8 min 69 462 Sandia 4 Percussion 8 min 66 474
LITERATURE CITED
Ashby WC Vogel WG Rogers NF 1985 Black locust in the reclamation equation General Technical Report Northeastern Forest Experiment Station USDA Forest Service NE-I05 12 p
Ballard LAT 1973 Physical barriers to gennination Seed Sci Technoll 285-303
___ 1976 Strophiolar water conduction in the seeds ofTrifolieae induced by action on the testa at nonstrophiolar sites Austr J Plant Physiol3 465-469
Barton LV 1947 Special studies on seed coat impenneability Contrib Boyce Thompson Inst 14 355-362
Baskin CC Baskin JM 1998 Seeds Ecology biogeography and evolution of donnancy and gennination Academic Press London UK 666 p
Bewley JD 1997 Seed germination and donnancy Plant Ce119 1055-1066
Bewley JD Black M 1994 Seeds physiology of development and germination 2nd ed Plenum Press New York NY 445 p
Briscoe CB 1996 Growing Nitrogen Fixing Trees in Acid Soils In MH Powell (ed) Nitrogen Fixing Trees for Acid Soils- A Field ManuaL Winrock International Morrilton AR 22 p
Copeland LO McDonald MB 1995 Principles of Seed Science and Technology 3rded Chapman amp Hall New York NY 409 p
Cox RE Klett JE 1984 Seed gennination requirements of native Colorado plants for use in the landscape Plant Propag 30(2) 6-10
Dell B 1980 Structure and function ofthe strophiolar plug in seeds ofAlbizia Lophantha Am J Bot 67 556-563
Dick-Peddie WA 1993 New Mexico Vegetation Past Present and Future University ofNew Mexico Press Albuquerque NM 244 p
Dreesen DR 2001 Propagation protocol for production ofcontainer Robinia neomexicana plants Los Lunas Plant Materials Center Los Lunas New Mexico In Native Plant Network URL httpwwwnativeplantnetworkorg (accessed 9 January 2002) Moscow (ID) University ofIdaho CoIl Natural Resources For Res Nursery
72
Dreesen DR Harrington JT 1997 Propagation of native plants for restoration projects in the Southwestern US- Preliminary Investigations In Landis TD Thompson JR tech coords National Proceedings Forest and Conservation Nursery Assocs Gen Tech Rep PNW-GTR-419 Portland OR USDA For Ser PNW Res Sta 77-88
Gottfried GJ 1980 Control ofNew Mexican locust and the effect on planted ponderosa pine in central Arizona US For Ser Res Note RM-386 Rocky Mountain For Range ExpStn Fort Collins CO
Gosling PG Samuel YK Jones SK 1995 A systematic examination of germination temperature chipping and water temperaturesoak duration pretreatments on the seeds ofLeucaena leucocephala Seed Sci amp Technol 23521-532
Hamly DH 1932 Softening of the seeds of Melilotus alba Bot Gazette 93 345shy375
Hanna PJ 1984 Anatomical features of the seed coat ofAcacia kempeana (Mueller) which relate to increased germination rate induced by heat treatment New Phytol 96 23-29
Hanks JP Dick-Peddie WA 1974 Vegetation patterns of the White Mountains New Mexico Southwest Naturalist 18(4) 371-382
Hine S Harrington JT Dreesen DR 1997 Seed scarification requirements for Robinia neomexicana In Harrington JTtech coord Proc Third SouthwestemContainer Growers Conf Feb 12-13 1997 Ft Collins CO
Hopkinson JM 1993 The strophiole of leguminous seeds Newsletter- IntI Herbage Seed Production Res Grp 18 7-8
---- 1997 Differences in hardseededness between Cunningham and Tarramba leucaena In Leucnet News The Newsletter of the International LeucaenaRes and Devlt Netwk No4
ISTA (International Seed Testing Association) 1999 International rules for seed testing rules Seed Sci amp Technol27 Supplement
Kelly KM van Staden J 1987 The lens as the site of permeability in the papilionoid seed Aspalathus linearis J Plant Physiol 128(45) 395-404
73
Keresztesi B 1988 Black locust the tree of agriculture Outlook on Agr 17(2) 77~85
Kigel 1 and Galili G 1995 Seed development and germination Marcel Dekker New York 853 p
Klemmedson 10 1994 New Mexico locust and parent material Influence on macronutrients of forest floor and soil Soil Sci Soc Am 1 58 974~980
Kuo WH Tam AW 1988 The pathway of water absorption of mung bean seeds Seed Sci amp Technol16(1) 139~144
Leadem CL 1997 Dormancy--unlocking seed secrets In Landis TD Thompson lR tech coords National Proceedings Forestand Conservation Nursery Assocs Gen Tech Rep PNW-G TR-419 Portland OR USDA For Ser PNW Res Sta 43-52
Lin Y Wagner MR Seweyestewa A Honahni MD DeGomez M McMillin JD 1996 Influence of cultural treatments on seed germination and seedling growth of Robinia neomexicana Forest Farm and Community Tree Res Reports Volume 1
Marunda CT 1990 Effects of seed pretreatment on the development ofAcacia auriculforinis and A holosericea seedlings ACIAR Proceedings Series No 28
Meginnis HG 1937 Sulphuric acid treatment to increase germination of black locust seed USDA Circ 453 34 p
Miklas PN Towensend CE Ladd SL 1987 Seedcoat anatomy and the scarification ofcicer milkvetch seed Crop Sci 27 766-772
Monsen SB 1984 Use of shrubs on mine spoils In The Challenge ofProducing Native Plants for the Intermountain Area Proceedings Intermountain Nurserymans Association 1983 conference Las Vegas NV compo PM Murphy 26-31 Ogden UT USDA For Ser Gen Tech Report INT-l68
Olson DF 1974 Robinia L Locust In Seeds of Woody Plants in the US tech coord CS Schopmeyer 728-731 USDA For Ser Agr Handbk No450
Rehman S Loescher RNl Harris P1C 1998 Dormancy breaking and germination ofAcacia salicina Lindl seeds Seed Sci amp Techno127 553-557
Rolston MP 1978 Water impermeable seed dormancy Bot Rev 44 365-396
74
SAS Institute Inc 1989 SASSTAT Users Guide Version 6 4th Edition Volume 1 Cary NC SAS Institute Inc 943 p
SerratoValenti G Conara L Ferrando M Modenesi P 1993 Structural and histochemical features ofStylosanthes scabra (Leguminosae Papilionoidae) seed coat as related to water entry Can J Bot 71 834-840
Simpson BJ 1988 A field guide to Texas trees Austin TX Texas Monthly Press 372p
Tran VN Cavanagh AK 1984 Structural aspects ofdormancy In Seed Physiology Vol 2 Murray DR (ed) p1-44 Academic Press Australia
USDA Forest Service 1948 Woody-plant seed manual USDA Misc Pub1654 416p
Vines RA 1960 Trees Shrubs and Woody Vines of the Southwest Austin Univ ofTexas Press 1104 p
Wagner MR Larson RE Craig TP 1992 Ecology and silvics ofNew Mexico locust (Robinia neomexicana) with an emphasis on the arboreal form p53-57 In JW Hanover K Miller and S Plesko (eds) Proc Intemat Confon Black Locust Biology Culture and Utilization Mich State Univ USA
Wilson JK 1944 Immersing seeds of species of Robinia in boiling water hastens germination J For 42 453-454
Zimmerman RW Carpenter SB 1980 First-year coppice production from a 5shyyear old black locust stand on surface mine spoil p309-314 Centr Hardwd For ConfProc University ofMissouri Columbia
genninating New Mexico locust artificially would be to loosen the strophiolar cells
and allow water to pass through to the embryo
Evolutionary Role of Physical Dormancy
Why is physical dormancy a mechanism that blocks germination
ecologically advantageous When a plant produc~s seeds with different degrees of
dormancy seeds that are dispersed at one time will germinate over a range of time
(Bewley and Black 1994) Through variability of dormancy within a seed crop seeds
are exposed to changing environmental conditions improving the overall chances of
survival of the seed line (Bewley and Black 1994) Varying expression of dormancy
is under genetic control which indicates that this may be an evolved survival strategy
(Kigel and Galili 1995) A range of dormancy levels may be a particular advantage in
the southwestern US where extreme environmental variations exist
Methods for Breaking Dormancy
There are three common approaches to breaking dormancy in New Mexico
locust seed acid scarification hot water soaks and mechanical treatments In each
case the objective of the treatment is to scarify or abrade the seed coat to make it
permeable to water
Acid Scarification
Acid scarification consists of soaking seeds in concentrated sulfuric acid
rinsing thoroughly with water upon removal followed by subsequent drying before
PlaJlltmg Olson (1974) reports that this scarification method produces the highest
middot~~jllalUll results for all Robinia species Most literature refers to a seminal 1937
6
study conducted by HG Meginnis This stud~ t~ough conducted on black locust
seed is regularly cited in the suggested use of acid to treat New Mexico locust seed
as well (Olson 1974 Vines 1960)
Thee published studies document the specific use of sulfuric acid to treat
New Mexico locust seed Cox and Klett (1984) report that soaking seeds in
concentrated sulfuric acid for thirty minutes improved germination to 34 over an
untreated control of4 Lin et al (1996) found that for two seed lots acid
scarification for twenty-five to eighty minutes produced germination of 80 A third
lot however resulted in less than 20 germination at all durations of 10 to 80
minutes Hine et al (1997) showed that acid treatment levels of 1 2 5 and 10
minutes resulted in less than 20 germination across five sources However there
was an increasing trend in germination with acid soak duration and acid treatments
were not imposed for as long a time as the previous two studies (30 to 80 minutes)
The mixed results obtained by these investigators were probably due to the
widely varying germination response of seed lots to acid treatment For this reason
Olson (1974) recommends correlating duration of acid treatment to each seed lot If
treatment duration is too long the embryo will be permanently damaged If soak
duration is too short the seed will remain impermeable to water and fail to germinate
Compounding the problem of treatment duration is the negative effect acid
have on seedling growth Any type of scarification can injure seeds to some
by disrupting cells essential for growth leading to fungal invasion and
middot-uuu~w injury (Copeland and MacDonald 1995) Some legume seeds subjected
7
to 60 minutes of sulfuric acid scarification (to achieve maximum gennination results)
showed more than 50 seedling mortality after gennination (Kelly and van Staden
1987) Bewley and Black (1994) noted that fungal growth on seeds is an indication
of leakage of sugars organic acids amino acids and proteins
Perhaps the greatest drawback to acid scarification is the dangerous nature of
the treatment itself Difficulties associated with storage and trained use of
concentrated acid may discourage many propagators from using this option (Dreesen
and Harrington 1997) Consequently hot water and to a lesser extent mechanical
scarification are more commonly used and practical treatments for legume seeds
Most southwestern container growers use either of these two treatments to scarify
New Mexico locust seed in particular (Hine et al 1997)
Hot Water Scarification
The widespread use ofhot water scarification to treat locust seed is probably
due to its ease of application and efficacy in comparison to other scarification
treatments Although this practice had been in use for some time Wilson (1944) was
one of the first to report on its systematic application for black locust The treatment
consists of placing seeds in boiling water (4 parts water tomiddot 1 part seed by volume)
removing the vessel from the heat source and allowing the water to cool and seeds to
soak for 24 hours
Hine et al (1997) Lin et al (1996) and Cox and Klett (1984) have examined
the specific application ofhot water scarification on New Mexico locust seed Each
these authors compared hot water to acid scarification Lin et aL (1996) reported
8
60 gennination for two hot water-treated seed lots while a third lot germinated
around 50 (as reported above acid scarification germination ranged from 20 to
80 for these lots) It should be noted that the authors only soaked the seeds in water
from 10 to 80 minutes for this experiment as opposed to the standard 24 hours Cox
and Klett (1984) stated that using the standard 24-hour hot water treatment improved
germination over an acid pre-treatment (which produced less than 20 germination)
but they did not give specific gennination numbers for the hot water treatment Hine
et al (1997) found that across five seed lots germination was 66 for the standard
hot water treatment (as compared to less than 20 germination for acid scarification)
Variations in treatment intensity and duration and their subsequent effects on seed lot
appear to contribute to conflicting conclusions as to whether acid scarification or hot
water treatment is a more successful treatment
Given mixed results ease ofapplication is an important argument for use of
hot water application over acid soaks Safety concerns and material costs for hot
water treatments are low (Dreesen and Harrington 1997) An extension of this is the
practicality ofhot water for treating large batches of seed where a relative increase in
water volume is all that is needed However increases in water and seed even when
inthe same 4 1 proportion can result in dramatically different rates of cooling than in
atest tube experiment Different cooling rates can lead to contrasting results for the
treatment temperature duration and seed lot (USDA Forest Service 1948)
As with acid scarification parameters ofhot water treatment application may
to be optimized to maximize germination Hine et al (1997) found that as
9
temperature was increased from 70degC to 100degC overall germination in New Mexico
locust seed increased The 100degC treatment maximized germination for each of five
sources tested with germination usually above 60
Prolonged exposure of seeds ofmany woody legumes to boiling temperatures
also may improve germination (Briscoe 1996) This approach has not been tested on
New Mexico locust seed Exposing seed to prolonged boiling temperatures may
result in fungal infection and subsequent seedling mortality Marunda (1990) noted
severe fungal infections on both acid and hot water treated Acacia holoserica Cunn
ex G Don seeds attributing the infections to seed damage resulting from the
treatments Identifying an optimal hot water treatment level that does not result in a
high rate of seedling mortality is a logical goal
With hot water treatments as with acid there is also a danger of overtreating
Seeds and reducing germination (Gosling et al 1995) The legume Leucaena
leucocephala (Lam) de Wit germinated equally well after 7 seconds of exposure to
water at 100degC 7 seconds at 90degC 30 seconds at 80degC and 4 minutes at 70degC In
addition acceptably high germination was obtained from treatment at 70degC from 3
minutes to 1 hour whereas no seeds germinated following treatment at 100degC for 2
minutes Perhaps along with a greater margin of error for treatment duration treating
~ew Mexico locust seeds at a lower temperature may result in a comparatively lower
of seedling mortality In practice however nursery growers tend to use the
and quickest treatment available The short treatment length at near-boiling
whether recommended or not may be what is used most frequently
10
Fine-tuning a treatment in this range of treatment temperature and duration may be
the most logical approach
Mechanical Scarification
This treatment involves scratching the seed coat with an abrasive material
(USDA Forest Service 1948) One system employs a rotating drum lined with
sandpaper A paddle spinning at a given number ofrevolutions per minute throws the
seed against the sandpaper (Hine et al 1997) A second method dry tumbling
tumbles seed in a mixer with pea gravel or sharp sand for 5 to 7 days (Dreesen 2001)
These approaches have the advantage that control of temperature is not required as in
water treatments nor is safety an issue as with add scarification
Hine et al (1997) mechanically treated New Mexico locust seed using 100shy
grit sandpaper in a Forsberg (rotating drum) scarifier Gennination was less than
10 They concluded that the seed coats were too thin for their apparatus and
proposed that a finer grit sandpaper would produce more even abrasion Although
germination varied by seed lot Dreesen (2001) reported that using a slow-speed dry
tumbling technique for 5 to 7 days produced gennination rates as high as 90
As with acid and hot water treatments treatment duration must be specifically
shy
correlated to seed lot Treated seeds are also susceptible to fungal infection
iHopkinson 1997) In addition equipment is specialized and may not always be
ltU~CUJl~ to a nursery (USDA Forest Service 1948) Several studies have confinned
degrading ofstrophiolar cells due to acid and hot water scarification (Miklas et al
11
1987 Dell 1980 Hanna 1984) but the effect ofmechanical scarification on the
strophiole has not been studied
Alternative Method for Breaking Dormancy Percussion
Strophiole degradation has been examined in depth using an impaction
treatment more commonly known as percussion Hamly (1932) first proposed that
shaking or impacting seeds could split the macrosc1ereid cells at the strophiole
Hamly successfully released tension ofbent strophiolar cells of impermeable
Melilotus alba Medik (White sweet clover) seeds by delivering physical blows
resulting in cells that had split apart and were only slightly bent Ninety-two percent
oftreated seeds became permeable as compared to 05 of control seeds Barton
(1947) showed that percussion significantly increases permeability in black locust
seed Ballard (1976) reasoned that the integrated nature of the strophiole within the
seed coat means a blow on any part of the seed weakens the strophiole There are no
published studies on the use of percussion on New Mexico locust
Percussion methods vary and often seem SUbjective or ill-defined Hamly
(1932) placed seeds in a glass bottle and shook them three times a second for 20
minutes resulting in 180 oscillations per minute Ballard (1973) describes shaking
seeds in a closed metal container with at least fifty seeds per container at 850-900
JS(~l11alIOIlS per minute for 1 minute Kelly and van Staden (1987) placed seeds into
sealed glass flasks Flasks were shaken by a wrist-shaker at a rate of 250
~UUVJ~ per minute Subsequent germination rates were 45 after 2 hours of
55 after 3 hours 60 after 6 hours and 100 after 12 hours
12
There are several potential advantages of percussion over acid hot water and
mechanical scarification Kelly and van Staden (1987) note that a percussion
treatment only alters macrosclereid cells of the strophiole by loosening separating
and raising them They emphasize that minimal damage is done to the overall
anatomical structure of the seed In acid scarification of the same seed lot radicle
and cotyledon damage occurred Based on photographic evidence the authors
believe that this morphological damage was due to uneven pressure during
imbibition with water entering haphazardly wherever the acid had sufficiently
dissolved the coat This ultimately led to uneven hydration of the embryo In
percussed seeds water only entered through the s~ophiole when the entire seed coat
had been saturated resulting in controlled and even embryo hydration (Kelly and van
Staden 1987)
Although all successful scarification treatments may focus at least in part on
the strophiole for papilionoid legumes (Baskin and Baskin 1998) percussion may be
less damaging to seed coat structures particularly when compared with acid Kelly
and van Staden did not compare hot water or mechanical scarification methods
against percussion However the fungal infection associated with hot water and
mechanical scarification as with acid scarificatio~ have been directly associated with
seed damage (Rehman et al 1998 USDA Forest Service 1948) Embryo damage in
has been observed in hot-water scarified legume seeds (Rehman et al 1998
13
By limiting embryo damage percussion scarification reduces seedling
mortality compared to other treatments Kelly and van Staden (1987) noted that
seedlings from the percussion treatment were morphologically sound and vigorous
when compared to acid treatment seedlings The authors attribute this improved
seedling vigor to the controlled water uptake that limited anatomical damage
Despite a 12-hour percussion treatment morphological damage was not substantially
greater than untreated seeds In addition seedling mass measured 10 days following
germination was not significantly different between percussed and untreated seeds
A final advantage ofpercussion may be the ability to adequately treat the most
dormant seeds while avoiding harm to seeds that have already been made permeable
by the treatment or that were permeable to begin with Instead ofcorroding scalding
or scratching the seed coat percussion appears to be just a loosening of the
strophiole spring (Ballard 1973) Kelly and van Staden (1987) were able to
increase germination with a lengthy treatment (12 hours) Figuring out whether a
substantial cushion oftreatment durations exist for percussion ofNew Mexico locust
seed would be a significant improvement to current scarification methods where seed
damage and seedling health is an issue
The greatest challenge for applying percussion to New Mexico locust seeds is
standardizing a treatment that is both effective and readily reproducible by nurseries
with mechanical scarification this may require the development of specific
~UllDment to properly quantify and execute the treatment
14
f
Objectives
The first objective of the study is to detennine which scarification treatment
results in optimal gennination speed and percentage ofNew Mexico locust The
second objective is to describe treatment interactions with nine seed sources collected
in New Mexico
Hypothesis
A percussion treatment acts exclusively on the main area ofwater uptake in
New Mexico locust seed loosening the strophiole so that imbibition can occur This
direct mechanism ofdonnancy release will result in improved gennination rate and
overall gennination percentage for New Mexico locust seed compared to standard
treatments Standard treatments including acid hot water and mechanical
scarification while eventually weakening the strophiolar cleft cause excessive
morphological damage through generalized seed coat degradation These treatments
are therefore prone to inconsistency due to over and under-treatment
15
MATERIALS AND METHODS
Seed Collection and Cleaning
Seeds were collected from August 25 through September 15 2000 from nine
locations along a latitudinal gradient in New Mexico Seed source locations and
descriptive information are given in Figure 1 and Tables I and 2 Stands were
separated by at least 45 km
Pods were hand picked as they began to ripen and dehisce (open) Collections
were made from ground level to approximately 3 m from several trees at each site
Pods were dried in open-ended brown paper bags for two to four weeks at room
temperature (21-230C) to facilitate cleaning Dried pods were placed in a closed cloth
sack which was pounded against the ground to separate seeds from pods For pods
still containing seeds hand maceration (crunching pods with hands) completed seed
separation Debris was removed by threshing with the aid of a greenhouse fan A
South Dakota Seed Blower (Seedbureau Chicago Illinois) was used to remove
remaining chaff and light seeds by air density separation Seeds were placed in dry
storage in sealed quart-size zip-lock bags within a sealed plastic container Storage
temperatures ranged from 2-4oC Seeds remained in storage for approximately 7
months before the experiment
Experimental Design and Treatment Structure
The experiment compared the impact ofhot water bath and percussion
l11aLl11 on germination properties ofNew Mexico locust seed A single
16
-l
Figure 1 Seed source collection map All sources were collected within New Mexico USA separated by at least 4S lan
IIIRZltItW lIJPIirmiddot rllll1lbullbull_~~~_II
Table 1 Latitude longitude location elevation and collection date ofNew Mexico locust seed sources
Lot title Latitude (N) Longitude (W) Location Elevation (m) Collection Date
Raton 36deg5846 104deg2872 Raton Pass NM 2295 82500
Holman 36deg0306 105deg2429 HolmanNM 2476 911500
Jemez 35deg4500 106deg4100 Jemez Springs NM 1976 9900 00
Sandia 35deg1083 106deg2341 Cedar Crest NM 2432 82600
Manzano 34deg3689 106deg2494 ManzanoNM 2465 9200
Magdalena 33deg5953 107deg0903 Magdalena NM 2544 82900
Capitan 33deg2728 105deg4325 Alto NM 2189 911200
Sacramento 32deg5798 105deg4496 Cloudcroft NM 2630 91400 Gila 32deg5400 107deg4600 Kingston NM 2432 9700
f
Table 2 Mean seed weight and standard error by source
a
Source Mean seed weight (g) for Standard error 100 seeds n = 5
Raton Holman Jemez Sandia Manzano Magdalena Capitan Sacramento Gila
2872 b c 2522 d 1826 e 2753 c 2413 d 2559 d 2443 d 3130 a 3029 ab
00103 00403 00276 00301 00370 00374 00458 00279 00348
a Mean seed weights followed by the same letter are not significantly
different
19
experiment was conducted utilizing a randomized complete block design blocked by
time with a factorial treatment structure Experimental factors were seed source and
scarification treatment Nine seed sources from throughout New Mexico were used
(Figure 1 Table 1) Scarification treatments included a control three levels ofhot
water and four levels of percussion Hot water treatments included immersion at
100degC and boiling for periods of 30 seconds and 60 seconds Percussion treatment
levels were 1 2 4 and 8 minutes One hundred seeds per block were used to test
each treatment combination Four blocks of seed were tested one at a time (all seed
sources included in each run) in close chronological sequence (within 6 weeks) A
total of four 100-seed samples were used to test each source by scarification treatment
combination
Treatments
Hot water treatment of seeds utilized 50 ml test tubes filled with 30 ml
distilled water Each test tube was used to treat one 100-seed sample at a time
resulting in a ratio of4 parts water to 1 part seed by volume Test tubes were placed
in a beaker with the beaker filled with tap water to the same level as the water within
test tubes Nine test tubes (one per seed source) were placed in the beaker
simultaneously so that one replication of each seed sourcescarification treatment
cotnblnatlon was conducted simultaneously Hot water treatments include immersion
boiling For immersion treatments seeds were added to test tubes with the water
(boiling) and the test tubes were immediately removed from the beaker to
For the boiling treatments test tubes remained in the beaker with water at
20
boiling temperature for a set duration after the addition of seeds Following all
treatments test tubes with seeds cooled 24 hours in a test tube rack
Percussion treatment utilized a pneumatic paint shaker (Central Pneumatic
model 00422 Camarillo CA) Compressed air was maintained at 530 kPa plusmn30 kPa
(80 psi plusmn 5 psi) resulting in approximately 350 oscillations per minute Seeds were
placed in 118 ml (4 oz) soil tin cans (US Can Lobard IL) and the lids were secured
with duct tape Each can was used to treat one 100-seed sample To take advantage
ofgreatest lateral movement of the paint shaker a spacer (paint can) was used to
position the soil tin at the end of the shaking arm This allowed shaking to occur at
the greatest distance from the pivot point (fulcrum) Tins were placed perpendicular
to the direction of shaking motion allowing seeds to impact at greatest force by
hitting flat surfaces of the soil tin
Germination Testing
Before gennination testing all seeds were soaked 24 hours in reverse
osmosis-treated water (this was included in the process ofhot water treatment
jU~SCIlOea above) Following soaking seeds were placed in 100 cm Petri dishes lined
90 cm Whatman qualitative 1 filter paper circles (MG Scientific Pleasant
WI) One Petri dish was used for each treatment by source combination
papers were initially moistened with 5 mL reverse osmosis-treated water then
~VU1CUlJl moistened as needed to ensure adequate conditions for gennination
were monitored daily (at 300 PM) for gennination for 14 days in accordance
~UIUlnJlVUa Seed Testing Association (ISTA) standards for Robinia species
21
(1STA 1999) Tests were carried out at room temperature (22degC plusmnOSoC)
Germination was classified as protrusion of the radicle from the embryo by at least
1mm as viewed with the naked eye
X-ray Radiography
On August 132001 three lOO-seed samples from the Sacramento seed source
were treated for 14 or 8-minute percussion durations The seeds were sent
immediately by overnight delivery to the Faxitron X-Ray Corporation in Wheeling
Illinois where Dr Bob Karrfalt ofthe USDA Forest Service oversaw their imaging
Radiographs were made at SX with a Faxitron Specimen Radiography System Model
MX-20
Scanning Electron Microscope Photography
On February 192002 a Hitachi S-3200N Variable Pressure Scanning
Electron Microscope (Mountain View CA) was used to examine strophiolar cells of
untreated seeds and seeds percussed for 4 minutes Seeds were cut longitudinally
igt their entire length using a razor blade splitting the strophiolar region roughly in
Cross sections of seed samples magnifying the strophiolar region were viewed
600X under the microscope
Analysis
Categorical Analysis of Variance was used to determine treatment source and
(Le time) main effects and interactions The response variable was total
~vlI This procedure is a Chi-square test of homogeneity using the natural log
of germinated to non-germinated seed for each treatment Maximumshy
22
likelihood analysis was used to calculate Chi-square test statistics with their observed
significance levels A single contrast was conducted to cempare treatment means
against the control Observed significance levels less than or equal to 005 were
considered significant Pairwise comparisons were conducted between non-control
treatment mean percents using a conservative alp~a value of 005 divided by the
number of comparisons (Bonferroni approach) and approximate pairwise z statistics
Categorical analyses were performed using Proc Catmod (SAS Institute 1989)
RESULTS
Scarification seed source block and all interactions between factors
influenced germination (Table 3) The main effects are reported in the presence of
these interactions because the overall robustness of treatments and seed sources are of
interest Background levels ofnaturally occurring variation in seed sources are
seen in control germination percentages in Figure 2 In particular time variability
is unlikely to be under the control of the producer Appendix B details experimental
data for source by treatment gennination percents for each of the four blocks
Averaged over all treatments germination by source ranged from 40 to
79 (Figure 2) Averaged over all sources (Figure 3) and for each source (Figure 4
5) percussion scarification durations of4 or 8 minutes yielded greatest germination
Averaged over all sources germination increased with increasing durations of
percussion then leveled off for the 4- and 8-minute treatments (Figure 3) Immersion
and 30-second boiling scarification maximized germination for the hot water
treatments with a decrease in germination when seeds were boiled for 60 seconds
For all but two sources there is a similar ipcreasing trend in germination from
1to 4 minutes ofpercussion (Figure 4) The Holman source increased in germination
1 to 2 minutes then leveled off at 4 minutes The Jemez source changed little
1 to 4 minutes of treatment As treatment duration increased from 4 to 8
mutes five sources decreased in germination (Figure 4a) whereas three sources
24
Table 3 Categorical Analysis of Variance table for germination response to seed treatment seed source block and the interaction of the factors
Source of DF Chi-Square Observed Variability Significance Level
Source 8 98370 lt00001 Trt 7 233876 lt00001
contrast 1 16976 lt00001 SourceTrt 56 97077 lt00001 Block 3 5538 lt00001
SourceBlock 24 10162 lt00001 TrtBlock 21 7564 lt00001
SourceTrtBlock 168 34283 lt00001
S 4)
8 ~
0
sectg ~ 40 ~
N 0 0
20
o f0ltgt ~ ~4 jybshy ((iltgto ~~ K1ltgt 6~b-~ ~~ 0b-~~ ~~ ~b-~ ~b-~b
80
60 -I-- - ~- middotmiddotmiddotmiddotmiddot1
r -] Northern NM Sources
Central NM Sources
Southern NM Sources
sect-o ~
~b-v(i
SOURCE
Figure 2 Effect ofNew Mexico locust seed source on germination for data averaged over all scarification treatments Control germination is shown by solid line within bar
80
~ cd ~ ltl) () 60 ~ ~
N
t 0shyrti tshySltl)
40
0
20
o lmmersion Boiling 30 sec Boiling 60 sec Control 1 min 2min 4 min 8 min
Hot Water Treatments Percussion Treatments
Figure 3 Effect of scarification on germination ofNew Mexico locust for data averaged over all seed sources Means labelled with the same letter are not significantly different
100~----------------------~--~----------------~
80
60
ltU
~
40 IJ
sectgsI
l100
80
60
40
a I middotmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot middotImiddotmiddotmiddotmiddotmiddotmiddot E
_~iii-bull _ _ bull f --~--
t-- -~ ---- -shy ---- shyI ~ ---- - shy
L
fiII
~
b
- shy ---r
- Holman
Sacramento Sandia Gila Jemez
c ~
ltgt ~
Tmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot-I
j~=~=-~~-1
I middotmiddot ~ bull r
t tr
Y -------M-a-gd-a-Ie-n-a--
1 _ _
fCapitan
---- Manzano ~y -_ Raton
2 4 8
Time interval ofpercussion scarification (min)
Figure 4 Effect of seed source by treatment interaction on New Mexico locust germination for percussion scarification pattern a and pattern b
28
tv 0
~ d
~ A s o
1 c3
80
60
40
20
o
TIT
t------=middotfmiddotmiddot-middotmiddot-middotmiddot-middotmiddot 1- --- - - --=- - -shy--cE= - _--shy --shy ----shy ~ ~ ---shy ~ -~- _ --shy-------r 1
I
Raton Holman
-_ Jemez
--- Sandia Manzano Magdalena Capitan Sacramento Gila
Immersion Boiling 30 sec Boiling 60 sec
Hot Water Scarification
Figure 5 Effect of seed source by treatment interaction on New Mexico locust germination for hot water scarification
continued to increase in germination (Figure 4b) There was no difference between
the two treatment levels for the Manzano source (Figure 4b)
The effect ofhot water scarification also varied among sources with few
overall differences between hot water treatments (Figure 5) However boiling for 60
seconds yielded highest germination for the Jemez source whereas increasing
durations ofboiling decreased germination for the Raton source
In addition to consistently high germination 4- and 8-minute percussionshy
treated seeds germinated as fast or faster than all other treatments (Figure 6 Table 4)
050 measured as the number of days needed to complete 50 ofeventual
germination averaged 2 days for both ofthese treatments
X-ray radiographs were taken of seeds collected from the Sacramento source
percussed for 1 4 or 8 minutes (Figure 7) Seeds show signs of damage at 8 minutes
ofpercussion (Figure 8) Compared to an undamaged seed percussed for only 4
minutes (Figure 8a) damage at the 8-minute level includes fractured radicles pitting
of the seed coat and chips in seed coat structure (Figure 8b-d)
Scanning electron microscope (SEM) images at 600X reveal the direct effect
of 4 minutes of percussion on cells of the strophiolar region (Figure 9) Whereas
untreated seeds retain cellular organization in the strophiolar region strophiolar cells
seeds treated for 4 minutes ofpercussion separate and raise resulting in a
~-~e~U appearance
30
Umiddot-~middot--l-~---middotymiddotmiddotmiddot-----middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot--middot--middotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middotmiddotmiddotmiddotmiddotmiddotmiddot- -
050= gt1
-e-- Percussion 8 min -0- Percussion 4 min ---T- Percussion 2 min -----ltil- Percussion 1 min -- Immersion --0- Boiling 30 sec -+- Boiling 60 sec -ltgt- Control
2 4 6 8 10 12
Day Figure 6 Timecourse ofNew Mexico locust gennination following scarification
treatments G50 is the number of days to 50 of eventual germination
31
14
Table 4 Effect of seed source and treatment on G50 (days to 50 germination)
Seed Source Control Immersion B30sec B60sec Plmin P2min P4min P8min mean Raton 3 3 5 7 4 3 2 2 4 Holman 2 2 2 2 2 2 2 2 2
Jemez 4 6 6 5 3 2 2 2 4 Sandia 2 2 2 2 2 2 2 2 2
w tv Manzano 5 2 2 3 3 2 2 2 3
Magdalena 4 2 2 3 4 3 2 1 3
Capitan 5 4 2 2 3 2 2 2 3
Sacramento 3 2 2 2 4 3 2 2 3 Gila 3 2 2 2 2 2 2 2 2 mean 3 3 3 3 3 2 2 2 3 standard 11 14 16 18 09 05 0 03 08 deviation
Note Columns and rows may not agree due to rounding
Figure 7 X-ray radiographs ofSacramento seed source percussed for 1 4 or 8 minutes
33
Figure 8a-d Seed damage close-ups Figure a shows undamaged seed percussed for 4 minutes Figures b-d show seeds percussed for 8 minutes Damage includes fracturing ofradicle in b complete removal ofseed coat in c and pitting ofseed coat in d
34
Untreated Treated
Figure 9 SEM images ofuntreated vs 4-minute percussion-treated strophiolar cells ofNew Mexico locust seeds Treated cells are loosened separated and raised
DISCUSSION
Determining the Optimal Treatment
Averaging results of scarification treatmems across seed sources is a useful
tool to predict which treatment is most likely to be successful for an unknown seed
source Averaged over all sources four- and eight-minute percussion treatments
resulted in the highest germination 815 and 790 respectively and produced
germination that was as fast or faster than all other treatments Although there was no
difference between these two treatments on average an argument for 4 minutes of
percussion as a standard protocol for treating New Mexico locust seeds can be made
Descriptively 4 minutes of percussion improved germination over 8 minutes
ofpercussion for a majority of sources (five out of nine) 8 minutes of percussion
improved germination for three ofnine sources with no difference for a fmal source
For individual sources 4 minutes ofpercussion was always the best or secondmiddot best
treatment and was always equal to or better than all hot water treatments
Germination at 8 minutes ofpercussion was lower than all other percussion durations
for the Jemez and Gila sources and was lower than at least one hot water treatment for
these sources as well
The x-ray radiographs of the Sacramento seed source illustrate the excessive
internal and external seed damage that can occur with 8 minutes of percussion
germination counts 8-minute percussed seeds from Holman and Capitan
ClUll signs of damage that were visible to the naked eye (see below)
r~evertheless some of these seeds still fit the criteria for germination Neither x-ray
36
photography nor visible observations during seed counts revealed seed damage at the
4-minute percussion level In operational trials seeds percussed for 4 minutes
developed into healthy seedlings (Khadduri and Harrington unpublished data)
Strophiole and Seed Coat Behavior
Four minutes ofpercussion successfully breaks seed dormancy in a large
percentage ofNew Mexico locust seeds without excessively damaging the
surrounding seed coat or internal seed coat structures Most papilionoid legume
seeds including seeds ofNew Mexico locust have a specific region of the seed coat
known as the strophiole or lens (Hamly 1932) The strophiole is located on the
cotyledonary lobe of a locust seed (Figure 10 a-c) If seeds are not indiscriminately
damaged the strophiole is the first point of water entry to the embryo (Hopkinson
1997) When a papilionoid seed is percussed for the appropriate amount of time
repeated hits on the integrated seed coat loosen the constrained cells of the strophiolar
region without excessively damaging the rest of the seed coat (Ballard 1976) SEM
photos illuStrate that 4 minutes of percussion loosened and separated strophiolar cells
ofNew Mexico locust seeds when compared with constrained organized strophiolar
cells of untreated seeds (Figure 9) Research-has shown that water passes to the
embryo through the treated strophiole in a controlled manner and regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues
(Kelly and van Staden 1987)
37
10 Strophiole ofunimbibed New Mexico locust seed (a) and following uptake (bc)
38
In contrast hot-water treatments can excessively degrade the seed coat
(Rebman et al 1998 Marunda 1990) Random cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rebman et aI 1998 Marunda 1990) Along with a
proportionately large amount ofun-imbibed (thus under-treated) seeds (Figure l1a)
damage was observed in hot-water treated seeds from six sources during germination
counts Damage included seeds where the radicle failed to elongate (Figure 11 b)
seeds where the radicle elongated but was delayed in freeing itself from the seed coat
(Figure 11c) and seeds where the radicle completely broke off from the embryo
(Figure lId) In addition some seeds germinated in reverse (the cotyledon emerged
fIrst) If the radicle emerged completely from the seed coat they were counted as
germinated
While nearly all seeds treated for 8 minutes ofpercussion imbibed water
damage was evident in x-ray evaluations (Sacramento seed source) and in
germination counts (Holman and Capitan) at this treatment level Seed damage
during germination counts was similar to levels found in hot-water treated seeds
suggesting that over-treatment by both hot water and longer durations of percussion
may result in similar damage to seed structure
The Sacramento and Holman seed sources were two of the fIve sources that
opcrplltlpn in germination from the 4- to 8-minute durations of percussion
the benefIts ofloosening the strophiole as evidenced by imbibition of
all 8-minute treated seeds were outweighed by external and internal seed coat
39
a
b c d
11 Damage to New Mexico locust seeds observed during germination cotmts following hot water scarification 9a Hot water replicate including ~t seedlings and numerous unimbibed seeds 9b Close-up of seed where raallCle failed to elongate 9c Close-up ofseed where radicle elongated but was
-gtIn in freeing itself from the seed coat 9d Close-up ofseed where radicle broke off from embryo
40
damage (which also may be contributing to high imbibition) For the three sources
for which 8 minutes of percussion resulted in maximum germination extended
percussion of seeds necessary to loosen the strophiole may not have excessively
damaged the seed anatomy The Manzano source did not significantly change in
germination from 4 to 8 minutes of percussion suggesting that an increase in
permeable seeds at the 8-minute treatment duration was balanced by the number of
seeds that failed to germinate due to seed damage To summarize the 4-minute
percussion treatment improved germination over 8 minutes ofpercussion for a
majority of sources (five ofnine) the 8-minute percussion treatment improved
germination over 4-minutes ofpercussion for three sources and there was no
difference with the Manzano source Notably for the Raton source 8 minutes of
percussion increased germination by 235 percentage points over 4-minutes of
percussion (7875 vs 5525 respectively) the next best treatment Raton was the
only source to result in germination less than 75 for 4 minutes ofpercussion so a
grower may wish to apply longer percussion durations to lots that do not respond well
to this treatment level
Finally there was an interesting trend in the Jemez and Raton sources seed
that significantly contributed to the source by treatment interaction (Figure 12) A
~u test showed that the Jemez source had a significantly lighter seed weight than
other sources while the Raton source had an average seed weight For the Jemez
the longest duration of boiling increased germination Conversely only short
U4UVll) of percussion were needed to achieve high germination and the longest
41
80 _ Jemez a Raton
iD
~ C iD e iD
Q c 0 ~
c ~ iD 0
20
0------shy
Immersion Boiling 30 sec Boiling 60 sec
Hot Water Scarification
10degTr========~--------~------------------------------
80
60
40
20
_ Jemez Raton
0------shy
1 min 2 min 4 min 8 min
Percussion Scarification
Figure 12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources
42
b
--~----~~~~========~---- duration ofpercussion reduced germination For the Raton source longer durations
ofboiling consistently decreased germination whereas longer percussion treatments
consistently increased germination
A possible explanation is that a thick seed coat is associated with a loose
strophiole structure and that a thin seed coat is associated with a constrained
strophiole This contrast merits further study and may suggest an ecological
adaptation Further investigation ofthis phenomenon may contribute to successful
pre-screening ofunknown seed sources to determine optimal treatment leveL
43
CONCLUSION
Based on results obtained in pilot study (see Appendix) and thesis
experiments 4 minutes ofpercussion is recommended as a standard protocol for
treating an unknown source ofNew Mexico locust seed In addition to laboratory
results the 4-minute percussion treatment has resulted in germination greater than
80 and healthy seedling grow-outs for production purposes (unpublished data) In
laboratory experiments only one hundred seeds per 118 ml (40z) soil tin were
percussed at a time whereas for production we have treated up to five hundred seeds
per tin For large-scale production ofNew Mexico locust seedlings growers may
wish to experiment with larger quantities of seed and larger-size soil tins
Seed storage trials following percussion treatments were not conducted
However germination greater than 80 and healthy seedlings were obtained when
seeds were percussion-treated and subsequently stored at room-temperature (22degC)
for one week (unpublished data) Seed storage trials merit further study as the
standard hot water treatment leaves seeds in a softened condition that is not
conducive to storage
Hydroseeding is an effective method for direct seeding in reclamation
situations Percussion treatment may be conducive to hydro seeding as seeds remain
a dry relatively undamaged yet permeable condition following treatment Sulfuric
seeds also remain dry following treatment but extensive damage to seed coat
poor seedling vigor have been reported with this treatment (Kelly and van Staden
A grow-out study using a hydro seeder on a reclamation site testing percussion
44
against other treatments such as sulfuric acid and mechanical scarification also
merits further study
Papilionoids are the largest subfamily oflegumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust these legumes often play an integral role in the revegetation of
disturbed lands The pilot study (Appendix) indicates that percussion scarification
successfully treats the widely used black locust as well Developing superior
scarification methods to the standard hot water mechanical scarification and acid
treatments may facilitate the use ofpapilionoid legumes in reclamation projects
45
APPENDICES
APPENDIX A
PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR
NEW MEXICO LOCUST AND BLACK LOCUST SEEDS
Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4
Hot water and sulfuric acid soaks are traditional treatments for seeds of many
temperate woody legwnes including locusts However these scarification techniques
often produce inconsistent germination Percussion scarification where seeds are
repeatedly propelled against a hard surface was compared with hot water
scarification to evaluate treatment efficacy for New Mexico locust (Robinia
neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment
seeds were placed in a 98degC water bath which was immediately removed from the
heat source For percussion scarification seeds were placed in a soil sample tin and
agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the
control were followed by 24-hour water soaks Hot water baths resulted in 56 and
41 germination for New Mexico locust and black locust respectively For both
species nearly all durations ofpercussion increased germination over the hot water
treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3
4 and 5 minutes for black locust resulted in at least 90 germination Traditional
scarification treatments randomly degrade the entire seed coat which can lead to
tissue damage during water uptake Percussion scarification specifically weakens the
strophiole the natural source ofwater entry to the seed in papilionoid legwnes
INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom
2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031
48
Following percussion imbibition is controlled through the strophiole and underlying
tissue is protected
Keywords New Mexico locust black locust scarification percussion germination
Introduction
Locust trees (Robinia spp) are aggressive pioneer species that quickly
colonize disturbed land fix atmospheric nitrogen in the soil and stabilize slopes that
are prone to erosion (Klemmedson 1994 Ashby et al 1985) (Figure A1ab)
Recognition of these attributes has contributed to increased demand for locusts in
reclamation projects Locust seeds however exhibit physical dormancy where a
thick seed coat prevents the movement of water and gasses to the embryo (Leadem
1997)
Sulfuric acid or hot water soaks have traditionally been used to break seed
dormancy in macro-propagation of locusts Many growers are moving away from the
use of su1furic acid in treating locust seeds (Dreesen and Harrington 1997) In
addition to being dangerous acid soak durations must be specifically correlated to
seed lot (Olson 1974) Collections of native seed can vary tremendously in seed size
seed weight and hard seededness etc This variability limits the utility of sulfuric
acid soaks as even within a given lot some seeds may be damaged whereas others
are under-treated and fail to imbibe (take up water) A survey of Southwestern
container growers indicates that hot water baths are currently the most common
scarification method for locust seeds (Hine et al 1997) However hot water baths
also produce inconsistent germination (Lin et al 1996)
49
b
Figure AI New Mexico locust a primary invader following disturbance Ala Crown-sprouting at edge ofburn near Los Alamos NM--one year after Cerro Grande fire Alb Colonizing road cut Gila National Forest NM
50
Percussion scarification where seeds are repeatedly propelled against a hard
surface is an alternative dormancy-breaking method that has proven successful in
legumes related to locusts (Hamly 1932 Barton 1947) We compared this alternative
treatment to hot water scarification to evaluate treatment efficacy for New Mexico
locust and black locust seeds
Materials and Methods
Commercial New Mexico locust seeds (Western Native Seed Coaldale CO)
were collected fall 2000 in Huerfano County CO Black locust seeds were collected
September 2000 from Taos County NM by harvesting ripe pods from several trees at
a distance of up to 3 m from ground level Black locust seeds were allowed to air-dry
for 3 weeks and were threshed and separated from large chaffusing a greenhouse
fan A South Dakota Seed Blower (Chicago Illinltois) was used to remove fine chaff
from both seed lots Seeds were storedmiddotat 2-4C until the germination study was
conducted in March 2001
This study compared hot water scarification with percussion scarification
using treatment and species as experimental factors Along with a control
scarification treatments included immersion of seed in boiling water as well as
percussion durations of 1 2 3 45 or 10 minutes Four 100-seed samples were used
to test each species by scarification treatment combination
For the hot water treatment test tubes were filled with 30 mL water and
placed in a water-filled beaker (Figure A2a) The bath was raised to boiling (-98C at
51
b
c
Figure A2 Implementation ofhot water and percussion scarification A2a Hot water bath A2b Paint-shaker set-up A2c Placing soil tin containing seeds at end ofpaint shaker ann
52
Las Cruces NM elevation of 1300 meters) Each 100-seed replicate was placed in a
test tube and each test tube was immediately removed to cool at room temperature
Percussion scarification was implemented using a pneumatic paint shaker
(Central Pneumatic model 00422 Camarillo CA) (Figure A2b) This allowed
standardization of treatment intensity Compressed air pressure was maintained at
530kPa plusmn 30kPa (80psi plusmn 5psi) resulting in approximately 350 oscillations per
minute Each lOO-seed sample was placed in a 118 ml (4 oz) soil tin for shaking To
maximize lateral movement of paint shaker a paint can was used as a spacer to place
soil tins at the end of the shaking arm (Figure A2c) This allowed shaking to occur at
the greatest distance from the pivot point (fulcrum) Following all treatments
including the control seeds were soaked for twenty-four hours in distilled water prior
to plating out
Gennination was evaluated on lab benches at room temperature Each
replicate of seeds was placed in a 100 cm petri dish on moistened filter paper with
humidity maintained by enclosing the petri dishes in ziplock bags Gennination was
monitored daily for 14 days in accordance with International Seed Testing
Association standards for Robinia species (ISTA 1999) Gennination was defined as
protrusion of the radicle from the embryo by at least 1mm as viewed by the naked
eye
Categorical Analysis of Variance using Prpc Catmod (SAS Institute 1989)
was used to detennine treatment differences in germination percentages for each
source This procedure is an extension of a Chi-square test of homogeneity using the
53
natural log of the ratio of germinated to non-germinated seeds for each treatment Pshy
values less than 005 were considered significant A limited set of pairwiseshy
comparisons was conducted to compare treatment mean percents using a
conservative alpha value of 005 divided by the number of comparisons
Results
Percussion maximized germination for both New Mexico locust and black
locust There were three germination response levels for New Mexico locust (Figure
A3) First there was a low response in the control indicating a dormant seed lot
The hot water treatment and I-minute duration of percussion share a second
intermediate response Germination was highest at percussion levels from 2 to 10
minutes
A selected percussion treatment intermediate in time (5 minutes) produced the
steepest germination curve indicating the fastest germination rate (Figure A4) The
hot water treatment produced an intermediate curve or relatively slower germination
rate The control curve was very flat indicating the slowest germination rate for New
Mexico locust
Control seeds germinated poorly for black locust as well (Figure A5) Hot
water scarification improved germination over the control but all levels of percussion
improved germination over hot water The optimal durations of percussion treatment
were bracketed with a significant increase from the 1 to 2 minute percussion level a
high germination response from 2 to 5 minutes of percussion and finally a significant
54
~ d 25 CL)
p t1 0 ~ t1
g Vl ~ Vl
100~---------------------------------------------------------------
80
60
40
20
o Control Hot Water Percussion
I min Percussion
2 min Percussion
3 min
Scarification
Percussion 4 min
Figure A3 Effect of scarification on germination response for New Mexico locust
Percussion 5 min
=significant difference between adjacent treatment bars ns = no significant difference
Percussion 10 min
~ ~ () u agt
Il-t d o ~
~ ~ c3
100middotlr~~~------------------------------------e- Percussion 5min middot 0middot Hot Water -- Control
80-J=middot======
6b-middot-lmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middot--middotmiddot-middot~-middotmiddot -middotmiddot middot middotmiddot-middot 1
40
20
-----~-----------
0 0 0 middotmiddotmiddotmiddotmiddot0middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot0
0 --~-----y-----~------_---~--- T-----~
o shy -TDay 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A4 Effect of selected scarification treatments on germination speed for New Mexico locust First seven days of fourteen-day experiment are shown
Day 7
80
~ cd
5 60 ()
[) Cl d 0 1t1 d
40sect dUl
-J
20
o
ns
Scarification
Figure A5 Effect of scarification on germination response for black locust = significant difference between adjacent treatment bars ns = no significant difference
decrease at the 10-minute level This drop-off suggests that 10 minutes of percussion
over-treats seeds from this lot of-black locust
A selected percussion treatment intermediate in time (5 minutes) also had the
fastest germination rate for black locust (Figure A6)_ The hot water curve is
somewhat flatter indicating a slower germination rate The control curve is very flat
indicating a slow germination rate
A qualitative difference between hot water and percussion-treated seeds was
also observed Along with healthy germinants the hot water treatment produced
over-treated seeds (Figure A 7a) Examples of damaged germinants include seeds
where the radicle did not elongate (Figure A 7b) seeds where the radicle elongated
but was delayed in freeing itself from the seed coat (Figure A7c) and seeds where the
radicle broke off completely from the embryo (Figure A7d) In addition to these
examples of over-treatment there were numerous small dark seeds that failed to
imbibe water For percussion treatment nearly all seeds took up water (Figure A8a)
Germination was rapid and uniform with little damage evident (Figure A8b)
To summarize both New Mexico locust and black locust seed lots were
dormant The standard hot water scarification treatment improved germination but
only to 56 and 41 for New Mexico locust and black locust respectively Nearly
all percussion durations improved total germination as well as germination rate over
the hot water treatment Germination was greater than 90 for percussion durations
of 4 5 and 10 minutes for New Mexico locust and 3 4 and 5 minutes for black locust
58
CI)
sgt t CI) 0
igt 0
sect ttl t E
Vl 0
c3
100r==~==~~=r------------------------------------~ ___ Percussion 5 min
middotmiddotmiddot0middotmiddotmiddot Hot Water -lIf- Control
80r=========~--~~----------------------------~
60
40
20 -~-~---~---------- ---~------------I
omiddot
O
_~_----------lIf-----~bull -----_ shy---shy I
Day 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A6 Effect of selected scarification treatments on germination speed for black locust First seven days of fourteen-day experiment are shown
Day 7
a
b c d
Figure A7 Damage to New Mexico locust seeds observed during germination counts following hot water scarification A7a Hot water replicate including damaged seedlings and numerous unimbibed seeds A7b Close-up ofseed where radicle failed to elongate A7c Close-up ofseed where radicle elongated but was delayed in freeing itself from the seed coat A7d Close-up ofseed where radicle completely broke offfrom embryo
60
a
b
Figure A8 New Mexico locust seeds following percussion treatment at time of germination counts A8a Percussion replicate with uniformly healthy seedlings and near complete imbibition of seeds A8b Close-up ofpercussion-treated seedling
61
Discussion
Papilionoid seeds have a specific anatomical feature known as the strophiole
or lens-the natural site of water entry to the seed (Hamly 1932) The strophiole is
located on the cotyledonary lobe of a locust seed (Figure A9) When a papilionoid
legume seed is percussed for the appropriate amount of time repeated hits on the
integrated seed coat loosen the constrained cells of the strophiolar region without
excessively damaging the rest of the seed coat (Ballard 1976)
When a percussed seed is soaked water enters exclusively through the
strophiole in a controlled manner (Kelly and Staden 1987) This regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues This
contrasts with seeds that have been hot water or acid scarified treatments that can
randomly degrade the seed coat Unlocalized cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rehman et al 1998 Marunda 1990)
Papilionoids are the largest subfamily of legumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust and black locust these legumes often play an integral role in the
revegetation ofdisturbed lands Developing superior scarification methods to the
standard hot water and acid treatments should fadlitate the use ofthese legumes in
restoration projects
62
Figure A9 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (bc)
63
APPENDIXB
GERMINATION PERCENTS AND STANDARD ERRORS FOR SOURCE
BLOCK AND TREATMENT FOR NEW MEXICO LOCUST
Table B 1 Gennination percents and standard errors for source block and treatment for New Mexico locust
Source Block Treatment Germination Percentage Standard Error
Capitan 1 Immersion 74 437 Capitan 2 Immersion 69 462 Capitan 3 Immersion 71 454 Capitan 4 Immersion 67 470 Capitan Boiling 30 sec 79 407 Capitan 2 Boiling 30 sec 76 427 Capitan 3 Boiling 30 sec 72 449 Capitan 4 Boiling 30 sec 76 427 Capitan Boiling 60 sec 71 454 Capitan 2 Boiling 60 sec 54 498 Capitan 3 Boiling 60 sec 67 470 Capitan 4 Boiling 60 sec 76 427 Capitan I Control 5 218 Capitan 2 Control 7 255 Capitan 3 Control 4 196 Capitan 4 Control 5 218 Capitan 1 Percussion I min 47 499 Capitan 2 Percussion I min
470
Capitan Percussion I min 39 488
Capitan 4 Percussion I min 38 485 Capitan I Percussion 2 min 55 497 Capitan Capitan
2
Percussion 2 min Percussion 2 min
71 65
454 477
Capitan 4 Percussion 2 min 56 496 Capitan I Percussion 4 min 86 347 Capitan 2 Percussion 4 min 79 407 Capitan 3 Percussion 4 min 81 392 Capitan 4 Percussion 4 min 82 384 Capitan I Percussion 8 min 90 300 Capitan 2 Percussion 8 min 88 325 Capitan 3 Percussion 8 min 90 300 Capitan 4 Percussion 8 min 90 300 Gila I Immersion 71 454 Gila 2 Immersion 77 421 Gila
Immersion 75 433
Gila 4 Immersion 72 449
Gila I Boiling 30 sec 68 466
Gila 2 Boiling 30 sec 81 392
Gila 3 Boiling 30 sec 54 498
Gila 4 Boiling 30 sec 63 483
Gila I Boiling 60 sec 55 497
Gila 2 Boiling 60 sec 68 466
Gila 3 Boiling 60 sec 57 495
Gila 4 Boiling 60 sec 70 458
65
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Gila 2 Control 13 336 Gila 3 Control 13 336 Gila 4 Control 20 400 Gila 1 Percussion 1 min 66 474 Gila 2 Percussion 1 min 57 495 Gila 3 Percussion 1 min 73 444 Gila 4 Percussion 1 min 82 384 Gila 1 Percussion 2 min 81 392 Gila 2 Percussion 2 min 86 347 Gila 3 Percussion 2 min 85 357 Gila 4 Percussion 2 min 54 498 Gila 1 Percussion 4 min 79 407 Gila 2 Percussion 4 min 83 376 Gila 3 Percussion 4 min 84 367 Gila 4 Percussion 4 min 79 407 Gila 1 Percussion 8 min 51 500 Gila 2 Percussion 8 min 76 427 Gila 3 Percussion 8 min 52 500 Gila 4 Percussion 8 min 74 439 Holman 1 Immersion 83 376 Holman 2 Immersion 86 347 Holman 3 Immersion 81 392 Holman 4 Immersion 90 300 Holman 1 Boiling 30 sec 90 300 Holman 2 Boiling 30 sec 85 357 Holman 3 Boiling 30 sec 87 336 Holman 4 Boiling 30 sec 83 376 Holman 1 Boiling 60 sec 79 407 Holman 2 Boiling 60 sec 80 400 Holman 3 Boiling 60 sec 86 347 Holman 4 Boiling 60 sec 93 255 Holman Control 25 433 Holman 2 Control 19 392 Holman Control 21 407
Holman 4 Control 24 427
Holman Percussion 1 min 77 421
Holman 2 Percussion 1 min 85 357
Holman 3 Percussion 1 min 80 400
Holman 4 Percussion 1 min 89 313
Holman 1 Percussion 2 min 88 325
Holman 2 Percussion 2 min 96 196
Holman 3 Percussion 2 min 93 255
Holman 4 Percussion 2 min 96 196
Holman 1 Percussion 4 min 92 271
Holman 2 Percussion 4 min 97 171
66
I
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Holman 3 Percussion 4 min 91 286 Holman 4 Percussion 4 min 96 196 Holman 1 Percussion 8 min 88 325 Holman 2 Percussion 8 min 85 357 Holman 3 Percussion 8 min 83 376 Holman 4 Percussion 8 min 82 384 Jemez 1 Immersion 53 499 Jemez 2 Immersion 44 496 Jemez 3 Immersion 52 500 Jemez 4 Immersion 70 458 Jemez 1 Boiling 30 sec 58 494 Jemez 2 Boiling 30 sec 55 497 Jemez 3 Boiling 30 sec 48 500 Jemez 4 Boiling 30 sec 65 477 Jemez 1 Boiling 60 sec 68 466 Jemez 2 Boiling 60 sec 63 483 Jemez 3 Boiling 60 sec 73 444 Jemez 4 Boiling 60 sec 89 313 Jemez 1 Control 2 140 Jemez 2 Control 9 286 Jemez 3 Control 6 237 Jemez 4 Control 8 271 Jemez 1 Percussion 1 min 58 494 Jemez 2 Percussion 1 min 63 483 Jemez 3 Percussion 1 min 77 421 Jemez 4 Percussion 1 min 89 313 Jemez 1 Percussion 2 min 68 466 Jemez 2 Percussion 2 min 64 480 Jemez 3 Percussion 2 min 80 400 Jemez 4 Percussion 2 min 83 376 Jemez Percussion 4 min 68 466 Jemez 2 Percussion 4 min 79 407 Jemez 3 Percussion 4 min 70 458 Jemez 4 Percussion 4 min 83 376 Jemez 1 Percussion 8 min 52 500 Jemez 2 Percussion 8 min 59 492 Jemez 3 Percussion 8 min 61 488 Jemez 4 Percussion 8 min 70 458 Magdalena 1 Immersion 69 462 Magdalena 2 Immersion 69 462 Magdalena J Immersion 70 458 Magdalena 4 Immersion 85 357 Magdalena 1 Boiling 30 sec 65 477 Magdalena 2 Boiling 30 sec 71 454 Magdalena 3 Boiling 30 sec 68 466
67
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Magdalena 4 Boiling 30 sec 83 376 Magdalena 1 Boiling 60 sec 63 483 Magdalena 2 Boiling 60 sec 68 466 Magdalena 3 Boiling 60 sec 63 483 Magdalena 4 Boiling 60 sec 82 384 Magdalena 1 Control 11 313 Magdalena 2 Control 6 237 Magdalena 3 Control 5 218 Magdalena 4 Control 4 196 Magdalena Percussion 1 min 32 466 Magdalena 2 Percussion 1 min 45 497 Magdalena 3 Percussion 1 min 46 498 Magdalena 4 Percussion I min 36 480 Magdalena 1 Percussion 2 min 67 470 Magdalena 2 Percussion 2 min 68 466 Magdalena 3 Percussion 2 min 65 477 Magdalena 4 Percussion 2 min 81 392 Magdalena 1 Percussion 4 min 87 336 Magdalena 2 Percussion 4 min 97 171 Magdalena 3 Percussion 4 min 83 376 Magdalena 4 Percussion 4 min 97 171 Magdalena 1 Percussion 8 min 98 lAO Magdalena 2 Percussion 8 min 100 000 Magdalena 3 Percussion 8 min 96 196 Magdalena 4 Percussion 8 min 98 lAO Manzano 1 Immersion 67 470 Manzano 2 Immersion 57 495 Manzano 3 Immersion 72 4049 Manzano 4 Immersion 73 4044 Manzano 1 Boiling 30 sec 70 458 Manzano 2 Boiling 30 sec 64 480 Manzano 3 Boiling 30 sec 71 454 Manzano 4 Boiling 30 sec 80 400 Manzano I Boiling 60 sec 50 500 Manzano 2 Boiling 60 sec 53 499 Manzano 3 Boiling 60 sec 59 492 Manzano 4 Boiling 60 sec 74 439 Manzano I Control 4 196 Manzano 2 Control 8 271 Manzano 3 Control 1 099 Manzano 4 Control 6 237 Manzano 1 Percussion 1 min 52 500 Manzano 2 Percussion 1 min 39 488 Manzano 3 Percussion 1 min 36 480 Manzano 4 Percussion 1 min 51 500
68
Table B 1 Continued
Source Block Treatment Gerrni~ation Percentage Standard Error
Manzano Percussion 2 min 53 499 Manzano 2 Percussion 2 min 58 494 Manzano 3 Percussion 2 min 61 488 Manzano 4 Percussion 2 min 62 485 Manzano 1 Percussion 4 min 76 427 Manzano 2 Percussion 4 min 85 357 Manzano 3 Percussion 4 min 79 407 Manzano 4 Percussion 4 min 83 376 Manzano 1 Percussion 8 min 87 336 Manzano 2 Percussion 8 min 83 376 Manzano 3 Percussion 8 min 83 376 Manzano 4 Percussion 8 min 75 433 Raton 1 Immersion 54 498 Raton 2 Immersion 37 483 Raton 3 Immersion 39 488 Raton 4 Immersion 69 462 Raton 1 Boiling 30 sec 36 480 Raton 2 Boiling 30 sec 20 400 Raton 3 Boiling 30 sec 28 449 Raton 4 Boiling 30 sec 60 490 Raton 1 Boiling 60 sec 15 357 Raton 2 Boiling 60 sec 171 Raton 3 Boiling 60 sec 4 196 Raton 4 Boiling 60 sec 35 477 Raton 1 Control 4 196 Raton 2 Control 12 325 Raton 3 Control 2 140 Raton 4 Control 6 237 Raton 1 Percussion 1 min 52 500 Raton 2 Percussion I min 29 454 Raton 3 Percussion I min 35 477 Raton 4 Percussion 1 min 31 462 Raton 1 Percussion 2 min 55 497 Raton 2 Percussion 2 min 35 477 Raton 3 Percussion 2 min 33 470 Raton 4 Percussion 2 min 42 494 Raton 1 Percussion 4 min 65 477 Raton 2 Percussion 4 min 52 500 Raton Percussion 4 min 51 500 Raton 4 Percussion 4 min 53 499 Raton 1 Percussion 8 min 88 325 Raton 2 Percussion 8 min 80 400 Raton 3 Percussion 8 min 73 444 Raton 4 Percussion 8 min 74 439 Sacramento 1 Immersion 43 495
69
Table B 1 Continued
-Source Block Treatment Germination Percentage Standard Error
Sacramento 2 Immersion 30 458 Sacramento 3 Immersion 38 485 Sacramento 4 Immersion 56 496 Sacramento 1 Boiling 30 sec 47 499 Sacramento 2 Boiling 30 sec 48 500 Sacramento 3 Boiling 30 sec 48 500 Sacramento 4 Boiling 30 sec 56 496 Sacramento 1 Boiling 60 sec 52 500 Sacramento 2 Boiling 60 sec 67 470 Sacramento 3 Boiling 60 sec 43 495 Sacramento 4 Boiling 60 sec 48 500 Sacramento 1 Control 0 000 Sacramento 2 Control 3 171 Sacramento 3 Control 3 171 Sacramento 4 Control 099 Sacramento 1 Percussion 1 min 57 495 Sacramento 2 Percussion 1 min 61 488 Sacramento 3 Percussion 1 min 62 485 Sacramento 4 Percussion 1 min 55 497 Sacramento 1 Percussion 2 min 84 367 Sacramento 2 Percussion 2 min 70 458 Sacramento 3 Percussion 2 min 81 392 Sacramento 4 Percussion 2 min 69 462 Sacramento 1 Percussion 4 min 91 286 Sacramento 2 Percussion 4 min 84 367 Sacramento 3 Percussion 4 min 84 367 Sacramento 4 Percussion 4 min 94 237 Sacramento 1 Percussion 8 min 82 384 Sacramento 2 Percussion 8 min 81 392 Sacramento 3 Percussion 8 min 82 384 Sacramento 4 Percussion 8 min 81 392 Sandia 1 Immersion 76 427 Sandia 2 Immersion 70 458 Sandia 3 Immersion 70 458 Sandia 4 Immersion 81 392 Sandia 1 Boiling 30 sec 78 414 Sandia 2 Boiling 30 sec 71 454 Sandia 3 Boiling 30 sec 63 483 Sandia 4 Boiling 30 sec 78 414 Sandia 1 Boiling 60 sec 67 470 Sandia 2 Boiling 60 sec 65 477 Sandia 3 Boiling 60 sec 74 439 Sandia 4 Boiling 60 sec 66 474 Sandia 1 Control 16 367 Sandia 2 Control 7 255
70
Table B 1 Continued
Source Block Treatment Gennination Percentage Standard Error
Sandia 3 Control 8 271 Sandia 4 Control 16 367 Sandia 1 Percussion 1 min 67 470 Sandia 2 Percussion 1 min 61 488 Sandia 3 Percussion 1 min 66 474 Sandia 4 Percussion 1 min 59 492 Sandia I Percussion 2 min 82 384 Sandia 2 Percussion 2 min 83 376 Sandia 3 Percussion 2 min 81 392 Sandia 4 Percussion 2 min 84 367 Sandia 1 Percussion 4 min 89 313 Sandia 2 Percussion 4 min 86 347 Sandia 3 Percussion 4 min 89 313 Sandia 4 Percussion 4 min 85 357 Sandia 1 Percussion 8 min 80 400 Sandia 2 Percussion 8 min 77 421 Sandia 3 Percussion 8 min 69 462 Sandia 4 Percussion 8 min 66 474
LITERATURE CITED
Ashby WC Vogel WG Rogers NF 1985 Black locust in the reclamation equation General Technical Report Northeastern Forest Experiment Station USDA Forest Service NE-I05 12 p
Ballard LAT 1973 Physical barriers to gennination Seed Sci Technoll 285-303
___ 1976 Strophiolar water conduction in the seeds ofTrifolieae induced by action on the testa at nonstrophiolar sites Austr J Plant Physiol3 465-469
Barton LV 1947 Special studies on seed coat impenneability Contrib Boyce Thompson Inst 14 355-362
Baskin CC Baskin JM 1998 Seeds Ecology biogeography and evolution of donnancy and gennination Academic Press London UK 666 p
Bewley JD 1997 Seed germination and donnancy Plant Ce119 1055-1066
Bewley JD Black M 1994 Seeds physiology of development and germination 2nd ed Plenum Press New York NY 445 p
Briscoe CB 1996 Growing Nitrogen Fixing Trees in Acid Soils In MH Powell (ed) Nitrogen Fixing Trees for Acid Soils- A Field ManuaL Winrock International Morrilton AR 22 p
Copeland LO McDonald MB 1995 Principles of Seed Science and Technology 3rded Chapman amp Hall New York NY 409 p
Cox RE Klett JE 1984 Seed gennination requirements of native Colorado plants for use in the landscape Plant Propag 30(2) 6-10
Dell B 1980 Structure and function ofthe strophiolar plug in seeds ofAlbizia Lophantha Am J Bot 67 556-563
Dick-Peddie WA 1993 New Mexico Vegetation Past Present and Future University ofNew Mexico Press Albuquerque NM 244 p
Dreesen DR 2001 Propagation protocol for production ofcontainer Robinia neomexicana plants Los Lunas Plant Materials Center Los Lunas New Mexico In Native Plant Network URL httpwwwnativeplantnetworkorg (accessed 9 January 2002) Moscow (ID) University ofIdaho CoIl Natural Resources For Res Nursery
72
Dreesen DR Harrington JT 1997 Propagation of native plants for restoration projects in the Southwestern US- Preliminary Investigations In Landis TD Thompson JR tech coords National Proceedings Forest and Conservation Nursery Assocs Gen Tech Rep PNW-GTR-419 Portland OR USDA For Ser PNW Res Sta 77-88
Gottfried GJ 1980 Control ofNew Mexican locust and the effect on planted ponderosa pine in central Arizona US For Ser Res Note RM-386 Rocky Mountain For Range ExpStn Fort Collins CO
Gosling PG Samuel YK Jones SK 1995 A systematic examination of germination temperature chipping and water temperaturesoak duration pretreatments on the seeds ofLeucaena leucocephala Seed Sci amp Technol 23521-532
Hamly DH 1932 Softening of the seeds of Melilotus alba Bot Gazette 93 345shy375
Hanna PJ 1984 Anatomical features of the seed coat ofAcacia kempeana (Mueller) which relate to increased germination rate induced by heat treatment New Phytol 96 23-29
Hanks JP Dick-Peddie WA 1974 Vegetation patterns of the White Mountains New Mexico Southwest Naturalist 18(4) 371-382
Hine S Harrington JT Dreesen DR 1997 Seed scarification requirements for Robinia neomexicana In Harrington JTtech coord Proc Third SouthwestemContainer Growers Conf Feb 12-13 1997 Ft Collins CO
Hopkinson JM 1993 The strophiole of leguminous seeds Newsletter- IntI Herbage Seed Production Res Grp 18 7-8
---- 1997 Differences in hardseededness between Cunningham and Tarramba leucaena In Leucnet News The Newsletter of the International LeucaenaRes and Devlt Netwk No4
ISTA (International Seed Testing Association) 1999 International rules for seed testing rules Seed Sci amp Technol27 Supplement
Kelly KM van Staden J 1987 The lens as the site of permeability in the papilionoid seed Aspalathus linearis J Plant Physiol 128(45) 395-404
73
Keresztesi B 1988 Black locust the tree of agriculture Outlook on Agr 17(2) 77~85
Kigel 1 and Galili G 1995 Seed development and germination Marcel Dekker New York 853 p
Klemmedson 10 1994 New Mexico locust and parent material Influence on macronutrients of forest floor and soil Soil Sci Soc Am 1 58 974~980
Kuo WH Tam AW 1988 The pathway of water absorption of mung bean seeds Seed Sci amp Technol16(1) 139~144
Leadem CL 1997 Dormancy--unlocking seed secrets In Landis TD Thompson lR tech coords National Proceedings Forestand Conservation Nursery Assocs Gen Tech Rep PNW-G TR-419 Portland OR USDA For Ser PNW Res Sta 43-52
Lin Y Wagner MR Seweyestewa A Honahni MD DeGomez M McMillin JD 1996 Influence of cultural treatments on seed germination and seedling growth of Robinia neomexicana Forest Farm and Community Tree Res Reports Volume 1
Marunda CT 1990 Effects of seed pretreatment on the development ofAcacia auriculforinis and A holosericea seedlings ACIAR Proceedings Series No 28
Meginnis HG 1937 Sulphuric acid treatment to increase germination of black locust seed USDA Circ 453 34 p
Miklas PN Towensend CE Ladd SL 1987 Seedcoat anatomy and the scarification ofcicer milkvetch seed Crop Sci 27 766-772
Monsen SB 1984 Use of shrubs on mine spoils In The Challenge ofProducing Native Plants for the Intermountain Area Proceedings Intermountain Nurserymans Association 1983 conference Las Vegas NV compo PM Murphy 26-31 Ogden UT USDA For Ser Gen Tech Report INT-l68
Olson DF 1974 Robinia L Locust In Seeds of Woody Plants in the US tech coord CS Schopmeyer 728-731 USDA For Ser Agr Handbk No450
Rehman S Loescher RNl Harris P1C 1998 Dormancy breaking and germination ofAcacia salicina Lindl seeds Seed Sci amp Techno127 553-557
Rolston MP 1978 Water impermeable seed dormancy Bot Rev 44 365-396
74
SAS Institute Inc 1989 SASSTAT Users Guide Version 6 4th Edition Volume 1 Cary NC SAS Institute Inc 943 p
SerratoValenti G Conara L Ferrando M Modenesi P 1993 Structural and histochemical features ofStylosanthes scabra (Leguminosae Papilionoidae) seed coat as related to water entry Can J Bot 71 834-840
Simpson BJ 1988 A field guide to Texas trees Austin TX Texas Monthly Press 372p
Tran VN Cavanagh AK 1984 Structural aspects ofdormancy In Seed Physiology Vol 2 Murray DR (ed) p1-44 Academic Press Australia
USDA Forest Service 1948 Woody-plant seed manual USDA Misc Pub1654 416p
Vines RA 1960 Trees Shrubs and Woody Vines of the Southwest Austin Univ ofTexas Press 1104 p
Wagner MR Larson RE Craig TP 1992 Ecology and silvics ofNew Mexico locust (Robinia neomexicana) with an emphasis on the arboreal form p53-57 In JW Hanover K Miller and S Plesko (eds) Proc Intemat Confon Black Locust Biology Culture and Utilization Mich State Univ USA
Wilson JK 1944 Immersing seeds of species of Robinia in boiling water hastens germination J For 42 453-454
Zimmerman RW Carpenter SB 1980 First-year coppice production from a 5shyyear old black locust stand on surface mine spoil p309-314 Centr Hardwd For ConfProc University ofMissouri Columbia
study conducted by HG Meginnis This stud~ t~ough conducted on black locust
seed is regularly cited in the suggested use of acid to treat New Mexico locust seed
as well (Olson 1974 Vines 1960)
Thee published studies document the specific use of sulfuric acid to treat
New Mexico locust seed Cox and Klett (1984) report that soaking seeds in
concentrated sulfuric acid for thirty minutes improved germination to 34 over an
untreated control of4 Lin et al (1996) found that for two seed lots acid
scarification for twenty-five to eighty minutes produced germination of 80 A third
lot however resulted in less than 20 germination at all durations of 10 to 80
minutes Hine et al (1997) showed that acid treatment levels of 1 2 5 and 10
minutes resulted in less than 20 germination across five sources However there
was an increasing trend in germination with acid soak duration and acid treatments
were not imposed for as long a time as the previous two studies (30 to 80 minutes)
The mixed results obtained by these investigators were probably due to the
widely varying germination response of seed lots to acid treatment For this reason
Olson (1974) recommends correlating duration of acid treatment to each seed lot If
treatment duration is too long the embryo will be permanently damaged If soak
duration is too short the seed will remain impermeable to water and fail to germinate
Compounding the problem of treatment duration is the negative effect acid
have on seedling growth Any type of scarification can injure seeds to some
by disrupting cells essential for growth leading to fungal invasion and
middot-uuu~w injury (Copeland and MacDonald 1995) Some legume seeds subjected
7
to 60 minutes of sulfuric acid scarification (to achieve maximum gennination results)
showed more than 50 seedling mortality after gennination (Kelly and van Staden
1987) Bewley and Black (1994) noted that fungal growth on seeds is an indication
of leakage of sugars organic acids amino acids and proteins
Perhaps the greatest drawback to acid scarification is the dangerous nature of
the treatment itself Difficulties associated with storage and trained use of
concentrated acid may discourage many propagators from using this option (Dreesen
and Harrington 1997) Consequently hot water and to a lesser extent mechanical
scarification are more commonly used and practical treatments for legume seeds
Most southwestern container growers use either of these two treatments to scarify
New Mexico locust seed in particular (Hine et al 1997)
Hot Water Scarification
The widespread use ofhot water scarification to treat locust seed is probably
due to its ease of application and efficacy in comparison to other scarification
treatments Although this practice had been in use for some time Wilson (1944) was
one of the first to report on its systematic application for black locust The treatment
consists of placing seeds in boiling water (4 parts water tomiddot 1 part seed by volume)
removing the vessel from the heat source and allowing the water to cool and seeds to
soak for 24 hours
Hine et al (1997) Lin et al (1996) and Cox and Klett (1984) have examined
the specific application ofhot water scarification on New Mexico locust seed Each
these authors compared hot water to acid scarification Lin et aL (1996) reported
8
60 gennination for two hot water-treated seed lots while a third lot germinated
around 50 (as reported above acid scarification germination ranged from 20 to
80 for these lots) It should be noted that the authors only soaked the seeds in water
from 10 to 80 minutes for this experiment as opposed to the standard 24 hours Cox
and Klett (1984) stated that using the standard 24-hour hot water treatment improved
germination over an acid pre-treatment (which produced less than 20 germination)
but they did not give specific gennination numbers for the hot water treatment Hine
et al (1997) found that across five seed lots germination was 66 for the standard
hot water treatment (as compared to less than 20 germination for acid scarification)
Variations in treatment intensity and duration and their subsequent effects on seed lot
appear to contribute to conflicting conclusions as to whether acid scarification or hot
water treatment is a more successful treatment
Given mixed results ease ofapplication is an important argument for use of
hot water application over acid soaks Safety concerns and material costs for hot
water treatments are low (Dreesen and Harrington 1997) An extension of this is the
practicality ofhot water for treating large batches of seed where a relative increase in
water volume is all that is needed However increases in water and seed even when
inthe same 4 1 proportion can result in dramatically different rates of cooling than in
atest tube experiment Different cooling rates can lead to contrasting results for the
treatment temperature duration and seed lot (USDA Forest Service 1948)
As with acid scarification parameters ofhot water treatment application may
to be optimized to maximize germination Hine et al (1997) found that as
9
temperature was increased from 70degC to 100degC overall germination in New Mexico
locust seed increased The 100degC treatment maximized germination for each of five
sources tested with germination usually above 60
Prolonged exposure of seeds ofmany woody legumes to boiling temperatures
also may improve germination (Briscoe 1996) This approach has not been tested on
New Mexico locust seed Exposing seed to prolonged boiling temperatures may
result in fungal infection and subsequent seedling mortality Marunda (1990) noted
severe fungal infections on both acid and hot water treated Acacia holoserica Cunn
ex G Don seeds attributing the infections to seed damage resulting from the
treatments Identifying an optimal hot water treatment level that does not result in a
high rate of seedling mortality is a logical goal
With hot water treatments as with acid there is also a danger of overtreating
Seeds and reducing germination (Gosling et al 1995) The legume Leucaena
leucocephala (Lam) de Wit germinated equally well after 7 seconds of exposure to
water at 100degC 7 seconds at 90degC 30 seconds at 80degC and 4 minutes at 70degC In
addition acceptably high germination was obtained from treatment at 70degC from 3
minutes to 1 hour whereas no seeds germinated following treatment at 100degC for 2
minutes Perhaps along with a greater margin of error for treatment duration treating
~ew Mexico locust seeds at a lower temperature may result in a comparatively lower
of seedling mortality In practice however nursery growers tend to use the
and quickest treatment available The short treatment length at near-boiling
whether recommended or not may be what is used most frequently
10
Fine-tuning a treatment in this range of treatment temperature and duration may be
the most logical approach
Mechanical Scarification
This treatment involves scratching the seed coat with an abrasive material
(USDA Forest Service 1948) One system employs a rotating drum lined with
sandpaper A paddle spinning at a given number ofrevolutions per minute throws the
seed against the sandpaper (Hine et al 1997) A second method dry tumbling
tumbles seed in a mixer with pea gravel or sharp sand for 5 to 7 days (Dreesen 2001)
These approaches have the advantage that control of temperature is not required as in
water treatments nor is safety an issue as with add scarification
Hine et al (1997) mechanically treated New Mexico locust seed using 100shy
grit sandpaper in a Forsberg (rotating drum) scarifier Gennination was less than
10 They concluded that the seed coats were too thin for their apparatus and
proposed that a finer grit sandpaper would produce more even abrasion Although
germination varied by seed lot Dreesen (2001) reported that using a slow-speed dry
tumbling technique for 5 to 7 days produced gennination rates as high as 90
As with acid and hot water treatments treatment duration must be specifically
shy
correlated to seed lot Treated seeds are also susceptible to fungal infection
iHopkinson 1997) In addition equipment is specialized and may not always be
ltU~CUJl~ to a nursery (USDA Forest Service 1948) Several studies have confinned
degrading ofstrophiolar cells due to acid and hot water scarification (Miklas et al
11
1987 Dell 1980 Hanna 1984) but the effect ofmechanical scarification on the
strophiole has not been studied
Alternative Method for Breaking Dormancy Percussion
Strophiole degradation has been examined in depth using an impaction
treatment more commonly known as percussion Hamly (1932) first proposed that
shaking or impacting seeds could split the macrosc1ereid cells at the strophiole
Hamly successfully released tension ofbent strophiolar cells of impermeable
Melilotus alba Medik (White sweet clover) seeds by delivering physical blows
resulting in cells that had split apart and were only slightly bent Ninety-two percent
oftreated seeds became permeable as compared to 05 of control seeds Barton
(1947) showed that percussion significantly increases permeability in black locust
seed Ballard (1976) reasoned that the integrated nature of the strophiole within the
seed coat means a blow on any part of the seed weakens the strophiole There are no
published studies on the use of percussion on New Mexico locust
Percussion methods vary and often seem SUbjective or ill-defined Hamly
(1932) placed seeds in a glass bottle and shook them three times a second for 20
minutes resulting in 180 oscillations per minute Ballard (1973) describes shaking
seeds in a closed metal container with at least fifty seeds per container at 850-900
JS(~l11alIOIlS per minute for 1 minute Kelly and van Staden (1987) placed seeds into
sealed glass flasks Flasks were shaken by a wrist-shaker at a rate of 250
~UUVJ~ per minute Subsequent germination rates were 45 after 2 hours of
55 after 3 hours 60 after 6 hours and 100 after 12 hours
12
There are several potential advantages of percussion over acid hot water and
mechanical scarification Kelly and van Staden (1987) note that a percussion
treatment only alters macrosclereid cells of the strophiole by loosening separating
and raising them They emphasize that minimal damage is done to the overall
anatomical structure of the seed In acid scarification of the same seed lot radicle
and cotyledon damage occurred Based on photographic evidence the authors
believe that this morphological damage was due to uneven pressure during
imbibition with water entering haphazardly wherever the acid had sufficiently
dissolved the coat This ultimately led to uneven hydration of the embryo In
percussed seeds water only entered through the s~ophiole when the entire seed coat
had been saturated resulting in controlled and even embryo hydration (Kelly and van
Staden 1987)
Although all successful scarification treatments may focus at least in part on
the strophiole for papilionoid legumes (Baskin and Baskin 1998) percussion may be
less damaging to seed coat structures particularly when compared with acid Kelly
and van Staden did not compare hot water or mechanical scarification methods
against percussion However the fungal infection associated with hot water and
mechanical scarification as with acid scarificatio~ have been directly associated with
seed damage (Rehman et al 1998 USDA Forest Service 1948) Embryo damage in
has been observed in hot-water scarified legume seeds (Rehman et al 1998
13
By limiting embryo damage percussion scarification reduces seedling
mortality compared to other treatments Kelly and van Staden (1987) noted that
seedlings from the percussion treatment were morphologically sound and vigorous
when compared to acid treatment seedlings The authors attribute this improved
seedling vigor to the controlled water uptake that limited anatomical damage
Despite a 12-hour percussion treatment morphological damage was not substantially
greater than untreated seeds In addition seedling mass measured 10 days following
germination was not significantly different between percussed and untreated seeds
A final advantage ofpercussion may be the ability to adequately treat the most
dormant seeds while avoiding harm to seeds that have already been made permeable
by the treatment or that were permeable to begin with Instead ofcorroding scalding
or scratching the seed coat percussion appears to be just a loosening of the
strophiole spring (Ballard 1973) Kelly and van Staden (1987) were able to
increase germination with a lengthy treatment (12 hours) Figuring out whether a
substantial cushion oftreatment durations exist for percussion ofNew Mexico locust
seed would be a significant improvement to current scarification methods where seed
damage and seedling health is an issue
The greatest challenge for applying percussion to New Mexico locust seeds is
standardizing a treatment that is both effective and readily reproducible by nurseries
with mechanical scarification this may require the development of specific
~UllDment to properly quantify and execute the treatment
14
f
Objectives
The first objective of the study is to detennine which scarification treatment
results in optimal gennination speed and percentage ofNew Mexico locust The
second objective is to describe treatment interactions with nine seed sources collected
in New Mexico
Hypothesis
A percussion treatment acts exclusively on the main area ofwater uptake in
New Mexico locust seed loosening the strophiole so that imbibition can occur This
direct mechanism ofdonnancy release will result in improved gennination rate and
overall gennination percentage for New Mexico locust seed compared to standard
treatments Standard treatments including acid hot water and mechanical
scarification while eventually weakening the strophiolar cleft cause excessive
morphological damage through generalized seed coat degradation These treatments
are therefore prone to inconsistency due to over and under-treatment
15
MATERIALS AND METHODS
Seed Collection and Cleaning
Seeds were collected from August 25 through September 15 2000 from nine
locations along a latitudinal gradient in New Mexico Seed source locations and
descriptive information are given in Figure 1 and Tables I and 2 Stands were
separated by at least 45 km
Pods were hand picked as they began to ripen and dehisce (open) Collections
were made from ground level to approximately 3 m from several trees at each site
Pods were dried in open-ended brown paper bags for two to four weeks at room
temperature (21-230C) to facilitate cleaning Dried pods were placed in a closed cloth
sack which was pounded against the ground to separate seeds from pods For pods
still containing seeds hand maceration (crunching pods with hands) completed seed
separation Debris was removed by threshing with the aid of a greenhouse fan A
South Dakota Seed Blower (Seedbureau Chicago Illinois) was used to remove
remaining chaff and light seeds by air density separation Seeds were placed in dry
storage in sealed quart-size zip-lock bags within a sealed plastic container Storage
temperatures ranged from 2-4oC Seeds remained in storage for approximately 7
months before the experiment
Experimental Design and Treatment Structure
The experiment compared the impact ofhot water bath and percussion
l11aLl11 on germination properties ofNew Mexico locust seed A single
16
-l
Figure 1 Seed source collection map All sources were collected within New Mexico USA separated by at least 4S lan
IIIRZltItW lIJPIirmiddot rllll1lbullbull_~~~_II
Table 1 Latitude longitude location elevation and collection date ofNew Mexico locust seed sources
Lot title Latitude (N) Longitude (W) Location Elevation (m) Collection Date
Raton 36deg5846 104deg2872 Raton Pass NM 2295 82500
Holman 36deg0306 105deg2429 HolmanNM 2476 911500
Jemez 35deg4500 106deg4100 Jemez Springs NM 1976 9900 00
Sandia 35deg1083 106deg2341 Cedar Crest NM 2432 82600
Manzano 34deg3689 106deg2494 ManzanoNM 2465 9200
Magdalena 33deg5953 107deg0903 Magdalena NM 2544 82900
Capitan 33deg2728 105deg4325 Alto NM 2189 911200
Sacramento 32deg5798 105deg4496 Cloudcroft NM 2630 91400 Gila 32deg5400 107deg4600 Kingston NM 2432 9700
f
Table 2 Mean seed weight and standard error by source
a
Source Mean seed weight (g) for Standard error 100 seeds n = 5
Raton Holman Jemez Sandia Manzano Magdalena Capitan Sacramento Gila
2872 b c 2522 d 1826 e 2753 c 2413 d 2559 d 2443 d 3130 a 3029 ab
00103 00403 00276 00301 00370 00374 00458 00279 00348
a Mean seed weights followed by the same letter are not significantly
different
19
experiment was conducted utilizing a randomized complete block design blocked by
time with a factorial treatment structure Experimental factors were seed source and
scarification treatment Nine seed sources from throughout New Mexico were used
(Figure 1 Table 1) Scarification treatments included a control three levels ofhot
water and four levels of percussion Hot water treatments included immersion at
100degC and boiling for periods of 30 seconds and 60 seconds Percussion treatment
levels were 1 2 4 and 8 minutes One hundred seeds per block were used to test
each treatment combination Four blocks of seed were tested one at a time (all seed
sources included in each run) in close chronological sequence (within 6 weeks) A
total of four 100-seed samples were used to test each source by scarification treatment
combination
Treatments
Hot water treatment of seeds utilized 50 ml test tubes filled with 30 ml
distilled water Each test tube was used to treat one 100-seed sample at a time
resulting in a ratio of4 parts water to 1 part seed by volume Test tubes were placed
in a beaker with the beaker filled with tap water to the same level as the water within
test tubes Nine test tubes (one per seed source) were placed in the beaker
simultaneously so that one replication of each seed sourcescarification treatment
cotnblnatlon was conducted simultaneously Hot water treatments include immersion
boiling For immersion treatments seeds were added to test tubes with the water
(boiling) and the test tubes were immediately removed from the beaker to
For the boiling treatments test tubes remained in the beaker with water at
20
boiling temperature for a set duration after the addition of seeds Following all
treatments test tubes with seeds cooled 24 hours in a test tube rack
Percussion treatment utilized a pneumatic paint shaker (Central Pneumatic
model 00422 Camarillo CA) Compressed air was maintained at 530 kPa plusmn30 kPa
(80 psi plusmn 5 psi) resulting in approximately 350 oscillations per minute Seeds were
placed in 118 ml (4 oz) soil tin cans (US Can Lobard IL) and the lids were secured
with duct tape Each can was used to treat one 100-seed sample To take advantage
ofgreatest lateral movement of the paint shaker a spacer (paint can) was used to
position the soil tin at the end of the shaking arm This allowed shaking to occur at
the greatest distance from the pivot point (fulcrum) Tins were placed perpendicular
to the direction of shaking motion allowing seeds to impact at greatest force by
hitting flat surfaces of the soil tin
Germination Testing
Before gennination testing all seeds were soaked 24 hours in reverse
osmosis-treated water (this was included in the process ofhot water treatment
jU~SCIlOea above) Following soaking seeds were placed in 100 cm Petri dishes lined
90 cm Whatman qualitative 1 filter paper circles (MG Scientific Pleasant
WI) One Petri dish was used for each treatment by source combination
papers were initially moistened with 5 mL reverse osmosis-treated water then
~VU1CUlJl moistened as needed to ensure adequate conditions for gennination
were monitored daily (at 300 PM) for gennination for 14 days in accordance
~UIUlnJlVUa Seed Testing Association (ISTA) standards for Robinia species
21
(1STA 1999) Tests were carried out at room temperature (22degC plusmnOSoC)
Germination was classified as protrusion of the radicle from the embryo by at least
1mm as viewed with the naked eye
X-ray Radiography
On August 132001 three lOO-seed samples from the Sacramento seed source
were treated for 14 or 8-minute percussion durations The seeds were sent
immediately by overnight delivery to the Faxitron X-Ray Corporation in Wheeling
Illinois where Dr Bob Karrfalt ofthe USDA Forest Service oversaw their imaging
Radiographs were made at SX with a Faxitron Specimen Radiography System Model
MX-20
Scanning Electron Microscope Photography
On February 192002 a Hitachi S-3200N Variable Pressure Scanning
Electron Microscope (Mountain View CA) was used to examine strophiolar cells of
untreated seeds and seeds percussed for 4 minutes Seeds were cut longitudinally
igt their entire length using a razor blade splitting the strophiolar region roughly in
Cross sections of seed samples magnifying the strophiolar region were viewed
600X under the microscope
Analysis
Categorical Analysis of Variance was used to determine treatment source and
(Le time) main effects and interactions The response variable was total
~vlI This procedure is a Chi-square test of homogeneity using the natural log
of germinated to non-germinated seed for each treatment Maximumshy
22
likelihood analysis was used to calculate Chi-square test statistics with their observed
significance levels A single contrast was conducted to cempare treatment means
against the control Observed significance levels less than or equal to 005 were
considered significant Pairwise comparisons were conducted between non-control
treatment mean percents using a conservative alp~a value of 005 divided by the
number of comparisons (Bonferroni approach) and approximate pairwise z statistics
Categorical analyses were performed using Proc Catmod (SAS Institute 1989)
RESULTS
Scarification seed source block and all interactions between factors
influenced germination (Table 3) The main effects are reported in the presence of
these interactions because the overall robustness of treatments and seed sources are of
interest Background levels ofnaturally occurring variation in seed sources are
seen in control germination percentages in Figure 2 In particular time variability
is unlikely to be under the control of the producer Appendix B details experimental
data for source by treatment gennination percents for each of the four blocks
Averaged over all treatments germination by source ranged from 40 to
79 (Figure 2) Averaged over all sources (Figure 3) and for each source (Figure 4
5) percussion scarification durations of4 or 8 minutes yielded greatest germination
Averaged over all sources germination increased with increasing durations of
percussion then leveled off for the 4- and 8-minute treatments (Figure 3) Immersion
and 30-second boiling scarification maximized germination for the hot water
treatments with a decrease in germination when seeds were boiled for 60 seconds
For all but two sources there is a similar ipcreasing trend in germination from
1to 4 minutes ofpercussion (Figure 4) The Holman source increased in germination
1 to 2 minutes then leveled off at 4 minutes The Jemez source changed little
1 to 4 minutes of treatment As treatment duration increased from 4 to 8
mutes five sources decreased in germination (Figure 4a) whereas three sources
24
Table 3 Categorical Analysis of Variance table for germination response to seed treatment seed source block and the interaction of the factors
Source of DF Chi-Square Observed Variability Significance Level
Source 8 98370 lt00001 Trt 7 233876 lt00001
contrast 1 16976 lt00001 SourceTrt 56 97077 lt00001 Block 3 5538 lt00001
SourceBlock 24 10162 lt00001 TrtBlock 21 7564 lt00001
SourceTrtBlock 168 34283 lt00001
S 4)
8 ~
0
sectg ~ 40 ~
N 0 0
20
o f0ltgt ~ ~4 jybshy ((iltgto ~~ K1ltgt 6~b-~ ~~ 0b-~~ ~~ ~b-~ ~b-~b
80
60 -I-- - ~- middotmiddotmiddotmiddotmiddot1
r -] Northern NM Sources
Central NM Sources
Southern NM Sources
sect-o ~
~b-v(i
SOURCE
Figure 2 Effect ofNew Mexico locust seed source on germination for data averaged over all scarification treatments Control germination is shown by solid line within bar
80
~ cd ~ ltl) () 60 ~ ~
N
t 0shyrti tshySltl)
40
0
20
o lmmersion Boiling 30 sec Boiling 60 sec Control 1 min 2min 4 min 8 min
Hot Water Treatments Percussion Treatments
Figure 3 Effect of scarification on germination ofNew Mexico locust for data averaged over all seed sources Means labelled with the same letter are not significantly different
100~----------------------~--~----------------~
80
60
ltU
~
40 IJ
sectgsI
l100
80
60
40
a I middotmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot middotImiddotmiddotmiddotmiddotmiddotmiddot E
_~iii-bull _ _ bull f --~--
t-- -~ ---- -shy ---- shyI ~ ---- - shy
L
fiII
~
b
- shy ---r
- Holman
Sacramento Sandia Gila Jemez
c ~
ltgt ~
Tmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot-I
j~=~=-~~-1
I middotmiddot ~ bull r
t tr
Y -------M-a-gd-a-Ie-n-a--
1 _ _
fCapitan
---- Manzano ~y -_ Raton
2 4 8
Time interval ofpercussion scarification (min)
Figure 4 Effect of seed source by treatment interaction on New Mexico locust germination for percussion scarification pattern a and pattern b
28
tv 0
~ d
~ A s o
1 c3
80
60
40
20
o
TIT
t------=middotfmiddotmiddot-middotmiddot-middotmiddot-middotmiddot 1- --- - - --=- - -shy--cE= - _--shy --shy ----shy ~ ~ ---shy ~ -~- _ --shy-------r 1
I
Raton Holman
-_ Jemez
--- Sandia Manzano Magdalena Capitan Sacramento Gila
Immersion Boiling 30 sec Boiling 60 sec
Hot Water Scarification
Figure 5 Effect of seed source by treatment interaction on New Mexico locust germination for hot water scarification
continued to increase in germination (Figure 4b) There was no difference between
the two treatment levels for the Manzano source (Figure 4b)
The effect ofhot water scarification also varied among sources with few
overall differences between hot water treatments (Figure 5) However boiling for 60
seconds yielded highest germination for the Jemez source whereas increasing
durations ofboiling decreased germination for the Raton source
In addition to consistently high germination 4- and 8-minute percussionshy
treated seeds germinated as fast or faster than all other treatments (Figure 6 Table 4)
050 measured as the number of days needed to complete 50 ofeventual
germination averaged 2 days for both ofthese treatments
X-ray radiographs were taken of seeds collected from the Sacramento source
percussed for 1 4 or 8 minutes (Figure 7) Seeds show signs of damage at 8 minutes
ofpercussion (Figure 8) Compared to an undamaged seed percussed for only 4
minutes (Figure 8a) damage at the 8-minute level includes fractured radicles pitting
of the seed coat and chips in seed coat structure (Figure 8b-d)
Scanning electron microscope (SEM) images at 600X reveal the direct effect
of 4 minutes of percussion on cells of the strophiolar region (Figure 9) Whereas
untreated seeds retain cellular organization in the strophiolar region strophiolar cells
seeds treated for 4 minutes ofpercussion separate and raise resulting in a
~-~e~U appearance
30
Umiddot-~middot--l-~---middotymiddotmiddotmiddot-----middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot--middot--middotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middotmiddotmiddotmiddotmiddotmiddotmiddot- -
050= gt1
-e-- Percussion 8 min -0- Percussion 4 min ---T- Percussion 2 min -----ltil- Percussion 1 min -- Immersion --0- Boiling 30 sec -+- Boiling 60 sec -ltgt- Control
2 4 6 8 10 12
Day Figure 6 Timecourse ofNew Mexico locust gennination following scarification
treatments G50 is the number of days to 50 of eventual germination
31
14
Table 4 Effect of seed source and treatment on G50 (days to 50 germination)
Seed Source Control Immersion B30sec B60sec Plmin P2min P4min P8min mean Raton 3 3 5 7 4 3 2 2 4 Holman 2 2 2 2 2 2 2 2 2
Jemez 4 6 6 5 3 2 2 2 4 Sandia 2 2 2 2 2 2 2 2 2
w tv Manzano 5 2 2 3 3 2 2 2 3
Magdalena 4 2 2 3 4 3 2 1 3
Capitan 5 4 2 2 3 2 2 2 3
Sacramento 3 2 2 2 4 3 2 2 3 Gila 3 2 2 2 2 2 2 2 2 mean 3 3 3 3 3 2 2 2 3 standard 11 14 16 18 09 05 0 03 08 deviation
Note Columns and rows may not agree due to rounding
Figure 7 X-ray radiographs ofSacramento seed source percussed for 1 4 or 8 minutes
33
Figure 8a-d Seed damage close-ups Figure a shows undamaged seed percussed for 4 minutes Figures b-d show seeds percussed for 8 minutes Damage includes fracturing ofradicle in b complete removal ofseed coat in c and pitting ofseed coat in d
34
Untreated Treated
Figure 9 SEM images ofuntreated vs 4-minute percussion-treated strophiolar cells ofNew Mexico locust seeds Treated cells are loosened separated and raised
DISCUSSION
Determining the Optimal Treatment
Averaging results of scarification treatmems across seed sources is a useful
tool to predict which treatment is most likely to be successful for an unknown seed
source Averaged over all sources four- and eight-minute percussion treatments
resulted in the highest germination 815 and 790 respectively and produced
germination that was as fast or faster than all other treatments Although there was no
difference between these two treatments on average an argument for 4 minutes of
percussion as a standard protocol for treating New Mexico locust seeds can be made
Descriptively 4 minutes of percussion improved germination over 8 minutes
ofpercussion for a majority of sources (five out of nine) 8 minutes of percussion
improved germination for three ofnine sources with no difference for a fmal source
For individual sources 4 minutes ofpercussion was always the best or secondmiddot best
treatment and was always equal to or better than all hot water treatments
Germination at 8 minutes ofpercussion was lower than all other percussion durations
for the Jemez and Gila sources and was lower than at least one hot water treatment for
these sources as well
The x-ray radiographs of the Sacramento seed source illustrate the excessive
internal and external seed damage that can occur with 8 minutes of percussion
germination counts 8-minute percussed seeds from Holman and Capitan
ClUll signs of damage that were visible to the naked eye (see below)
r~evertheless some of these seeds still fit the criteria for germination Neither x-ray
36
photography nor visible observations during seed counts revealed seed damage at the
4-minute percussion level In operational trials seeds percussed for 4 minutes
developed into healthy seedlings (Khadduri and Harrington unpublished data)
Strophiole and Seed Coat Behavior
Four minutes ofpercussion successfully breaks seed dormancy in a large
percentage ofNew Mexico locust seeds without excessively damaging the
surrounding seed coat or internal seed coat structures Most papilionoid legume
seeds including seeds ofNew Mexico locust have a specific region of the seed coat
known as the strophiole or lens (Hamly 1932) The strophiole is located on the
cotyledonary lobe of a locust seed (Figure 10 a-c) If seeds are not indiscriminately
damaged the strophiole is the first point of water entry to the embryo (Hopkinson
1997) When a papilionoid seed is percussed for the appropriate amount of time
repeated hits on the integrated seed coat loosen the constrained cells of the strophiolar
region without excessively damaging the rest of the seed coat (Ballard 1976) SEM
photos illuStrate that 4 minutes of percussion loosened and separated strophiolar cells
ofNew Mexico locust seeds when compared with constrained organized strophiolar
cells of untreated seeds (Figure 9) Research-has shown that water passes to the
embryo through the treated strophiole in a controlled manner and regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues
(Kelly and van Staden 1987)
37
10 Strophiole ofunimbibed New Mexico locust seed (a) and following uptake (bc)
38
In contrast hot-water treatments can excessively degrade the seed coat
(Rebman et al 1998 Marunda 1990) Random cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rebman et aI 1998 Marunda 1990) Along with a
proportionately large amount ofun-imbibed (thus under-treated) seeds (Figure l1a)
damage was observed in hot-water treated seeds from six sources during germination
counts Damage included seeds where the radicle failed to elongate (Figure 11 b)
seeds where the radicle elongated but was delayed in freeing itself from the seed coat
(Figure 11c) and seeds where the radicle completely broke off from the embryo
(Figure lId) In addition some seeds germinated in reverse (the cotyledon emerged
fIrst) If the radicle emerged completely from the seed coat they were counted as
germinated
While nearly all seeds treated for 8 minutes ofpercussion imbibed water
damage was evident in x-ray evaluations (Sacramento seed source) and in
germination counts (Holman and Capitan) at this treatment level Seed damage
during germination counts was similar to levels found in hot-water treated seeds
suggesting that over-treatment by both hot water and longer durations of percussion
may result in similar damage to seed structure
The Sacramento and Holman seed sources were two of the fIve sources that
opcrplltlpn in germination from the 4- to 8-minute durations of percussion
the benefIts ofloosening the strophiole as evidenced by imbibition of
all 8-minute treated seeds were outweighed by external and internal seed coat
39
a
b c d
11 Damage to New Mexico locust seeds observed during germination cotmts following hot water scarification 9a Hot water replicate including ~t seedlings and numerous unimbibed seeds 9b Close-up of seed where raallCle failed to elongate 9c Close-up ofseed where radicle elongated but was
-gtIn in freeing itself from the seed coat 9d Close-up ofseed where radicle broke off from embryo
40
damage (which also may be contributing to high imbibition) For the three sources
for which 8 minutes of percussion resulted in maximum germination extended
percussion of seeds necessary to loosen the strophiole may not have excessively
damaged the seed anatomy The Manzano source did not significantly change in
germination from 4 to 8 minutes of percussion suggesting that an increase in
permeable seeds at the 8-minute treatment duration was balanced by the number of
seeds that failed to germinate due to seed damage To summarize the 4-minute
percussion treatment improved germination over 8 minutes ofpercussion for a
majority of sources (five ofnine) the 8-minute percussion treatment improved
germination over 4-minutes ofpercussion for three sources and there was no
difference with the Manzano source Notably for the Raton source 8 minutes of
percussion increased germination by 235 percentage points over 4-minutes of
percussion (7875 vs 5525 respectively) the next best treatment Raton was the
only source to result in germination less than 75 for 4 minutes ofpercussion so a
grower may wish to apply longer percussion durations to lots that do not respond well
to this treatment level
Finally there was an interesting trend in the Jemez and Raton sources seed
that significantly contributed to the source by treatment interaction (Figure 12) A
~u test showed that the Jemez source had a significantly lighter seed weight than
other sources while the Raton source had an average seed weight For the Jemez
the longest duration of boiling increased germination Conversely only short
U4UVll) of percussion were needed to achieve high germination and the longest
41
80 _ Jemez a Raton
iD
~ C iD e iD
Q c 0 ~
c ~ iD 0
20
0------shy
Immersion Boiling 30 sec Boiling 60 sec
Hot Water Scarification
10degTr========~--------~------------------------------
80
60
40
20
_ Jemez Raton
0------shy
1 min 2 min 4 min 8 min
Percussion Scarification
Figure 12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources
42
b
--~----~~~~========~---- duration ofpercussion reduced germination For the Raton source longer durations
ofboiling consistently decreased germination whereas longer percussion treatments
consistently increased germination
A possible explanation is that a thick seed coat is associated with a loose
strophiole structure and that a thin seed coat is associated with a constrained
strophiole This contrast merits further study and may suggest an ecological
adaptation Further investigation ofthis phenomenon may contribute to successful
pre-screening ofunknown seed sources to determine optimal treatment leveL
43
CONCLUSION
Based on results obtained in pilot study (see Appendix) and thesis
experiments 4 minutes ofpercussion is recommended as a standard protocol for
treating an unknown source ofNew Mexico locust seed In addition to laboratory
results the 4-minute percussion treatment has resulted in germination greater than
80 and healthy seedling grow-outs for production purposes (unpublished data) In
laboratory experiments only one hundred seeds per 118 ml (40z) soil tin were
percussed at a time whereas for production we have treated up to five hundred seeds
per tin For large-scale production ofNew Mexico locust seedlings growers may
wish to experiment with larger quantities of seed and larger-size soil tins
Seed storage trials following percussion treatments were not conducted
However germination greater than 80 and healthy seedlings were obtained when
seeds were percussion-treated and subsequently stored at room-temperature (22degC)
for one week (unpublished data) Seed storage trials merit further study as the
standard hot water treatment leaves seeds in a softened condition that is not
conducive to storage
Hydroseeding is an effective method for direct seeding in reclamation
situations Percussion treatment may be conducive to hydro seeding as seeds remain
a dry relatively undamaged yet permeable condition following treatment Sulfuric
seeds also remain dry following treatment but extensive damage to seed coat
poor seedling vigor have been reported with this treatment (Kelly and van Staden
A grow-out study using a hydro seeder on a reclamation site testing percussion
44
against other treatments such as sulfuric acid and mechanical scarification also
merits further study
Papilionoids are the largest subfamily oflegumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust these legumes often play an integral role in the revegetation of
disturbed lands The pilot study (Appendix) indicates that percussion scarification
successfully treats the widely used black locust as well Developing superior
scarification methods to the standard hot water mechanical scarification and acid
treatments may facilitate the use ofpapilionoid legumes in reclamation projects
45
APPENDICES
APPENDIX A
PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR
NEW MEXICO LOCUST AND BLACK LOCUST SEEDS
Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4
Hot water and sulfuric acid soaks are traditional treatments for seeds of many
temperate woody legwnes including locusts However these scarification techniques
often produce inconsistent germination Percussion scarification where seeds are
repeatedly propelled against a hard surface was compared with hot water
scarification to evaluate treatment efficacy for New Mexico locust (Robinia
neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment
seeds were placed in a 98degC water bath which was immediately removed from the
heat source For percussion scarification seeds were placed in a soil sample tin and
agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the
control were followed by 24-hour water soaks Hot water baths resulted in 56 and
41 germination for New Mexico locust and black locust respectively For both
species nearly all durations ofpercussion increased germination over the hot water
treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3
4 and 5 minutes for black locust resulted in at least 90 germination Traditional
scarification treatments randomly degrade the entire seed coat which can lead to
tissue damage during water uptake Percussion scarification specifically weakens the
strophiole the natural source ofwater entry to the seed in papilionoid legwnes
INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom
2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031
48
Following percussion imbibition is controlled through the strophiole and underlying
tissue is protected
Keywords New Mexico locust black locust scarification percussion germination
Introduction
Locust trees (Robinia spp) are aggressive pioneer species that quickly
colonize disturbed land fix atmospheric nitrogen in the soil and stabilize slopes that
are prone to erosion (Klemmedson 1994 Ashby et al 1985) (Figure A1ab)
Recognition of these attributes has contributed to increased demand for locusts in
reclamation projects Locust seeds however exhibit physical dormancy where a
thick seed coat prevents the movement of water and gasses to the embryo (Leadem
1997)
Sulfuric acid or hot water soaks have traditionally been used to break seed
dormancy in macro-propagation of locusts Many growers are moving away from the
use of su1furic acid in treating locust seeds (Dreesen and Harrington 1997) In
addition to being dangerous acid soak durations must be specifically correlated to
seed lot (Olson 1974) Collections of native seed can vary tremendously in seed size
seed weight and hard seededness etc This variability limits the utility of sulfuric
acid soaks as even within a given lot some seeds may be damaged whereas others
are under-treated and fail to imbibe (take up water) A survey of Southwestern
container growers indicates that hot water baths are currently the most common
scarification method for locust seeds (Hine et al 1997) However hot water baths
also produce inconsistent germination (Lin et al 1996)
49
b
Figure AI New Mexico locust a primary invader following disturbance Ala Crown-sprouting at edge ofburn near Los Alamos NM--one year after Cerro Grande fire Alb Colonizing road cut Gila National Forest NM
50
Percussion scarification where seeds are repeatedly propelled against a hard
surface is an alternative dormancy-breaking method that has proven successful in
legumes related to locusts (Hamly 1932 Barton 1947) We compared this alternative
treatment to hot water scarification to evaluate treatment efficacy for New Mexico
locust and black locust seeds
Materials and Methods
Commercial New Mexico locust seeds (Western Native Seed Coaldale CO)
were collected fall 2000 in Huerfano County CO Black locust seeds were collected
September 2000 from Taos County NM by harvesting ripe pods from several trees at
a distance of up to 3 m from ground level Black locust seeds were allowed to air-dry
for 3 weeks and were threshed and separated from large chaffusing a greenhouse
fan A South Dakota Seed Blower (Chicago Illinltois) was used to remove fine chaff
from both seed lots Seeds were storedmiddotat 2-4C until the germination study was
conducted in March 2001
This study compared hot water scarification with percussion scarification
using treatment and species as experimental factors Along with a control
scarification treatments included immersion of seed in boiling water as well as
percussion durations of 1 2 3 45 or 10 minutes Four 100-seed samples were used
to test each species by scarification treatment combination
For the hot water treatment test tubes were filled with 30 mL water and
placed in a water-filled beaker (Figure A2a) The bath was raised to boiling (-98C at
51
b
c
Figure A2 Implementation ofhot water and percussion scarification A2a Hot water bath A2b Paint-shaker set-up A2c Placing soil tin containing seeds at end ofpaint shaker ann
52
Las Cruces NM elevation of 1300 meters) Each 100-seed replicate was placed in a
test tube and each test tube was immediately removed to cool at room temperature
Percussion scarification was implemented using a pneumatic paint shaker
(Central Pneumatic model 00422 Camarillo CA) (Figure A2b) This allowed
standardization of treatment intensity Compressed air pressure was maintained at
530kPa plusmn 30kPa (80psi plusmn 5psi) resulting in approximately 350 oscillations per
minute Each lOO-seed sample was placed in a 118 ml (4 oz) soil tin for shaking To
maximize lateral movement of paint shaker a paint can was used as a spacer to place
soil tins at the end of the shaking arm (Figure A2c) This allowed shaking to occur at
the greatest distance from the pivot point (fulcrum) Following all treatments
including the control seeds were soaked for twenty-four hours in distilled water prior
to plating out
Gennination was evaluated on lab benches at room temperature Each
replicate of seeds was placed in a 100 cm petri dish on moistened filter paper with
humidity maintained by enclosing the petri dishes in ziplock bags Gennination was
monitored daily for 14 days in accordance with International Seed Testing
Association standards for Robinia species (ISTA 1999) Gennination was defined as
protrusion of the radicle from the embryo by at least 1mm as viewed by the naked
eye
Categorical Analysis of Variance using Prpc Catmod (SAS Institute 1989)
was used to detennine treatment differences in germination percentages for each
source This procedure is an extension of a Chi-square test of homogeneity using the
53
natural log of the ratio of germinated to non-germinated seeds for each treatment Pshy
values less than 005 were considered significant A limited set of pairwiseshy
comparisons was conducted to compare treatment mean percents using a
conservative alpha value of 005 divided by the number of comparisons
Results
Percussion maximized germination for both New Mexico locust and black
locust There were three germination response levels for New Mexico locust (Figure
A3) First there was a low response in the control indicating a dormant seed lot
The hot water treatment and I-minute duration of percussion share a second
intermediate response Germination was highest at percussion levels from 2 to 10
minutes
A selected percussion treatment intermediate in time (5 minutes) produced the
steepest germination curve indicating the fastest germination rate (Figure A4) The
hot water treatment produced an intermediate curve or relatively slower germination
rate The control curve was very flat indicating the slowest germination rate for New
Mexico locust
Control seeds germinated poorly for black locust as well (Figure A5) Hot
water scarification improved germination over the control but all levels of percussion
improved germination over hot water The optimal durations of percussion treatment
were bracketed with a significant increase from the 1 to 2 minute percussion level a
high germination response from 2 to 5 minutes of percussion and finally a significant
54
~ d 25 CL)
p t1 0 ~ t1
g Vl ~ Vl
100~---------------------------------------------------------------
80
60
40
20
o Control Hot Water Percussion
I min Percussion
2 min Percussion
3 min
Scarification
Percussion 4 min
Figure A3 Effect of scarification on germination response for New Mexico locust
Percussion 5 min
=significant difference between adjacent treatment bars ns = no significant difference
Percussion 10 min
~ ~ () u agt
Il-t d o ~
~ ~ c3
100middotlr~~~------------------------------------e- Percussion 5min middot 0middot Hot Water -- Control
80-J=middot======
6b-middot-lmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middot--middotmiddot-middot~-middotmiddot -middotmiddot middot middotmiddot-middot 1
40
20
-----~-----------
0 0 0 middotmiddotmiddotmiddotmiddot0middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot0
0 --~-----y-----~------_---~--- T-----~
o shy -TDay 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A4 Effect of selected scarification treatments on germination speed for New Mexico locust First seven days of fourteen-day experiment are shown
Day 7
80
~ cd
5 60 ()
[) Cl d 0 1t1 d
40sect dUl
-J
20
o
ns
Scarification
Figure A5 Effect of scarification on germination response for black locust = significant difference between adjacent treatment bars ns = no significant difference
decrease at the 10-minute level This drop-off suggests that 10 minutes of percussion
over-treats seeds from this lot of-black locust
A selected percussion treatment intermediate in time (5 minutes) also had the
fastest germination rate for black locust (Figure A6)_ The hot water curve is
somewhat flatter indicating a slower germination rate The control curve is very flat
indicating a slow germination rate
A qualitative difference between hot water and percussion-treated seeds was
also observed Along with healthy germinants the hot water treatment produced
over-treated seeds (Figure A 7a) Examples of damaged germinants include seeds
where the radicle did not elongate (Figure A 7b) seeds where the radicle elongated
but was delayed in freeing itself from the seed coat (Figure A7c) and seeds where the
radicle broke off completely from the embryo (Figure A7d) In addition to these
examples of over-treatment there were numerous small dark seeds that failed to
imbibe water For percussion treatment nearly all seeds took up water (Figure A8a)
Germination was rapid and uniform with little damage evident (Figure A8b)
To summarize both New Mexico locust and black locust seed lots were
dormant The standard hot water scarification treatment improved germination but
only to 56 and 41 for New Mexico locust and black locust respectively Nearly
all percussion durations improved total germination as well as germination rate over
the hot water treatment Germination was greater than 90 for percussion durations
of 4 5 and 10 minutes for New Mexico locust and 3 4 and 5 minutes for black locust
58
CI)
sgt t CI) 0
igt 0
sect ttl t E
Vl 0
c3
100r==~==~~=r------------------------------------~ ___ Percussion 5 min
middotmiddotmiddot0middotmiddotmiddot Hot Water -lIf- Control
80r=========~--~~----------------------------~
60
40
20 -~-~---~---------- ---~------------I
omiddot
O
_~_----------lIf-----~bull -----_ shy---shy I
Day 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A6 Effect of selected scarification treatments on germination speed for black locust First seven days of fourteen-day experiment are shown
Day 7
a
b c d
Figure A7 Damage to New Mexico locust seeds observed during germination counts following hot water scarification A7a Hot water replicate including damaged seedlings and numerous unimbibed seeds A7b Close-up ofseed where radicle failed to elongate A7c Close-up ofseed where radicle elongated but was delayed in freeing itself from the seed coat A7d Close-up ofseed where radicle completely broke offfrom embryo
60
a
b
Figure A8 New Mexico locust seeds following percussion treatment at time of germination counts A8a Percussion replicate with uniformly healthy seedlings and near complete imbibition of seeds A8b Close-up ofpercussion-treated seedling
61
Discussion
Papilionoid seeds have a specific anatomical feature known as the strophiole
or lens-the natural site of water entry to the seed (Hamly 1932) The strophiole is
located on the cotyledonary lobe of a locust seed (Figure A9) When a papilionoid
legume seed is percussed for the appropriate amount of time repeated hits on the
integrated seed coat loosen the constrained cells of the strophiolar region without
excessively damaging the rest of the seed coat (Ballard 1976)
When a percussed seed is soaked water enters exclusively through the
strophiole in a controlled manner (Kelly and Staden 1987) This regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues This
contrasts with seeds that have been hot water or acid scarified treatments that can
randomly degrade the seed coat Unlocalized cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rehman et al 1998 Marunda 1990)
Papilionoids are the largest subfamily of legumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust and black locust these legumes often play an integral role in the
revegetation ofdisturbed lands Developing superior scarification methods to the
standard hot water and acid treatments should fadlitate the use ofthese legumes in
restoration projects
62
Figure A9 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (bc)
63
APPENDIXB
GERMINATION PERCENTS AND STANDARD ERRORS FOR SOURCE
BLOCK AND TREATMENT FOR NEW MEXICO LOCUST
Table B 1 Gennination percents and standard errors for source block and treatment for New Mexico locust
Source Block Treatment Germination Percentage Standard Error
Capitan 1 Immersion 74 437 Capitan 2 Immersion 69 462 Capitan 3 Immersion 71 454 Capitan 4 Immersion 67 470 Capitan Boiling 30 sec 79 407 Capitan 2 Boiling 30 sec 76 427 Capitan 3 Boiling 30 sec 72 449 Capitan 4 Boiling 30 sec 76 427 Capitan Boiling 60 sec 71 454 Capitan 2 Boiling 60 sec 54 498 Capitan 3 Boiling 60 sec 67 470 Capitan 4 Boiling 60 sec 76 427 Capitan I Control 5 218 Capitan 2 Control 7 255 Capitan 3 Control 4 196 Capitan 4 Control 5 218 Capitan 1 Percussion I min 47 499 Capitan 2 Percussion I min
470
Capitan Percussion I min 39 488
Capitan 4 Percussion I min 38 485 Capitan I Percussion 2 min 55 497 Capitan Capitan
2
Percussion 2 min Percussion 2 min
71 65
454 477
Capitan 4 Percussion 2 min 56 496 Capitan I Percussion 4 min 86 347 Capitan 2 Percussion 4 min 79 407 Capitan 3 Percussion 4 min 81 392 Capitan 4 Percussion 4 min 82 384 Capitan I Percussion 8 min 90 300 Capitan 2 Percussion 8 min 88 325 Capitan 3 Percussion 8 min 90 300 Capitan 4 Percussion 8 min 90 300 Gila I Immersion 71 454 Gila 2 Immersion 77 421 Gila
Immersion 75 433
Gila 4 Immersion 72 449
Gila I Boiling 30 sec 68 466
Gila 2 Boiling 30 sec 81 392
Gila 3 Boiling 30 sec 54 498
Gila 4 Boiling 30 sec 63 483
Gila I Boiling 60 sec 55 497
Gila 2 Boiling 60 sec 68 466
Gila 3 Boiling 60 sec 57 495
Gila 4 Boiling 60 sec 70 458
65
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Gila 2 Control 13 336 Gila 3 Control 13 336 Gila 4 Control 20 400 Gila 1 Percussion 1 min 66 474 Gila 2 Percussion 1 min 57 495 Gila 3 Percussion 1 min 73 444 Gila 4 Percussion 1 min 82 384 Gila 1 Percussion 2 min 81 392 Gila 2 Percussion 2 min 86 347 Gila 3 Percussion 2 min 85 357 Gila 4 Percussion 2 min 54 498 Gila 1 Percussion 4 min 79 407 Gila 2 Percussion 4 min 83 376 Gila 3 Percussion 4 min 84 367 Gila 4 Percussion 4 min 79 407 Gila 1 Percussion 8 min 51 500 Gila 2 Percussion 8 min 76 427 Gila 3 Percussion 8 min 52 500 Gila 4 Percussion 8 min 74 439 Holman 1 Immersion 83 376 Holman 2 Immersion 86 347 Holman 3 Immersion 81 392 Holman 4 Immersion 90 300 Holman 1 Boiling 30 sec 90 300 Holman 2 Boiling 30 sec 85 357 Holman 3 Boiling 30 sec 87 336 Holman 4 Boiling 30 sec 83 376 Holman 1 Boiling 60 sec 79 407 Holman 2 Boiling 60 sec 80 400 Holman 3 Boiling 60 sec 86 347 Holman 4 Boiling 60 sec 93 255 Holman Control 25 433 Holman 2 Control 19 392 Holman Control 21 407
Holman 4 Control 24 427
Holman Percussion 1 min 77 421
Holman 2 Percussion 1 min 85 357
Holman 3 Percussion 1 min 80 400
Holman 4 Percussion 1 min 89 313
Holman 1 Percussion 2 min 88 325
Holman 2 Percussion 2 min 96 196
Holman 3 Percussion 2 min 93 255
Holman 4 Percussion 2 min 96 196
Holman 1 Percussion 4 min 92 271
Holman 2 Percussion 4 min 97 171
66
I
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Holman 3 Percussion 4 min 91 286 Holman 4 Percussion 4 min 96 196 Holman 1 Percussion 8 min 88 325 Holman 2 Percussion 8 min 85 357 Holman 3 Percussion 8 min 83 376 Holman 4 Percussion 8 min 82 384 Jemez 1 Immersion 53 499 Jemez 2 Immersion 44 496 Jemez 3 Immersion 52 500 Jemez 4 Immersion 70 458 Jemez 1 Boiling 30 sec 58 494 Jemez 2 Boiling 30 sec 55 497 Jemez 3 Boiling 30 sec 48 500 Jemez 4 Boiling 30 sec 65 477 Jemez 1 Boiling 60 sec 68 466 Jemez 2 Boiling 60 sec 63 483 Jemez 3 Boiling 60 sec 73 444 Jemez 4 Boiling 60 sec 89 313 Jemez 1 Control 2 140 Jemez 2 Control 9 286 Jemez 3 Control 6 237 Jemez 4 Control 8 271 Jemez 1 Percussion 1 min 58 494 Jemez 2 Percussion 1 min 63 483 Jemez 3 Percussion 1 min 77 421 Jemez 4 Percussion 1 min 89 313 Jemez 1 Percussion 2 min 68 466 Jemez 2 Percussion 2 min 64 480 Jemez 3 Percussion 2 min 80 400 Jemez 4 Percussion 2 min 83 376 Jemez Percussion 4 min 68 466 Jemez 2 Percussion 4 min 79 407 Jemez 3 Percussion 4 min 70 458 Jemez 4 Percussion 4 min 83 376 Jemez 1 Percussion 8 min 52 500 Jemez 2 Percussion 8 min 59 492 Jemez 3 Percussion 8 min 61 488 Jemez 4 Percussion 8 min 70 458 Magdalena 1 Immersion 69 462 Magdalena 2 Immersion 69 462 Magdalena J Immersion 70 458 Magdalena 4 Immersion 85 357 Magdalena 1 Boiling 30 sec 65 477 Magdalena 2 Boiling 30 sec 71 454 Magdalena 3 Boiling 30 sec 68 466
67
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Magdalena 4 Boiling 30 sec 83 376 Magdalena 1 Boiling 60 sec 63 483 Magdalena 2 Boiling 60 sec 68 466 Magdalena 3 Boiling 60 sec 63 483 Magdalena 4 Boiling 60 sec 82 384 Magdalena 1 Control 11 313 Magdalena 2 Control 6 237 Magdalena 3 Control 5 218 Magdalena 4 Control 4 196 Magdalena Percussion 1 min 32 466 Magdalena 2 Percussion 1 min 45 497 Magdalena 3 Percussion 1 min 46 498 Magdalena 4 Percussion I min 36 480 Magdalena 1 Percussion 2 min 67 470 Magdalena 2 Percussion 2 min 68 466 Magdalena 3 Percussion 2 min 65 477 Magdalena 4 Percussion 2 min 81 392 Magdalena 1 Percussion 4 min 87 336 Magdalena 2 Percussion 4 min 97 171 Magdalena 3 Percussion 4 min 83 376 Magdalena 4 Percussion 4 min 97 171 Magdalena 1 Percussion 8 min 98 lAO Magdalena 2 Percussion 8 min 100 000 Magdalena 3 Percussion 8 min 96 196 Magdalena 4 Percussion 8 min 98 lAO Manzano 1 Immersion 67 470 Manzano 2 Immersion 57 495 Manzano 3 Immersion 72 4049 Manzano 4 Immersion 73 4044 Manzano 1 Boiling 30 sec 70 458 Manzano 2 Boiling 30 sec 64 480 Manzano 3 Boiling 30 sec 71 454 Manzano 4 Boiling 30 sec 80 400 Manzano I Boiling 60 sec 50 500 Manzano 2 Boiling 60 sec 53 499 Manzano 3 Boiling 60 sec 59 492 Manzano 4 Boiling 60 sec 74 439 Manzano I Control 4 196 Manzano 2 Control 8 271 Manzano 3 Control 1 099 Manzano 4 Control 6 237 Manzano 1 Percussion 1 min 52 500 Manzano 2 Percussion 1 min 39 488 Manzano 3 Percussion 1 min 36 480 Manzano 4 Percussion 1 min 51 500
68
Table B 1 Continued
Source Block Treatment Gerrni~ation Percentage Standard Error
Manzano Percussion 2 min 53 499 Manzano 2 Percussion 2 min 58 494 Manzano 3 Percussion 2 min 61 488 Manzano 4 Percussion 2 min 62 485 Manzano 1 Percussion 4 min 76 427 Manzano 2 Percussion 4 min 85 357 Manzano 3 Percussion 4 min 79 407 Manzano 4 Percussion 4 min 83 376 Manzano 1 Percussion 8 min 87 336 Manzano 2 Percussion 8 min 83 376 Manzano 3 Percussion 8 min 83 376 Manzano 4 Percussion 8 min 75 433 Raton 1 Immersion 54 498 Raton 2 Immersion 37 483 Raton 3 Immersion 39 488 Raton 4 Immersion 69 462 Raton 1 Boiling 30 sec 36 480 Raton 2 Boiling 30 sec 20 400 Raton 3 Boiling 30 sec 28 449 Raton 4 Boiling 30 sec 60 490 Raton 1 Boiling 60 sec 15 357 Raton 2 Boiling 60 sec 171 Raton 3 Boiling 60 sec 4 196 Raton 4 Boiling 60 sec 35 477 Raton 1 Control 4 196 Raton 2 Control 12 325 Raton 3 Control 2 140 Raton 4 Control 6 237 Raton 1 Percussion 1 min 52 500 Raton 2 Percussion I min 29 454 Raton 3 Percussion I min 35 477 Raton 4 Percussion 1 min 31 462 Raton 1 Percussion 2 min 55 497 Raton 2 Percussion 2 min 35 477 Raton 3 Percussion 2 min 33 470 Raton 4 Percussion 2 min 42 494 Raton 1 Percussion 4 min 65 477 Raton 2 Percussion 4 min 52 500 Raton Percussion 4 min 51 500 Raton 4 Percussion 4 min 53 499 Raton 1 Percussion 8 min 88 325 Raton 2 Percussion 8 min 80 400 Raton 3 Percussion 8 min 73 444 Raton 4 Percussion 8 min 74 439 Sacramento 1 Immersion 43 495
69
Table B 1 Continued
-Source Block Treatment Germination Percentage Standard Error
Sacramento 2 Immersion 30 458 Sacramento 3 Immersion 38 485 Sacramento 4 Immersion 56 496 Sacramento 1 Boiling 30 sec 47 499 Sacramento 2 Boiling 30 sec 48 500 Sacramento 3 Boiling 30 sec 48 500 Sacramento 4 Boiling 30 sec 56 496 Sacramento 1 Boiling 60 sec 52 500 Sacramento 2 Boiling 60 sec 67 470 Sacramento 3 Boiling 60 sec 43 495 Sacramento 4 Boiling 60 sec 48 500 Sacramento 1 Control 0 000 Sacramento 2 Control 3 171 Sacramento 3 Control 3 171 Sacramento 4 Control 099 Sacramento 1 Percussion 1 min 57 495 Sacramento 2 Percussion 1 min 61 488 Sacramento 3 Percussion 1 min 62 485 Sacramento 4 Percussion 1 min 55 497 Sacramento 1 Percussion 2 min 84 367 Sacramento 2 Percussion 2 min 70 458 Sacramento 3 Percussion 2 min 81 392 Sacramento 4 Percussion 2 min 69 462 Sacramento 1 Percussion 4 min 91 286 Sacramento 2 Percussion 4 min 84 367 Sacramento 3 Percussion 4 min 84 367 Sacramento 4 Percussion 4 min 94 237 Sacramento 1 Percussion 8 min 82 384 Sacramento 2 Percussion 8 min 81 392 Sacramento 3 Percussion 8 min 82 384 Sacramento 4 Percussion 8 min 81 392 Sandia 1 Immersion 76 427 Sandia 2 Immersion 70 458 Sandia 3 Immersion 70 458 Sandia 4 Immersion 81 392 Sandia 1 Boiling 30 sec 78 414 Sandia 2 Boiling 30 sec 71 454 Sandia 3 Boiling 30 sec 63 483 Sandia 4 Boiling 30 sec 78 414 Sandia 1 Boiling 60 sec 67 470 Sandia 2 Boiling 60 sec 65 477 Sandia 3 Boiling 60 sec 74 439 Sandia 4 Boiling 60 sec 66 474 Sandia 1 Control 16 367 Sandia 2 Control 7 255
70
Table B 1 Continued
Source Block Treatment Gennination Percentage Standard Error
Sandia 3 Control 8 271 Sandia 4 Control 16 367 Sandia 1 Percussion 1 min 67 470 Sandia 2 Percussion 1 min 61 488 Sandia 3 Percussion 1 min 66 474 Sandia 4 Percussion 1 min 59 492 Sandia I Percussion 2 min 82 384 Sandia 2 Percussion 2 min 83 376 Sandia 3 Percussion 2 min 81 392 Sandia 4 Percussion 2 min 84 367 Sandia 1 Percussion 4 min 89 313 Sandia 2 Percussion 4 min 86 347 Sandia 3 Percussion 4 min 89 313 Sandia 4 Percussion 4 min 85 357 Sandia 1 Percussion 8 min 80 400 Sandia 2 Percussion 8 min 77 421 Sandia 3 Percussion 8 min 69 462 Sandia 4 Percussion 8 min 66 474
LITERATURE CITED
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Ballard LAT 1973 Physical barriers to gennination Seed Sci Technoll 285-303
___ 1976 Strophiolar water conduction in the seeds ofTrifolieae induced by action on the testa at nonstrophiolar sites Austr J Plant Physiol3 465-469
Barton LV 1947 Special studies on seed coat impenneability Contrib Boyce Thompson Inst 14 355-362
Baskin CC Baskin JM 1998 Seeds Ecology biogeography and evolution of donnancy and gennination Academic Press London UK 666 p
Bewley JD 1997 Seed germination and donnancy Plant Ce119 1055-1066
Bewley JD Black M 1994 Seeds physiology of development and germination 2nd ed Plenum Press New York NY 445 p
Briscoe CB 1996 Growing Nitrogen Fixing Trees in Acid Soils In MH Powell (ed) Nitrogen Fixing Trees for Acid Soils- A Field ManuaL Winrock International Morrilton AR 22 p
Copeland LO McDonald MB 1995 Principles of Seed Science and Technology 3rded Chapman amp Hall New York NY 409 p
Cox RE Klett JE 1984 Seed gennination requirements of native Colorado plants for use in the landscape Plant Propag 30(2) 6-10
Dell B 1980 Structure and function ofthe strophiolar plug in seeds ofAlbizia Lophantha Am J Bot 67 556-563
Dick-Peddie WA 1993 New Mexico Vegetation Past Present and Future University ofNew Mexico Press Albuquerque NM 244 p
Dreesen DR 2001 Propagation protocol for production ofcontainer Robinia neomexicana plants Los Lunas Plant Materials Center Los Lunas New Mexico In Native Plant Network URL httpwwwnativeplantnetworkorg (accessed 9 January 2002) Moscow (ID) University ofIdaho CoIl Natural Resources For Res Nursery
72
Dreesen DR Harrington JT 1997 Propagation of native plants for restoration projects in the Southwestern US- Preliminary Investigations In Landis TD Thompson JR tech coords National Proceedings Forest and Conservation Nursery Assocs Gen Tech Rep PNW-GTR-419 Portland OR USDA For Ser PNW Res Sta 77-88
Gottfried GJ 1980 Control ofNew Mexican locust and the effect on planted ponderosa pine in central Arizona US For Ser Res Note RM-386 Rocky Mountain For Range ExpStn Fort Collins CO
Gosling PG Samuel YK Jones SK 1995 A systematic examination of germination temperature chipping and water temperaturesoak duration pretreatments on the seeds ofLeucaena leucocephala Seed Sci amp Technol 23521-532
Hamly DH 1932 Softening of the seeds of Melilotus alba Bot Gazette 93 345shy375
Hanna PJ 1984 Anatomical features of the seed coat ofAcacia kempeana (Mueller) which relate to increased germination rate induced by heat treatment New Phytol 96 23-29
Hanks JP Dick-Peddie WA 1974 Vegetation patterns of the White Mountains New Mexico Southwest Naturalist 18(4) 371-382
Hine S Harrington JT Dreesen DR 1997 Seed scarification requirements for Robinia neomexicana In Harrington JTtech coord Proc Third SouthwestemContainer Growers Conf Feb 12-13 1997 Ft Collins CO
Hopkinson JM 1993 The strophiole of leguminous seeds Newsletter- IntI Herbage Seed Production Res Grp 18 7-8
---- 1997 Differences in hardseededness between Cunningham and Tarramba leucaena In Leucnet News The Newsletter of the International LeucaenaRes and Devlt Netwk No4
ISTA (International Seed Testing Association) 1999 International rules for seed testing rules Seed Sci amp Technol27 Supplement
Kelly KM van Staden J 1987 The lens as the site of permeability in the papilionoid seed Aspalathus linearis J Plant Physiol 128(45) 395-404
73
Keresztesi B 1988 Black locust the tree of agriculture Outlook on Agr 17(2) 77~85
Kigel 1 and Galili G 1995 Seed development and germination Marcel Dekker New York 853 p
Klemmedson 10 1994 New Mexico locust and parent material Influence on macronutrients of forest floor and soil Soil Sci Soc Am 1 58 974~980
Kuo WH Tam AW 1988 The pathway of water absorption of mung bean seeds Seed Sci amp Technol16(1) 139~144
Leadem CL 1997 Dormancy--unlocking seed secrets In Landis TD Thompson lR tech coords National Proceedings Forestand Conservation Nursery Assocs Gen Tech Rep PNW-G TR-419 Portland OR USDA For Ser PNW Res Sta 43-52
Lin Y Wagner MR Seweyestewa A Honahni MD DeGomez M McMillin JD 1996 Influence of cultural treatments on seed germination and seedling growth of Robinia neomexicana Forest Farm and Community Tree Res Reports Volume 1
Marunda CT 1990 Effects of seed pretreatment on the development ofAcacia auriculforinis and A holosericea seedlings ACIAR Proceedings Series No 28
Meginnis HG 1937 Sulphuric acid treatment to increase germination of black locust seed USDA Circ 453 34 p
Miklas PN Towensend CE Ladd SL 1987 Seedcoat anatomy and the scarification ofcicer milkvetch seed Crop Sci 27 766-772
Monsen SB 1984 Use of shrubs on mine spoils In The Challenge ofProducing Native Plants for the Intermountain Area Proceedings Intermountain Nurserymans Association 1983 conference Las Vegas NV compo PM Murphy 26-31 Ogden UT USDA For Ser Gen Tech Report INT-l68
Olson DF 1974 Robinia L Locust In Seeds of Woody Plants in the US tech coord CS Schopmeyer 728-731 USDA For Ser Agr Handbk No450
Rehman S Loescher RNl Harris P1C 1998 Dormancy breaking and germination ofAcacia salicina Lindl seeds Seed Sci amp Techno127 553-557
Rolston MP 1978 Water impermeable seed dormancy Bot Rev 44 365-396
74
SAS Institute Inc 1989 SASSTAT Users Guide Version 6 4th Edition Volume 1 Cary NC SAS Institute Inc 943 p
SerratoValenti G Conara L Ferrando M Modenesi P 1993 Structural and histochemical features ofStylosanthes scabra (Leguminosae Papilionoidae) seed coat as related to water entry Can J Bot 71 834-840
Simpson BJ 1988 A field guide to Texas trees Austin TX Texas Monthly Press 372p
Tran VN Cavanagh AK 1984 Structural aspects ofdormancy In Seed Physiology Vol 2 Murray DR (ed) p1-44 Academic Press Australia
USDA Forest Service 1948 Woody-plant seed manual USDA Misc Pub1654 416p
Vines RA 1960 Trees Shrubs and Woody Vines of the Southwest Austin Univ ofTexas Press 1104 p
Wagner MR Larson RE Craig TP 1992 Ecology and silvics ofNew Mexico locust (Robinia neomexicana) with an emphasis on the arboreal form p53-57 In JW Hanover K Miller and S Plesko (eds) Proc Intemat Confon Black Locust Biology Culture and Utilization Mich State Univ USA
Wilson JK 1944 Immersing seeds of species of Robinia in boiling water hastens germination J For 42 453-454
Zimmerman RW Carpenter SB 1980 First-year coppice production from a 5shyyear old black locust stand on surface mine spoil p309-314 Centr Hardwd For ConfProc University ofMissouri Columbia
to 60 minutes of sulfuric acid scarification (to achieve maximum gennination results)
showed more than 50 seedling mortality after gennination (Kelly and van Staden
1987) Bewley and Black (1994) noted that fungal growth on seeds is an indication
of leakage of sugars organic acids amino acids and proteins
Perhaps the greatest drawback to acid scarification is the dangerous nature of
the treatment itself Difficulties associated with storage and trained use of
concentrated acid may discourage many propagators from using this option (Dreesen
and Harrington 1997) Consequently hot water and to a lesser extent mechanical
scarification are more commonly used and practical treatments for legume seeds
Most southwestern container growers use either of these two treatments to scarify
New Mexico locust seed in particular (Hine et al 1997)
Hot Water Scarification
The widespread use ofhot water scarification to treat locust seed is probably
due to its ease of application and efficacy in comparison to other scarification
treatments Although this practice had been in use for some time Wilson (1944) was
one of the first to report on its systematic application for black locust The treatment
consists of placing seeds in boiling water (4 parts water tomiddot 1 part seed by volume)
removing the vessel from the heat source and allowing the water to cool and seeds to
soak for 24 hours
Hine et al (1997) Lin et al (1996) and Cox and Klett (1984) have examined
the specific application ofhot water scarification on New Mexico locust seed Each
these authors compared hot water to acid scarification Lin et aL (1996) reported
8
60 gennination for two hot water-treated seed lots while a third lot germinated
around 50 (as reported above acid scarification germination ranged from 20 to
80 for these lots) It should be noted that the authors only soaked the seeds in water
from 10 to 80 minutes for this experiment as opposed to the standard 24 hours Cox
and Klett (1984) stated that using the standard 24-hour hot water treatment improved
germination over an acid pre-treatment (which produced less than 20 germination)
but they did not give specific gennination numbers for the hot water treatment Hine
et al (1997) found that across five seed lots germination was 66 for the standard
hot water treatment (as compared to less than 20 germination for acid scarification)
Variations in treatment intensity and duration and their subsequent effects on seed lot
appear to contribute to conflicting conclusions as to whether acid scarification or hot
water treatment is a more successful treatment
Given mixed results ease ofapplication is an important argument for use of
hot water application over acid soaks Safety concerns and material costs for hot
water treatments are low (Dreesen and Harrington 1997) An extension of this is the
practicality ofhot water for treating large batches of seed where a relative increase in
water volume is all that is needed However increases in water and seed even when
inthe same 4 1 proportion can result in dramatically different rates of cooling than in
atest tube experiment Different cooling rates can lead to contrasting results for the
treatment temperature duration and seed lot (USDA Forest Service 1948)
As with acid scarification parameters ofhot water treatment application may
to be optimized to maximize germination Hine et al (1997) found that as
9
temperature was increased from 70degC to 100degC overall germination in New Mexico
locust seed increased The 100degC treatment maximized germination for each of five
sources tested with germination usually above 60
Prolonged exposure of seeds ofmany woody legumes to boiling temperatures
also may improve germination (Briscoe 1996) This approach has not been tested on
New Mexico locust seed Exposing seed to prolonged boiling temperatures may
result in fungal infection and subsequent seedling mortality Marunda (1990) noted
severe fungal infections on both acid and hot water treated Acacia holoserica Cunn
ex G Don seeds attributing the infections to seed damage resulting from the
treatments Identifying an optimal hot water treatment level that does not result in a
high rate of seedling mortality is a logical goal
With hot water treatments as with acid there is also a danger of overtreating
Seeds and reducing germination (Gosling et al 1995) The legume Leucaena
leucocephala (Lam) de Wit germinated equally well after 7 seconds of exposure to
water at 100degC 7 seconds at 90degC 30 seconds at 80degC and 4 minutes at 70degC In
addition acceptably high germination was obtained from treatment at 70degC from 3
minutes to 1 hour whereas no seeds germinated following treatment at 100degC for 2
minutes Perhaps along with a greater margin of error for treatment duration treating
~ew Mexico locust seeds at a lower temperature may result in a comparatively lower
of seedling mortality In practice however nursery growers tend to use the
and quickest treatment available The short treatment length at near-boiling
whether recommended or not may be what is used most frequently
10
Fine-tuning a treatment in this range of treatment temperature and duration may be
the most logical approach
Mechanical Scarification
This treatment involves scratching the seed coat with an abrasive material
(USDA Forest Service 1948) One system employs a rotating drum lined with
sandpaper A paddle spinning at a given number ofrevolutions per minute throws the
seed against the sandpaper (Hine et al 1997) A second method dry tumbling
tumbles seed in a mixer with pea gravel or sharp sand for 5 to 7 days (Dreesen 2001)
These approaches have the advantage that control of temperature is not required as in
water treatments nor is safety an issue as with add scarification
Hine et al (1997) mechanically treated New Mexico locust seed using 100shy
grit sandpaper in a Forsberg (rotating drum) scarifier Gennination was less than
10 They concluded that the seed coats were too thin for their apparatus and
proposed that a finer grit sandpaper would produce more even abrasion Although
germination varied by seed lot Dreesen (2001) reported that using a slow-speed dry
tumbling technique for 5 to 7 days produced gennination rates as high as 90
As with acid and hot water treatments treatment duration must be specifically
shy
correlated to seed lot Treated seeds are also susceptible to fungal infection
iHopkinson 1997) In addition equipment is specialized and may not always be
ltU~CUJl~ to a nursery (USDA Forest Service 1948) Several studies have confinned
degrading ofstrophiolar cells due to acid and hot water scarification (Miklas et al
11
1987 Dell 1980 Hanna 1984) but the effect ofmechanical scarification on the
strophiole has not been studied
Alternative Method for Breaking Dormancy Percussion
Strophiole degradation has been examined in depth using an impaction
treatment more commonly known as percussion Hamly (1932) first proposed that
shaking or impacting seeds could split the macrosc1ereid cells at the strophiole
Hamly successfully released tension ofbent strophiolar cells of impermeable
Melilotus alba Medik (White sweet clover) seeds by delivering physical blows
resulting in cells that had split apart and were only slightly bent Ninety-two percent
oftreated seeds became permeable as compared to 05 of control seeds Barton
(1947) showed that percussion significantly increases permeability in black locust
seed Ballard (1976) reasoned that the integrated nature of the strophiole within the
seed coat means a blow on any part of the seed weakens the strophiole There are no
published studies on the use of percussion on New Mexico locust
Percussion methods vary and often seem SUbjective or ill-defined Hamly
(1932) placed seeds in a glass bottle and shook them three times a second for 20
minutes resulting in 180 oscillations per minute Ballard (1973) describes shaking
seeds in a closed metal container with at least fifty seeds per container at 850-900
JS(~l11alIOIlS per minute for 1 minute Kelly and van Staden (1987) placed seeds into
sealed glass flasks Flasks were shaken by a wrist-shaker at a rate of 250
~UUVJ~ per minute Subsequent germination rates were 45 after 2 hours of
55 after 3 hours 60 after 6 hours and 100 after 12 hours
12
There are several potential advantages of percussion over acid hot water and
mechanical scarification Kelly and van Staden (1987) note that a percussion
treatment only alters macrosclereid cells of the strophiole by loosening separating
and raising them They emphasize that minimal damage is done to the overall
anatomical structure of the seed In acid scarification of the same seed lot radicle
and cotyledon damage occurred Based on photographic evidence the authors
believe that this morphological damage was due to uneven pressure during
imbibition with water entering haphazardly wherever the acid had sufficiently
dissolved the coat This ultimately led to uneven hydration of the embryo In
percussed seeds water only entered through the s~ophiole when the entire seed coat
had been saturated resulting in controlled and even embryo hydration (Kelly and van
Staden 1987)
Although all successful scarification treatments may focus at least in part on
the strophiole for papilionoid legumes (Baskin and Baskin 1998) percussion may be
less damaging to seed coat structures particularly when compared with acid Kelly
and van Staden did not compare hot water or mechanical scarification methods
against percussion However the fungal infection associated with hot water and
mechanical scarification as with acid scarificatio~ have been directly associated with
seed damage (Rehman et al 1998 USDA Forest Service 1948) Embryo damage in
has been observed in hot-water scarified legume seeds (Rehman et al 1998
13
By limiting embryo damage percussion scarification reduces seedling
mortality compared to other treatments Kelly and van Staden (1987) noted that
seedlings from the percussion treatment were morphologically sound and vigorous
when compared to acid treatment seedlings The authors attribute this improved
seedling vigor to the controlled water uptake that limited anatomical damage
Despite a 12-hour percussion treatment morphological damage was not substantially
greater than untreated seeds In addition seedling mass measured 10 days following
germination was not significantly different between percussed and untreated seeds
A final advantage ofpercussion may be the ability to adequately treat the most
dormant seeds while avoiding harm to seeds that have already been made permeable
by the treatment or that were permeable to begin with Instead ofcorroding scalding
or scratching the seed coat percussion appears to be just a loosening of the
strophiole spring (Ballard 1973) Kelly and van Staden (1987) were able to
increase germination with a lengthy treatment (12 hours) Figuring out whether a
substantial cushion oftreatment durations exist for percussion ofNew Mexico locust
seed would be a significant improvement to current scarification methods where seed
damage and seedling health is an issue
The greatest challenge for applying percussion to New Mexico locust seeds is
standardizing a treatment that is both effective and readily reproducible by nurseries
with mechanical scarification this may require the development of specific
~UllDment to properly quantify and execute the treatment
14
f
Objectives
The first objective of the study is to detennine which scarification treatment
results in optimal gennination speed and percentage ofNew Mexico locust The
second objective is to describe treatment interactions with nine seed sources collected
in New Mexico
Hypothesis
A percussion treatment acts exclusively on the main area ofwater uptake in
New Mexico locust seed loosening the strophiole so that imbibition can occur This
direct mechanism ofdonnancy release will result in improved gennination rate and
overall gennination percentage for New Mexico locust seed compared to standard
treatments Standard treatments including acid hot water and mechanical
scarification while eventually weakening the strophiolar cleft cause excessive
morphological damage through generalized seed coat degradation These treatments
are therefore prone to inconsistency due to over and under-treatment
15
MATERIALS AND METHODS
Seed Collection and Cleaning
Seeds were collected from August 25 through September 15 2000 from nine
locations along a latitudinal gradient in New Mexico Seed source locations and
descriptive information are given in Figure 1 and Tables I and 2 Stands were
separated by at least 45 km
Pods were hand picked as they began to ripen and dehisce (open) Collections
were made from ground level to approximately 3 m from several trees at each site
Pods were dried in open-ended brown paper bags for two to four weeks at room
temperature (21-230C) to facilitate cleaning Dried pods were placed in a closed cloth
sack which was pounded against the ground to separate seeds from pods For pods
still containing seeds hand maceration (crunching pods with hands) completed seed
separation Debris was removed by threshing with the aid of a greenhouse fan A
South Dakota Seed Blower (Seedbureau Chicago Illinois) was used to remove
remaining chaff and light seeds by air density separation Seeds were placed in dry
storage in sealed quart-size zip-lock bags within a sealed plastic container Storage
temperatures ranged from 2-4oC Seeds remained in storage for approximately 7
months before the experiment
Experimental Design and Treatment Structure
The experiment compared the impact ofhot water bath and percussion
l11aLl11 on germination properties ofNew Mexico locust seed A single
16
-l
Figure 1 Seed source collection map All sources were collected within New Mexico USA separated by at least 4S lan
IIIRZltItW lIJPIirmiddot rllll1lbullbull_~~~_II
Table 1 Latitude longitude location elevation and collection date ofNew Mexico locust seed sources
Lot title Latitude (N) Longitude (W) Location Elevation (m) Collection Date
Raton 36deg5846 104deg2872 Raton Pass NM 2295 82500
Holman 36deg0306 105deg2429 HolmanNM 2476 911500
Jemez 35deg4500 106deg4100 Jemez Springs NM 1976 9900 00
Sandia 35deg1083 106deg2341 Cedar Crest NM 2432 82600
Manzano 34deg3689 106deg2494 ManzanoNM 2465 9200
Magdalena 33deg5953 107deg0903 Magdalena NM 2544 82900
Capitan 33deg2728 105deg4325 Alto NM 2189 911200
Sacramento 32deg5798 105deg4496 Cloudcroft NM 2630 91400 Gila 32deg5400 107deg4600 Kingston NM 2432 9700
f
Table 2 Mean seed weight and standard error by source
a
Source Mean seed weight (g) for Standard error 100 seeds n = 5
Raton Holman Jemez Sandia Manzano Magdalena Capitan Sacramento Gila
2872 b c 2522 d 1826 e 2753 c 2413 d 2559 d 2443 d 3130 a 3029 ab
00103 00403 00276 00301 00370 00374 00458 00279 00348
a Mean seed weights followed by the same letter are not significantly
different
19
experiment was conducted utilizing a randomized complete block design blocked by
time with a factorial treatment structure Experimental factors were seed source and
scarification treatment Nine seed sources from throughout New Mexico were used
(Figure 1 Table 1) Scarification treatments included a control three levels ofhot
water and four levels of percussion Hot water treatments included immersion at
100degC and boiling for periods of 30 seconds and 60 seconds Percussion treatment
levels were 1 2 4 and 8 minutes One hundred seeds per block were used to test
each treatment combination Four blocks of seed were tested one at a time (all seed
sources included in each run) in close chronological sequence (within 6 weeks) A
total of four 100-seed samples were used to test each source by scarification treatment
combination
Treatments
Hot water treatment of seeds utilized 50 ml test tubes filled with 30 ml
distilled water Each test tube was used to treat one 100-seed sample at a time
resulting in a ratio of4 parts water to 1 part seed by volume Test tubes were placed
in a beaker with the beaker filled with tap water to the same level as the water within
test tubes Nine test tubes (one per seed source) were placed in the beaker
simultaneously so that one replication of each seed sourcescarification treatment
cotnblnatlon was conducted simultaneously Hot water treatments include immersion
boiling For immersion treatments seeds were added to test tubes with the water
(boiling) and the test tubes were immediately removed from the beaker to
For the boiling treatments test tubes remained in the beaker with water at
20
boiling temperature for a set duration after the addition of seeds Following all
treatments test tubes with seeds cooled 24 hours in a test tube rack
Percussion treatment utilized a pneumatic paint shaker (Central Pneumatic
model 00422 Camarillo CA) Compressed air was maintained at 530 kPa plusmn30 kPa
(80 psi plusmn 5 psi) resulting in approximately 350 oscillations per minute Seeds were
placed in 118 ml (4 oz) soil tin cans (US Can Lobard IL) and the lids were secured
with duct tape Each can was used to treat one 100-seed sample To take advantage
ofgreatest lateral movement of the paint shaker a spacer (paint can) was used to
position the soil tin at the end of the shaking arm This allowed shaking to occur at
the greatest distance from the pivot point (fulcrum) Tins were placed perpendicular
to the direction of shaking motion allowing seeds to impact at greatest force by
hitting flat surfaces of the soil tin
Germination Testing
Before gennination testing all seeds were soaked 24 hours in reverse
osmosis-treated water (this was included in the process ofhot water treatment
jU~SCIlOea above) Following soaking seeds were placed in 100 cm Petri dishes lined
90 cm Whatman qualitative 1 filter paper circles (MG Scientific Pleasant
WI) One Petri dish was used for each treatment by source combination
papers were initially moistened with 5 mL reverse osmosis-treated water then
~VU1CUlJl moistened as needed to ensure adequate conditions for gennination
were monitored daily (at 300 PM) for gennination for 14 days in accordance
~UIUlnJlVUa Seed Testing Association (ISTA) standards for Robinia species
21
(1STA 1999) Tests were carried out at room temperature (22degC plusmnOSoC)
Germination was classified as protrusion of the radicle from the embryo by at least
1mm as viewed with the naked eye
X-ray Radiography
On August 132001 three lOO-seed samples from the Sacramento seed source
were treated for 14 or 8-minute percussion durations The seeds were sent
immediately by overnight delivery to the Faxitron X-Ray Corporation in Wheeling
Illinois where Dr Bob Karrfalt ofthe USDA Forest Service oversaw their imaging
Radiographs were made at SX with a Faxitron Specimen Radiography System Model
MX-20
Scanning Electron Microscope Photography
On February 192002 a Hitachi S-3200N Variable Pressure Scanning
Electron Microscope (Mountain View CA) was used to examine strophiolar cells of
untreated seeds and seeds percussed for 4 minutes Seeds were cut longitudinally
igt their entire length using a razor blade splitting the strophiolar region roughly in
Cross sections of seed samples magnifying the strophiolar region were viewed
600X under the microscope
Analysis
Categorical Analysis of Variance was used to determine treatment source and
(Le time) main effects and interactions The response variable was total
~vlI This procedure is a Chi-square test of homogeneity using the natural log
of germinated to non-germinated seed for each treatment Maximumshy
22
likelihood analysis was used to calculate Chi-square test statistics with their observed
significance levels A single contrast was conducted to cempare treatment means
against the control Observed significance levels less than or equal to 005 were
considered significant Pairwise comparisons were conducted between non-control
treatment mean percents using a conservative alp~a value of 005 divided by the
number of comparisons (Bonferroni approach) and approximate pairwise z statistics
Categorical analyses were performed using Proc Catmod (SAS Institute 1989)
RESULTS
Scarification seed source block and all interactions between factors
influenced germination (Table 3) The main effects are reported in the presence of
these interactions because the overall robustness of treatments and seed sources are of
interest Background levels ofnaturally occurring variation in seed sources are
seen in control germination percentages in Figure 2 In particular time variability
is unlikely to be under the control of the producer Appendix B details experimental
data for source by treatment gennination percents for each of the four blocks
Averaged over all treatments germination by source ranged from 40 to
79 (Figure 2) Averaged over all sources (Figure 3) and for each source (Figure 4
5) percussion scarification durations of4 or 8 minutes yielded greatest germination
Averaged over all sources germination increased with increasing durations of
percussion then leveled off for the 4- and 8-minute treatments (Figure 3) Immersion
and 30-second boiling scarification maximized germination for the hot water
treatments with a decrease in germination when seeds were boiled for 60 seconds
For all but two sources there is a similar ipcreasing trend in germination from
1to 4 minutes ofpercussion (Figure 4) The Holman source increased in germination
1 to 2 minutes then leveled off at 4 minutes The Jemez source changed little
1 to 4 minutes of treatment As treatment duration increased from 4 to 8
mutes five sources decreased in germination (Figure 4a) whereas three sources
24
Table 3 Categorical Analysis of Variance table for germination response to seed treatment seed source block and the interaction of the factors
Source of DF Chi-Square Observed Variability Significance Level
Source 8 98370 lt00001 Trt 7 233876 lt00001
contrast 1 16976 lt00001 SourceTrt 56 97077 lt00001 Block 3 5538 lt00001
SourceBlock 24 10162 lt00001 TrtBlock 21 7564 lt00001
SourceTrtBlock 168 34283 lt00001
S 4)
8 ~
0
sectg ~ 40 ~
N 0 0
20
o f0ltgt ~ ~4 jybshy ((iltgto ~~ K1ltgt 6~b-~ ~~ 0b-~~ ~~ ~b-~ ~b-~b
80
60 -I-- - ~- middotmiddotmiddotmiddotmiddot1
r -] Northern NM Sources
Central NM Sources
Southern NM Sources
sect-o ~
~b-v(i
SOURCE
Figure 2 Effect ofNew Mexico locust seed source on germination for data averaged over all scarification treatments Control germination is shown by solid line within bar
80
~ cd ~ ltl) () 60 ~ ~
N
t 0shyrti tshySltl)
40
0
20
o lmmersion Boiling 30 sec Boiling 60 sec Control 1 min 2min 4 min 8 min
Hot Water Treatments Percussion Treatments
Figure 3 Effect of scarification on germination ofNew Mexico locust for data averaged over all seed sources Means labelled with the same letter are not significantly different
100~----------------------~--~----------------~
80
60
ltU
~
40 IJ
sectgsI
l100
80
60
40
a I middotmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot middotImiddotmiddotmiddotmiddotmiddotmiddot E
_~iii-bull _ _ bull f --~--
t-- -~ ---- -shy ---- shyI ~ ---- - shy
L
fiII
~
b
- shy ---r
- Holman
Sacramento Sandia Gila Jemez
c ~
ltgt ~
Tmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot-I
j~=~=-~~-1
I middotmiddot ~ bull r
t tr
Y -------M-a-gd-a-Ie-n-a--
1 _ _
fCapitan
---- Manzano ~y -_ Raton
2 4 8
Time interval ofpercussion scarification (min)
Figure 4 Effect of seed source by treatment interaction on New Mexico locust germination for percussion scarification pattern a and pattern b
28
tv 0
~ d
~ A s o
1 c3
80
60
40
20
o
TIT
t------=middotfmiddotmiddot-middotmiddot-middotmiddot-middotmiddot 1- --- - - --=- - -shy--cE= - _--shy --shy ----shy ~ ~ ---shy ~ -~- _ --shy-------r 1
I
Raton Holman
-_ Jemez
--- Sandia Manzano Magdalena Capitan Sacramento Gila
Immersion Boiling 30 sec Boiling 60 sec
Hot Water Scarification
Figure 5 Effect of seed source by treatment interaction on New Mexico locust germination for hot water scarification
continued to increase in germination (Figure 4b) There was no difference between
the two treatment levels for the Manzano source (Figure 4b)
The effect ofhot water scarification also varied among sources with few
overall differences between hot water treatments (Figure 5) However boiling for 60
seconds yielded highest germination for the Jemez source whereas increasing
durations ofboiling decreased germination for the Raton source
In addition to consistently high germination 4- and 8-minute percussionshy
treated seeds germinated as fast or faster than all other treatments (Figure 6 Table 4)
050 measured as the number of days needed to complete 50 ofeventual
germination averaged 2 days for both ofthese treatments
X-ray radiographs were taken of seeds collected from the Sacramento source
percussed for 1 4 or 8 minutes (Figure 7) Seeds show signs of damage at 8 minutes
ofpercussion (Figure 8) Compared to an undamaged seed percussed for only 4
minutes (Figure 8a) damage at the 8-minute level includes fractured radicles pitting
of the seed coat and chips in seed coat structure (Figure 8b-d)
Scanning electron microscope (SEM) images at 600X reveal the direct effect
of 4 minutes of percussion on cells of the strophiolar region (Figure 9) Whereas
untreated seeds retain cellular organization in the strophiolar region strophiolar cells
seeds treated for 4 minutes ofpercussion separate and raise resulting in a
~-~e~U appearance
30
Umiddot-~middot--l-~---middotymiddotmiddotmiddot-----middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot--middot--middotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middotmiddotmiddotmiddotmiddotmiddotmiddot- -
050= gt1
-e-- Percussion 8 min -0- Percussion 4 min ---T- Percussion 2 min -----ltil- Percussion 1 min -- Immersion --0- Boiling 30 sec -+- Boiling 60 sec -ltgt- Control
2 4 6 8 10 12
Day Figure 6 Timecourse ofNew Mexico locust gennination following scarification
treatments G50 is the number of days to 50 of eventual germination
31
14
Table 4 Effect of seed source and treatment on G50 (days to 50 germination)
Seed Source Control Immersion B30sec B60sec Plmin P2min P4min P8min mean Raton 3 3 5 7 4 3 2 2 4 Holman 2 2 2 2 2 2 2 2 2
Jemez 4 6 6 5 3 2 2 2 4 Sandia 2 2 2 2 2 2 2 2 2
w tv Manzano 5 2 2 3 3 2 2 2 3
Magdalena 4 2 2 3 4 3 2 1 3
Capitan 5 4 2 2 3 2 2 2 3
Sacramento 3 2 2 2 4 3 2 2 3 Gila 3 2 2 2 2 2 2 2 2 mean 3 3 3 3 3 2 2 2 3 standard 11 14 16 18 09 05 0 03 08 deviation
Note Columns and rows may not agree due to rounding
Figure 7 X-ray radiographs ofSacramento seed source percussed for 1 4 or 8 minutes
33
Figure 8a-d Seed damage close-ups Figure a shows undamaged seed percussed for 4 minutes Figures b-d show seeds percussed for 8 minutes Damage includes fracturing ofradicle in b complete removal ofseed coat in c and pitting ofseed coat in d
34
Untreated Treated
Figure 9 SEM images ofuntreated vs 4-minute percussion-treated strophiolar cells ofNew Mexico locust seeds Treated cells are loosened separated and raised
DISCUSSION
Determining the Optimal Treatment
Averaging results of scarification treatmems across seed sources is a useful
tool to predict which treatment is most likely to be successful for an unknown seed
source Averaged over all sources four- and eight-minute percussion treatments
resulted in the highest germination 815 and 790 respectively and produced
germination that was as fast or faster than all other treatments Although there was no
difference between these two treatments on average an argument for 4 minutes of
percussion as a standard protocol for treating New Mexico locust seeds can be made
Descriptively 4 minutes of percussion improved germination over 8 minutes
ofpercussion for a majority of sources (five out of nine) 8 minutes of percussion
improved germination for three ofnine sources with no difference for a fmal source
For individual sources 4 minutes ofpercussion was always the best or secondmiddot best
treatment and was always equal to or better than all hot water treatments
Germination at 8 minutes ofpercussion was lower than all other percussion durations
for the Jemez and Gila sources and was lower than at least one hot water treatment for
these sources as well
The x-ray radiographs of the Sacramento seed source illustrate the excessive
internal and external seed damage that can occur with 8 minutes of percussion
germination counts 8-minute percussed seeds from Holman and Capitan
ClUll signs of damage that were visible to the naked eye (see below)
r~evertheless some of these seeds still fit the criteria for germination Neither x-ray
36
photography nor visible observations during seed counts revealed seed damage at the
4-minute percussion level In operational trials seeds percussed for 4 minutes
developed into healthy seedlings (Khadduri and Harrington unpublished data)
Strophiole and Seed Coat Behavior
Four minutes ofpercussion successfully breaks seed dormancy in a large
percentage ofNew Mexico locust seeds without excessively damaging the
surrounding seed coat or internal seed coat structures Most papilionoid legume
seeds including seeds ofNew Mexico locust have a specific region of the seed coat
known as the strophiole or lens (Hamly 1932) The strophiole is located on the
cotyledonary lobe of a locust seed (Figure 10 a-c) If seeds are not indiscriminately
damaged the strophiole is the first point of water entry to the embryo (Hopkinson
1997) When a papilionoid seed is percussed for the appropriate amount of time
repeated hits on the integrated seed coat loosen the constrained cells of the strophiolar
region without excessively damaging the rest of the seed coat (Ballard 1976) SEM
photos illuStrate that 4 minutes of percussion loosened and separated strophiolar cells
ofNew Mexico locust seeds when compared with constrained organized strophiolar
cells of untreated seeds (Figure 9) Research-has shown that water passes to the
embryo through the treated strophiole in a controlled manner and regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues
(Kelly and van Staden 1987)
37
10 Strophiole ofunimbibed New Mexico locust seed (a) and following uptake (bc)
38
In contrast hot-water treatments can excessively degrade the seed coat
(Rebman et al 1998 Marunda 1990) Random cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rebman et aI 1998 Marunda 1990) Along with a
proportionately large amount ofun-imbibed (thus under-treated) seeds (Figure l1a)
damage was observed in hot-water treated seeds from six sources during germination
counts Damage included seeds where the radicle failed to elongate (Figure 11 b)
seeds where the radicle elongated but was delayed in freeing itself from the seed coat
(Figure 11c) and seeds where the radicle completely broke off from the embryo
(Figure lId) In addition some seeds germinated in reverse (the cotyledon emerged
fIrst) If the radicle emerged completely from the seed coat they were counted as
germinated
While nearly all seeds treated for 8 minutes ofpercussion imbibed water
damage was evident in x-ray evaluations (Sacramento seed source) and in
germination counts (Holman and Capitan) at this treatment level Seed damage
during germination counts was similar to levels found in hot-water treated seeds
suggesting that over-treatment by both hot water and longer durations of percussion
may result in similar damage to seed structure
The Sacramento and Holman seed sources were two of the fIve sources that
opcrplltlpn in germination from the 4- to 8-minute durations of percussion
the benefIts ofloosening the strophiole as evidenced by imbibition of
all 8-minute treated seeds were outweighed by external and internal seed coat
39
a
b c d
11 Damage to New Mexico locust seeds observed during germination cotmts following hot water scarification 9a Hot water replicate including ~t seedlings and numerous unimbibed seeds 9b Close-up of seed where raallCle failed to elongate 9c Close-up ofseed where radicle elongated but was
-gtIn in freeing itself from the seed coat 9d Close-up ofseed where radicle broke off from embryo
40
damage (which also may be contributing to high imbibition) For the three sources
for which 8 minutes of percussion resulted in maximum germination extended
percussion of seeds necessary to loosen the strophiole may not have excessively
damaged the seed anatomy The Manzano source did not significantly change in
germination from 4 to 8 minutes of percussion suggesting that an increase in
permeable seeds at the 8-minute treatment duration was balanced by the number of
seeds that failed to germinate due to seed damage To summarize the 4-minute
percussion treatment improved germination over 8 minutes ofpercussion for a
majority of sources (five ofnine) the 8-minute percussion treatment improved
germination over 4-minutes ofpercussion for three sources and there was no
difference with the Manzano source Notably for the Raton source 8 minutes of
percussion increased germination by 235 percentage points over 4-minutes of
percussion (7875 vs 5525 respectively) the next best treatment Raton was the
only source to result in germination less than 75 for 4 minutes ofpercussion so a
grower may wish to apply longer percussion durations to lots that do not respond well
to this treatment level
Finally there was an interesting trend in the Jemez and Raton sources seed
that significantly contributed to the source by treatment interaction (Figure 12) A
~u test showed that the Jemez source had a significantly lighter seed weight than
other sources while the Raton source had an average seed weight For the Jemez
the longest duration of boiling increased germination Conversely only short
U4UVll) of percussion were needed to achieve high germination and the longest
41
80 _ Jemez a Raton
iD
~ C iD e iD
Q c 0 ~
c ~ iD 0
20
0------shy
Immersion Boiling 30 sec Boiling 60 sec
Hot Water Scarification
10degTr========~--------~------------------------------
80
60
40
20
_ Jemez Raton
0------shy
1 min 2 min 4 min 8 min
Percussion Scarification
Figure 12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources
42
b
--~----~~~~========~---- duration ofpercussion reduced germination For the Raton source longer durations
ofboiling consistently decreased germination whereas longer percussion treatments
consistently increased germination
A possible explanation is that a thick seed coat is associated with a loose
strophiole structure and that a thin seed coat is associated with a constrained
strophiole This contrast merits further study and may suggest an ecological
adaptation Further investigation ofthis phenomenon may contribute to successful
pre-screening ofunknown seed sources to determine optimal treatment leveL
43
CONCLUSION
Based on results obtained in pilot study (see Appendix) and thesis
experiments 4 minutes ofpercussion is recommended as a standard protocol for
treating an unknown source ofNew Mexico locust seed In addition to laboratory
results the 4-minute percussion treatment has resulted in germination greater than
80 and healthy seedling grow-outs for production purposes (unpublished data) In
laboratory experiments only one hundred seeds per 118 ml (40z) soil tin were
percussed at a time whereas for production we have treated up to five hundred seeds
per tin For large-scale production ofNew Mexico locust seedlings growers may
wish to experiment with larger quantities of seed and larger-size soil tins
Seed storage trials following percussion treatments were not conducted
However germination greater than 80 and healthy seedlings were obtained when
seeds were percussion-treated and subsequently stored at room-temperature (22degC)
for one week (unpublished data) Seed storage trials merit further study as the
standard hot water treatment leaves seeds in a softened condition that is not
conducive to storage
Hydroseeding is an effective method for direct seeding in reclamation
situations Percussion treatment may be conducive to hydro seeding as seeds remain
a dry relatively undamaged yet permeable condition following treatment Sulfuric
seeds also remain dry following treatment but extensive damage to seed coat
poor seedling vigor have been reported with this treatment (Kelly and van Staden
A grow-out study using a hydro seeder on a reclamation site testing percussion
44
against other treatments such as sulfuric acid and mechanical scarification also
merits further study
Papilionoids are the largest subfamily oflegumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust these legumes often play an integral role in the revegetation of
disturbed lands The pilot study (Appendix) indicates that percussion scarification
successfully treats the widely used black locust as well Developing superior
scarification methods to the standard hot water mechanical scarification and acid
treatments may facilitate the use ofpapilionoid legumes in reclamation projects
45
APPENDICES
APPENDIX A
PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR
NEW MEXICO LOCUST AND BLACK LOCUST SEEDS
Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4
Hot water and sulfuric acid soaks are traditional treatments for seeds of many
temperate woody legwnes including locusts However these scarification techniques
often produce inconsistent germination Percussion scarification where seeds are
repeatedly propelled against a hard surface was compared with hot water
scarification to evaluate treatment efficacy for New Mexico locust (Robinia
neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment
seeds were placed in a 98degC water bath which was immediately removed from the
heat source For percussion scarification seeds were placed in a soil sample tin and
agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the
control were followed by 24-hour water soaks Hot water baths resulted in 56 and
41 germination for New Mexico locust and black locust respectively For both
species nearly all durations ofpercussion increased germination over the hot water
treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3
4 and 5 minutes for black locust resulted in at least 90 germination Traditional
scarification treatments randomly degrade the entire seed coat which can lead to
tissue damage during water uptake Percussion scarification specifically weakens the
strophiole the natural source ofwater entry to the seed in papilionoid legwnes
INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom
2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031
48
Following percussion imbibition is controlled through the strophiole and underlying
tissue is protected
Keywords New Mexico locust black locust scarification percussion germination
Introduction
Locust trees (Robinia spp) are aggressive pioneer species that quickly
colonize disturbed land fix atmospheric nitrogen in the soil and stabilize slopes that
are prone to erosion (Klemmedson 1994 Ashby et al 1985) (Figure A1ab)
Recognition of these attributes has contributed to increased demand for locusts in
reclamation projects Locust seeds however exhibit physical dormancy where a
thick seed coat prevents the movement of water and gasses to the embryo (Leadem
1997)
Sulfuric acid or hot water soaks have traditionally been used to break seed
dormancy in macro-propagation of locusts Many growers are moving away from the
use of su1furic acid in treating locust seeds (Dreesen and Harrington 1997) In
addition to being dangerous acid soak durations must be specifically correlated to
seed lot (Olson 1974) Collections of native seed can vary tremendously in seed size
seed weight and hard seededness etc This variability limits the utility of sulfuric
acid soaks as even within a given lot some seeds may be damaged whereas others
are under-treated and fail to imbibe (take up water) A survey of Southwestern
container growers indicates that hot water baths are currently the most common
scarification method for locust seeds (Hine et al 1997) However hot water baths
also produce inconsistent germination (Lin et al 1996)
49
b
Figure AI New Mexico locust a primary invader following disturbance Ala Crown-sprouting at edge ofburn near Los Alamos NM--one year after Cerro Grande fire Alb Colonizing road cut Gila National Forest NM
50
Percussion scarification where seeds are repeatedly propelled against a hard
surface is an alternative dormancy-breaking method that has proven successful in
legumes related to locusts (Hamly 1932 Barton 1947) We compared this alternative
treatment to hot water scarification to evaluate treatment efficacy for New Mexico
locust and black locust seeds
Materials and Methods
Commercial New Mexico locust seeds (Western Native Seed Coaldale CO)
were collected fall 2000 in Huerfano County CO Black locust seeds were collected
September 2000 from Taos County NM by harvesting ripe pods from several trees at
a distance of up to 3 m from ground level Black locust seeds were allowed to air-dry
for 3 weeks and were threshed and separated from large chaffusing a greenhouse
fan A South Dakota Seed Blower (Chicago Illinltois) was used to remove fine chaff
from both seed lots Seeds were storedmiddotat 2-4C until the germination study was
conducted in March 2001
This study compared hot water scarification with percussion scarification
using treatment and species as experimental factors Along with a control
scarification treatments included immersion of seed in boiling water as well as
percussion durations of 1 2 3 45 or 10 minutes Four 100-seed samples were used
to test each species by scarification treatment combination
For the hot water treatment test tubes were filled with 30 mL water and
placed in a water-filled beaker (Figure A2a) The bath was raised to boiling (-98C at
51
b
c
Figure A2 Implementation ofhot water and percussion scarification A2a Hot water bath A2b Paint-shaker set-up A2c Placing soil tin containing seeds at end ofpaint shaker ann
52
Las Cruces NM elevation of 1300 meters) Each 100-seed replicate was placed in a
test tube and each test tube was immediately removed to cool at room temperature
Percussion scarification was implemented using a pneumatic paint shaker
(Central Pneumatic model 00422 Camarillo CA) (Figure A2b) This allowed
standardization of treatment intensity Compressed air pressure was maintained at
530kPa plusmn 30kPa (80psi plusmn 5psi) resulting in approximately 350 oscillations per
minute Each lOO-seed sample was placed in a 118 ml (4 oz) soil tin for shaking To
maximize lateral movement of paint shaker a paint can was used as a spacer to place
soil tins at the end of the shaking arm (Figure A2c) This allowed shaking to occur at
the greatest distance from the pivot point (fulcrum) Following all treatments
including the control seeds were soaked for twenty-four hours in distilled water prior
to plating out
Gennination was evaluated on lab benches at room temperature Each
replicate of seeds was placed in a 100 cm petri dish on moistened filter paper with
humidity maintained by enclosing the petri dishes in ziplock bags Gennination was
monitored daily for 14 days in accordance with International Seed Testing
Association standards for Robinia species (ISTA 1999) Gennination was defined as
protrusion of the radicle from the embryo by at least 1mm as viewed by the naked
eye
Categorical Analysis of Variance using Prpc Catmod (SAS Institute 1989)
was used to detennine treatment differences in germination percentages for each
source This procedure is an extension of a Chi-square test of homogeneity using the
53
natural log of the ratio of germinated to non-germinated seeds for each treatment Pshy
values less than 005 were considered significant A limited set of pairwiseshy
comparisons was conducted to compare treatment mean percents using a
conservative alpha value of 005 divided by the number of comparisons
Results
Percussion maximized germination for both New Mexico locust and black
locust There were three germination response levels for New Mexico locust (Figure
A3) First there was a low response in the control indicating a dormant seed lot
The hot water treatment and I-minute duration of percussion share a second
intermediate response Germination was highest at percussion levels from 2 to 10
minutes
A selected percussion treatment intermediate in time (5 minutes) produced the
steepest germination curve indicating the fastest germination rate (Figure A4) The
hot water treatment produced an intermediate curve or relatively slower germination
rate The control curve was very flat indicating the slowest germination rate for New
Mexico locust
Control seeds germinated poorly for black locust as well (Figure A5) Hot
water scarification improved germination over the control but all levels of percussion
improved germination over hot water The optimal durations of percussion treatment
were bracketed with a significant increase from the 1 to 2 minute percussion level a
high germination response from 2 to 5 minutes of percussion and finally a significant
54
~ d 25 CL)
p t1 0 ~ t1
g Vl ~ Vl
100~---------------------------------------------------------------
80
60
40
20
o Control Hot Water Percussion
I min Percussion
2 min Percussion
3 min
Scarification
Percussion 4 min
Figure A3 Effect of scarification on germination response for New Mexico locust
Percussion 5 min
=significant difference between adjacent treatment bars ns = no significant difference
Percussion 10 min
~ ~ () u agt
Il-t d o ~
~ ~ c3
100middotlr~~~------------------------------------e- Percussion 5min middot 0middot Hot Water -- Control
80-J=middot======
6b-middot-lmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middot--middotmiddot-middot~-middotmiddot -middotmiddot middot middotmiddot-middot 1
40
20
-----~-----------
0 0 0 middotmiddotmiddotmiddotmiddot0middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot0
0 --~-----y-----~------_---~--- T-----~
o shy -TDay 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A4 Effect of selected scarification treatments on germination speed for New Mexico locust First seven days of fourteen-day experiment are shown
Day 7
80
~ cd
5 60 ()
[) Cl d 0 1t1 d
40sect dUl
-J
20
o
ns
Scarification
Figure A5 Effect of scarification on germination response for black locust = significant difference between adjacent treatment bars ns = no significant difference
decrease at the 10-minute level This drop-off suggests that 10 minutes of percussion
over-treats seeds from this lot of-black locust
A selected percussion treatment intermediate in time (5 minutes) also had the
fastest germination rate for black locust (Figure A6)_ The hot water curve is
somewhat flatter indicating a slower germination rate The control curve is very flat
indicating a slow germination rate
A qualitative difference between hot water and percussion-treated seeds was
also observed Along with healthy germinants the hot water treatment produced
over-treated seeds (Figure A 7a) Examples of damaged germinants include seeds
where the radicle did not elongate (Figure A 7b) seeds where the radicle elongated
but was delayed in freeing itself from the seed coat (Figure A7c) and seeds where the
radicle broke off completely from the embryo (Figure A7d) In addition to these
examples of over-treatment there were numerous small dark seeds that failed to
imbibe water For percussion treatment nearly all seeds took up water (Figure A8a)
Germination was rapid and uniform with little damage evident (Figure A8b)
To summarize both New Mexico locust and black locust seed lots were
dormant The standard hot water scarification treatment improved germination but
only to 56 and 41 for New Mexico locust and black locust respectively Nearly
all percussion durations improved total germination as well as germination rate over
the hot water treatment Germination was greater than 90 for percussion durations
of 4 5 and 10 minutes for New Mexico locust and 3 4 and 5 minutes for black locust
58
CI)
sgt t CI) 0
igt 0
sect ttl t E
Vl 0
c3
100r==~==~~=r------------------------------------~ ___ Percussion 5 min
middotmiddotmiddot0middotmiddotmiddot Hot Water -lIf- Control
80r=========~--~~----------------------------~
60
40
20 -~-~---~---------- ---~------------I
omiddot
O
_~_----------lIf-----~bull -----_ shy---shy I
Day 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A6 Effect of selected scarification treatments on germination speed for black locust First seven days of fourteen-day experiment are shown
Day 7
a
b c d
Figure A7 Damage to New Mexico locust seeds observed during germination counts following hot water scarification A7a Hot water replicate including damaged seedlings and numerous unimbibed seeds A7b Close-up ofseed where radicle failed to elongate A7c Close-up ofseed where radicle elongated but was delayed in freeing itself from the seed coat A7d Close-up ofseed where radicle completely broke offfrom embryo
60
a
b
Figure A8 New Mexico locust seeds following percussion treatment at time of germination counts A8a Percussion replicate with uniformly healthy seedlings and near complete imbibition of seeds A8b Close-up ofpercussion-treated seedling
61
Discussion
Papilionoid seeds have a specific anatomical feature known as the strophiole
or lens-the natural site of water entry to the seed (Hamly 1932) The strophiole is
located on the cotyledonary lobe of a locust seed (Figure A9) When a papilionoid
legume seed is percussed for the appropriate amount of time repeated hits on the
integrated seed coat loosen the constrained cells of the strophiolar region without
excessively damaging the rest of the seed coat (Ballard 1976)
When a percussed seed is soaked water enters exclusively through the
strophiole in a controlled manner (Kelly and Staden 1987) This regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues This
contrasts with seeds that have been hot water or acid scarified treatments that can
randomly degrade the seed coat Unlocalized cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rehman et al 1998 Marunda 1990)
Papilionoids are the largest subfamily of legumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust and black locust these legumes often play an integral role in the
revegetation ofdisturbed lands Developing superior scarification methods to the
standard hot water and acid treatments should fadlitate the use ofthese legumes in
restoration projects
62
Figure A9 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (bc)
63
APPENDIXB
GERMINATION PERCENTS AND STANDARD ERRORS FOR SOURCE
BLOCK AND TREATMENT FOR NEW MEXICO LOCUST
Table B 1 Gennination percents and standard errors for source block and treatment for New Mexico locust
Source Block Treatment Germination Percentage Standard Error
Capitan 1 Immersion 74 437 Capitan 2 Immersion 69 462 Capitan 3 Immersion 71 454 Capitan 4 Immersion 67 470 Capitan Boiling 30 sec 79 407 Capitan 2 Boiling 30 sec 76 427 Capitan 3 Boiling 30 sec 72 449 Capitan 4 Boiling 30 sec 76 427 Capitan Boiling 60 sec 71 454 Capitan 2 Boiling 60 sec 54 498 Capitan 3 Boiling 60 sec 67 470 Capitan 4 Boiling 60 sec 76 427 Capitan I Control 5 218 Capitan 2 Control 7 255 Capitan 3 Control 4 196 Capitan 4 Control 5 218 Capitan 1 Percussion I min 47 499 Capitan 2 Percussion I min
470
Capitan Percussion I min 39 488
Capitan 4 Percussion I min 38 485 Capitan I Percussion 2 min 55 497 Capitan Capitan
2
Percussion 2 min Percussion 2 min
71 65
454 477
Capitan 4 Percussion 2 min 56 496 Capitan I Percussion 4 min 86 347 Capitan 2 Percussion 4 min 79 407 Capitan 3 Percussion 4 min 81 392 Capitan 4 Percussion 4 min 82 384 Capitan I Percussion 8 min 90 300 Capitan 2 Percussion 8 min 88 325 Capitan 3 Percussion 8 min 90 300 Capitan 4 Percussion 8 min 90 300 Gila I Immersion 71 454 Gila 2 Immersion 77 421 Gila
Immersion 75 433
Gila 4 Immersion 72 449
Gila I Boiling 30 sec 68 466
Gila 2 Boiling 30 sec 81 392
Gila 3 Boiling 30 sec 54 498
Gila 4 Boiling 30 sec 63 483
Gila I Boiling 60 sec 55 497
Gila 2 Boiling 60 sec 68 466
Gila 3 Boiling 60 sec 57 495
Gila 4 Boiling 60 sec 70 458
65
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Gila 2 Control 13 336 Gila 3 Control 13 336 Gila 4 Control 20 400 Gila 1 Percussion 1 min 66 474 Gila 2 Percussion 1 min 57 495 Gila 3 Percussion 1 min 73 444 Gila 4 Percussion 1 min 82 384 Gila 1 Percussion 2 min 81 392 Gila 2 Percussion 2 min 86 347 Gila 3 Percussion 2 min 85 357 Gila 4 Percussion 2 min 54 498 Gila 1 Percussion 4 min 79 407 Gila 2 Percussion 4 min 83 376 Gila 3 Percussion 4 min 84 367 Gila 4 Percussion 4 min 79 407 Gila 1 Percussion 8 min 51 500 Gila 2 Percussion 8 min 76 427 Gila 3 Percussion 8 min 52 500 Gila 4 Percussion 8 min 74 439 Holman 1 Immersion 83 376 Holman 2 Immersion 86 347 Holman 3 Immersion 81 392 Holman 4 Immersion 90 300 Holman 1 Boiling 30 sec 90 300 Holman 2 Boiling 30 sec 85 357 Holman 3 Boiling 30 sec 87 336 Holman 4 Boiling 30 sec 83 376 Holman 1 Boiling 60 sec 79 407 Holman 2 Boiling 60 sec 80 400 Holman 3 Boiling 60 sec 86 347 Holman 4 Boiling 60 sec 93 255 Holman Control 25 433 Holman 2 Control 19 392 Holman Control 21 407
Holman 4 Control 24 427
Holman Percussion 1 min 77 421
Holman 2 Percussion 1 min 85 357
Holman 3 Percussion 1 min 80 400
Holman 4 Percussion 1 min 89 313
Holman 1 Percussion 2 min 88 325
Holman 2 Percussion 2 min 96 196
Holman 3 Percussion 2 min 93 255
Holman 4 Percussion 2 min 96 196
Holman 1 Percussion 4 min 92 271
Holman 2 Percussion 4 min 97 171
66
I
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Holman 3 Percussion 4 min 91 286 Holman 4 Percussion 4 min 96 196 Holman 1 Percussion 8 min 88 325 Holman 2 Percussion 8 min 85 357 Holman 3 Percussion 8 min 83 376 Holman 4 Percussion 8 min 82 384 Jemez 1 Immersion 53 499 Jemez 2 Immersion 44 496 Jemez 3 Immersion 52 500 Jemez 4 Immersion 70 458 Jemez 1 Boiling 30 sec 58 494 Jemez 2 Boiling 30 sec 55 497 Jemez 3 Boiling 30 sec 48 500 Jemez 4 Boiling 30 sec 65 477 Jemez 1 Boiling 60 sec 68 466 Jemez 2 Boiling 60 sec 63 483 Jemez 3 Boiling 60 sec 73 444 Jemez 4 Boiling 60 sec 89 313 Jemez 1 Control 2 140 Jemez 2 Control 9 286 Jemez 3 Control 6 237 Jemez 4 Control 8 271 Jemez 1 Percussion 1 min 58 494 Jemez 2 Percussion 1 min 63 483 Jemez 3 Percussion 1 min 77 421 Jemez 4 Percussion 1 min 89 313 Jemez 1 Percussion 2 min 68 466 Jemez 2 Percussion 2 min 64 480 Jemez 3 Percussion 2 min 80 400 Jemez 4 Percussion 2 min 83 376 Jemez Percussion 4 min 68 466 Jemez 2 Percussion 4 min 79 407 Jemez 3 Percussion 4 min 70 458 Jemez 4 Percussion 4 min 83 376 Jemez 1 Percussion 8 min 52 500 Jemez 2 Percussion 8 min 59 492 Jemez 3 Percussion 8 min 61 488 Jemez 4 Percussion 8 min 70 458 Magdalena 1 Immersion 69 462 Magdalena 2 Immersion 69 462 Magdalena J Immersion 70 458 Magdalena 4 Immersion 85 357 Magdalena 1 Boiling 30 sec 65 477 Magdalena 2 Boiling 30 sec 71 454 Magdalena 3 Boiling 30 sec 68 466
67
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Magdalena 4 Boiling 30 sec 83 376 Magdalena 1 Boiling 60 sec 63 483 Magdalena 2 Boiling 60 sec 68 466 Magdalena 3 Boiling 60 sec 63 483 Magdalena 4 Boiling 60 sec 82 384 Magdalena 1 Control 11 313 Magdalena 2 Control 6 237 Magdalena 3 Control 5 218 Magdalena 4 Control 4 196 Magdalena Percussion 1 min 32 466 Magdalena 2 Percussion 1 min 45 497 Magdalena 3 Percussion 1 min 46 498 Magdalena 4 Percussion I min 36 480 Magdalena 1 Percussion 2 min 67 470 Magdalena 2 Percussion 2 min 68 466 Magdalena 3 Percussion 2 min 65 477 Magdalena 4 Percussion 2 min 81 392 Magdalena 1 Percussion 4 min 87 336 Magdalena 2 Percussion 4 min 97 171 Magdalena 3 Percussion 4 min 83 376 Magdalena 4 Percussion 4 min 97 171 Magdalena 1 Percussion 8 min 98 lAO Magdalena 2 Percussion 8 min 100 000 Magdalena 3 Percussion 8 min 96 196 Magdalena 4 Percussion 8 min 98 lAO Manzano 1 Immersion 67 470 Manzano 2 Immersion 57 495 Manzano 3 Immersion 72 4049 Manzano 4 Immersion 73 4044 Manzano 1 Boiling 30 sec 70 458 Manzano 2 Boiling 30 sec 64 480 Manzano 3 Boiling 30 sec 71 454 Manzano 4 Boiling 30 sec 80 400 Manzano I Boiling 60 sec 50 500 Manzano 2 Boiling 60 sec 53 499 Manzano 3 Boiling 60 sec 59 492 Manzano 4 Boiling 60 sec 74 439 Manzano I Control 4 196 Manzano 2 Control 8 271 Manzano 3 Control 1 099 Manzano 4 Control 6 237 Manzano 1 Percussion 1 min 52 500 Manzano 2 Percussion 1 min 39 488 Manzano 3 Percussion 1 min 36 480 Manzano 4 Percussion 1 min 51 500
68
Table B 1 Continued
Source Block Treatment Gerrni~ation Percentage Standard Error
Manzano Percussion 2 min 53 499 Manzano 2 Percussion 2 min 58 494 Manzano 3 Percussion 2 min 61 488 Manzano 4 Percussion 2 min 62 485 Manzano 1 Percussion 4 min 76 427 Manzano 2 Percussion 4 min 85 357 Manzano 3 Percussion 4 min 79 407 Manzano 4 Percussion 4 min 83 376 Manzano 1 Percussion 8 min 87 336 Manzano 2 Percussion 8 min 83 376 Manzano 3 Percussion 8 min 83 376 Manzano 4 Percussion 8 min 75 433 Raton 1 Immersion 54 498 Raton 2 Immersion 37 483 Raton 3 Immersion 39 488 Raton 4 Immersion 69 462 Raton 1 Boiling 30 sec 36 480 Raton 2 Boiling 30 sec 20 400 Raton 3 Boiling 30 sec 28 449 Raton 4 Boiling 30 sec 60 490 Raton 1 Boiling 60 sec 15 357 Raton 2 Boiling 60 sec 171 Raton 3 Boiling 60 sec 4 196 Raton 4 Boiling 60 sec 35 477 Raton 1 Control 4 196 Raton 2 Control 12 325 Raton 3 Control 2 140 Raton 4 Control 6 237 Raton 1 Percussion 1 min 52 500 Raton 2 Percussion I min 29 454 Raton 3 Percussion I min 35 477 Raton 4 Percussion 1 min 31 462 Raton 1 Percussion 2 min 55 497 Raton 2 Percussion 2 min 35 477 Raton 3 Percussion 2 min 33 470 Raton 4 Percussion 2 min 42 494 Raton 1 Percussion 4 min 65 477 Raton 2 Percussion 4 min 52 500 Raton Percussion 4 min 51 500 Raton 4 Percussion 4 min 53 499 Raton 1 Percussion 8 min 88 325 Raton 2 Percussion 8 min 80 400 Raton 3 Percussion 8 min 73 444 Raton 4 Percussion 8 min 74 439 Sacramento 1 Immersion 43 495
69
Table B 1 Continued
-Source Block Treatment Germination Percentage Standard Error
Sacramento 2 Immersion 30 458 Sacramento 3 Immersion 38 485 Sacramento 4 Immersion 56 496 Sacramento 1 Boiling 30 sec 47 499 Sacramento 2 Boiling 30 sec 48 500 Sacramento 3 Boiling 30 sec 48 500 Sacramento 4 Boiling 30 sec 56 496 Sacramento 1 Boiling 60 sec 52 500 Sacramento 2 Boiling 60 sec 67 470 Sacramento 3 Boiling 60 sec 43 495 Sacramento 4 Boiling 60 sec 48 500 Sacramento 1 Control 0 000 Sacramento 2 Control 3 171 Sacramento 3 Control 3 171 Sacramento 4 Control 099 Sacramento 1 Percussion 1 min 57 495 Sacramento 2 Percussion 1 min 61 488 Sacramento 3 Percussion 1 min 62 485 Sacramento 4 Percussion 1 min 55 497 Sacramento 1 Percussion 2 min 84 367 Sacramento 2 Percussion 2 min 70 458 Sacramento 3 Percussion 2 min 81 392 Sacramento 4 Percussion 2 min 69 462 Sacramento 1 Percussion 4 min 91 286 Sacramento 2 Percussion 4 min 84 367 Sacramento 3 Percussion 4 min 84 367 Sacramento 4 Percussion 4 min 94 237 Sacramento 1 Percussion 8 min 82 384 Sacramento 2 Percussion 8 min 81 392 Sacramento 3 Percussion 8 min 82 384 Sacramento 4 Percussion 8 min 81 392 Sandia 1 Immersion 76 427 Sandia 2 Immersion 70 458 Sandia 3 Immersion 70 458 Sandia 4 Immersion 81 392 Sandia 1 Boiling 30 sec 78 414 Sandia 2 Boiling 30 sec 71 454 Sandia 3 Boiling 30 sec 63 483 Sandia 4 Boiling 30 sec 78 414 Sandia 1 Boiling 60 sec 67 470 Sandia 2 Boiling 60 sec 65 477 Sandia 3 Boiling 60 sec 74 439 Sandia 4 Boiling 60 sec 66 474 Sandia 1 Control 16 367 Sandia 2 Control 7 255
70
Table B 1 Continued
Source Block Treatment Gennination Percentage Standard Error
Sandia 3 Control 8 271 Sandia 4 Control 16 367 Sandia 1 Percussion 1 min 67 470 Sandia 2 Percussion 1 min 61 488 Sandia 3 Percussion 1 min 66 474 Sandia 4 Percussion 1 min 59 492 Sandia I Percussion 2 min 82 384 Sandia 2 Percussion 2 min 83 376 Sandia 3 Percussion 2 min 81 392 Sandia 4 Percussion 2 min 84 367 Sandia 1 Percussion 4 min 89 313 Sandia 2 Percussion 4 min 86 347 Sandia 3 Percussion 4 min 89 313 Sandia 4 Percussion 4 min 85 357 Sandia 1 Percussion 8 min 80 400 Sandia 2 Percussion 8 min 77 421 Sandia 3 Percussion 8 min 69 462 Sandia 4 Percussion 8 min 66 474
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60 gennination for two hot water-treated seed lots while a third lot germinated
around 50 (as reported above acid scarification germination ranged from 20 to
80 for these lots) It should be noted that the authors only soaked the seeds in water
from 10 to 80 minutes for this experiment as opposed to the standard 24 hours Cox
and Klett (1984) stated that using the standard 24-hour hot water treatment improved
germination over an acid pre-treatment (which produced less than 20 germination)
but they did not give specific gennination numbers for the hot water treatment Hine
et al (1997) found that across five seed lots germination was 66 for the standard
hot water treatment (as compared to less than 20 germination for acid scarification)
Variations in treatment intensity and duration and their subsequent effects on seed lot
appear to contribute to conflicting conclusions as to whether acid scarification or hot
water treatment is a more successful treatment
Given mixed results ease ofapplication is an important argument for use of
hot water application over acid soaks Safety concerns and material costs for hot
water treatments are low (Dreesen and Harrington 1997) An extension of this is the
practicality ofhot water for treating large batches of seed where a relative increase in
water volume is all that is needed However increases in water and seed even when
inthe same 4 1 proportion can result in dramatically different rates of cooling than in
atest tube experiment Different cooling rates can lead to contrasting results for the
treatment temperature duration and seed lot (USDA Forest Service 1948)
As with acid scarification parameters ofhot water treatment application may
to be optimized to maximize germination Hine et al (1997) found that as
9
temperature was increased from 70degC to 100degC overall germination in New Mexico
locust seed increased The 100degC treatment maximized germination for each of five
sources tested with germination usually above 60
Prolonged exposure of seeds ofmany woody legumes to boiling temperatures
also may improve germination (Briscoe 1996) This approach has not been tested on
New Mexico locust seed Exposing seed to prolonged boiling temperatures may
result in fungal infection and subsequent seedling mortality Marunda (1990) noted
severe fungal infections on both acid and hot water treated Acacia holoserica Cunn
ex G Don seeds attributing the infections to seed damage resulting from the
treatments Identifying an optimal hot water treatment level that does not result in a
high rate of seedling mortality is a logical goal
With hot water treatments as with acid there is also a danger of overtreating
Seeds and reducing germination (Gosling et al 1995) The legume Leucaena
leucocephala (Lam) de Wit germinated equally well after 7 seconds of exposure to
water at 100degC 7 seconds at 90degC 30 seconds at 80degC and 4 minutes at 70degC In
addition acceptably high germination was obtained from treatment at 70degC from 3
minutes to 1 hour whereas no seeds germinated following treatment at 100degC for 2
minutes Perhaps along with a greater margin of error for treatment duration treating
~ew Mexico locust seeds at a lower temperature may result in a comparatively lower
of seedling mortality In practice however nursery growers tend to use the
and quickest treatment available The short treatment length at near-boiling
whether recommended or not may be what is used most frequently
10
Fine-tuning a treatment in this range of treatment temperature and duration may be
the most logical approach
Mechanical Scarification
This treatment involves scratching the seed coat with an abrasive material
(USDA Forest Service 1948) One system employs a rotating drum lined with
sandpaper A paddle spinning at a given number ofrevolutions per minute throws the
seed against the sandpaper (Hine et al 1997) A second method dry tumbling
tumbles seed in a mixer with pea gravel or sharp sand for 5 to 7 days (Dreesen 2001)
These approaches have the advantage that control of temperature is not required as in
water treatments nor is safety an issue as with add scarification
Hine et al (1997) mechanically treated New Mexico locust seed using 100shy
grit sandpaper in a Forsberg (rotating drum) scarifier Gennination was less than
10 They concluded that the seed coats were too thin for their apparatus and
proposed that a finer grit sandpaper would produce more even abrasion Although
germination varied by seed lot Dreesen (2001) reported that using a slow-speed dry
tumbling technique for 5 to 7 days produced gennination rates as high as 90
As with acid and hot water treatments treatment duration must be specifically
shy
correlated to seed lot Treated seeds are also susceptible to fungal infection
iHopkinson 1997) In addition equipment is specialized and may not always be
ltU~CUJl~ to a nursery (USDA Forest Service 1948) Several studies have confinned
degrading ofstrophiolar cells due to acid and hot water scarification (Miklas et al
11
1987 Dell 1980 Hanna 1984) but the effect ofmechanical scarification on the
strophiole has not been studied
Alternative Method for Breaking Dormancy Percussion
Strophiole degradation has been examined in depth using an impaction
treatment more commonly known as percussion Hamly (1932) first proposed that
shaking or impacting seeds could split the macrosc1ereid cells at the strophiole
Hamly successfully released tension ofbent strophiolar cells of impermeable
Melilotus alba Medik (White sweet clover) seeds by delivering physical blows
resulting in cells that had split apart and were only slightly bent Ninety-two percent
oftreated seeds became permeable as compared to 05 of control seeds Barton
(1947) showed that percussion significantly increases permeability in black locust
seed Ballard (1976) reasoned that the integrated nature of the strophiole within the
seed coat means a blow on any part of the seed weakens the strophiole There are no
published studies on the use of percussion on New Mexico locust
Percussion methods vary and often seem SUbjective or ill-defined Hamly
(1932) placed seeds in a glass bottle and shook them three times a second for 20
minutes resulting in 180 oscillations per minute Ballard (1973) describes shaking
seeds in a closed metal container with at least fifty seeds per container at 850-900
JS(~l11alIOIlS per minute for 1 minute Kelly and van Staden (1987) placed seeds into
sealed glass flasks Flasks were shaken by a wrist-shaker at a rate of 250
~UUVJ~ per minute Subsequent germination rates were 45 after 2 hours of
55 after 3 hours 60 after 6 hours and 100 after 12 hours
12
There are several potential advantages of percussion over acid hot water and
mechanical scarification Kelly and van Staden (1987) note that a percussion
treatment only alters macrosclereid cells of the strophiole by loosening separating
and raising them They emphasize that minimal damage is done to the overall
anatomical structure of the seed In acid scarification of the same seed lot radicle
and cotyledon damage occurred Based on photographic evidence the authors
believe that this morphological damage was due to uneven pressure during
imbibition with water entering haphazardly wherever the acid had sufficiently
dissolved the coat This ultimately led to uneven hydration of the embryo In
percussed seeds water only entered through the s~ophiole when the entire seed coat
had been saturated resulting in controlled and even embryo hydration (Kelly and van
Staden 1987)
Although all successful scarification treatments may focus at least in part on
the strophiole for papilionoid legumes (Baskin and Baskin 1998) percussion may be
less damaging to seed coat structures particularly when compared with acid Kelly
and van Staden did not compare hot water or mechanical scarification methods
against percussion However the fungal infection associated with hot water and
mechanical scarification as with acid scarificatio~ have been directly associated with
seed damage (Rehman et al 1998 USDA Forest Service 1948) Embryo damage in
has been observed in hot-water scarified legume seeds (Rehman et al 1998
13
By limiting embryo damage percussion scarification reduces seedling
mortality compared to other treatments Kelly and van Staden (1987) noted that
seedlings from the percussion treatment were morphologically sound and vigorous
when compared to acid treatment seedlings The authors attribute this improved
seedling vigor to the controlled water uptake that limited anatomical damage
Despite a 12-hour percussion treatment morphological damage was not substantially
greater than untreated seeds In addition seedling mass measured 10 days following
germination was not significantly different between percussed and untreated seeds
A final advantage ofpercussion may be the ability to adequately treat the most
dormant seeds while avoiding harm to seeds that have already been made permeable
by the treatment or that were permeable to begin with Instead ofcorroding scalding
or scratching the seed coat percussion appears to be just a loosening of the
strophiole spring (Ballard 1973) Kelly and van Staden (1987) were able to
increase germination with a lengthy treatment (12 hours) Figuring out whether a
substantial cushion oftreatment durations exist for percussion ofNew Mexico locust
seed would be a significant improvement to current scarification methods where seed
damage and seedling health is an issue
The greatest challenge for applying percussion to New Mexico locust seeds is
standardizing a treatment that is both effective and readily reproducible by nurseries
with mechanical scarification this may require the development of specific
~UllDment to properly quantify and execute the treatment
14
f
Objectives
The first objective of the study is to detennine which scarification treatment
results in optimal gennination speed and percentage ofNew Mexico locust The
second objective is to describe treatment interactions with nine seed sources collected
in New Mexico
Hypothesis
A percussion treatment acts exclusively on the main area ofwater uptake in
New Mexico locust seed loosening the strophiole so that imbibition can occur This
direct mechanism ofdonnancy release will result in improved gennination rate and
overall gennination percentage for New Mexico locust seed compared to standard
treatments Standard treatments including acid hot water and mechanical
scarification while eventually weakening the strophiolar cleft cause excessive
morphological damage through generalized seed coat degradation These treatments
are therefore prone to inconsistency due to over and under-treatment
15
MATERIALS AND METHODS
Seed Collection and Cleaning
Seeds were collected from August 25 through September 15 2000 from nine
locations along a latitudinal gradient in New Mexico Seed source locations and
descriptive information are given in Figure 1 and Tables I and 2 Stands were
separated by at least 45 km
Pods were hand picked as they began to ripen and dehisce (open) Collections
were made from ground level to approximately 3 m from several trees at each site
Pods were dried in open-ended brown paper bags for two to four weeks at room
temperature (21-230C) to facilitate cleaning Dried pods were placed in a closed cloth
sack which was pounded against the ground to separate seeds from pods For pods
still containing seeds hand maceration (crunching pods with hands) completed seed
separation Debris was removed by threshing with the aid of a greenhouse fan A
South Dakota Seed Blower (Seedbureau Chicago Illinois) was used to remove
remaining chaff and light seeds by air density separation Seeds were placed in dry
storage in sealed quart-size zip-lock bags within a sealed plastic container Storage
temperatures ranged from 2-4oC Seeds remained in storage for approximately 7
months before the experiment
Experimental Design and Treatment Structure
The experiment compared the impact ofhot water bath and percussion
l11aLl11 on germination properties ofNew Mexico locust seed A single
16
-l
Figure 1 Seed source collection map All sources were collected within New Mexico USA separated by at least 4S lan
IIIRZltItW lIJPIirmiddot rllll1lbullbull_~~~_II
Table 1 Latitude longitude location elevation and collection date ofNew Mexico locust seed sources
Lot title Latitude (N) Longitude (W) Location Elevation (m) Collection Date
Raton 36deg5846 104deg2872 Raton Pass NM 2295 82500
Holman 36deg0306 105deg2429 HolmanNM 2476 911500
Jemez 35deg4500 106deg4100 Jemez Springs NM 1976 9900 00
Sandia 35deg1083 106deg2341 Cedar Crest NM 2432 82600
Manzano 34deg3689 106deg2494 ManzanoNM 2465 9200
Magdalena 33deg5953 107deg0903 Magdalena NM 2544 82900
Capitan 33deg2728 105deg4325 Alto NM 2189 911200
Sacramento 32deg5798 105deg4496 Cloudcroft NM 2630 91400 Gila 32deg5400 107deg4600 Kingston NM 2432 9700
f
Table 2 Mean seed weight and standard error by source
a
Source Mean seed weight (g) for Standard error 100 seeds n = 5
Raton Holman Jemez Sandia Manzano Magdalena Capitan Sacramento Gila
2872 b c 2522 d 1826 e 2753 c 2413 d 2559 d 2443 d 3130 a 3029 ab
00103 00403 00276 00301 00370 00374 00458 00279 00348
a Mean seed weights followed by the same letter are not significantly
different
19
experiment was conducted utilizing a randomized complete block design blocked by
time with a factorial treatment structure Experimental factors were seed source and
scarification treatment Nine seed sources from throughout New Mexico were used
(Figure 1 Table 1) Scarification treatments included a control three levels ofhot
water and four levels of percussion Hot water treatments included immersion at
100degC and boiling for periods of 30 seconds and 60 seconds Percussion treatment
levels were 1 2 4 and 8 minutes One hundred seeds per block were used to test
each treatment combination Four blocks of seed were tested one at a time (all seed
sources included in each run) in close chronological sequence (within 6 weeks) A
total of four 100-seed samples were used to test each source by scarification treatment
combination
Treatments
Hot water treatment of seeds utilized 50 ml test tubes filled with 30 ml
distilled water Each test tube was used to treat one 100-seed sample at a time
resulting in a ratio of4 parts water to 1 part seed by volume Test tubes were placed
in a beaker with the beaker filled with tap water to the same level as the water within
test tubes Nine test tubes (one per seed source) were placed in the beaker
simultaneously so that one replication of each seed sourcescarification treatment
cotnblnatlon was conducted simultaneously Hot water treatments include immersion
boiling For immersion treatments seeds were added to test tubes with the water
(boiling) and the test tubes were immediately removed from the beaker to
For the boiling treatments test tubes remained in the beaker with water at
20
boiling temperature for a set duration after the addition of seeds Following all
treatments test tubes with seeds cooled 24 hours in a test tube rack
Percussion treatment utilized a pneumatic paint shaker (Central Pneumatic
model 00422 Camarillo CA) Compressed air was maintained at 530 kPa plusmn30 kPa
(80 psi plusmn 5 psi) resulting in approximately 350 oscillations per minute Seeds were
placed in 118 ml (4 oz) soil tin cans (US Can Lobard IL) and the lids were secured
with duct tape Each can was used to treat one 100-seed sample To take advantage
ofgreatest lateral movement of the paint shaker a spacer (paint can) was used to
position the soil tin at the end of the shaking arm This allowed shaking to occur at
the greatest distance from the pivot point (fulcrum) Tins were placed perpendicular
to the direction of shaking motion allowing seeds to impact at greatest force by
hitting flat surfaces of the soil tin
Germination Testing
Before gennination testing all seeds were soaked 24 hours in reverse
osmosis-treated water (this was included in the process ofhot water treatment
jU~SCIlOea above) Following soaking seeds were placed in 100 cm Petri dishes lined
90 cm Whatman qualitative 1 filter paper circles (MG Scientific Pleasant
WI) One Petri dish was used for each treatment by source combination
papers were initially moistened with 5 mL reverse osmosis-treated water then
~VU1CUlJl moistened as needed to ensure adequate conditions for gennination
were monitored daily (at 300 PM) for gennination for 14 days in accordance
~UIUlnJlVUa Seed Testing Association (ISTA) standards for Robinia species
21
(1STA 1999) Tests were carried out at room temperature (22degC plusmnOSoC)
Germination was classified as protrusion of the radicle from the embryo by at least
1mm as viewed with the naked eye
X-ray Radiography
On August 132001 three lOO-seed samples from the Sacramento seed source
were treated for 14 or 8-minute percussion durations The seeds were sent
immediately by overnight delivery to the Faxitron X-Ray Corporation in Wheeling
Illinois where Dr Bob Karrfalt ofthe USDA Forest Service oversaw their imaging
Radiographs were made at SX with a Faxitron Specimen Radiography System Model
MX-20
Scanning Electron Microscope Photography
On February 192002 a Hitachi S-3200N Variable Pressure Scanning
Electron Microscope (Mountain View CA) was used to examine strophiolar cells of
untreated seeds and seeds percussed for 4 minutes Seeds were cut longitudinally
igt their entire length using a razor blade splitting the strophiolar region roughly in
Cross sections of seed samples magnifying the strophiolar region were viewed
600X under the microscope
Analysis
Categorical Analysis of Variance was used to determine treatment source and
(Le time) main effects and interactions The response variable was total
~vlI This procedure is a Chi-square test of homogeneity using the natural log
of germinated to non-germinated seed for each treatment Maximumshy
22
likelihood analysis was used to calculate Chi-square test statistics with their observed
significance levels A single contrast was conducted to cempare treatment means
against the control Observed significance levels less than or equal to 005 were
considered significant Pairwise comparisons were conducted between non-control
treatment mean percents using a conservative alp~a value of 005 divided by the
number of comparisons (Bonferroni approach) and approximate pairwise z statistics
Categorical analyses were performed using Proc Catmod (SAS Institute 1989)
RESULTS
Scarification seed source block and all interactions between factors
influenced germination (Table 3) The main effects are reported in the presence of
these interactions because the overall robustness of treatments and seed sources are of
interest Background levels ofnaturally occurring variation in seed sources are
seen in control germination percentages in Figure 2 In particular time variability
is unlikely to be under the control of the producer Appendix B details experimental
data for source by treatment gennination percents for each of the four blocks
Averaged over all treatments germination by source ranged from 40 to
79 (Figure 2) Averaged over all sources (Figure 3) and for each source (Figure 4
5) percussion scarification durations of4 or 8 minutes yielded greatest germination
Averaged over all sources germination increased with increasing durations of
percussion then leveled off for the 4- and 8-minute treatments (Figure 3) Immersion
and 30-second boiling scarification maximized germination for the hot water
treatments with a decrease in germination when seeds were boiled for 60 seconds
For all but two sources there is a similar ipcreasing trend in germination from
1to 4 minutes ofpercussion (Figure 4) The Holman source increased in germination
1 to 2 minutes then leveled off at 4 minutes The Jemez source changed little
1 to 4 minutes of treatment As treatment duration increased from 4 to 8
mutes five sources decreased in germination (Figure 4a) whereas three sources
24
Table 3 Categorical Analysis of Variance table for germination response to seed treatment seed source block and the interaction of the factors
Source of DF Chi-Square Observed Variability Significance Level
Source 8 98370 lt00001 Trt 7 233876 lt00001
contrast 1 16976 lt00001 SourceTrt 56 97077 lt00001 Block 3 5538 lt00001
SourceBlock 24 10162 lt00001 TrtBlock 21 7564 lt00001
SourceTrtBlock 168 34283 lt00001
S 4)
8 ~
0
sectg ~ 40 ~
N 0 0
20
o f0ltgt ~ ~4 jybshy ((iltgto ~~ K1ltgt 6~b-~ ~~ 0b-~~ ~~ ~b-~ ~b-~b
80
60 -I-- - ~- middotmiddotmiddotmiddotmiddot1
r -] Northern NM Sources
Central NM Sources
Southern NM Sources
sect-o ~
~b-v(i
SOURCE
Figure 2 Effect ofNew Mexico locust seed source on germination for data averaged over all scarification treatments Control germination is shown by solid line within bar
80
~ cd ~ ltl) () 60 ~ ~
N
t 0shyrti tshySltl)
40
0
20
o lmmersion Boiling 30 sec Boiling 60 sec Control 1 min 2min 4 min 8 min
Hot Water Treatments Percussion Treatments
Figure 3 Effect of scarification on germination ofNew Mexico locust for data averaged over all seed sources Means labelled with the same letter are not significantly different
100~----------------------~--~----------------~
80
60
ltU
~
40 IJ
sectgsI
l100
80
60
40
a I middotmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot middotImiddotmiddotmiddotmiddotmiddotmiddot E
_~iii-bull _ _ bull f --~--
t-- -~ ---- -shy ---- shyI ~ ---- - shy
L
fiII
~
b
- shy ---r
- Holman
Sacramento Sandia Gila Jemez
c ~
ltgt ~
Tmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot-I
j~=~=-~~-1
I middotmiddot ~ bull r
t tr
Y -------M-a-gd-a-Ie-n-a--
1 _ _
fCapitan
---- Manzano ~y -_ Raton
2 4 8
Time interval ofpercussion scarification (min)
Figure 4 Effect of seed source by treatment interaction on New Mexico locust germination for percussion scarification pattern a and pattern b
28
tv 0
~ d
~ A s o
1 c3
80
60
40
20
o
TIT
t------=middotfmiddotmiddot-middotmiddot-middotmiddot-middotmiddot 1- --- - - --=- - -shy--cE= - _--shy --shy ----shy ~ ~ ---shy ~ -~- _ --shy-------r 1
I
Raton Holman
-_ Jemez
--- Sandia Manzano Magdalena Capitan Sacramento Gila
Immersion Boiling 30 sec Boiling 60 sec
Hot Water Scarification
Figure 5 Effect of seed source by treatment interaction on New Mexico locust germination for hot water scarification
continued to increase in germination (Figure 4b) There was no difference between
the two treatment levels for the Manzano source (Figure 4b)
The effect ofhot water scarification also varied among sources with few
overall differences between hot water treatments (Figure 5) However boiling for 60
seconds yielded highest germination for the Jemez source whereas increasing
durations ofboiling decreased germination for the Raton source
In addition to consistently high germination 4- and 8-minute percussionshy
treated seeds germinated as fast or faster than all other treatments (Figure 6 Table 4)
050 measured as the number of days needed to complete 50 ofeventual
germination averaged 2 days for both ofthese treatments
X-ray radiographs were taken of seeds collected from the Sacramento source
percussed for 1 4 or 8 minutes (Figure 7) Seeds show signs of damage at 8 minutes
ofpercussion (Figure 8) Compared to an undamaged seed percussed for only 4
minutes (Figure 8a) damage at the 8-minute level includes fractured radicles pitting
of the seed coat and chips in seed coat structure (Figure 8b-d)
Scanning electron microscope (SEM) images at 600X reveal the direct effect
of 4 minutes of percussion on cells of the strophiolar region (Figure 9) Whereas
untreated seeds retain cellular organization in the strophiolar region strophiolar cells
seeds treated for 4 minutes ofpercussion separate and raise resulting in a
~-~e~U appearance
30
Umiddot-~middot--l-~---middotymiddotmiddotmiddot-----middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot--middot--middotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middotmiddotmiddotmiddotmiddotmiddotmiddot- -
050= gt1
-e-- Percussion 8 min -0- Percussion 4 min ---T- Percussion 2 min -----ltil- Percussion 1 min -- Immersion --0- Boiling 30 sec -+- Boiling 60 sec -ltgt- Control
2 4 6 8 10 12
Day Figure 6 Timecourse ofNew Mexico locust gennination following scarification
treatments G50 is the number of days to 50 of eventual germination
31
14
Table 4 Effect of seed source and treatment on G50 (days to 50 germination)
Seed Source Control Immersion B30sec B60sec Plmin P2min P4min P8min mean Raton 3 3 5 7 4 3 2 2 4 Holman 2 2 2 2 2 2 2 2 2
Jemez 4 6 6 5 3 2 2 2 4 Sandia 2 2 2 2 2 2 2 2 2
w tv Manzano 5 2 2 3 3 2 2 2 3
Magdalena 4 2 2 3 4 3 2 1 3
Capitan 5 4 2 2 3 2 2 2 3
Sacramento 3 2 2 2 4 3 2 2 3 Gila 3 2 2 2 2 2 2 2 2 mean 3 3 3 3 3 2 2 2 3 standard 11 14 16 18 09 05 0 03 08 deviation
Note Columns and rows may not agree due to rounding
Figure 7 X-ray radiographs ofSacramento seed source percussed for 1 4 or 8 minutes
33
Figure 8a-d Seed damage close-ups Figure a shows undamaged seed percussed for 4 minutes Figures b-d show seeds percussed for 8 minutes Damage includes fracturing ofradicle in b complete removal ofseed coat in c and pitting ofseed coat in d
34
Untreated Treated
Figure 9 SEM images ofuntreated vs 4-minute percussion-treated strophiolar cells ofNew Mexico locust seeds Treated cells are loosened separated and raised
DISCUSSION
Determining the Optimal Treatment
Averaging results of scarification treatmems across seed sources is a useful
tool to predict which treatment is most likely to be successful for an unknown seed
source Averaged over all sources four- and eight-minute percussion treatments
resulted in the highest germination 815 and 790 respectively and produced
germination that was as fast or faster than all other treatments Although there was no
difference between these two treatments on average an argument for 4 minutes of
percussion as a standard protocol for treating New Mexico locust seeds can be made
Descriptively 4 minutes of percussion improved germination over 8 minutes
ofpercussion for a majority of sources (five out of nine) 8 minutes of percussion
improved germination for three ofnine sources with no difference for a fmal source
For individual sources 4 minutes ofpercussion was always the best or secondmiddot best
treatment and was always equal to or better than all hot water treatments
Germination at 8 minutes ofpercussion was lower than all other percussion durations
for the Jemez and Gila sources and was lower than at least one hot water treatment for
these sources as well
The x-ray radiographs of the Sacramento seed source illustrate the excessive
internal and external seed damage that can occur with 8 minutes of percussion
germination counts 8-minute percussed seeds from Holman and Capitan
ClUll signs of damage that were visible to the naked eye (see below)
r~evertheless some of these seeds still fit the criteria for germination Neither x-ray
36
photography nor visible observations during seed counts revealed seed damage at the
4-minute percussion level In operational trials seeds percussed for 4 minutes
developed into healthy seedlings (Khadduri and Harrington unpublished data)
Strophiole and Seed Coat Behavior
Four minutes ofpercussion successfully breaks seed dormancy in a large
percentage ofNew Mexico locust seeds without excessively damaging the
surrounding seed coat or internal seed coat structures Most papilionoid legume
seeds including seeds ofNew Mexico locust have a specific region of the seed coat
known as the strophiole or lens (Hamly 1932) The strophiole is located on the
cotyledonary lobe of a locust seed (Figure 10 a-c) If seeds are not indiscriminately
damaged the strophiole is the first point of water entry to the embryo (Hopkinson
1997) When a papilionoid seed is percussed for the appropriate amount of time
repeated hits on the integrated seed coat loosen the constrained cells of the strophiolar
region without excessively damaging the rest of the seed coat (Ballard 1976) SEM
photos illuStrate that 4 minutes of percussion loosened and separated strophiolar cells
ofNew Mexico locust seeds when compared with constrained organized strophiolar
cells of untreated seeds (Figure 9) Research-has shown that water passes to the
embryo through the treated strophiole in a controlled manner and regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues
(Kelly and van Staden 1987)
37
10 Strophiole ofunimbibed New Mexico locust seed (a) and following uptake (bc)
38
In contrast hot-water treatments can excessively degrade the seed coat
(Rebman et al 1998 Marunda 1990) Random cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rebman et aI 1998 Marunda 1990) Along with a
proportionately large amount ofun-imbibed (thus under-treated) seeds (Figure l1a)
damage was observed in hot-water treated seeds from six sources during germination
counts Damage included seeds where the radicle failed to elongate (Figure 11 b)
seeds where the radicle elongated but was delayed in freeing itself from the seed coat
(Figure 11c) and seeds where the radicle completely broke off from the embryo
(Figure lId) In addition some seeds germinated in reverse (the cotyledon emerged
fIrst) If the radicle emerged completely from the seed coat they were counted as
germinated
While nearly all seeds treated for 8 minutes ofpercussion imbibed water
damage was evident in x-ray evaluations (Sacramento seed source) and in
germination counts (Holman and Capitan) at this treatment level Seed damage
during germination counts was similar to levels found in hot-water treated seeds
suggesting that over-treatment by both hot water and longer durations of percussion
may result in similar damage to seed structure
The Sacramento and Holman seed sources were two of the fIve sources that
opcrplltlpn in germination from the 4- to 8-minute durations of percussion
the benefIts ofloosening the strophiole as evidenced by imbibition of
all 8-minute treated seeds were outweighed by external and internal seed coat
39
a
b c d
11 Damage to New Mexico locust seeds observed during germination cotmts following hot water scarification 9a Hot water replicate including ~t seedlings and numerous unimbibed seeds 9b Close-up of seed where raallCle failed to elongate 9c Close-up ofseed where radicle elongated but was
-gtIn in freeing itself from the seed coat 9d Close-up ofseed where radicle broke off from embryo
40
damage (which also may be contributing to high imbibition) For the three sources
for which 8 minutes of percussion resulted in maximum germination extended
percussion of seeds necessary to loosen the strophiole may not have excessively
damaged the seed anatomy The Manzano source did not significantly change in
germination from 4 to 8 minutes of percussion suggesting that an increase in
permeable seeds at the 8-minute treatment duration was balanced by the number of
seeds that failed to germinate due to seed damage To summarize the 4-minute
percussion treatment improved germination over 8 minutes ofpercussion for a
majority of sources (five ofnine) the 8-minute percussion treatment improved
germination over 4-minutes ofpercussion for three sources and there was no
difference with the Manzano source Notably for the Raton source 8 minutes of
percussion increased germination by 235 percentage points over 4-minutes of
percussion (7875 vs 5525 respectively) the next best treatment Raton was the
only source to result in germination less than 75 for 4 minutes ofpercussion so a
grower may wish to apply longer percussion durations to lots that do not respond well
to this treatment level
Finally there was an interesting trend in the Jemez and Raton sources seed
that significantly contributed to the source by treatment interaction (Figure 12) A
~u test showed that the Jemez source had a significantly lighter seed weight than
other sources while the Raton source had an average seed weight For the Jemez
the longest duration of boiling increased germination Conversely only short
U4UVll) of percussion were needed to achieve high germination and the longest
41
80 _ Jemez a Raton
iD
~ C iD e iD
Q c 0 ~
c ~ iD 0
20
0------shy
Immersion Boiling 30 sec Boiling 60 sec
Hot Water Scarification
10degTr========~--------~------------------------------
80
60
40
20
_ Jemez Raton
0------shy
1 min 2 min 4 min 8 min
Percussion Scarification
Figure 12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources
42
b
--~----~~~~========~---- duration ofpercussion reduced germination For the Raton source longer durations
ofboiling consistently decreased germination whereas longer percussion treatments
consistently increased germination
A possible explanation is that a thick seed coat is associated with a loose
strophiole structure and that a thin seed coat is associated with a constrained
strophiole This contrast merits further study and may suggest an ecological
adaptation Further investigation ofthis phenomenon may contribute to successful
pre-screening ofunknown seed sources to determine optimal treatment leveL
43
CONCLUSION
Based on results obtained in pilot study (see Appendix) and thesis
experiments 4 minutes ofpercussion is recommended as a standard protocol for
treating an unknown source ofNew Mexico locust seed In addition to laboratory
results the 4-minute percussion treatment has resulted in germination greater than
80 and healthy seedling grow-outs for production purposes (unpublished data) In
laboratory experiments only one hundred seeds per 118 ml (40z) soil tin were
percussed at a time whereas for production we have treated up to five hundred seeds
per tin For large-scale production ofNew Mexico locust seedlings growers may
wish to experiment with larger quantities of seed and larger-size soil tins
Seed storage trials following percussion treatments were not conducted
However germination greater than 80 and healthy seedlings were obtained when
seeds were percussion-treated and subsequently stored at room-temperature (22degC)
for one week (unpublished data) Seed storage trials merit further study as the
standard hot water treatment leaves seeds in a softened condition that is not
conducive to storage
Hydroseeding is an effective method for direct seeding in reclamation
situations Percussion treatment may be conducive to hydro seeding as seeds remain
a dry relatively undamaged yet permeable condition following treatment Sulfuric
seeds also remain dry following treatment but extensive damage to seed coat
poor seedling vigor have been reported with this treatment (Kelly and van Staden
A grow-out study using a hydro seeder on a reclamation site testing percussion
44
against other treatments such as sulfuric acid and mechanical scarification also
merits further study
Papilionoids are the largest subfamily oflegumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust these legumes often play an integral role in the revegetation of
disturbed lands The pilot study (Appendix) indicates that percussion scarification
successfully treats the widely used black locust as well Developing superior
scarification methods to the standard hot water mechanical scarification and acid
treatments may facilitate the use ofpapilionoid legumes in reclamation projects
45
APPENDICES
APPENDIX A
PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR
NEW MEXICO LOCUST AND BLACK LOCUST SEEDS
Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4
Hot water and sulfuric acid soaks are traditional treatments for seeds of many
temperate woody legwnes including locusts However these scarification techniques
often produce inconsistent germination Percussion scarification where seeds are
repeatedly propelled against a hard surface was compared with hot water
scarification to evaluate treatment efficacy for New Mexico locust (Robinia
neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment
seeds were placed in a 98degC water bath which was immediately removed from the
heat source For percussion scarification seeds were placed in a soil sample tin and
agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the
control were followed by 24-hour water soaks Hot water baths resulted in 56 and
41 germination for New Mexico locust and black locust respectively For both
species nearly all durations ofpercussion increased germination over the hot water
treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3
4 and 5 minutes for black locust resulted in at least 90 germination Traditional
scarification treatments randomly degrade the entire seed coat which can lead to
tissue damage during water uptake Percussion scarification specifically weakens the
strophiole the natural source ofwater entry to the seed in papilionoid legwnes
INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom
2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031
48
Following percussion imbibition is controlled through the strophiole and underlying
tissue is protected
Keywords New Mexico locust black locust scarification percussion germination
Introduction
Locust trees (Robinia spp) are aggressive pioneer species that quickly
colonize disturbed land fix atmospheric nitrogen in the soil and stabilize slopes that
are prone to erosion (Klemmedson 1994 Ashby et al 1985) (Figure A1ab)
Recognition of these attributes has contributed to increased demand for locusts in
reclamation projects Locust seeds however exhibit physical dormancy where a
thick seed coat prevents the movement of water and gasses to the embryo (Leadem
1997)
Sulfuric acid or hot water soaks have traditionally been used to break seed
dormancy in macro-propagation of locusts Many growers are moving away from the
use of su1furic acid in treating locust seeds (Dreesen and Harrington 1997) In
addition to being dangerous acid soak durations must be specifically correlated to
seed lot (Olson 1974) Collections of native seed can vary tremendously in seed size
seed weight and hard seededness etc This variability limits the utility of sulfuric
acid soaks as even within a given lot some seeds may be damaged whereas others
are under-treated and fail to imbibe (take up water) A survey of Southwestern
container growers indicates that hot water baths are currently the most common
scarification method for locust seeds (Hine et al 1997) However hot water baths
also produce inconsistent germination (Lin et al 1996)
49
b
Figure AI New Mexico locust a primary invader following disturbance Ala Crown-sprouting at edge ofburn near Los Alamos NM--one year after Cerro Grande fire Alb Colonizing road cut Gila National Forest NM
50
Percussion scarification where seeds are repeatedly propelled against a hard
surface is an alternative dormancy-breaking method that has proven successful in
legumes related to locusts (Hamly 1932 Barton 1947) We compared this alternative
treatment to hot water scarification to evaluate treatment efficacy for New Mexico
locust and black locust seeds
Materials and Methods
Commercial New Mexico locust seeds (Western Native Seed Coaldale CO)
were collected fall 2000 in Huerfano County CO Black locust seeds were collected
September 2000 from Taos County NM by harvesting ripe pods from several trees at
a distance of up to 3 m from ground level Black locust seeds were allowed to air-dry
for 3 weeks and were threshed and separated from large chaffusing a greenhouse
fan A South Dakota Seed Blower (Chicago Illinltois) was used to remove fine chaff
from both seed lots Seeds were storedmiddotat 2-4C until the germination study was
conducted in March 2001
This study compared hot water scarification with percussion scarification
using treatment and species as experimental factors Along with a control
scarification treatments included immersion of seed in boiling water as well as
percussion durations of 1 2 3 45 or 10 minutes Four 100-seed samples were used
to test each species by scarification treatment combination
For the hot water treatment test tubes were filled with 30 mL water and
placed in a water-filled beaker (Figure A2a) The bath was raised to boiling (-98C at
51
b
c
Figure A2 Implementation ofhot water and percussion scarification A2a Hot water bath A2b Paint-shaker set-up A2c Placing soil tin containing seeds at end ofpaint shaker ann
52
Las Cruces NM elevation of 1300 meters) Each 100-seed replicate was placed in a
test tube and each test tube was immediately removed to cool at room temperature
Percussion scarification was implemented using a pneumatic paint shaker
(Central Pneumatic model 00422 Camarillo CA) (Figure A2b) This allowed
standardization of treatment intensity Compressed air pressure was maintained at
530kPa plusmn 30kPa (80psi plusmn 5psi) resulting in approximately 350 oscillations per
minute Each lOO-seed sample was placed in a 118 ml (4 oz) soil tin for shaking To
maximize lateral movement of paint shaker a paint can was used as a spacer to place
soil tins at the end of the shaking arm (Figure A2c) This allowed shaking to occur at
the greatest distance from the pivot point (fulcrum) Following all treatments
including the control seeds were soaked for twenty-four hours in distilled water prior
to plating out
Gennination was evaluated on lab benches at room temperature Each
replicate of seeds was placed in a 100 cm petri dish on moistened filter paper with
humidity maintained by enclosing the petri dishes in ziplock bags Gennination was
monitored daily for 14 days in accordance with International Seed Testing
Association standards for Robinia species (ISTA 1999) Gennination was defined as
protrusion of the radicle from the embryo by at least 1mm as viewed by the naked
eye
Categorical Analysis of Variance using Prpc Catmod (SAS Institute 1989)
was used to detennine treatment differences in germination percentages for each
source This procedure is an extension of a Chi-square test of homogeneity using the
53
natural log of the ratio of germinated to non-germinated seeds for each treatment Pshy
values less than 005 were considered significant A limited set of pairwiseshy
comparisons was conducted to compare treatment mean percents using a
conservative alpha value of 005 divided by the number of comparisons
Results
Percussion maximized germination for both New Mexico locust and black
locust There were three germination response levels for New Mexico locust (Figure
A3) First there was a low response in the control indicating a dormant seed lot
The hot water treatment and I-minute duration of percussion share a second
intermediate response Germination was highest at percussion levels from 2 to 10
minutes
A selected percussion treatment intermediate in time (5 minutes) produced the
steepest germination curve indicating the fastest germination rate (Figure A4) The
hot water treatment produced an intermediate curve or relatively slower germination
rate The control curve was very flat indicating the slowest germination rate for New
Mexico locust
Control seeds germinated poorly for black locust as well (Figure A5) Hot
water scarification improved germination over the control but all levels of percussion
improved germination over hot water The optimal durations of percussion treatment
were bracketed with a significant increase from the 1 to 2 minute percussion level a
high germination response from 2 to 5 minutes of percussion and finally a significant
54
~ d 25 CL)
p t1 0 ~ t1
g Vl ~ Vl
100~---------------------------------------------------------------
80
60
40
20
o Control Hot Water Percussion
I min Percussion
2 min Percussion
3 min
Scarification
Percussion 4 min
Figure A3 Effect of scarification on germination response for New Mexico locust
Percussion 5 min
=significant difference between adjacent treatment bars ns = no significant difference
Percussion 10 min
~ ~ () u agt
Il-t d o ~
~ ~ c3
100middotlr~~~------------------------------------e- Percussion 5min middot 0middot Hot Water -- Control
80-J=middot======
6b-middot-lmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middot--middotmiddot-middot~-middotmiddot -middotmiddot middot middotmiddot-middot 1
40
20
-----~-----------
0 0 0 middotmiddotmiddotmiddotmiddot0middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot0
0 --~-----y-----~------_---~--- T-----~
o shy -TDay 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A4 Effect of selected scarification treatments on germination speed for New Mexico locust First seven days of fourteen-day experiment are shown
Day 7
80
~ cd
5 60 ()
[) Cl d 0 1t1 d
40sect dUl
-J
20
o
ns
Scarification
Figure A5 Effect of scarification on germination response for black locust = significant difference between adjacent treatment bars ns = no significant difference
decrease at the 10-minute level This drop-off suggests that 10 minutes of percussion
over-treats seeds from this lot of-black locust
A selected percussion treatment intermediate in time (5 minutes) also had the
fastest germination rate for black locust (Figure A6)_ The hot water curve is
somewhat flatter indicating a slower germination rate The control curve is very flat
indicating a slow germination rate
A qualitative difference between hot water and percussion-treated seeds was
also observed Along with healthy germinants the hot water treatment produced
over-treated seeds (Figure A 7a) Examples of damaged germinants include seeds
where the radicle did not elongate (Figure A 7b) seeds where the radicle elongated
but was delayed in freeing itself from the seed coat (Figure A7c) and seeds where the
radicle broke off completely from the embryo (Figure A7d) In addition to these
examples of over-treatment there were numerous small dark seeds that failed to
imbibe water For percussion treatment nearly all seeds took up water (Figure A8a)
Germination was rapid and uniform with little damage evident (Figure A8b)
To summarize both New Mexico locust and black locust seed lots were
dormant The standard hot water scarification treatment improved germination but
only to 56 and 41 for New Mexico locust and black locust respectively Nearly
all percussion durations improved total germination as well as germination rate over
the hot water treatment Germination was greater than 90 for percussion durations
of 4 5 and 10 minutes for New Mexico locust and 3 4 and 5 minutes for black locust
58
CI)
sgt t CI) 0
igt 0
sect ttl t E
Vl 0
c3
100r==~==~~=r------------------------------------~ ___ Percussion 5 min
middotmiddotmiddot0middotmiddotmiddot Hot Water -lIf- Control
80r=========~--~~----------------------------~
60
40
20 -~-~---~---------- ---~------------I
omiddot
O
_~_----------lIf-----~bull -----_ shy---shy I
Day 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A6 Effect of selected scarification treatments on germination speed for black locust First seven days of fourteen-day experiment are shown
Day 7
a
b c d
Figure A7 Damage to New Mexico locust seeds observed during germination counts following hot water scarification A7a Hot water replicate including damaged seedlings and numerous unimbibed seeds A7b Close-up ofseed where radicle failed to elongate A7c Close-up ofseed where radicle elongated but was delayed in freeing itself from the seed coat A7d Close-up ofseed where radicle completely broke offfrom embryo
60
a
b
Figure A8 New Mexico locust seeds following percussion treatment at time of germination counts A8a Percussion replicate with uniformly healthy seedlings and near complete imbibition of seeds A8b Close-up ofpercussion-treated seedling
61
Discussion
Papilionoid seeds have a specific anatomical feature known as the strophiole
or lens-the natural site of water entry to the seed (Hamly 1932) The strophiole is
located on the cotyledonary lobe of a locust seed (Figure A9) When a papilionoid
legume seed is percussed for the appropriate amount of time repeated hits on the
integrated seed coat loosen the constrained cells of the strophiolar region without
excessively damaging the rest of the seed coat (Ballard 1976)
When a percussed seed is soaked water enters exclusively through the
strophiole in a controlled manner (Kelly and Staden 1987) This regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues This
contrasts with seeds that have been hot water or acid scarified treatments that can
randomly degrade the seed coat Unlocalized cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rehman et al 1998 Marunda 1990)
Papilionoids are the largest subfamily of legumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust and black locust these legumes often play an integral role in the
revegetation ofdisturbed lands Developing superior scarification methods to the
standard hot water and acid treatments should fadlitate the use ofthese legumes in
restoration projects
62
Figure A9 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (bc)
63
APPENDIXB
GERMINATION PERCENTS AND STANDARD ERRORS FOR SOURCE
BLOCK AND TREATMENT FOR NEW MEXICO LOCUST
Table B 1 Gennination percents and standard errors for source block and treatment for New Mexico locust
Source Block Treatment Germination Percentage Standard Error
Capitan 1 Immersion 74 437 Capitan 2 Immersion 69 462 Capitan 3 Immersion 71 454 Capitan 4 Immersion 67 470 Capitan Boiling 30 sec 79 407 Capitan 2 Boiling 30 sec 76 427 Capitan 3 Boiling 30 sec 72 449 Capitan 4 Boiling 30 sec 76 427 Capitan Boiling 60 sec 71 454 Capitan 2 Boiling 60 sec 54 498 Capitan 3 Boiling 60 sec 67 470 Capitan 4 Boiling 60 sec 76 427 Capitan I Control 5 218 Capitan 2 Control 7 255 Capitan 3 Control 4 196 Capitan 4 Control 5 218 Capitan 1 Percussion I min 47 499 Capitan 2 Percussion I min
470
Capitan Percussion I min 39 488
Capitan 4 Percussion I min 38 485 Capitan I Percussion 2 min 55 497 Capitan Capitan
2
Percussion 2 min Percussion 2 min
71 65
454 477
Capitan 4 Percussion 2 min 56 496 Capitan I Percussion 4 min 86 347 Capitan 2 Percussion 4 min 79 407 Capitan 3 Percussion 4 min 81 392 Capitan 4 Percussion 4 min 82 384 Capitan I Percussion 8 min 90 300 Capitan 2 Percussion 8 min 88 325 Capitan 3 Percussion 8 min 90 300 Capitan 4 Percussion 8 min 90 300 Gila I Immersion 71 454 Gila 2 Immersion 77 421 Gila
Immersion 75 433
Gila 4 Immersion 72 449
Gila I Boiling 30 sec 68 466
Gila 2 Boiling 30 sec 81 392
Gila 3 Boiling 30 sec 54 498
Gila 4 Boiling 30 sec 63 483
Gila I Boiling 60 sec 55 497
Gila 2 Boiling 60 sec 68 466
Gila 3 Boiling 60 sec 57 495
Gila 4 Boiling 60 sec 70 458
65
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Gila 2 Control 13 336 Gila 3 Control 13 336 Gila 4 Control 20 400 Gila 1 Percussion 1 min 66 474 Gila 2 Percussion 1 min 57 495 Gila 3 Percussion 1 min 73 444 Gila 4 Percussion 1 min 82 384 Gila 1 Percussion 2 min 81 392 Gila 2 Percussion 2 min 86 347 Gila 3 Percussion 2 min 85 357 Gila 4 Percussion 2 min 54 498 Gila 1 Percussion 4 min 79 407 Gila 2 Percussion 4 min 83 376 Gila 3 Percussion 4 min 84 367 Gila 4 Percussion 4 min 79 407 Gila 1 Percussion 8 min 51 500 Gila 2 Percussion 8 min 76 427 Gila 3 Percussion 8 min 52 500 Gila 4 Percussion 8 min 74 439 Holman 1 Immersion 83 376 Holman 2 Immersion 86 347 Holman 3 Immersion 81 392 Holman 4 Immersion 90 300 Holman 1 Boiling 30 sec 90 300 Holman 2 Boiling 30 sec 85 357 Holman 3 Boiling 30 sec 87 336 Holman 4 Boiling 30 sec 83 376 Holman 1 Boiling 60 sec 79 407 Holman 2 Boiling 60 sec 80 400 Holman 3 Boiling 60 sec 86 347 Holman 4 Boiling 60 sec 93 255 Holman Control 25 433 Holman 2 Control 19 392 Holman Control 21 407
Holman 4 Control 24 427
Holman Percussion 1 min 77 421
Holman 2 Percussion 1 min 85 357
Holman 3 Percussion 1 min 80 400
Holman 4 Percussion 1 min 89 313
Holman 1 Percussion 2 min 88 325
Holman 2 Percussion 2 min 96 196
Holman 3 Percussion 2 min 93 255
Holman 4 Percussion 2 min 96 196
Holman 1 Percussion 4 min 92 271
Holman 2 Percussion 4 min 97 171
66
I
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Holman 3 Percussion 4 min 91 286 Holman 4 Percussion 4 min 96 196 Holman 1 Percussion 8 min 88 325 Holman 2 Percussion 8 min 85 357 Holman 3 Percussion 8 min 83 376 Holman 4 Percussion 8 min 82 384 Jemez 1 Immersion 53 499 Jemez 2 Immersion 44 496 Jemez 3 Immersion 52 500 Jemez 4 Immersion 70 458 Jemez 1 Boiling 30 sec 58 494 Jemez 2 Boiling 30 sec 55 497 Jemez 3 Boiling 30 sec 48 500 Jemez 4 Boiling 30 sec 65 477 Jemez 1 Boiling 60 sec 68 466 Jemez 2 Boiling 60 sec 63 483 Jemez 3 Boiling 60 sec 73 444 Jemez 4 Boiling 60 sec 89 313 Jemez 1 Control 2 140 Jemez 2 Control 9 286 Jemez 3 Control 6 237 Jemez 4 Control 8 271 Jemez 1 Percussion 1 min 58 494 Jemez 2 Percussion 1 min 63 483 Jemez 3 Percussion 1 min 77 421 Jemez 4 Percussion 1 min 89 313 Jemez 1 Percussion 2 min 68 466 Jemez 2 Percussion 2 min 64 480 Jemez 3 Percussion 2 min 80 400 Jemez 4 Percussion 2 min 83 376 Jemez Percussion 4 min 68 466 Jemez 2 Percussion 4 min 79 407 Jemez 3 Percussion 4 min 70 458 Jemez 4 Percussion 4 min 83 376 Jemez 1 Percussion 8 min 52 500 Jemez 2 Percussion 8 min 59 492 Jemez 3 Percussion 8 min 61 488 Jemez 4 Percussion 8 min 70 458 Magdalena 1 Immersion 69 462 Magdalena 2 Immersion 69 462 Magdalena J Immersion 70 458 Magdalena 4 Immersion 85 357 Magdalena 1 Boiling 30 sec 65 477 Magdalena 2 Boiling 30 sec 71 454 Magdalena 3 Boiling 30 sec 68 466
67
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Magdalena 4 Boiling 30 sec 83 376 Magdalena 1 Boiling 60 sec 63 483 Magdalena 2 Boiling 60 sec 68 466 Magdalena 3 Boiling 60 sec 63 483 Magdalena 4 Boiling 60 sec 82 384 Magdalena 1 Control 11 313 Magdalena 2 Control 6 237 Magdalena 3 Control 5 218 Magdalena 4 Control 4 196 Magdalena Percussion 1 min 32 466 Magdalena 2 Percussion 1 min 45 497 Magdalena 3 Percussion 1 min 46 498 Magdalena 4 Percussion I min 36 480 Magdalena 1 Percussion 2 min 67 470 Magdalena 2 Percussion 2 min 68 466 Magdalena 3 Percussion 2 min 65 477 Magdalena 4 Percussion 2 min 81 392 Magdalena 1 Percussion 4 min 87 336 Magdalena 2 Percussion 4 min 97 171 Magdalena 3 Percussion 4 min 83 376 Magdalena 4 Percussion 4 min 97 171 Magdalena 1 Percussion 8 min 98 lAO Magdalena 2 Percussion 8 min 100 000 Magdalena 3 Percussion 8 min 96 196 Magdalena 4 Percussion 8 min 98 lAO Manzano 1 Immersion 67 470 Manzano 2 Immersion 57 495 Manzano 3 Immersion 72 4049 Manzano 4 Immersion 73 4044 Manzano 1 Boiling 30 sec 70 458 Manzano 2 Boiling 30 sec 64 480 Manzano 3 Boiling 30 sec 71 454 Manzano 4 Boiling 30 sec 80 400 Manzano I Boiling 60 sec 50 500 Manzano 2 Boiling 60 sec 53 499 Manzano 3 Boiling 60 sec 59 492 Manzano 4 Boiling 60 sec 74 439 Manzano I Control 4 196 Manzano 2 Control 8 271 Manzano 3 Control 1 099 Manzano 4 Control 6 237 Manzano 1 Percussion 1 min 52 500 Manzano 2 Percussion 1 min 39 488 Manzano 3 Percussion 1 min 36 480 Manzano 4 Percussion 1 min 51 500
68
Table B 1 Continued
Source Block Treatment Gerrni~ation Percentage Standard Error
Manzano Percussion 2 min 53 499 Manzano 2 Percussion 2 min 58 494 Manzano 3 Percussion 2 min 61 488 Manzano 4 Percussion 2 min 62 485 Manzano 1 Percussion 4 min 76 427 Manzano 2 Percussion 4 min 85 357 Manzano 3 Percussion 4 min 79 407 Manzano 4 Percussion 4 min 83 376 Manzano 1 Percussion 8 min 87 336 Manzano 2 Percussion 8 min 83 376 Manzano 3 Percussion 8 min 83 376 Manzano 4 Percussion 8 min 75 433 Raton 1 Immersion 54 498 Raton 2 Immersion 37 483 Raton 3 Immersion 39 488 Raton 4 Immersion 69 462 Raton 1 Boiling 30 sec 36 480 Raton 2 Boiling 30 sec 20 400 Raton 3 Boiling 30 sec 28 449 Raton 4 Boiling 30 sec 60 490 Raton 1 Boiling 60 sec 15 357 Raton 2 Boiling 60 sec 171 Raton 3 Boiling 60 sec 4 196 Raton 4 Boiling 60 sec 35 477 Raton 1 Control 4 196 Raton 2 Control 12 325 Raton 3 Control 2 140 Raton 4 Control 6 237 Raton 1 Percussion 1 min 52 500 Raton 2 Percussion I min 29 454 Raton 3 Percussion I min 35 477 Raton 4 Percussion 1 min 31 462 Raton 1 Percussion 2 min 55 497 Raton 2 Percussion 2 min 35 477 Raton 3 Percussion 2 min 33 470 Raton 4 Percussion 2 min 42 494 Raton 1 Percussion 4 min 65 477 Raton 2 Percussion 4 min 52 500 Raton Percussion 4 min 51 500 Raton 4 Percussion 4 min 53 499 Raton 1 Percussion 8 min 88 325 Raton 2 Percussion 8 min 80 400 Raton 3 Percussion 8 min 73 444 Raton 4 Percussion 8 min 74 439 Sacramento 1 Immersion 43 495
69
Table B 1 Continued
-Source Block Treatment Germination Percentage Standard Error
Sacramento 2 Immersion 30 458 Sacramento 3 Immersion 38 485 Sacramento 4 Immersion 56 496 Sacramento 1 Boiling 30 sec 47 499 Sacramento 2 Boiling 30 sec 48 500 Sacramento 3 Boiling 30 sec 48 500 Sacramento 4 Boiling 30 sec 56 496 Sacramento 1 Boiling 60 sec 52 500 Sacramento 2 Boiling 60 sec 67 470 Sacramento 3 Boiling 60 sec 43 495 Sacramento 4 Boiling 60 sec 48 500 Sacramento 1 Control 0 000 Sacramento 2 Control 3 171 Sacramento 3 Control 3 171 Sacramento 4 Control 099 Sacramento 1 Percussion 1 min 57 495 Sacramento 2 Percussion 1 min 61 488 Sacramento 3 Percussion 1 min 62 485 Sacramento 4 Percussion 1 min 55 497 Sacramento 1 Percussion 2 min 84 367 Sacramento 2 Percussion 2 min 70 458 Sacramento 3 Percussion 2 min 81 392 Sacramento 4 Percussion 2 min 69 462 Sacramento 1 Percussion 4 min 91 286 Sacramento 2 Percussion 4 min 84 367 Sacramento 3 Percussion 4 min 84 367 Sacramento 4 Percussion 4 min 94 237 Sacramento 1 Percussion 8 min 82 384 Sacramento 2 Percussion 8 min 81 392 Sacramento 3 Percussion 8 min 82 384 Sacramento 4 Percussion 8 min 81 392 Sandia 1 Immersion 76 427 Sandia 2 Immersion 70 458 Sandia 3 Immersion 70 458 Sandia 4 Immersion 81 392 Sandia 1 Boiling 30 sec 78 414 Sandia 2 Boiling 30 sec 71 454 Sandia 3 Boiling 30 sec 63 483 Sandia 4 Boiling 30 sec 78 414 Sandia 1 Boiling 60 sec 67 470 Sandia 2 Boiling 60 sec 65 477 Sandia 3 Boiling 60 sec 74 439 Sandia 4 Boiling 60 sec 66 474 Sandia 1 Control 16 367 Sandia 2 Control 7 255
70
Table B 1 Continued
Source Block Treatment Gennination Percentage Standard Error
Sandia 3 Control 8 271 Sandia 4 Control 16 367 Sandia 1 Percussion 1 min 67 470 Sandia 2 Percussion 1 min 61 488 Sandia 3 Percussion 1 min 66 474 Sandia 4 Percussion 1 min 59 492 Sandia I Percussion 2 min 82 384 Sandia 2 Percussion 2 min 83 376 Sandia 3 Percussion 2 min 81 392 Sandia 4 Percussion 2 min 84 367 Sandia 1 Percussion 4 min 89 313 Sandia 2 Percussion 4 min 86 347 Sandia 3 Percussion 4 min 89 313 Sandia 4 Percussion 4 min 85 357 Sandia 1 Percussion 8 min 80 400 Sandia 2 Percussion 8 min 77 421 Sandia 3 Percussion 8 min 69 462 Sandia 4 Percussion 8 min 66 474
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Marunda CT 1990 Effects of seed pretreatment on the development ofAcacia auriculforinis and A holosericea seedlings ACIAR Proceedings Series No 28
Meginnis HG 1937 Sulphuric acid treatment to increase germination of black locust seed USDA Circ 453 34 p
Miklas PN Towensend CE Ladd SL 1987 Seedcoat anatomy and the scarification ofcicer milkvetch seed Crop Sci 27 766-772
Monsen SB 1984 Use of shrubs on mine spoils In The Challenge ofProducing Native Plants for the Intermountain Area Proceedings Intermountain Nurserymans Association 1983 conference Las Vegas NV compo PM Murphy 26-31 Ogden UT USDA For Ser Gen Tech Report INT-l68
Olson DF 1974 Robinia L Locust In Seeds of Woody Plants in the US tech coord CS Schopmeyer 728-731 USDA For Ser Agr Handbk No450
Rehman S Loescher RNl Harris P1C 1998 Dormancy breaking and germination ofAcacia salicina Lindl seeds Seed Sci amp Techno127 553-557
Rolston MP 1978 Water impermeable seed dormancy Bot Rev 44 365-396
74
SAS Institute Inc 1989 SASSTAT Users Guide Version 6 4th Edition Volume 1 Cary NC SAS Institute Inc 943 p
SerratoValenti G Conara L Ferrando M Modenesi P 1993 Structural and histochemical features ofStylosanthes scabra (Leguminosae Papilionoidae) seed coat as related to water entry Can J Bot 71 834-840
Simpson BJ 1988 A field guide to Texas trees Austin TX Texas Monthly Press 372p
Tran VN Cavanagh AK 1984 Structural aspects ofdormancy In Seed Physiology Vol 2 Murray DR (ed) p1-44 Academic Press Australia
USDA Forest Service 1948 Woody-plant seed manual USDA Misc Pub1654 416p
Vines RA 1960 Trees Shrubs and Woody Vines of the Southwest Austin Univ ofTexas Press 1104 p
Wagner MR Larson RE Craig TP 1992 Ecology and silvics ofNew Mexico locust (Robinia neomexicana) with an emphasis on the arboreal form p53-57 In JW Hanover K Miller and S Plesko (eds) Proc Intemat Confon Black Locust Biology Culture and Utilization Mich State Univ USA
Wilson JK 1944 Immersing seeds of species of Robinia in boiling water hastens germination J For 42 453-454
Zimmerman RW Carpenter SB 1980 First-year coppice production from a 5shyyear old black locust stand on surface mine spoil p309-314 Centr Hardwd For ConfProc University ofMissouri Columbia
temperature was increased from 70degC to 100degC overall germination in New Mexico
locust seed increased The 100degC treatment maximized germination for each of five
sources tested with germination usually above 60
Prolonged exposure of seeds ofmany woody legumes to boiling temperatures
also may improve germination (Briscoe 1996) This approach has not been tested on
New Mexico locust seed Exposing seed to prolonged boiling temperatures may
result in fungal infection and subsequent seedling mortality Marunda (1990) noted
severe fungal infections on both acid and hot water treated Acacia holoserica Cunn
ex G Don seeds attributing the infections to seed damage resulting from the
treatments Identifying an optimal hot water treatment level that does not result in a
high rate of seedling mortality is a logical goal
With hot water treatments as with acid there is also a danger of overtreating
Seeds and reducing germination (Gosling et al 1995) The legume Leucaena
leucocephala (Lam) de Wit germinated equally well after 7 seconds of exposure to
water at 100degC 7 seconds at 90degC 30 seconds at 80degC and 4 minutes at 70degC In
addition acceptably high germination was obtained from treatment at 70degC from 3
minutes to 1 hour whereas no seeds germinated following treatment at 100degC for 2
minutes Perhaps along with a greater margin of error for treatment duration treating
~ew Mexico locust seeds at a lower temperature may result in a comparatively lower
of seedling mortality In practice however nursery growers tend to use the
and quickest treatment available The short treatment length at near-boiling
whether recommended or not may be what is used most frequently
10
Fine-tuning a treatment in this range of treatment temperature and duration may be
the most logical approach
Mechanical Scarification
This treatment involves scratching the seed coat with an abrasive material
(USDA Forest Service 1948) One system employs a rotating drum lined with
sandpaper A paddle spinning at a given number ofrevolutions per minute throws the
seed against the sandpaper (Hine et al 1997) A second method dry tumbling
tumbles seed in a mixer with pea gravel or sharp sand for 5 to 7 days (Dreesen 2001)
These approaches have the advantage that control of temperature is not required as in
water treatments nor is safety an issue as with add scarification
Hine et al (1997) mechanically treated New Mexico locust seed using 100shy
grit sandpaper in a Forsberg (rotating drum) scarifier Gennination was less than
10 They concluded that the seed coats were too thin for their apparatus and
proposed that a finer grit sandpaper would produce more even abrasion Although
germination varied by seed lot Dreesen (2001) reported that using a slow-speed dry
tumbling technique for 5 to 7 days produced gennination rates as high as 90
As with acid and hot water treatments treatment duration must be specifically
shy
correlated to seed lot Treated seeds are also susceptible to fungal infection
iHopkinson 1997) In addition equipment is specialized and may not always be
ltU~CUJl~ to a nursery (USDA Forest Service 1948) Several studies have confinned
degrading ofstrophiolar cells due to acid and hot water scarification (Miklas et al
11
1987 Dell 1980 Hanna 1984) but the effect ofmechanical scarification on the
strophiole has not been studied
Alternative Method for Breaking Dormancy Percussion
Strophiole degradation has been examined in depth using an impaction
treatment more commonly known as percussion Hamly (1932) first proposed that
shaking or impacting seeds could split the macrosc1ereid cells at the strophiole
Hamly successfully released tension ofbent strophiolar cells of impermeable
Melilotus alba Medik (White sweet clover) seeds by delivering physical blows
resulting in cells that had split apart and were only slightly bent Ninety-two percent
oftreated seeds became permeable as compared to 05 of control seeds Barton
(1947) showed that percussion significantly increases permeability in black locust
seed Ballard (1976) reasoned that the integrated nature of the strophiole within the
seed coat means a blow on any part of the seed weakens the strophiole There are no
published studies on the use of percussion on New Mexico locust
Percussion methods vary and often seem SUbjective or ill-defined Hamly
(1932) placed seeds in a glass bottle and shook them three times a second for 20
minutes resulting in 180 oscillations per minute Ballard (1973) describes shaking
seeds in a closed metal container with at least fifty seeds per container at 850-900
JS(~l11alIOIlS per minute for 1 minute Kelly and van Staden (1987) placed seeds into
sealed glass flasks Flasks were shaken by a wrist-shaker at a rate of 250
~UUVJ~ per minute Subsequent germination rates were 45 after 2 hours of
55 after 3 hours 60 after 6 hours and 100 after 12 hours
12
There are several potential advantages of percussion over acid hot water and
mechanical scarification Kelly and van Staden (1987) note that a percussion
treatment only alters macrosclereid cells of the strophiole by loosening separating
and raising them They emphasize that minimal damage is done to the overall
anatomical structure of the seed In acid scarification of the same seed lot radicle
and cotyledon damage occurred Based on photographic evidence the authors
believe that this morphological damage was due to uneven pressure during
imbibition with water entering haphazardly wherever the acid had sufficiently
dissolved the coat This ultimately led to uneven hydration of the embryo In
percussed seeds water only entered through the s~ophiole when the entire seed coat
had been saturated resulting in controlled and even embryo hydration (Kelly and van
Staden 1987)
Although all successful scarification treatments may focus at least in part on
the strophiole for papilionoid legumes (Baskin and Baskin 1998) percussion may be
less damaging to seed coat structures particularly when compared with acid Kelly
and van Staden did not compare hot water or mechanical scarification methods
against percussion However the fungal infection associated with hot water and
mechanical scarification as with acid scarificatio~ have been directly associated with
seed damage (Rehman et al 1998 USDA Forest Service 1948) Embryo damage in
has been observed in hot-water scarified legume seeds (Rehman et al 1998
13
By limiting embryo damage percussion scarification reduces seedling
mortality compared to other treatments Kelly and van Staden (1987) noted that
seedlings from the percussion treatment were morphologically sound and vigorous
when compared to acid treatment seedlings The authors attribute this improved
seedling vigor to the controlled water uptake that limited anatomical damage
Despite a 12-hour percussion treatment morphological damage was not substantially
greater than untreated seeds In addition seedling mass measured 10 days following
germination was not significantly different between percussed and untreated seeds
A final advantage ofpercussion may be the ability to adequately treat the most
dormant seeds while avoiding harm to seeds that have already been made permeable
by the treatment or that were permeable to begin with Instead ofcorroding scalding
or scratching the seed coat percussion appears to be just a loosening of the
strophiole spring (Ballard 1973) Kelly and van Staden (1987) were able to
increase germination with a lengthy treatment (12 hours) Figuring out whether a
substantial cushion oftreatment durations exist for percussion ofNew Mexico locust
seed would be a significant improvement to current scarification methods where seed
damage and seedling health is an issue
The greatest challenge for applying percussion to New Mexico locust seeds is
standardizing a treatment that is both effective and readily reproducible by nurseries
with mechanical scarification this may require the development of specific
~UllDment to properly quantify and execute the treatment
14
f
Objectives
The first objective of the study is to detennine which scarification treatment
results in optimal gennination speed and percentage ofNew Mexico locust The
second objective is to describe treatment interactions with nine seed sources collected
in New Mexico
Hypothesis
A percussion treatment acts exclusively on the main area ofwater uptake in
New Mexico locust seed loosening the strophiole so that imbibition can occur This
direct mechanism ofdonnancy release will result in improved gennination rate and
overall gennination percentage for New Mexico locust seed compared to standard
treatments Standard treatments including acid hot water and mechanical
scarification while eventually weakening the strophiolar cleft cause excessive
morphological damage through generalized seed coat degradation These treatments
are therefore prone to inconsistency due to over and under-treatment
15
MATERIALS AND METHODS
Seed Collection and Cleaning
Seeds were collected from August 25 through September 15 2000 from nine
locations along a latitudinal gradient in New Mexico Seed source locations and
descriptive information are given in Figure 1 and Tables I and 2 Stands were
separated by at least 45 km
Pods were hand picked as they began to ripen and dehisce (open) Collections
were made from ground level to approximately 3 m from several trees at each site
Pods were dried in open-ended brown paper bags for two to four weeks at room
temperature (21-230C) to facilitate cleaning Dried pods were placed in a closed cloth
sack which was pounded against the ground to separate seeds from pods For pods
still containing seeds hand maceration (crunching pods with hands) completed seed
separation Debris was removed by threshing with the aid of a greenhouse fan A
South Dakota Seed Blower (Seedbureau Chicago Illinois) was used to remove
remaining chaff and light seeds by air density separation Seeds were placed in dry
storage in sealed quart-size zip-lock bags within a sealed plastic container Storage
temperatures ranged from 2-4oC Seeds remained in storage for approximately 7
months before the experiment
Experimental Design and Treatment Structure
The experiment compared the impact ofhot water bath and percussion
l11aLl11 on germination properties ofNew Mexico locust seed A single
16
-l
Figure 1 Seed source collection map All sources were collected within New Mexico USA separated by at least 4S lan
IIIRZltItW lIJPIirmiddot rllll1lbullbull_~~~_II
Table 1 Latitude longitude location elevation and collection date ofNew Mexico locust seed sources
Lot title Latitude (N) Longitude (W) Location Elevation (m) Collection Date
Raton 36deg5846 104deg2872 Raton Pass NM 2295 82500
Holman 36deg0306 105deg2429 HolmanNM 2476 911500
Jemez 35deg4500 106deg4100 Jemez Springs NM 1976 9900 00
Sandia 35deg1083 106deg2341 Cedar Crest NM 2432 82600
Manzano 34deg3689 106deg2494 ManzanoNM 2465 9200
Magdalena 33deg5953 107deg0903 Magdalena NM 2544 82900
Capitan 33deg2728 105deg4325 Alto NM 2189 911200
Sacramento 32deg5798 105deg4496 Cloudcroft NM 2630 91400 Gila 32deg5400 107deg4600 Kingston NM 2432 9700
f
Table 2 Mean seed weight and standard error by source
a
Source Mean seed weight (g) for Standard error 100 seeds n = 5
Raton Holman Jemez Sandia Manzano Magdalena Capitan Sacramento Gila
2872 b c 2522 d 1826 e 2753 c 2413 d 2559 d 2443 d 3130 a 3029 ab
00103 00403 00276 00301 00370 00374 00458 00279 00348
a Mean seed weights followed by the same letter are not significantly
different
19
experiment was conducted utilizing a randomized complete block design blocked by
time with a factorial treatment structure Experimental factors were seed source and
scarification treatment Nine seed sources from throughout New Mexico were used
(Figure 1 Table 1) Scarification treatments included a control three levels ofhot
water and four levels of percussion Hot water treatments included immersion at
100degC and boiling for periods of 30 seconds and 60 seconds Percussion treatment
levels were 1 2 4 and 8 minutes One hundred seeds per block were used to test
each treatment combination Four blocks of seed were tested one at a time (all seed
sources included in each run) in close chronological sequence (within 6 weeks) A
total of four 100-seed samples were used to test each source by scarification treatment
combination
Treatments
Hot water treatment of seeds utilized 50 ml test tubes filled with 30 ml
distilled water Each test tube was used to treat one 100-seed sample at a time
resulting in a ratio of4 parts water to 1 part seed by volume Test tubes were placed
in a beaker with the beaker filled with tap water to the same level as the water within
test tubes Nine test tubes (one per seed source) were placed in the beaker
simultaneously so that one replication of each seed sourcescarification treatment
cotnblnatlon was conducted simultaneously Hot water treatments include immersion
boiling For immersion treatments seeds were added to test tubes with the water
(boiling) and the test tubes were immediately removed from the beaker to
For the boiling treatments test tubes remained in the beaker with water at
20
boiling temperature for a set duration after the addition of seeds Following all
treatments test tubes with seeds cooled 24 hours in a test tube rack
Percussion treatment utilized a pneumatic paint shaker (Central Pneumatic
model 00422 Camarillo CA) Compressed air was maintained at 530 kPa plusmn30 kPa
(80 psi plusmn 5 psi) resulting in approximately 350 oscillations per minute Seeds were
placed in 118 ml (4 oz) soil tin cans (US Can Lobard IL) and the lids were secured
with duct tape Each can was used to treat one 100-seed sample To take advantage
ofgreatest lateral movement of the paint shaker a spacer (paint can) was used to
position the soil tin at the end of the shaking arm This allowed shaking to occur at
the greatest distance from the pivot point (fulcrum) Tins were placed perpendicular
to the direction of shaking motion allowing seeds to impact at greatest force by
hitting flat surfaces of the soil tin
Germination Testing
Before gennination testing all seeds were soaked 24 hours in reverse
osmosis-treated water (this was included in the process ofhot water treatment
jU~SCIlOea above) Following soaking seeds were placed in 100 cm Petri dishes lined
90 cm Whatman qualitative 1 filter paper circles (MG Scientific Pleasant
WI) One Petri dish was used for each treatment by source combination
papers were initially moistened with 5 mL reverse osmosis-treated water then
~VU1CUlJl moistened as needed to ensure adequate conditions for gennination
were monitored daily (at 300 PM) for gennination for 14 days in accordance
~UIUlnJlVUa Seed Testing Association (ISTA) standards for Robinia species
21
(1STA 1999) Tests were carried out at room temperature (22degC plusmnOSoC)
Germination was classified as protrusion of the radicle from the embryo by at least
1mm as viewed with the naked eye
X-ray Radiography
On August 132001 three lOO-seed samples from the Sacramento seed source
were treated for 14 or 8-minute percussion durations The seeds were sent
immediately by overnight delivery to the Faxitron X-Ray Corporation in Wheeling
Illinois where Dr Bob Karrfalt ofthe USDA Forest Service oversaw their imaging
Radiographs were made at SX with a Faxitron Specimen Radiography System Model
MX-20
Scanning Electron Microscope Photography
On February 192002 a Hitachi S-3200N Variable Pressure Scanning
Electron Microscope (Mountain View CA) was used to examine strophiolar cells of
untreated seeds and seeds percussed for 4 minutes Seeds were cut longitudinally
igt their entire length using a razor blade splitting the strophiolar region roughly in
Cross sections of seed samples magnifying the strophiolar region were viewed
600X under the microscope
Analysis
Categorical Analysis of Variance was used to determine treatment source and
(Le time) main effects and interactions The response variable was total
~vlI This procedure is a Chi-square test of homogeneity using the natural log
of germinated to non-germinated seed for each treatment Maximumshy
22
likelihood analysis was used to calculate Chi-square test statistics with their observed
significance levels A single contrast was conducted to cempare treatment means
against the control Observed significance levels less than or equal to 005 were
considered significant Pairwise comparisons were conducted between non-control
treatment mean percents using a conservative alp~a value of 005 divided by the
number of comparisons (Bonferroni approach) and approximate pairwise z statistics
Categorical analyses were performed using Proc Catmod (SAS Institute 1989)
RESULTS
Scarification seed source block and all interactions between factors
influenced germination (Table 3) The main effects are reported in the presence of
these interactions because the overall robustness of treatments and seed sources are of
interest Background levels ofnaturally occurring variation in seed sources are
seen in control germination percentages in Figure 2 In particular time variability
is unlikely to be under the control of the producer Appendix B details experimental
data for source by treatment gennination percents for each of the four blocks
Averaged over all treatments germination by source ranged from 40 to
79 (Figure 2) Averaged over all sources (Figure 3) and for each source (Figure 4
5) percussion scarification durations of4 or 8 minutes yielded greatest germination
Averaged over all sources germination increased with increasing durations of
percussion then leveled off for the 4- and 8-minute treatments (Figure 3) Immersion
and 30-second boiling scarification maximized germination for the hot water
treatments with a decrease in germination when seeds were boiled for 60 seconds
For all but two sources there is a similar ipcreasing trend in germination from
1to 4 minutes ofpercussion (Figure 4) The Holman source increased in germination
1 to 2 minutes then leveled off at 4 minutes The Jemez source changed little
1 to 4 minutes of treatment As treatment duration increased from 4 to 8
mutes five sources decreased in germination (Figure 4a) whereas three sources
24
Table 3 Categorical Analysis of Variance table for germination response to seed treatment seed source block and the interaction of the factors
Source of DF Chi-Square Observed Variability Significance Level
Source 8 98370 lt00001 Trt 7 233876 lt00001
contrast 1 16976 lt00001 SourceTrt 56 97077 lt00001 Block 3 5538 lt00001
SourceBlock 24 10162 lt00001 TrtBlock 21 7564 lt00001
SourceTrtBlock 168 34283 lt00001
S 4)
8 ~
0
sectg ~ 40 ~
N 0 0
20
o f0ltgt ~ ~4 jybshy ((iltgto ~~ K1ltgt 6~b-~ ~~ 0b-~~ ~~ ~b-~ ~b-~b
80
60 -I-- - ~- middotmiddotmiddotmiddotmiddot1
r -] Northern NM Sources
Central NM Sources
Southern NM Sources
sect-o ~
~b-v(i
SOURCE
Figure 2 Effect ofNew Mexico locust seed source on germination for data averaged over all scarification treatments Control germination is shown by solid line within bar
80
~ cd ~ ltl) () 60 ~ ~
N
t 0shyrti tshySltl)
40
0
20
o lmmersion Boiling 30 sec Boiling 60 sec Control 1 min 2min 4 min 8 min
Hot Water Treatments Percussion Treatments
Figure 3 Effect of scarification on germination ofNew Mexico locust for data averaged over all seed sources Means labelled with the same letter are not significantly different
100~----------------------~--~----------------~
80
60
ltU
~
40 IJ
sectgsI
l100
80
60
40
a I middotmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot middotImiddotmiddotmiddotmiddotmiddotmiddot E
_~iii-bull _ _ bull f --~--
t-- -~ ---- -shy ---- shyI ~ ---- - shy
L
fiII
~
b
- shy ---r
- Holman
Sacramento Sandia Gila Jemez
c ~
ltgt ~
Tmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot-I
j~=~=-~~-1
I middotmiddot ~ bull r
t tr
Y -------M-a-gd-a-Ie-n-a--
1 _ _
fCapitan
---- Manzano ~y -_ Raton
2 4 8
Time interval ofpercussion scarification (min)
Figure 4 Effect of seed source by treatment interaction on New Mexico locust germination for percussion scarification pattern a and pattern b
28
tv 0
~ d
~ A s o
1 c3
80
60
40
20
o
TIT
t------=middotfmiddotmiddot-middotmiddot-middotmiddot-middotmiddot 1- --- - - --=- - -shy--cE= - _--shy --shy ----shy ~ ~ ---shy ~ -~- _ --shy-------r 1
I
Raton Holman
-_ Jemez
--- Sandia Manzano Magdalena Capitan Sacramento Gila
Immersion Boiling 30 sec Boiling 60 sec
Hot Water Scarification
Figure 5 Effect of seed source by treatment interaction on New Mexico locust germination for hot water scarification
continued to increase in germination (Figure 4b) There was no difference between
the two treatment levels for the Manzano source (Figure 4b)
The effect ofhot water scarification also varied among sources with few
overall differences between hot water treatments (Figure 5) However boiling for 60
seconds yielded highest germination for the Jemez source whereas increasing
durations ofboiling decreased germination for the Raton source
In addition to consistently high germination 4- and 8-minute percussionshy
treated seeds germinated as fast or faster than all other treatments (Figure 6 Table 4)
050 measured as the number of days needed to complete 50 ofeventual
germination averaged 2 days for both ofthese treatments
X-ray radiographs were taken of seeds collected from the Sacramento source
percussed for 1 4 or 8 minutes (Figure 7) Seeds show signs of damage at 8 minutes
ofpercussion (Figure 8) Compared to an undamaged seed percussed for only 4
minutes (Figure 8a) damage at the 8-minute level includes fractured radicles pitting
of the seed coat and chips in seed coat structure (Figure 8b-d)
Scanning electron microscope (SEM) images at 600X reveal the direct effect
of 4 minutes of percussion on cells of the strophiolar region (Figure 9) Whereas
untreated seeds retain cellular organization in the strophiolar region strophiolar cells
seeds treated for 4 minutes ofpercussion separate and raise resulting in a
~-~e~U appearance
30
Umiddot-~middot--l-~---middotymiddotmiddotmiddot-----middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot--middot--middotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middotmiddotmiddotmiddotmiddotmiddotmiddot- -
050= gt1
-e-- Percussion 8 min -0- Percussion 4 min ---T- Percussion 2 min -----ltil- Percussion 1 min -- Immersion --0- Boiling 30 sec -+- Boiling 60 sec -ltgt- Control
2 4 6 8 10 12
Day Figure 6 Timecourse ofNew Mexico locust gennination following scarification
treatments G50 is the number of days to 50 of eventual germination
31
14
Table 4 Effect of seed source and treatment on G50 (days to 50 germination)
Seed Source Control Immersion B30sec B60sec Plmin P2min P4min P8min mean Raton 3 3 5 7 4 3 2 2 4 Holman 2 2 2 2 2 2 2 2 2
Jemez 4 6 6 5 3 2 2 2 4 Sandia 2 2 2 2 2 2 2 2 2
w tv Manzano 5 2 2 3 3 2 2 2 3
Magdalena 4 2 2 3 4 3 2 1 3
Capitan 5 4 2 2 3 2 2 2 3
Sacramento 3 2 2 2 4 3 2 2 3 Gila 3 2 2 2 2 2 2 2 2 mean 3 3 3 3 3 2 2 2 3 standard 11 14 16 18 09 05 0 03 08 deviation
Note Columns and rows may not agree due to rounding
Figure 7 X-ray radiographs ofSacramento seed source percussed for 1 4 or 8 minutes
33
Figure 8a-d Seed damage close-ups Figure a shows undamaged seed percussed for 4 minutes Figures b-d show seeds percussed for 8 minutes Damage includes fracturing ofradicle in b complete removal ofseed coat in c and pitting ofseed coat in d
34
Untreated Treated
Figure 9 SEM images ofuntreated vs 4-minute percussion-treated strophiolar cells ofNew Mexico locust seeds Treated cells are loosened separated and raised
DISCUSSION
Determining the Optimal Treatment
Averaging results of scarification treatmems across seed sources is a useful
tool to predict which treatment is most likely to be successful for an unknown seed
source Averaged over all sources four- and eight-minute percussion treatments
resulted in the highest germination 815 and 790 respectively and produced
germination that was as fast or faster than all other treatments Although there was no
difference between these two treatments on average an argument for 4 minutes of
percussion as a standard protocol for treating New Mexico locust seeds can be made
Descriptively 4 minutes of percussion improved germination over 8 minutes
ofpercussion for a majority of sources (five out of nine) 8 minutes of percussion
improved germination for three ofnine sources with no difference for a fmal source
For individual sources 4 minutes ofpercussion was always the best or secondmiddot best
treatment and was always equal to or better than all hot water treatments
Germination at 8 minutes ofpercussion was lower than all other percussion durations
for the Jemez and Gila sources and was lower than at least one hot water treatment for
these sources as well
The x-ray radiographs of the Sacramento seed source illustrate the excessive
internal and external seed damage that can occur with 8 minutes of percussion
germination counts 8-minute percussed seeds from Holman and Capitan
ClUll signs of damage that were visible to the naked eye (see below)
r~evertheless some of these seeds still fit the criteria for germination Neither x-ray
36
photography nor visible observations during seed counts revealed seed damage at the
4-minute percussion level In operational trials seeds percussed for 4 minutes
developed into healthy seedlings (Khadduri and Harrington unpublished data)
Strophiole and Seed Coat Behavior
Four minutes ofpercussion successfully breaks seed dormancy in a large
percentage ofNew Mexico locust seeds without excessively damaging the
surrounding seed coat or internal seed coat structures Most papilionoid legume
seeds including seeds ofNew Mexico locust have a specific region of the seed coat
known as the strophiole or lens (Hamly 1932) The strophiole is located on the
cotyledonary lobe of a locust seed (Figure 10 a-c) If seeds are not indiscriminately
damaged the strophiole is the first point of water entry to the embryo (Hopkinson
1997) When a papilionoid seed is percussed for the appropriate amount of time
repeated hits on the integrated seed coat loosen the constrained cells of the strophiolar
region without excessively damaging the rest of the seed coat (Ballard 1976) SEM
photos illuStrate that 4 minutes of percussion loosened and separated strophiolar cells
ofNew Mexico locust seeds when compared with constrained organized strophiolar
cells of untreated seeds (Figure 9) Research-has shown that water passes to the
embryo through the treated strophiole in a controlled manner and regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues
(Kelly and van Staden 1987)
37
10 Strophiole ofunimbibed New Mexico locust seed (a) and following uptake (bc)
38
In contrast hot-water treatments can excessively degrade the seed coat
(Rebman et al 1998 Marunda 1990) Random cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rebman et aI 1998 Marunda 1990) Along with a
proportionately large amount ofun-imbibed (thus under-treated) seeds (Figure l1a)
damage was observed in hot-water treated seeds from six sources during germination
counts Damage included seeds where the radicle failed to elongate (Figure 11 b)
seeds where the radicle elongated but was delayed in freeing itself from the seed coat
(Figure 11c) and seeds where the radicle completely broke off from the embryo
(Figure lId) In addition some seeds germinated in reverse (the cotyledon emerged
fIrst) If the radicle emerged completely from the seed coat they were counted as
germinated
While nearly all seeds treated for 8 minutes ofpercussion imbibed water
damage was evident in x-ray evaluations (Sacramento seed source) and in
germination counts (Holman and Capitan) at this treatment level Seed damage
during germination counts was similar to levels found in hot-water treated seeds
suggesting that over-treatment by both hot water and longer durations of percussion
may result in similar damage to seed structure
The Sacramento and Holman seed sources were two of the fIve sources that
opcrplltlpn in germination from the 4- to 8-minute durations of percussion
the benefIts ofloosening the strophiole as evidenced by imbibition of
all 8-minute treated seeds were outweighed by external and internal seed coat
39
a
b c d
11 Damage to New Mexico locust seeds observed during germination cotmts following hot water scarification 9a Hot water replicate including ~t seedlings and numerous unimbibed seeds 9b Close-up of seed where raallCle failed to elongate 9c Close-up ofseed where radicle elongated but was
-gtIn in freeing itself from the seed coat 9d Close-up ofseed where radicle broke off from embryo
40
damage (which also may be contributing to high imbibition) For the three sources
for which 8 minutes of percussion resulted in maximum germination extended
percussion of seeds necessary to loosen the strophiole may not have excessively
damaged the seed anatomy The Manzano source did not significantly change in
germination from 4 to 8 minutes of percussion suggesting that an increase in
permeable seeds at the 8-minute treatment duration was balanced by the number of
seeds that failed to germinate due to seed damage To summarize the 4-minute
percussion treatment improved germination over 8 minutes ofpercussion for a
majority of sources (five ofnine) the 8-minute percussion treatment improved
germination over 4-minutes ofpercussion for three sources and there was no
difference with the Manzano source Notably for the Raton source 8 minutes of
percussion increased germination by 235 percentage points over 4-minutes of
percussion (7875 vs 5525 respectively) the next best treatment Raton was the
only source to result in germination less than 75 for 4 minutes ofpercussion so a
grower may wish to apply longer percussion durations to lots that do not respond well
to this treatment level
Finally there was an interesting trend in the Jemez and Raton sources seed
that significantly contributed to the source by treatment interaction (Figure 12) A
~u test showed that the Jemez source had a significantly lighter seed weight than
other sources while the Raton source had an average seed weight For the Jemez
the longest duration of boiling increased germination Conversely only short
U4UVll) of percussion were needed to achieve high germination and the longest
41
80 _ Jemez a Raton
iD
~ C iD e iD
Q c 0 ~
c ~ iD 0
20
0------shy
Immersion Boiling 30 sec Boiling 60 sec
Hot Water Scarification
10degTr========~--------~------------------------------
80
60
40
20
_ Jemez Raton
0------shy
1 min 2 min 4 min 8 min
Percussion Scarification
Figure 12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources
42
b
--~----~~~~========~---- duration ofpercussion reduced germination For the Raton source longer durations
ofboiling consistently decreased germination whereas longer percussion treatments
consistently increased germination
A possible explanation is that a thick seed coat is associated with a loose
strophiole structure and that a thin seed coat is associated with a constrained
strophiole This contrast merits further study and may suggest an ecological
adaptation Further investigation ofthis phenomenon may contribute to successful
pre-screening ofunknown seed sources to determine optimal treatment leveL
43
CONCLUSION
Based on results obtained in pilot study (see Appendix) and thesis
experiments 4 minutes ofpercussion is recommended as a standard protocol for
treating an unknown source ofNew Mexico locust seed In addition to laboratory
results the 4-minute percussion treatment has resulted in germination greater than
80 and healthy seedling grow-outs for production purposes (unpublished data) In
laboratory experiments only one hundred seeds per 118 ml (40z) soil tin were
percussed at a time whereas for production we have treated up to five hundred seeds
per tin For large-scale production ofNew Mexico locust seedlings growers may
wish to experiment with larger quantities of seed and larger-size soil tins
Seed storage trials following percussion treatments were not conducted
However germination greater than 80 and healthy seedlings were obtained when
seeds were percussion-treated and subsequently stored at room-temperature (22degC)
for one week (unpublished data) Seed storage trials merit further study as the
standard hot water treatment leaves seeds in a softened condition that is not
conducive to storage
Hydroseeding is an effective method for direct seeding in reclamation
situations Percussion treatment may be conducive to hydro seeding as seeds remain
a dry relatively undamaged yet permeable condition following treatment Sulfuric
seeds also remain dry following treatment but extensive damage to seed coat
poor seedling vigor have been reported with this treatment (Kelly and van Staden
A grow-out study using a hydro seeder on a reclamation site testing percussion
44
against other treatments such as sulfuric acid and mechanical scarification also
merits further study
Papilionoids are the largest subfamily oflegumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust these legumes often play an integral role in the revegetation of
disturbed lands The pilot study (Appendix) indicates that percussion scarification
successfully treats the widely used black locust as well Developing superior
scarification methods to the standard hot water mechanical scarification and acid
treatments may facilitate the use ofpapilionoid legumes in reclamation projects
45
APPENDICES
APPENDIX A
PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR
NEW MEXICO LOCUST AND BLACK LOCUST SEEDS
Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4
Hot water and sulfuric acid soaks are traditional treatments for seeds of many
temperate woody legwnes including locusts However these scarification techniques
often produce inconsistent germination Percussion scarification where seeds are
repeatedly propelled against a hard surface was compared with hot water
scarification to evaluate treatment efficacy for New Mexico locust (Robinia
neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment
seeds were placed in a 98degC water bath which was immediately removed from the
heat source For percussion scarification seeds were placed in a soil sample tin and
agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the
control were followed by 24-hour water soaks Hot water baths resulted in 56 and
41 germination for New Mexico locust and black locust respectively For both
species nearly all durations ofpercussion increased germination over the hot water
treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3
4 and 5 minutes for black locust resulted in at least 90 germination Traditional
scarification treatments randomly degrade the entire seed coat which can lead to
tissue damage during water uptake Percussion scarification specifically weakens the
strophiole the natural source ofwater entry to the seed in papilionoid legwnes
INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom
2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031
48
Following percussion imbibition is controlled through the strophiole and underlying
tissue is protected
Keywords New Mexico locust black locust scarification percussion germination
Introduction
Locust trees (Robinia spp) are aggressive pioneer species that quickly
colonize disturbed land fix atmospheric nitrogen in the soil and stabilize slopes that
are prone to erosion (Klemmedson 1994 Ashby et al 1985) (Figure A1ab)
Recognition of these attributes has contributed to increased demand for locusts in
reclamation projects Locust seeds however exhibit physical dormancy where a
thick seed coat prevents the movement of water and gasses to the embryo (Leadem
1997)
Sulfuric acid or hot water soaks have traditionally been used to break seed
dormancy in macro-propagation of locusts Many growers are moving away from the
use of su1furic acid in treating locust seeds (Dreesen and Harrington 1997) In
addition to being dangerous acid soak durations must be specifically correlated to
seed lot (Olson 1974) Collections of native seed can vary tremendously in seed size
seed weight and hard seededness etc This variability limits the utility of sulfuric
acid soaks as even within a given lot some seeds may be damaged whereas others
are under-treated and fail to imbibe (take up water) A survey of Southwestern
container growers indicates that hot water baths are currently the most common
scarification method for locust seeds (Hine et al 1997) However hot water baths
also produce inconsistent germination (Lin et al 1996)
49
b
Figure AI New Mexico locust a primary invader following disturbance Ala Crown-sprouting at edge ofburn near Los Alamos NM--one year after Cerro Grande fire Alb Colonizing road cut Gila National Forest NM
50
Percussion scarification where seeds are repeatedly propelled against a hard
surface is an alternative dormancy-breaking method that has proven successful in
legumes related to locusts (Hamly 1932 Barton 1947) We compared this alternative
treatment to hot water scarification to evaluate treatment efficacy for New Mexico
locust and black locust seeds
Materials and Methods
Commercial New Mexico locust seeds (Western Native Seed Coaldale CO)
were collected fall 2000 in Huerfano County CO Black locust seeds were collected
September 2000 from Taos County NM by harvesting ripe pods from several trees at
a distance of up to 3 m from ground level Black locust seeds were allowed to air-dry
for 3 weeks and were threshed and separated from large chaffusing a greenhouse
fan A South Dakota Seed Blower (Chicago Illinltois) was used to remove fine chaff
from both seed lots Seeds were storedmiddotat 2-4C until the germination study was
conducted in March 2001
This study compared hot water scarification with percussion scarification
using treatment and species as experimental factors Along with a control
scarification treatments included immersion of seed in boiling water as well as
percussion durations of 1 2 3 45 or 10 minutes Four 100-seed samples were used
to test each species by scarification treatment combination
For the hot water treatment test tubes were filled with 30 mL water and
placed in a water-filled beaker (Figure A2a) The bath was raised to boiling (-98C at
51
b
c
Figure A2 Implementation ofhot water and percussion scarification A2a Hot water bath A2b Paint-shaker set-up A2c Placing soil tin containing seeds at end ofpaint shaker ann
52
Las Cruces NM elevation of 1300 meters) Each 100-seed replicate was placed in a
test tube and each test tube was immediately removed to cool at room temperature
Percussion scarification was implemented using a pneumatic paint shaker
(Central Pneumatic model 00422 Camarillo CA) (Figure A2b) This allowed
standardization of treatment intensity Compressed air pressure was maintained at
530kPa plusmn 30kPa (80psi plusmn 5psi) resulting in approximately 350 oscillations per
minute Each lOO-seed sample was placed in a 118 ml (4 oz) soil tin for shaking To
maximize lateral movement of paint shaker a paint can was used as a spacer to place
soil tins at the end of the shaking arm (Figure A2c) This allowed shaking to occur at
the greatest distance from the pivot point (fulcrum) Following all treatments
including the control seeds were soaked for twenty-four hours in distilled water prior
to plating out
Gennination was evaluated on lab benches at room temperature Each
replicate of seeds was placed in a 100 cm petri dish on moistened filter paper with
humidity maintained by enclosing the petri dishes in ziplock bags Gennination was
monitored daily for 14 days in accordance with International Seed Testing
Association standards for Robinia species (ISTA 1999) Gennination was defined as
protrusion of the radicle from the embryo by at least 1mm as viewed by the naked
eye
Categorical Analysis of Variance using Prpc Catmod (SAS Institute 1989)
was used to detennine treatment differences in germination percentages for each
source This procedure is an extension of a Chi-square test of homogeneity using the
53
natural log of the ratio of germinated to non-germinated seeds for each treatment Pshy
values less than 005 were considered significant A limited set of pairwiseshy
comparisons was conducted to compare treatment mean percents using a
conservative alpha value of 005 divided by the number of comparisons
Results
Percussion maximized germination for both New Mexico locust and black
locust There were three germination response levels for New Mexico locust (Figure
A3) First there was a low response in the control indicating a dormant seed lot
The hot water treatment and I-minute duration of percussion share a second
intermediate response Germination was highest at percussion levels from 2 to 10
minutes
A selected percussion treatment intermediate in time (5 minutes) produced the
steepest germination curve indicating the fastest germination rate (Figure A4) The
hot water treatment produced an intermediate curve or relatively slower germination
rate The control curve was very flat indicating the slowest germination rate for New
Mexico locust
Control seeds germinated poorly for black locust as well (Figure A5) Hot
water scarification improved germination over the control but all levels of percussion
improved germination over hot water The optimal durations of percussion treatment
were bracketed with a significant increase from the 1 to 2 minute percussion level a
high germination response from 2 to 5 minutes of percussion and finally a significant
54
~ d 25 CL)
p t1 0 ~ t1
g Vl ~ Vl
100~---------------------------------------------------------------
80
60
40
20
o Control Hot Water Percussion
I min Percussion
2 min Percussion
3 min
Scarification
Percussion 4 min
Figure A3 Effect of scarification on germination response for New Mexico locust
Percussion 5 min
=significant difference between adjacent treatment bars ns = no significant difference
Percussion 10 min
~ ~ () u agt
Il-t d o ~
~ ~ c3
100middotlr~~~------------------------------------e- Percussion 5min middot 0middot Hot Water -- Control
80-J=middot======
6b-middot-lmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middot--middotmiddot-middot~-middotmiddot -middotmiddot middot middotmiddot-middot 1
40
20
-----~-----------
0 0 0 middotmiddotmiddotmiddotmiddot0middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot0
0 --~-----y-----~------_---~--- T-----~
o shy -TDay 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A4 Effect of selected scarification treatments on germination speed for New Mexico locust First seven days of fourteen-day experiment are shown
Day 7
80
~ cd
5 60 ()
[) Cl d 0 1t1 d
40sect dUl
-J
20
o
ns
Scarification
Figure A5 Effect of scarification on germination response for black locust = significant difference between adjacent treatment bars ns = no significant difference
decrease at the 10-minute level This drop-off suggests that 10 minutes of percussion
over-treats seeds from this lot of-black locust
A selected percussion treatment intermediate in time (5 minutes) also had the
fastest germination rate for black locust (Figure A6)_ The hot water curve is
somewhat flatter indicating a slower germination rate The control curve is very flat
indicating a slow germination rate
A qualitative difference between hot water and percussion-treated seeds was
also observed Along with healthy germinants the hot water treatment produced
over-treated seeds (Figure A 7a) Examples of damaged germinants include seeds
where the radicle did not elongate (Figure A 7b) seeds where the radicle elongated
but was delayed in freeing itself from the seed coat (Figure A7c) and seeds where the
radicle broke off completely from the embryo (Figure A7d) In addition to these
examples of over-treatment there were numerous small dark seeds that failed to
imbibe water For percussion treatment nearly all seeds took up water (Figure A8a)
Germination was rapid and uniform with little damage evident (Figure A8b)
To summarize both New Mexico locust and black locust seed lots were
dormant The standard hot water scarification treatment improved germination but
only to 56 and 41 for New Mexico locust and black locust respectively Nearly
all percussion durations improved total germination as well as germination rate over
the hot water treatment Germination was greater than 90 for percussion durations
of 4 5 and 10 minutes for New Mexico locust and 3 4 and 5 minutes for black locust
58
CI)
sgt t CI) 0
igt 0
sect ttl t E
Vl 0
c3
100r==~==~~=r------------------------------------~ ___ Percussion 5 min
middotmiddotmiddot0middotmiddotmiddot Hot Water -lIf- Control
80r=========~--~~----------------------------~
60
40
20 -~-~---~---------- ---~------------I
omiddot
O
_~_----------lIf-----~bull -----_ shy---shy I
Day 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A6 Effect of selected scarification treatments on germination speed for black locust First seven days of fourteen-day experiment are shown
Day 7
a
b c d
Figure A7 Damage to New Mexico locust seeds observed during germination counts following hot water scarification A7a Hot water replicate including damaged seedlings and numerous unimbibed seeds A7b Close-up ofseed where radicle failed to elongate A7c Close-up ofseed where radicle elongated but was delayed in freeing itself from the seed coat A7d Close-up ofseed where radicle completely broke offfrom embryo
60
a
b
Figure A8 New Mexico locust seeds following percussion treatment at time of germination counts A8a Percussion replicate with uniformly healthy seedlings and near complete imbibition of seeds A8b Close-up ofpercussion-treated seedling
61
Discussion
Papilionoid seeds have a specific anatomical feature known as the strophiole
or lens-the natural site of water entry to the seed (Hamly 1932) The strophiole is
located on the cotyledonary lobe of a locust seed (Figure A9) When a papilionoid
legume seed is percussed for the appropriate amount of time repeated hits on the
integrated seed coat loosen the constrained cells of the strophiolar region without
excessively damaging the rest of the seed coat (Ballard 1976)
When a percussed seed is soaked water enters exclusively through the
strophiole in a controlled manner (Kelly and Staden 1987) This regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues This
contrasts with seeds that have been hot water or acid scarified treatments that can
randomly degrade the seed coat Unlocalized cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rehman et al 1998 Marunda 1990)
Papilionoids are the largest subfamily of legumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust and black locust these legumes often play an integral role in the
revegetation ofdisturbed lands Developing superior scarification methods to the
standard hot water and acid treatments should fadlitate the use ofthese legumes in
restoration projects
62
Figure A9 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (bc)
63
APPENDIXB
GERMINATION PERCENTS AND STANDARD ERRORS FOR SOURCE
BLOCK AND TREATMENT FOR NEW MEXICO LOCUST
Table B 1 Gennination percents and standard errors for source block and treatment for New Mexico locust
Source Block Treatment Germination Percentage Standard Error
Capitan 1 Immersion 74 437 Capitan 2 Immersion 69 462 Capitan 3 Immersion 71 454 Capitan 4 Immersion 67 470 Capitan Boiling 30 sec 79 407 Capitan 2 Boiling 30 sec 76 427 Capitan 3 Boiling 30 sec 72 449 Capitan 4 Boiling 30 sec 76 427 Capitan Boiling 60 sec 71 454 Capitan 2 Boiling 60 sec 54 498 Capitan 3 Boiling 60 sec 67 470 Capitan 4 Boiling 60 sec 76 427 Capitan I Control 5 218 Capitan 2 Control 7 255 Capitan 3 Control 4 196 Capitan 4 Control 5 218 Capitan 1 Percussion I min 47 499 Capitan 2 Percussion I min
470
Capitan Percussion I min 39 488
Capitan 4 Percussion I min 38 485 Capitan I Percussion 2 min 55 497 Capitan Capitan
2
Percussion 2 min Percussion 2 min
71 65
454 477
Capitan 4 Percussion 2 min 56 496 Capitan I Percussion 4 min 86 347 Capitan 2 Percussion 4 min 79 407 Capitan 3 Percussion 4 min 81 392 Capitan 4 Percussion 4 min 82 384 Capitan I Percussion 8 min 90 300 Capitan 2 Percussion 8 min 88 325 Capitan 3 Percussion 8 min 90 300 Capitan 4 Percussion 8 min 90 300 Gila I Immersion 71 454 Gila 2 Immersion 77 421 Gila
Immersion 75 433
Gila 4 Immersion 72 449
Gila I Boiling 30 sec 68 466
Gila 2 Boiling 30 sec 81 392
Gila 3 Boiling 30 sec 54 498
Gila 4 Boiling 30 sec 63 483
Gila I Boiling 60 sec 55 497
Gila 2 Boiling 60 sec 68 466
Gila 3 Boiling 60 sec 57 495
Gila 4 Boiling 60 sec 70 458
65
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Gila 2 Control 13 336 Gila 3 Control 13 336 Gila 4 Control 20 400 Gila 1 Percussion 1 min 66 474 Gila 2 Percussion 1 min 57 495 Gila 3 Percussion 1 min 73 444 Gila 4 Percussion 1 min 82 384 Gila 1 Percussion 2 min 81 392 Gila 2 Percussion 2 min 86 347 Gila 3 Percussion 2 min 85 357 Gila 4 Percussion 2 min 54 498 Gila 1 Percussion 4 min 79 407 Gila 2 Percussion 4 min 83 376 Gila 3 Percussion 4 min 84 367 Gila 4 Percussion 4 min 79 407 Gila 1 Percussion 8 min 51 500 Gila 2 Percussion 8 min 76 427 Gila 3 Percussion 8 min 52 500 Gila 4 Percussion 8 min 74 439 Holman 1 Immersion 83 376 Holman 2 Immersion 86 347 Holman 3 Immersion 81 392 Holman 4 Immersion 90 300 Holman 1 Boiling 30 sec 90 300 Holman 2 Boiling 30 sec 85 357 Holman 3 Boiling 30 sec 87 336 Holman 4 Boiling 30 sec 83 376 Holman 1 Boiling 60 sec 79 407 Holman 2 Boiling 60 sec 80 400 Holman 3 Boiling 60 sec 86 347 Holman 4 Boiling 60 sec 93 255 Holman Control 25 433 Holman 2 Control 19 392 Holman Control 21 407
Holman 4 Control 24 427
Holman Percussion 1 min 77 421
Holman 2 Percussion 1 min 85 357
Holman 3 Percussion 1 min 80 400
Holman 4 Percussion 1 min 89 313
Holman 1 Percussion 2 min 88 325
Holman 2 Percussion 2 min 96 196
Holman 3 Percussion 2 min 93 255
Holman 4 Percussion 2 min 96 196
Holman 1 Percussion 4 min 92 271
Holman 2 Percussion 4 min 97 171
66
I
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Holman 3 Percussion 4 min 91 286 Holman 4 Percussion 4 min 96 196 Holman 1 Percussion 8 min 88 325 Holman 2 Percussion 8 min 85 357 Holman 3 Percussion 8 min 83 376 Holman 4 Percussion 8 min 82 384 Jemez 1 Immersion 53 499 Jemez 2 Immersion 44 496 Jemez 3 Immersion 52 500 Jemez 4 Immersion 70 458 Jemez 1 Boiling 30 sec 58 494 Jemez 2 Boiling 30 sec 55 497 Jemez 3 Boiling 30 sec 48 500 Jemez 4 Boiling 30 sec 65 477 Jemez 1 Boiling 60 sec 68 466 Jemez 2 Boiling 60 sec 63 483 Jemez 3 Boiling 60 sec 73 444 Jemez 4 Boiling 60 sec 89 313 Jemez 1 Control 2 140 Jemez 2 Control 9 286 Jemez 3 Control 6 237 Jemez 4 Control 8 271 Jemez 1 Percussion 1 min 58 494 Jemez 2 Percussion 1 min 63 483 Jemez 3 Percussion 1 min 77 421 Jemez 4 Percussion 1 min 89 313 Jemez 1 Percussion 2 min 68 466 Jemez 2 Percussion 2 min 64 480 Jemez 3 Percussion 2 min 80 400 Jemez 4 Percussion 2 min 83 376 Jemez Percussion 4 min 68 466 Jemez 2 Percussion 4 min 79 407 Jemez 3 Percussion 4 min 70 458 Jemez 4 Percussion 4 min 83 376 Jemez 1 Percussion 8 min 52 500 Jemez 2 Percussion 8 min 59 492 Jemez 3 Percussion 8 min 61 488 Jemez 4 Percussion 8 min 70 458 Magdalena 1 Immersion 69 462 Magdalena 2 Immersion 69 462 Magdalena J Immersion 70 458 Magdalena 4 Immersion 85 357 Magdalena 1 Boiling 30 sec 65 477 Magdalena 2 Boiling 30 sec 71 454 Magdalena 3 Boiling 30 sec 68 466
67
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Magdalena 4 Boiling 30 sec 83 376 Magdalena 1 Boiling 60 sec 63 483 Magdalena 2 Boiling 60 sec 68 466 Magdalena 3 Boiling 60 sec 63 483 Magdalena 4 Boiling 60 sec 82 384 Magdalena 1 Control 11 313 Magdalena 2 Control 6 237 Magdalena 3 Control 5 218 Magdalena 4 Control 4 196 Magdalena Percussion 1 min 32 466 Magdalena 2 Percussion 1 min 45 497 Magdalena 3 Percussion 1 min 46 498 Magdalena 4 Percussion I min 36 480 Magdalena 1 Percussion 2 min 67 470 Magdalena 2 Percussion 2 min 68 466 Magdalena 3 Percussion 2 min 65 477 Magdalena 4 Percussion 2 min 81 392 Magdalena 1 Percussion 4 min 87 336 Magdalena 2 Percussion 4 min 97 171 Magdalena 3 Percussion 4 min 83 376 Magdalena 4 Percussion 4 min 97 171 Magdalena 1 Percussion 8 min 98 lAO Magdalena 2 Percussion 8 min 100 000 Magdalena 3 Percussion 8 min 96 196 Magdalena 4 Percussion 8 min 98 lAO Manzano 1 Immersion 67 470 Manzano 2 Immersion 57 495 Manzano 3 Immersion 72 4049 Manzano 4 Immersion 73 4044 Manzano 1 Boiling 30 sec 70 458 Manzano 2 Boiling 30 sec 64 480 Manzano 3 Boiling 30 sec 71 454 Manzano 4 Boiling 30 sec 80 400 Manzano I Boiling 60 sec 50 500 Manzano 2 Boiling 60 sec 53 499 Manzano 3 Boiling 60 sec 59 492 Manzano 4 Boiling 60 sec 74 439 Manzano I Control 4 196 Manzano 2 Control 8 271 Manzano 3 Control 1 099 Manzano 4 Control 6 237 Manzano 1 Percussion 1 min 52 500 Manzano 2 Percussion 1 min 39 488 Manzano 3 Percussion 1 min 36 480 Manzano 4 Percussion 1 min 51 500
68
Table B 1 Continued
Source Block Treatment Gerrni~ation Percentage Standard Error
Manzano Percussion 2 min 53 499 Manzano 2 Percussion 2 min 58 494 Manzano 3 Percussion 2 min 61 488 Manzano 4 Percussion 2 min 62 485 Manzano 1 Percussion 4 min 76 427 Manzano 2 Percussion 4 min 85 357 Manzano 3 Percussion 4 min 79 407 Manzano 4 Percussion 4 min 83 376 Manzano 1 Percussion 8 min 87 336 Manzano 2 Percussion 8 min 83 376 Manzano 3 Percussion 8 min 83 376 Manzano 4 Percussion 8 min 75 433 Raton 1 Immersion 54 498 Raton 2 Immersion 37 483 Raton 3 Immersion 39 488 Raton 4 Immersion 69 462 Raton 1 Boiling 30 sec 36 480 Raton 2 Boiling 30 sec 20 400 Raton 3 Boiling 30 sec 28 449 Raton 4 Boiling 30 sec 60 490 Raton 1 Boiling 60 sec 15 357 Raton 2 Boiling 60 sec 171 Raton 3 Boiling 60 sec 4 196 Raton 4 Boiling 60 sec 35 477 Raton 1 Control 4 196 Raton 2 Control 12 325 Raton 3 Control 2 140 Raton 4 Control 6 237 Raton 1 Percussion 1 min 52 500 Raton 2 Percussion I min 29 454 Raton 3 Percussion I min 35 477 Raton 4 Percussion 1 min 31 462 Raton 1 Percussion 2 min 55 497 Raton 2 Percussion 2 min 35 477 Raton 3 Percussion 2 min 33 470 Raton 4 Percussion 2 min 42 494 Raton 1 Percussion 4 min 65 477 Raton 2 Percussion 4 min 52 500 Raton Percussion 4 min 51 500 Raton 4 Percussion 4 min 53 499 Raton 1 Percussion 8 min 88 325 Raton 2 Percussion 8 min 80 400 Raton 3 Percussion 8 min 73 444 Raton 4 Percussion 8 min 74 439 Sacramento 1 Immersion 43 495
69
Table B 1 Continued
-Source Block Treatment Germination Percentage Standard Error
Sacramento 2 Immersion 30 458 Sacramento 3 Immersion 38 485 Sacramento 4 Immersion 56 496 Sacramento 1 Boiling 30 sec 47 499 Sacramento 2 Boiling 30 sec 48 500 Sacramento 3 Boiling 30 sec 48 500 Sacramento 4 Boiling 30 sec 56 496 Sacramento 1 Boiling 60 sec 52 500 Sacramento 2 Boiling 60 sec 67 470 Sacramento 3 Boiling 60 sec 43 495 Sacramento 4 Boiling 60 sec 48 500 Sacramento 1 Control 0 000 Sacramento 2 Control 3 171 Sacramento 3 Control 3 171 Sacramento 4 Control 099 Sacramento 1 Percussion 1 min 57 495 Sacramento 2 Percussion 1 min 61 488 Sacramento 3 Percussion 1 min 62 485 Sacramento 4 Percussion 1 min 55 497 Sacramento 1 Percussion 2 min 84 367 Sacramento 2 Percussion 2 min 70 458 Sacramento 3 Percussion 2 min 81 392 Sacramento 4 Percussion 2 min 69 462 Sacramento 1 Percussion 4 min 91 286 Sacramento 2 Percussion 4 min 84 367 Sacramento 3 Percussion 4 min 84 367 Sacramento 4 Percussion 4 min 94 237 Sacramento 1 Percussion 8 min 82 384 Sacramento 2 Percussion 8 min 81 392 Sacramento 3 Percussion 8 min 82 384 Sacramento 4 Percussion 8 min 81 392 Sandia 1 Immersion 76 427 Sandia 2 Immersion 70 458 Sandia 3 Immersion 70 458 Sandia 4 Immersion 81 392 Sandia 1 Boiling 30 sec 78 414 Sandia 2 Boiling 30 sec 71 454 Sandia 3 Boiling 30 sec 63 483 Sandia 4 Boiling 30 sec 78 414 Sandia 1 Boiling 60 sec 67 470 Sandia 2 Boiling 60 sec 65 477 Sandia 3 Boiling 60 sec 74 439 Sandia 4 Boiling 60 sec 66 474 Sandia 1 Control 16 367 Sandia 2 Control 7 255
70
Table B 1 Continued
Source Block Treatment Gennination Percentage Standard Error
Sandia 3 Control 8 271 Sandia 4 Control 16 367 Sandia 1 Percussion 1 min 67 470 Sandia 2 Percussion 1 min 61 488 Sandia 3 Percussion 1 min 66 474 Sandia 4 Percussion 1 min 59 492 Sandia I Percussion 2 min 82 384 Sandia 2 Percussion 2 min 83 376 Sandia 3 Percussion 2 min 81 392 Sandia 4 Percussion 2 min 84 367 Sandia 1 Percussion 4 min 89 313 Sandia 2 Percussion 4 min 86 347 Sandia 3 Percussion 4 min 89 313 Sandia 4 Percussion 4 min 85 357 Sandia 1 Percussion 8 min 80 400 Sandia 2 Percussion 8 min 77 421 Sandia 3 Percussion 8 min 69 462 Sandia 4 Percussion 8 min 66 474
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Hamly DH 1932 Softening of the seeds of Melilotus alba Bot Gazette 93 345shy375
Hanna PJ 1984 Anatomical features of the seed coat ofAcacia kempeana (Mueller) which relate to increased germination rate induced by heat treatment New Phytol 96 23-29
Hanks JP Dick-Peddie WA 1974 Vegetation patterns of the White Mountains New Mexico Southwest Naturalist 18(4) 371-382
Hine S Harrington JT Dreesen DR 1997 Seed scarification requirements for Robinia neomexicana In Harrington JTtech coord Proc Third SouthwestemContainer Growers Conf Feb 12-13 1997 Ft Collins CO
Hopkinson JM 1993 The strophiole of leguminous seeds Newsletter- IntI Herbage Seed Production Res Grp 18 7-8
---- 1997 Differences in hardseededness between Cunningham and Tarramba leucaena In Leucnet News The Newsletter of the International LeucaenaRes and Devlt Netwk No4
ISTA (International Seed Testing Association) 1999 International rules for seed testing rules Seed Sci amp Technol27 Supplement
Kelly KM van Staden J 1987 The lens as the site of permeability in the papilionoid seed Aspalathus linearis J Plant Physiol 128(45) 395-404
73
Keresztesi B 1988 Black locust the tree of agriculture Outlook on Agr 17(2) 77~85
Kigel 1 and Galili G 1995 Seed development and germination Marcel Dekker New York 853 p
Klemmedson 10 1994 New Mexico locust and parent material Influence on macronutrients of forest floor and soil Soil Sci Soc Am 1 58 974~980
Kuo WH Tam AW 1988 The pathway of water absorption of mung bean seeds Seed Sci amp Technol16(1) 139~144
Leadem CL 1997 Dormancy--unlocking seed secrets In Landis TD Thompson lR tech coords National Proceedings Forestand Conservation Nursery Assocs Gen Tech Rep PNW-G TR-419 Portland OR USDA For Ser PNW Res Sta 43-52
Lin Y Wagner MR Seweyestewa A Honahni MD DeGomez M McMillin JD 1996 Influence of cultural treatments on seed germination and seedling growth of Robinia neomexicana Forest Farm and Community Tree Res Reports Volume 1
Marunda CT 1990 Effects of seed pretreatment on the development ofAcacia auriculforinis and A holosericea seedlings ACIAR Proceedings Series No 28
Meginnis HG 1937 Sulphuric acid treatment to increase germination of black locust seed USDA Circ 453 34 p
Miklas PN Towensend CE Ladd SL 1987 Seedcoat anatomy and the scarification ofcicer milkvetch seed Crop Sci 27 766-772
Monsen SB 1984 Use of shrubs on mine spoils In The Challenge ofProducing Native Plants for the Intermountain Area Proceedings Intermountain Nurserymans Association 1983 conference Las Vegas NV compo PM Murphy 26-31 Ogden UT USDA For Ser Gen Tech Report INT-l68
Olson DF 1974 Robinia L Locust In Seeds of Woody Plants in the US tech coord CS Schopmeyer 728-731 USDA For Ser Agr Handbk No450
Rehman S Loescher RNl Harris P1C 1998 Dormancy breaking and germination ofAcacia salicina Lindl seeds Seed Sci amp Techno127 553-557
Rolston MP 1978 Water impermeable seed dormancy Bot Rev 44 365-396
74
SAS Institute Inc 1989 SASSTAT Users Guide Version 6 4th Edition Volume 1 Cary NC SAS Institute Inc 943 p
SerratoValenti G Conara L Ferrando M Modenesi P 1993 Structural and histochemical features ofStylosanthes scabra (Leguminosae Papilionoidae) seed coat as related to water entry Can J Bot 71 834-840
Simpson BJ 1988 A field guide to Texas trees Austin TX Texas Monthly Press 372p
Tran VN Cavanagh AK 1984 Structural aspects ofdormancy In Seed Physiology Vol 2 Murray DR (ed) p1-44 Academic Press Australia
USDA Forest Service 1948 Woody-plant seed manual USDA Misc Pub1654 416p
Vines RA 1960 Trees Shrubs and Woody Vines of the Southwest Austin Univ ofTexas Press 1104 p
Wagner MR Larson RE Craig TP 1992 Ecology and silvics ofNew Mexico locust (Robinia neomexicana) with an emphasis on the arboreal form p53-57 In JW Hanover K Miller and S Plesko (eds) Proc Intemat Confon Black Locust Biology Culture and Utilization Mich State Univ USA
Wilson JK 1944 Immersing seeds of species of Robinia in boiling water hastens germination J For 42 453-454
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Fine-tuning a treatment in this range of treatment temperature and duration may be
the most logical approach
Mechanical Scarification
This treatment involves scratching the seed coat with an abrasive material
(USDA Forest Service 1948) One system employs a rotating drum lined with
sandpaper A paddle spinning at a given number ofrevolutions per minute throws the
seed against the sandpaper (Hine et al 1997) A second method dry tumbling
tumbles seed in a mixer with pea gravel or sharp sand for 5 to 7 days (Dreesen 2001)
These approaches have the advantage that control of temperature is not required as in
water treatments nor is safety an issue as with add scarification
Hine et al (1997) mechanically treated New Mexico locust seed using 100shy
grit sandpaper in a Forsberg (rotating drum) scarifier Gennination was less than
10 They concluded that the seed coats were too thin for their apparatus and
proposed that a finer grit sandpaper would produce more even abrasion Although
germination varied by seed lot Dreesen (2001) reported that using a slow-speed dry
tumbling technique for 5 to 7 days produced gennination rates as high as 90
As with acid and hot water treatments treatment duration must be specifically
shy
correlated to seed lot Treated seeds are also susceptible to fungal infection
iHopkinson 1997) In addition equipment is specialized and may not always be
ltU~CUJl~ to a nursery (USDA Forest Service 1948) Several studies have confinned
degrading ofstrophiolar cells due to acid and hot water scarification (Miklas et al
11
1987 Dell 1980 Hanna 1984) but the effect ofmechanical scarification on the
strophiole has not been studied
Alternative Method for Breaking Dormancy Percussion
Strophiole degradation has been examined in depth using an impaction
treatment more commonly known as percussion Hamly (1932) first proposed that
shaking or impacting seeds could split the macrosc1ereid cells at the strophiole
Hamly successfully released tension ofbent strophiolar cells of impermeable
Melilotus alba Medik (White sweet clover) seeds by delivering physical blows
resulting in cells that had split apart and were only slightly bent Ninety-two percent
oftreated seeds became permeable as compared to 05 of control seeds Barton
(1947) showed that percussion significantly increases permeability in black locust
seed Ballard (1976) reasoned that the integrated nature of the strophiole within the
seed coat means a blow on any part of the seed weakens the strophiole There are no
published studies on the use of percussion on New Mexico locust
Percussion methods vary and often seem SUbjective or ill-defined Hamly
(1932) placed seeds in a glass bottle and shook them three times a second for 20
minutes resulting in 180 oscillations per minute Ballard (1973) describes shaking
seeds in a closed metal container with at least fifty seeds per container at 850-900
JS(~l11alIOIlS per minute for 1 minute Kelly and van Staden (1987) placed seeds into
sealed glass flasks Flasks were shaken by a wrist-shaker at a rate of 250
~UUVJ~ per minute Subsequent germination rates were 45 after 2 hours of
55 after 3 hours 60 after 6 hours and 100 after 12 hours
12
There are several potential advantages of percussion over acid hot water and
mechanical scarification Kelly and van Staden (1987) note that a percussion
treatment only alters macrosclereid cells of the strophiole by loosening separating
and raising them They emphasize that minimal damage is done to the overall
anatomical structure of the seed In acid scarification of the same seed lot radicle
and cotyledon damage occurred Based on photographic evidence the authors
believe that this morphological damage was due to uneven pressure during
imbibition with water entering haphazardly wherever the acid had sufficiently
dissolved the coat This ultimately led to uneven hydration of the embryo In
percussed seeds water only entered through the s~ophiole when the entire seed coat
had been saturated resulting in controlled and even embryo hydration (Kelly and van
Staden 1987)
Although all successful scarification treatments may focus at least in part on
the strophiole for papilionoid legumes (Baskin and Baskin 1998) percussion may be
less damaging to seed coat structures particularly when compared with acid Kelly
and van Staden did not compare hot water or mechanical scarification methods
against percussion However the fungal infection associated with hot water and
mechanical scarification as with acid scarificatio~ have been directly associated with
seed damage (Rehman et al 1998 USDA Forest Service 1948) Embryo damage in
has been observed in hot-water scarified legume seeds (Rehman et al 1998
13
By limiting embryo damage percussion scarification reduces seedling
mortality compared to other treatments Kelly and van Staden (1987) noted that
seedlings from the percussion treatment were morphologically sound and vigorous
when compared to acid treatment seedlings The authors attribute this improved
seedling vigor to the controlled water uptake that limited anatomical damage
Despite a 12-hour percussion treatment morphological damage was not substantially
greater than untreated seeds In addition seedling mass measured 10 days following
germination was not significantly different between percussed and untreated seeds
A final advantage ofpercussion may be the ability to adequately treat the most
dormant seeds while avoiding harm to seeds that have already been made permeable
by the treatment or that were permeable to begin with Instead ofcorroding scalding
or scratching the seed coat percussion appears to be just a loosening of the
strophiole spring (Ballard 1973) Kelly and van Staden (1987) were able to
increase germination with a lengthy treatment (12 hours) Figuring out whether a
substantial cushion oftreatment durations exist for percussion ofNew Mexico locust
seed would be a significant improvement to current scarification methods where seed
damage and seedling health is an issue
The greatest challenge for applying percussion to New Mexico locust seeds is
standardizing a treatment that is both effective and readily reproducible by nurseries
with mechanical scarification this may require the development of specific
~UllDment to properly quantify and execute the treatment
14
f
Objectives
The first objective of the study is to detennine which scarification treatment
results in optimal gennination speed and percentage ofNew Mexico locust The
second objective is to describe treatment interactions with nine seed sources collected
in New Mexico
Hypothesis
A percussion treatment acts exclusively on the main area ofwater uptake in
New Mexico locust seed loosening the strophiole so that imbibition can occur This
direct mechanism ofdonnancy release will result in improved gennination rate and
overall gennination percentage for New Mexico locust seed compared to standard
treatments Standard treatments including acid hot water and mechanical
scarification while eventually weakening the strophiolar cleft cause excessive
morphological damage through generalized seed coat degradation These treatments
are therefore prone to inconsistency due to over and under-treatment
15
MATERIALS AND METHODS
Seed Collection and Cleaning
Seeds were collected from August 25 through September 15 2000 from nine
locations along a latitudinal gradient in New Mexico Seed source locations and
descriptive information are given in Figure 1 and Tables I and 2 Stands were
separated by at least 45 km
Pods were hand picked as they began to ripen and dehisce (open) Collections
were made from ground level to approximately 3 m from several trees at each site
Pods were dried in open-ended brown paper bags for two to four weeks at room
temperature (21-230C) to facilitate cleaning Dried pods were placed in a closed cloth
sack which was pounded against the ground to separate seeds from pods For pods
still containing seeds hand maceration (crunching pods with hands) completed seed
separation Debris was removed by threshing with the aid of a greenhouse fan A
South Dakota Seed Blower (Seedbureau Chicago Illinois) was used to remove
remaining chaff and light seeds by air density separation Seeds were placed in dry
storage in sealed quart-size zip-lock bags within a sealed plastic container Storage
temperatures ranged from 2-4oC Seeds remained in storage for approximately 7
months before the experiment
Experimental Design and Treatment Structure
The experiment compared the impact ofhot water bath and percussion
l11aLl11 on germination properties ofNew Mexico locust seed A single
16
-l
Figure 1 Seed source collection map All sources were collected within New Mexico USA separated by at least 4S lan
IIIRZltItW lIJPIirmiddot rllll1lbullbull_~~~_II
Table 1 Latitude longitude location elevation and collection date ofNew Mexico locust seed sources
Lot title Latitude (N) Longitude (W) Location Elevation (m) Collection Date
Raton 36deg5846 104deg2872 Raton Pass NM 2295 82500
Holman 36deg0306 105deg2429 HolmanNM 2476 911500
Jemez 35deg4500 106deg4100 Jemez Springs NM 1976 9900 00
Sandia 35deg1083 106deg2341 Cedar Crest NM 2432 82600
Manzano 34deg3689 106deg2494 ManzanoNM 2465 9200
Magdalena 33deg5953 107deg0903 Magdalena NM 2544 82900
Capitan 33deg2728 105deg4325 Alto NM 2189 911200
Sacramento 32deg5798 105deg4496 Cloudcroft NM 2630 91400 Gila 32deg5400 107deg4600 Kingston NM 2432 9700
f
Table 2 Mean seed weight and standard error by source
a
Source Mean seed weight (g) for Standard error 100 seeds n = 5
Raton Holman Jemez Sandia Manzano Magdalena Capitan Sacramento Gila
2872 b c 2522 d 1826 e 2753 c 2413 d 2559 d 2443 d 3130 a 3029 ab
00103 00403 00276 00301 00370 00374 00458 00279 00348
a Mean seed weights followed by the same letter are not significantly
different
19
experiment was conducted utilizing a randomized complete block design blocked by
time with a factorial treatment structure Experimental factors were seed source and
scarification treatment Nine seed sources from throughout New Mexico were used
(Figure 1 Table 1) Scarification treatments included a control three levels ofhot
water and four levels of percussion Hot water treatments included immersion at
100degC and boiling for periods of 30 seconds and 60 seconds Percussion treatment
levels were 1 2 4 and 8 minutes One hundred seeds per block were used to test
each treatment combination Four blocks of seed were tested one at a time (all seed
sources included in each run) in close chronological sequence (within 6 weeks) A
total of four 100-seed samples were used to test each source by scarification treatment
combination
Treatments
Hot water treatment of seeds utilized 50 ml test tubes filled with 30 ml
distilled water Each test tube was used to treat one 100-seed sample at a time
resulting in a ratio of4 parts water to 1 part seed by volume Test tubes were placed
in a beaker with the beaker filled with tap water to the same level as the water within
test tubes Nine test tubes (one per seed source) were placed in the beaker
simultaneously so that one replication of each seed sourcescarification treatment
cotnblnatlon was conducted simultaneously Hot water treatments include immersion
boiling For immersion treatments seeds were added to test tubes with the water
(boiling) and the test tubes were immediately removed from the beaker to
For the boiling treatments test tubes remained in the beaker with water at
20
boiling temperature for a set duration after the addition of seeds Following all
treatments test tubes with seeds cooled 24 hours in a test tube rack
Percussion treatment utilized a pneumatic paint shaker (Central Pneumatic
model 00422 Camarillo CA) Compressed air was maintained at 530 kPa plusmn30 kPa
(80 psi plusmn 5 psi) resulting in approximately 350 oscillations per minute Seeds were
placed in 118 ml (4 oz) soil tin cans (US Can Lobard IL) and the lids were secured
with duct tape Each can was used to treat one 100-seed sample To take advantage
ofgreatest lateral movement of the paint shaker a spacer (paint can) was used to
position the soil tin at the end of the shaking arm This allowed shaking to occur at
the greatest distance from the pivot point (fulcrum) Tins were placed perpendicular
to the direction of shaking motion allowing seeds to impact at greatest force by
hitting flat surfaces of the soil tin
Germination Testing
Before gennination testing all seeds were soaked 24 hours in reverse
osmosis-treated water (this was included in the process ofhot water treatment
jU~SCIlOea above) Following soaking seeds were placed in 100 cm Petri dishes lined
90 cm Whatman qualitative 1 filter paper circles (MG Scientific Pleasant
WI) One Petri dish was used for each treatment by source combination
papers were initially moistened with 5 mL reverse osmosis-treated water then
~VU1CUlJl moistened as needed to ensure adequate conditions for gennination
were monitored daily (at 300 PM) for gennination for 14 days in accordance
~UIUlnJlVUa Seed Testing Association (ISTA) standards for Robinia species
21
(1STA 1999) Tests were carried out at room temperature (22degC plusmnOSoC)
Germination was classified as protrusion of the radicle from the embryo by at least
1mm as viewed with the naked eye
X-ray Radiography
On August 132001 three lOO-seed samples from the Sacramento seed source
were treated for 14 or 8-minute percussion durations The seeds were sent
immediately by overnight delivery to the Faxitron X-Ray Corporation in Wheeling
Illinois where Dr Bob Karrfalt ofthe USDA Forest Service oversaw their imaging
Radiographs were made at SX with a Faxitron Specimen Radiography System Model
MX-20
Scanning Electron Microscope Photography
On February 192002 a Hitachi S-3200N Variable Pressure Scanning
Electron Microscope (Mountain View CA) was used to examine strophiolar cells of
untreated seeds and seeds percussed for 4 minutes Seeds were cut longitudinally
igt their entire length using a razor blade splitting the strophiolar region roughly in
Cross sections of seed samples magnifying the strophiolar region were viewed
600X under the microscope
Analysis
Categorical Analysis of Variance was used to determine treatment source and
(Le time) main effects and interactions The response variable was total
~vlI This procedure is a Chi-square test of homogeneity using the natural log
of germinated to non-germinated seed for each treatment Maximumshy
22
likelihood analysis was used to calculate Chi-square test statistics with their observed
significance levels A single contrast was conducted to cempare treatment means
against the control Observed significance levels less than or equal to 005 were
considered significant Pairwise comparisons were conducted between non-control
treatment mean percents using a conservative alp~a value of 005 divided by the
number of comparisons (Bonferroni approach) and approximate pairwise z statistics
Categorical analyses were performed using Proc Catmod (SAS Institute 1989)
RESULTS
Scarification seed source block and all interactions between factors
influenced germination (Table 3) The main effects are reported in the presence of
these interactions because the overall robustness of treatments and seed sources are of
interest Background levels ofnaturally occurring variation in seed sources are
seen in control germination percentages in Figure 2 In particular time variability
is unlikely to be under the control of the producer Appendix B details experimental
data for source by treatment gennination percents for each of the four blocks
Averaged over all treatments germination by source ranged from 40 to
79 (Figure 2) Averaged over all sources (Figure 3) and for each source (Figure 4
5) percussion scarification durations of4 or 8 minutes yielded greatest germination
Averaged over all sources germination increased with increasing durations of
percussion then leveled off for the 4- and 8-minute treatments (Figure 3) Immersion
and 30-second boiling scarification maximized germination for the hot water
treatments with a decrease in germination when seeds were boiled for 60 seconds
For all but two sources there is a similar ipcreasing trend in germination from
1to 4 minutes ofpercussion (Figure 4) The Holman source increased in germination
1 to 2 minutes then leveled off at 4 minutes The Jemez source changed little
1 to 4 minutes of treatment As treatment duration increased from 4 to 8
mutes five sources decreased in germination (Figure 4a) whereas three sources
24
Table 3 Categorical Analysis of Variance table for germination response to seed treatment seed source block and the interaction of the factors
Source of DF Chi-Square Observed Variability Significance Level
Source 8 98370 lt00001 Trt 7 233876 lt00001
contrast 1 16976 lt00001 SourceTrt 56 97077 lt00001 Block 3 5538 lt00001
SourceBlock 24 10162 lt00001 TrtBlock 21 7564 lt00001
SourceTrtBlock 168 34283 lt00001
S 4)
8 ~
0
sectg ~ 40 ~
N 0 0
20
o f0ltgt ~ ~4 jybshy ((iltgto ~~ K1ltgt 6~b-~ ~~ 0b-~~ ~~ ~b-~ ~b-~b
80
60 -I-- - ~- middotmiddotmiddotmiddotmiddot1
r -] Northern NM Sources
Central NM Sources
Southern NM Sources
sect-o ~
~b-v(i
SOURCE
Figure 2 Effect ofNew Mexico locust seed source on germination for data averaged over all scarification treatments Control germination is shown by solid line within bar
80
~ cd ~ ltl) () 60 ~ ~
N
t 0shyrti tshySltl)
40
0
20
o lmmersion Boiling 30 sec Boiling 60 sec Control 1 min 2min 4 min 8 min
Hot Water Treatments Percussion Treatments
Figure 3 Effect of scarification on germination ofNew Mexico locust for data averaged over all seed sources Means labelled with the same letter are not significantly different
100~----------------------~--~----------------~
80
60
ltU
~
40 IJ
sectgsI
l100
80
60
40
a I middotmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot middotImiddotmiddotmiddotmiddotmiddotmiddot E
_~iii-bull _ _ bull f --~--
t-- -~ ---- -shy ---- shyI ~ ---- - shy
L
fiII
~
b
- shy ---r
- Holman
Sacramento Sandia Gila Jemez
c ~
ltgt ~
Tmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot-I
j~=~=-~~-1
I middotmiddot ~ bull r
t tr
Y -------M-a-gd-a-Ie-n-a--
1 _ _
fCapitan
---- Manzano ~y -_ Raton
2 4 8
Time interval ofpercussion scarification (min)
Figure 4 Effect of seed source by treatment interaction on New Mexico locust germination for percussion scarification pattern a and pattern b
28
tv 0
~ d
~ A s o
1 c3
80
60
40
20
o
TIT
t------=middotfmiddotmiddot-middotmiddot-middotmiddot-middotmiddot 1- --- - - --=- - -shy--cE= - _--shy --shy ----shy ~ ~ ---shy ~ -~- _ --shy-------r 1
I
Raton Holman
-_ Jemez
--- Sandia Manzano Magdalena Capitan Sacramento Gila
Immersion Boiling 30 sec Boiling 60 sec
Hot Water Scarification
Figure 5 Effect of seed source by treatment interaction on New Mexico locust germination for hot water scarification
continued to increase in germination (Figure 4b) There was no difference between
the two treatment levels for the Manzano source (Figure 4b)
The effect ofhot water scarification also varied among sources with few
overall differences between hot water treatments (Figure 5) However boiling for 60
seconds yielded highest germination for the Jemez source whereas increasing
durations ofboiling decreased germination for the Raton source
In addition to consistently high germination 4- and 8-minute percussionshy
treated seeds germinated as fast or faster than all other treatments (Figure 6 Table 4)
050 measured as the number of days needed to complete 50 ofeventual
germination averaged 2 days for both ofthese treatments
X-ray radiographs were taken of seeds collected from the Sacramento source
percussed for 1 4 or 8 minutes (Figure 7) Seeds show signs of damage at 8 minutes
ofpercussion (Figure 8) Compared to an undamaged seed percussed for only 4
minutes (Figure 8a) damage at the 8-minute level includes fractured radicles pitting
of the seed coat and chips in seed coat structure (Figure 8b-d)
Scanning electron microscope (SEM) images at 600X reveal the direct effect
of 4 minutes of percussion on cells of the strophiolar region (Figure 9) Whereas
untreated seeds retain cellular organization in the strophiolar region strophiolar cells
seeds treated for 4 minutes ofpercussion separate and raise resulting in a
~-~e~U appearance
30
Umiddot-~middot--l-~---middotymiddotmiddotmiddot-----middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot--middot--middotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middotmiddotmiddotmiddotmiddotmiddotmiddot- -
050= gt1
-e-- Percussion 8 min -0- Percussion 4 min ---T- Percussion 2 min -----ltil- Percussion 1 min -- Immersion --0- Boiling 30 sec -+- Boiling 60 sec -ltgt- Control
2 4 6 8 10 12
Day Figure 6 Timecourse ofNew Mexico locust gennination following scarification
treatments G50 is the number of days to 50 of eventual germination
31
14
Table 4 Effect of seed source and treatment on G50 (days to 50 germination)
Seed Source Control Immersion B30sec B60sec Plmin P2min P4min P8min mean Raton 3 3 5 7 4 3 2 2 4 Holman 2 2 2 2 2 2 2 2 2
Jemez 4 6 6 5 3 2 2 2 4 Sandia 2 2 2 2 2 2 2 2 2
w tv Manzano 5 2 2 3 3 2 2 2 3
Magdalena 4 2 2 3 4 3 2 1 3
Capitan 5 4 2 2 3 2 2 2 3
Sacramento 3 2 2 2 4 3 2 2 3 Gila 3 2 2 2 2 2 2 2 2 mean 3 3 3 3 3 2 2 2 3 standard 11 14 16 18 09 05 0 03 08 deviation
Note Columns and rows may not agree due to rounding
Figure 7 X-ray radiographs ofSacramento seed source percussed for 1 4 or 8 minutes
33
Figure 8a-d Seed damage close-ups Figure a shows undamaged seed percussed for 4 minutes Figures b-d show seeds percussed for 8 minutes Damage includes fracturing ofradicle in b complete removal ofseed coat in c and pitting ofseed coat in d
34
Untreated Treated
Figure 9 SEM images ofuntreated vs 4-minute percussion-treated strophiolar cells ofNew Mexico locust seeds Treated cells are loosened separated and raised
DISCUSSION
Determining the Optimal Treatment
Averaging results of scarification treatmems across seed sources is a useful
tool to predict which treatment is most likely to be successful for an unknown seed
source Averaged over all sources four- and eight-minute percussion treatments
resulted in the highest germination 815 and 790 respectively and produced
germination that was as fast or faster than all other treatments Although there was no
difference between these two treatments on average an argument for 4 minutes of
percussion as a standard protocol for treating New Mexico locust seeds can be made
Descriptively 4 minutes of percussion improved germination over 8 minutes
ofpercussion for a majority of sources (five out of nine) 8 minutes of percussion
improved germination for three ofnine sources with no difference for a fmal source
For individual sources 4 minutes ofpercussion was always the best or secondmiddot best
treatment and was always equal to or better than all hot water treatments
Germination at 8 minutes ofpercussion was lower than all other percussion durations
for the Jemez and Gila sources and was lower than at least one hot water treatment for
these sources as well
The x-ray radiographs of the Sacramento seed source illustrate the excessive
internal and external seed damage that can occur with 8 minutes of percussion
germination counts 8-minute percussed seeds from Holman and Capitan
ClUll signs of damage that were visible to the naked eye (see below)
r~evertheless some of these seeds still fit the criteria for germination Neither x-ray
36
photography nor visible observations during seed counts revealed seed damage at the
4-minute percussion level In operational trials seeds percussed for 4 minutes
developed into healthy seedlings (Khadduri and Harrington unpublished data)
Strophiole and Seed Coat Behavior
Four minutes ofpercussion successfully breaks seed dormancy in a large
percentage ofNew Mexico locust seeds without excessively damaging the
surrounding seed coat or internal seed coat structures Most papilionoid legume
seeds including seeds ofNew Mexico locust have a specific region of the seed coat
known as the strophiole or lens (Hamly 1932) The strophiole is located on the
cotyledonary lobe of a locust seed (Figure 10 a-c) If seeds are not indiscriminately
damaged the strophiole is the first point of water entry to the embryo (Hopkinson
1997) When a papilionoid seed is percussed for the appropriate amount of time
repeated hits on the integrated seed coat loosen the constrained cells of the strophiolar
region without excessively damaging the rest of the seed coat (Ballard 1976) SEM
photos illuStrate that 4 minutes of percussion loosened and separated strophiolar cells
ofNew Mexico locust seeds when compared with constrained organized strophiolar
cells of untreated seeds (Figure 9) Research-has shown that water passes to the
embryo through the treated strophiole in a controlled manner and regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues
(Kelly and van Staden 1987)
37
10 Strophiole ofunimbibed New Mexico locust seed (a) and following uptake (bc)
38
In contrast hot-water treatments can excessively degrade the seed coat
(Rebman et al 1998 Marunda 1990) Random cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rebman et aI 1998 Marunda 1990) Along with a
proportionately large amount ofun-imbibed (thus under-treated) seeds (Figure l1a)
damage was observed in hot-water treated seeds from six sources during germination
counts Damage included seeds where the radicle failed to elongate (Figure 11 b)
seeds where the radicle elongated but was delayed in freeing itself from the seed coat
(Figure 11c) and seeds where the radicle completely broke off from the embryo
(Figure lId) In addition some seeds germinated in reverse (the cotyledon emerged
fIrst) If the radicle emerged completely from the seed coat they were counted as
germinated
While nearly all seeds treated for 8 minutes ofpercussion imbibed water
damage was evident in x-ray evaluations (Sacramento seed source) and in
germination counts (Holman and Capitan) at this treatment level Seed damage
during germination counts was similar to levels found in hot-water treated seeds
suggesting that over-treatment by both hot water and longer durations of percussion
may result in similar damage to seed structure
The Sacramento and Holman seed sources were two of the fIve sources that
opcrplltlpn in germination from the 4- to 8-minute durations of percussion
the benefIts ofloosening the strophiole as evidenced by imbibition of
all 8-minute treated seeds were outweighed by external and internal seed coat
39
a
b c d
11 Damage to New Mexico locust seeds observed during germination cotmts following hot water scarification 9a Hot water replicate including ~t seedlings and numerous unimbibed seeds 9b Close-up of seed where raallCle failed to elongate 9c Close-up ofseed where radicle elongated but was
-gtIn in freeing itself from the seed coat 9d Close-up ofseed where radicle broke off from embryo
40
damage (which also may be contributing to high imbibition) For the three sources
for which 8 minutes of percussion resulted in maximum germination extended
percussion of seeds necessary to loosen the strophiole may not have excessively
damaged the seed anatomy The Manzano source did not significantly change in
germination from 4 to 8 minutes of percussion suggesting that an increase in
permeable seeds at the 8-minute treatment duration was balanced by the number of
seeds that failed to germinate due to seed damage To summarize the 4-minute
percussion treatment improved germination over 8 minutes ofpercussion for a
majority of sources (five ofnine) the 8-minute percussion treatment improved
germination over 4-minutes ofpercussion for three sources and there was no
difference with the Manzano source Notably for the Raton source 8 minutes of
percussion increased germination by 235 percentage points over 4-minutes of
percussion (7875 vs 5525 respectively) the next best treatment Raton was the
only source to result in germination less than 75 for 4 minutes ofpercussion so a
grower may wish to apply longer percussion durations to lots that do not respond well
to this treatment level
Finally there was an interesting trend in the Jemez and Raton sources seed
that significantly contributed to the source by treatment interaction (Figure 12) A
~u test showed that the Jemez source had a significantly lighter seed weight than
other sources while the Raton source had an average seed weight For the Jemez
the longest duration of boiling increased germination Conversely only short
U4UVll) of percussion were needed to achieve high germination and the longest
41
80 _ Jemez a Raton
iD
~ C iD e iD
Q c 0 ~
c ~ iD 0
20
0------shy
Immersion Boiling 30 sec Boiling 60 sec
Hot Water Scarification
10degTr========~--------~------------------------------
80
60
40
20
_ Jemez Raton
0------shy
1 min 2 min 4 min 8 min
Percussion Scarification
Figure 12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources
42
b
--~----~~~~========~---- duration ofpercussion reduced germination For the Raton source longer durations
ofboiling consistently decreased germination whereas longer percussion treatments
consistently increased germination
A possible explanation is that a thick seed coat is associated with a loose
strophiole structure and that a thin seed coat is associated with a constrained
strophiole This contrast merits further study and may suggest an ecological
adaptation Further investigation ofthis phenomenon may contribute to successful
pre-screening ofunknown seed sources to determine optimal treatment leveL
43
CONCLUSION
Based on results obtained in pilot study (see Appendix) and thesis
experiments 4 minutes ofpercussion is recommended as a standard protocol for
treating an unknown source ofNew Mexico locust seed In addition to laboratory
results the 4-minute percussion treatment has resulted in germination greater than
80 and healthy seedling grow-outs for production purposes (unpublished data) In
laboratory experiments only one hundred seeds per 118 ml (40z) soil tin were
percussed at a time whereas for production we have treated up to five hundred seeds
per tin For large-scale production ofNew Mexico locust seedlings growers may
wish to experiment with larger quantities of seed and larger-size soil tins
Seed storage trials following percussion treatments were not conducted
However germination greater than 80 and healthy seedlings were obtained when
seeds were percussion-treated and subsequently stored at room-temperature (22degC)
for one week (unpublished data) Seed storage trials merit further study as the
standard hot water treatment leaves seeds in a softened condition that is not
conducive to storage
Hydroseeding is an effective method for direct seeding in reclamation
situations Percussion treatment may be conducive to hydro seeding as seeds remain
a dry relatively undamaged yet permeable condition following treatment Sulfuric
seeds also remain dry following treatment but extensive damage to seed coat
poor seedling vigor have been reported with this treatment (Kelly and van Staden
A grow-out study using a hydro seeder on a reclamation site testing percussion
44
against other treatments such as sulfuric acid and mechanical scarification also
merits further study
Papilionoids are the largest subfamily oflegumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust these legumes often play an integral role in the revegetation of
disturbed lands The pilot study (Appendix) indicates that percussion scarification
successfully treats the widely used black locust as well Developing superior
scarification methods to the standard hot water mechanical scarification and acid
treatments may facilitate the use ofpapilionoid legumes in reclamation projects
45
APPENDICES
APPENDIX A
PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR
NEW MEXICO LOCUST AND BLACK LOCUST SEEDS
Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4
Hot water and sulfuric acid soaks are traditional treatments for seeds of many
temperate woody legwnes including locusts However these scarification techniques
often produce inconsistent germination Percussion scarification where seeds are
repeatedly propelled against a hard surface was compared with hot water
scarification to evaluate treatment efficacy for New Mexico locust (Robinia
neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment
seeds were placed in a 98degC water bath which was immediately removed from the
heat source For percussion scarification seeds were placed in a soil sample tin and
agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the
control were followed by 24-hour water soaks Hot water baths resulted in 56 and
41 germination for New Mexico locust and black locust respectively For both
species nearly all durations ofpercussion increased germination over the hot water
treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3
4 and 5 minutes for black locust resulted in at least 90 germination Traditional
scarification treatments randomly degrade the entire seed coat which can lead to
tissue damage during water uptake Percussion scarification specifically weakens the
strophiole the natural source ofwater entry to the seed in papilionoid legwnes
INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom
2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031
48
Following percussion imbibition is controlled through the strophiole and underlying
tissue is protected
Keywords New Mexico locust black locust scarification percussion germination
Introduction
Locust trees (Robinia spp) are aggressive pioneer species that quickly
colonize disturbed land fix atmospheric nitrogen in the soil and stabilize slopes that
are prone to erosion (Klemmedson 1994 Ashby et al 1985) (Figure A1ab)
Recognition of these attributes has contributed to increased demand for locusts in
reclamation projects Locust seeds however exhibit physical dormancy where a
thick seed coat prevents the movement of water and gasses to the embryo (Leadem
1997)
Sulfuric acid or hot water soaks have traditionally been used to break seed
dormancy in macro-propagation of locusts Many growers are moving away from the
use of su1furic acid in treating locust seeds (Dreesen and Harrington 1997) In
addition to being dangerous acid soak durations must be specifically correlated to
seed lot (Olson 1974) Collections of native seed can vary tremendously in seed size
seed weight and hard seededness etc This variability limits the utility of sulfuric
acid soaks as even within a given lot some seeds may be damaged whereas others
are under-treated and fail to imbibe (take up water) A survey of Southwestern
container growers indicates that hot water baths are currently the most common
scarification method for locust seeds (Hine et al 1997) However hot water baths
also produce inconsistent germination (Lin et al 1996)
49
b
Figure AI New Mexico locust a primary invader following disturbance Ala Crown-sprouting at edge ofburn near Los Alamos NM--one year after Cerro Grande fire Alb Colonizing road cut Gila National Forest NM
50
Percussion scarification where seeds are repeatedly propelled against a hard
surface is an alternative dormancy-breaking method that has proven successful in
legumes related to locusts (Hamly 1932 Barton 1947) We compared this alternative
treatment to hot water scarification to evaluate treatment efficacy for New Mexico
locust and black locust seeds
Materials and Methods
Commercial New Mexico locust seeds (Western Native Seed Coaldale CO)
were collected fall 2000 in Huerfano County CO Black locust seeds were collected
September 2000 from Taos County NM by harvesting ripe pods from several trees at
a distance of up to 3 m from ground level Black locust seeds were allowed to air-dry
for 3 weeks and were threshed and separated from large chaffusing a greenhouse
fan A South Dakota Seed Blower (Chicago Illinltois) was used to remove fine chaff
from both seed lots Seeds were storedmiddotat 2-4C until the germination study was
conducted in March 2001
This study compared hot water scarification with percussion scarification
using treatment and species as experimental factors Along with a control
scarification treatments included immersion of seed in boiling water as well as
percussion durations of 1 2 3 45 or 10 minutes Four 100-seed samples were used
to test each species by scarification treatment combination
For the hot water treatment test tubes were filled with 30 mL water and
placed in a water-filled beaker (Figure A2a) The bath was raised to boiling (-98C at
51
b
c
Figure A2 Implementation ofhot water and percussion scarification A2a Hot water bath A2b Paint-shaker set-up A2c Placing soil tin containing seeds at end ofpaint shaker ann
52
Las Cruces NM elevation of 1300 meters) Each 100-seed replicate was placed in a
test tube and each test tube was immediately removed to cool at room temperature
Percussion scarification was implemented using a pneumatic paint shaker
(Central Pneumatic model 00422 Camarillo CA) (Figure A2b) This allowed
standardization of treatment intensity Compressed air pressure was maintained at
530kPa plusmn 30kPa (80psi plusmn 5psi) resulting in approximately 350 oscillations per
minute Each lOO-seed sample was placed in a 118 ml (4 oz) soil tin for shaking To
maximize lateral movement of paint shaker a paint can was used as a spacer to place
soil tins at the end of the shaking arm (Figure A2c) This allowed shaking to occur at
the greatest distance from the pivot point (fulcrum) Following all treatments
including the control seeds were soaked for twenty-four hours in distilled water prior
to plating out
Gennination was evaluated on lab benches at room temperature Each
replicate of seeds was placed in a 100 cm petri dish on moistened filter paper with
humidity maintained by enclosing the petri dishes in ziplock bags Gennination was
monitored daily for 14 days in accordance with International Seed Testing
Association standards for Robinia species (ISTA 1999) Gennination was defined as
protrusion of the radicle from the embryo by at least 1mm as viewed by the naked
eye
Categorical Analysis of Variance using Prpc Catmod (SAS Institute 1989)
was used to detennine treatment differences in germination percentages for each
source This procedure is an extension of a Chi-square test of homogeneity using the
53
natural log of the ratio of germinated to non-germinated seeds for each treatment Pshy
values less than 005 were considered significant A limited set of pairwiseshy
comparisons was conducted to compare treatment mean percents using a
conservative alpha value of 005 divided by the number of comparisons
Results
Percussion maximized germination for both New Mexico locust and black
locust There were three germination response levels for New Mexico locust (Figure
A3) First there was a low response in the control indicating a dormant seed lot
The hot water treatment and I-minute duration of percussion share a second
intermediate response Germination was highest at percussion levels from 2 to 10
minutes
A selected percussion treatment intermediate in time (5 minutes) produced the
steepest germination curve indicating the fastest germination rate (Figure A4) The
hot water treatment produced an intermediate curve or relatively slower germination
rate The control curve was very flat indicating the slowest germination rate for New
Mexico locust
Control seeds germinated poorly for black locust as well (Figure A5) Hot
water scarification improved germination over the control but all levels of percussion
improved germination over hot water The optimal durations of percussion treatment
were bracketed with a significant increase from the 1 to 2 minute percussion level a
high germination response from 2 to 5 minutes of percussion and finally a significant
54
~ d 25 CL)
p t1 0 ~ t1
g Vl ~ Vl
100~---------------------------------------------------------------
80
60
40
20
o Control Hot Water Percussion
I min Percussion
2 min Percussion
3 min
Scarification
Percussion 4 min
Figure A3 Effect of scarification on germination response for New Mexico locust
Percussion 5 min
=significant difference between adjacent treatment bars ns = no significant difference
Percussion 10 min
~ ~ () u agt
Il-t d o ~
~ ~ c3
100middotlr~~~------------------------------------e- Percussion 5min middot 0middot Hot Water -- Control
80-J=middot======
6b-middot-lmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middot--middotmiddot-middot~-middotmiddot -middotmiddot middot middotmiddot-middot 1
40
20
-----~-----------
0 0 0 middotmiddotmiddotmiddotmiddot0middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot0
0 --~-----y-----~------_---~--- T-----~
o shy -TDay 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A4 Effect of selected scarification treatments on germination speed for New Mexico locust First seven days of fourteen-day experiment are shown
Day 7
80
~ cd
5 60 ()
[) Cl d 0 1t1 d
40sect dUl
-J
20
o
ns
Scarification
Figure A5 Effect of scarification on germination response for black locust = significant difference between adjacent treatment bars ns = no significant difference
decrease at the 10-minute level This drop-off suggests that 10 minutes of percussion
over-treats seeds from this lot of-black locust
A selected percussion treatment intermediate in time (5 minutes) also had the
fastest germination rate for black locust (Figure A6)_ The hot water curve is
somewhat flatter indicating a slower germination rate The control curve is very flat
indicating a slow germination rate
A qualitative difference between hot water and percussion-treated seeds was
also observed Along with healthy germinants the hot water treatment produced
over-treated seeds (Figure A 7a) Examples of damaged germinants include seeds
where the radicle did not elongate (Figure A 7b) seeds where the radicle elongated
but was delayed in freeing itself from the seed coat (Figure A7c) and seeds where the
radicle broke off completely from the embryo (Figure A7d) In addition to these
examples of over-treatment there were numerous small dark seeds that failed to
imbibe water For percussion treatment nearly all seeds took up water (Figure A8a)
Germination was rapid and uniform with little damage evident (Figure A8b)
To summarize both New Mexico locust and black locust seed lots were
dormant The standard hot water scarification treatment improved germination but
only to 56 and 41 for New Mexico locust and black locust respectively Nearly
all percussion durations improved total germination as well as germination rate over
the hot water treatment Germination was greater than 90 for percussion durations
of 4 5 and 10 minutes for New Mexico locust and 3 4 and 5 minutes for black locust
58
CI)
sgt t CI) 0
igt 0
sect ttl t E
Vl 0
c3
100r==~==~~=r------------------------------------~ ___ Percussion 5 min
middotmiddotmiddot0middotmiddotmiddot Hot Water -lIf- Control
80r=========~--~~----------------------------~
60
40
20 -~-~---~---------- ---~------------I
omiddot
O
_~_----------lIf-----~bull -----_ shy---shy I
Day 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A6 Effect of selected scarification treatments on germination speed for black locust First seven days of fourteen-day experiment are shown
Day 7
a
b c d
Figure A7 Damage to New Mexico locust seeds observed during germination counts following hot water scarification A7a Hot water replicate including damaged seedlings and numerous unimbibed seeds A7b Close-up ofseed where radicle failed to elongate A7c Close-up ofseed where radicle elongated but was delayed in freeing itself from the seed coat A7d Close-up ofseed where radicle completely broke offfrom embryo
60
a
b
Figure A8 New Mexico locust seeds following percussion treatment at time of germination counts A8a Percussion replicate with uniformly healthy seedlings and near complete imbibition of seeds A8b Close-up ofpercussion-treated seedling
61
Discussion
Papilionoid seeds have a specific anatomical feature known as the strophiole
or lens-the natural site of water entry to the seed (Hamly 1932) The strophiole is
located on the cotyledonary lobe of a locust seed (Figure A9) When a papilionoid
legume seed is percussed for the appropriate amount of time repeated hits on the
integrated seed coat loosen the constrained cells of the strophiolar region without
excessively damaging the rest of the seed coat (Ballard 1976)
When a percussed seed is soaked water enters exclusively through the
strophiole in a controlled manner (Kelly and Staden 1987) This regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues This
contrasts with seeds that have been hot water or acid scarified treatments that can
randomly degrade the seed coat Unlocalized cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rehman et al 1998 Marunda 1990)
Papilionoids are the largest subfamily of legumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust and black locust these legumes often play an integral role in the
revegetation ofdisturbed lands Developing superior scarification methods to the
standard hot water and acid treatments should fadlitate the use ofthese legumes in
restoration projects
62
Figure A9 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (bc)
63
APPENDIXB
GERMINATION PERCENTS AND STANDARD ERRORS FOR SOURCE
BLOCK AND TREATMENT FOR NEW MEXICO LOCUST
Table B 1 Gennination percents and standard errors for source block and treatment for New Mexico locust
Source Block Treatment Germination Percentage Standard Error
Capitan 1 Immersion 74 437 Capitan 2 Immersion 69 462 Capitan 3 Immersion 71 454 Capitan 4 Immersion 67 470 Capitan Boiling 30 sec 79 407 Capitan 2 Boiling 30 sec 76 427 Capitan 3 Boiling 30 sec 72 449 Capitan 4 Boiling 30 sec 76 427 Capitan Boiling 60 sec 71 454 Capitan 2 Boiling 60 sec 54 498 Capitan 3 Boiling 60 sec 67 470 Capitan 4 Boiling 60 sec 76 427 Capitan I Control 5 218 Capitan 2 Control 7 255 Capitan 3 Control 4 196 Capitan 4 Control 5 218 Capitan 1 Percussion I min 47 499 Capitan 2 Percussion I min
470
Capitan Percussion I min 39 488
Capitan 4 Percussion I min 38 485 Capitan I Percussion 2 min 55 497 Capitan Capitan
2
Percussion 2 min Percussion 2 min
71 65
454 477
Capitan 4 Percussion 2 min 56 496 Capitan I Percussion 4 min 86 347 Capitan 2 Percussion 4 min 79 407 Capitan 3 Percussion 4 min 81 392 Capitan 4 Percussion 4 min 82 384 Capitan I Percussion 8 min 90 300 Capitan 2 Percussion 8 min 88 325 Capitan 3 Percussion 8 min 90 300 Capitan 4 Percussion 8 min 90 300 Gila I Immersion 71 454 Gila 2 Immersion 77 421 Gila
Immersion 75 433
Gila 4 Immersion 72 449
Gila I Boiling 30 sec 68 466
Gila 2 Boiling 30 sec 81 392
Gila 3 Boiling 30 sec 54 498
Gila 4 Boiling 30 sec 63 483
Gila I Boiling 60 sec 55 497
Gila 2 Boiling 60 sec 68 466
Gila 3 Boiling 60 sec 57 495
Gila 4 Boiling 60 sec 70 458
65
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Gila 2 Control 13 336 Gila 3 Control 13 336 Gila 4 Control 20 400 Gila 1 Percussion 1 min 66 474 Gila 2 Percussion 1 min 57 495 Gila 3 Percussion 1 min 73 444 Gila 4 Percussion 1 min 82 384 Gila 1 Percussion 2 min 81 392 Gila 2 Percussion 2 min 86 347 Gila 3 Percussion 2 min 85 357 Gila 4 Percussion 2 min 54 498 Gila 1 Percussion 4 min 79 407 Gila 2 Percussion 4 min 83 376 Gila 3 Percussion 4 min 84 367 Gila 4 Percussion 4 min 79 407 Gila 1 Percussion 8 min 51 500 Gila 2 Percussion 8 min 76 427 Gila 3 Percussion 8 min 52 500 Gila 4 Percussion 8 min 74 439 Holman 1 Immersion 83 376 Holman 2 Immersion 86 347 Holman 3 Immersion 81 392 Holman 4 Immersion 90 300 Holman 1 Boiling 30 sec 90 300 Holman 2 Boiling 30 sec 85 357 Holman 3 Boiling 30 sec 87 336 Holman 4 Boiling 30 sec 83 376 Holman 1 Boiling 60 sec 79 407 Holman 2 Boiling 60 sec 80 400 Holman 3 Boiling 60 sec 86 347 Holman 4 Boiling 60 sec 93 255 Holman Control 25 433 Holman 2 Control 19 392 Holman Control 21 407
Holman 4 Control 24 427
Holman Percussion 1 min 77 421
Holman 2 Percussion 1 min 85 357
Holman 3 Percussion 1 min 80 400
Holman 4 Percussion 1 min 89 313
Holman 1 Percussion 2 min 88 325
Holman 2 Percussion 2 min 96 196
Holman 3 Percussion 2 min 93 255
Holman 4 Percussion 2 min 96 196
Holman 1 Percussion 4 min 92 271
Holman 2 Percussion 4 min 97 171
66
I
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Holman 3 Percussion 4 min 91 286 Holman 4 Percussion 4 min 96 196 Holman 1 Percussion 8 min 88 325 Holman 2 Percussion 8 min 85 357 Holman 3 Percussion 8 min 83 376 Holman 4 Percussion 8 min 82 384 Jemez 1 Immersion 53 499 Jemez 2 Immersion 44 496 Jemez 3 Immersion 52 500 Jemez 4 Immersion 70 458 Jemez 1 Boiling 30 sec 58 494 Jemez 2 Boiling 30 sec 55 497 Jemez 3 Boiling 30 sec 48 500 Jemez 4 Boiling 30 sec 65 477 Jemez 1 Boiling 60 sec 68 466 Jemez 2 Boiling 60 sec 63 483 Jemez 3 Boiling 60 sec 73 444 Jemez 4 Boiling 60 sec 89 313 Jemez 1 Control 2 140 Jemez 2 Control 9 286 Jemez 3 Control 6 237 Jemez 4 Control 8 271 Jemez 1 Percussion 1 min 58 494 Jemez 2 Percussion 1 min 63 483 Jemez 3 Percussion 1 min 77 421 Jemez 4 Percussion 1 min 89 313 Jemez 1 Percussion 2 min 68 466 Jemez 2 Percussion 2 min 64 480 Jemez 3 Percussion 2 min 80 400 Jemez 4 Percussion 2 min 83 376 Jemez Percussion 4 min 68 466 Jemez 2 Percussion 4 min 79 407 Jemez 3 Percussion 4 min 70 458 Jemez 4 Percussion 4 min 83 376 Jemez 1 Percussion 8 min 52 500 Jemez 2 Percussion 8 min 59 492 Jemez 3 Percussion 8 min 61 488 Jemez 4 Percussion 8 min 70 458 Magdalena 1 Immersion 69 462 Magdalena 2 Immersion 69 462 Magdalena J Immersion 70 458 Magdalena 4 Immersion 85 357 Magdalena 1 Boiling 30 sec 65 477 Magdalena 2 Boiling 30 sec 71 454 Magdalena 3 Boiling 30 sec 68 466
67
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Magdalena 4 Boiling 30 sec 83 376 Magdalena 1 Boiling 60 sec 63 483 Magdalena 2 Boiling 60 sec 68 466 Magdalena 3 Boiling 60 sec 63 483 Magdalena 4 Boiling 60 sec 82 384 Magdalena 1 Control 11 313 Magdalena 2 Control 6 237 Magdalena 3 Control 5 218 Magdalena 4 Control 4 196 Magdalena Percussion 1 min 32 466 Magdalena 2 Percussion 1 min 45 497 Magdalena 3 Percussion 1 min 46 498 Magdalena 4 Percussion I min 36 480 Magdalena 1 Percussion 2 min 67 470 Magdalena 2 Percussion 2 min 68 466 Magdalena 3 Percussion 2 min 65 477 Magdalena 4 Percussion 2 min 81 392 Magdalena 1 Percussion 4 min 87 336 Magdalena 2 Percussion 4 min 97 171 Magdalena 3 Percussion 4 min 83 376 Magdalena 4 Percussion 4 min 97 171 Magdalena 1 Percussion 8 min 98 lAO Magdalena 2 Percussion 8 min 100 000 Magdalena 3 Percussion 8 min 96 196 Magdalena 4 Percussion 8 min 98 lAO Manzano 1 Immersion 67 470 Manzano 2 Immersion 57 495 Manzano 3 Immersion 72 4049 Manzano 4 Immersion 73 4044 Manzano 1 Boiling 30 sec 70 458 Manzano 2 Boiling 30 sec 64 480 Manzano 3 Boiling 30 sec 71 454 Manzano 4 Boiling 30 sec 80 400 Manzano I Boiling 60 sec 50 500 Manzano 2 Boiling 60 sec 53 499 Manzano 3 Boiling 60 sec 59 492 Manzano 4 Boiling 60 sec 74 439 Manzano I Control 4 196 Manzano 2 Control 8 271 Manzano 3 Control 1 099 Manzano 4 Control 6 237 Manzano 1 Percussion 1 min 52 500 Manzano 2 Percussion 1 min 39 488 Manzano 3 Percussion 1 min 36 480 Manzano 4 Percussion 1 min 51 500
68
Table B 1 Continued
Source Block Treatment Gerrni~ation Percentage Standard Error
Manzano Percussion 2 min 53 499 Manzano 2 Percussion 2 min 58 494 Manzano 3 Percussion 2 min 61 488 Manzano 4 Percussion 2 min 62 485 Manzano 1 Percussion 4 min 76 427 Manzano 2 Percussion 4 min 85 357 Manzano 3 Percussion 4 min 79 407 Manzano 4 Percussion 4 min 83 376 Manzano 1 Percussion 8 min 87 336 Manzano 2 Percussion 8 min 83 376 Manzano 3 Percussion 8 min 83 376 Manzano 4 Percussion 8 min 75 433 Raton 1 Immersion 54 498 Raton 2 Immersion 37 483 Raton 3 Immersion 39 488 Raton 4 Immersion 69 462 Raton 1 Boiling 30 sec 36 480 Raton 2 Boiling 30 sec 20 400 Raton 3 Boiling 30 sec 28 449 Raton 4 Boiling 30 sec 60 490 Raton 1 Boiling 60 sec 15 357 Raton 2 Boiling 60 sec 171 Raton 3 Boiling 60 sec 4 196 Raton 4 Boiling 60 sec 35 477 Raton 1 Control 4 196 Raton 2 Control 12 325 Raton 3 Control 2 140 Raton 4 Control 6 237 Raton 1 Percussion 1 min 52 500 Raton 2 Percussion I min 29 454 Raton 3 Percussion I min 35 477 Raton 4 Percussion 1 min 31 462 Raton 1 Percussion 2 min 55 497 Raton 2 Percussion 2 min 35 477 Raton 3 Percussion 2 min 33 470 Raton 4 Percussion 2 min 42 494 Raton 1 Percussion 4 min 65 477 Raton 2 Percussion 4 min 52 500 Raton Percussion 4 min 51 500 Raton 4 Percussion 4 min 53 499 Raton 1 Percussion 8 min 88 325 Raton 2 Percussion 8 min 80 400 Raton 3 Percussion 8 min 73 444 Raton 4 Percussion 8 min 74 439 Sacramento 1 Immersion 43 495
69
Table B 1 Continued
-Source Block Treatment Germination Percentage Standard Error
Sacramento 2 Immersion 30 458 Sacramento 3 Immersion 38 485 Sacramento 4 Immersion 56 496 Sacramento 1 Boiling 30 sec 47 499 Sacramento 2 Boiling 30 sec 48 500 Sacramento 3 Boiling 30 sec 48 500 Sacramento 4 Boiling 30 sec 56 496 Sacramento 1 Boiling 60 sec 52 500 Sacramento 2 Boiling 60 sec 67 470 Sacramento 3 Boiling 60 sec 43 495 Sacramento 4 Boiling 60 sec 48 500 Sacramento 1 Control 0 000 Sacramento 2 Control 3 171 Sacramento 3 Control 3 171 Sacramento 4 Control 099 Sacramento 1 Percussion 1 min 57 495 Sacramento 2 Percussion 1 min 61 488 Sacramento 3 Percussion 1 min 62 485 Sacramento 4 Percussion 1 min 55 497 Sacramento 1 Percussion 2 min 84 367 Sacramento 2 Percussion 2 min 70 458 Sacramento 3 Percussion 2 min 81 392 Sacramento 4 Percussion 2 min 69 462 Sacramento 1 Percussion 4 min 91 286 Sacramento 2 Percussion 4 min 84 367 Sacramento 3 Percussion 4 min 84 367 Sacramento 4 Percussion 4 min 94 237 Sacramento 1 Percussion 8 min 82 384 Sacramento 2 Percussion 8 min 81 392 Sacramento 3 Percussion 8 min 82 384 Sacramento 4 Percussion 8 min 81 392 Sandia 1 Immersion 76 427 Sandia 2 Immersion 70 458 Sandia 3 Immersion 70 458 Sandia 4 Immersion 81 392 Sandia 1 Boiling 30 sec 78 414 Sandia 2 Boiling 30 sec 71 454 Sandia 3 Boiling 30 sec 63 483 Sandia 4 Boiling 30 sec 78 414 Sandia 1 Boiling 60 sec 67 470 Sandia 2 Boiling 60 sec 65 477 Sandia 3 Boiling 60 sec 74 439 Sandia 4 Boiling 60 sec 66 474 Sandia 1 Control 16 367 Sandia 2 Control 7 255
70
Table B 1 Continued
Source Block Treatment Gennination Percentage Standard Error
Sandia 3 Control 8 271 Sandia 4 Control 16 367 Sandia 1 Percussion 1 min 67 470 Sandia 2 Percussion 1 min 61 488 Sandia 3 Percussion 1 min 66 474 Sandia 4 Percussion 1 min 59 492 Sandia I Percussion 2 min 82 384 Sandia 2 Percussion 2 min 83 376 Sandia 3 Percussion 2 min 81 392 Sandia 4 Percussion 2 min 84 367 Sandia 1 Percussion 4 min 89 313 Sandia 2 Percussion 4 min 86 347 Sandia 3 Percussion 4 min 89 313 Sandia 4 Percussion 4 min 85 357 Sandia 1 Percussion 8 min 80 400 Sandia 2 Percussion 8 min 77 421 Sandia 3 Percussion 8 min 69 462 Sandia 4 Percussion 8 min 66 474
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Dreesen DR Harrington JT 1997 Propagation of native plants for restoration projects in the Southwestern US- Preliminary Investigations In Landis TD Thompson JR tech coords National Proceedings Forest and Conservation Nursery Assocs Gen Tech Rep PNW-GTR-419 Portland OR USDA For Ser PNW Res Sta 77-88
Gottfried GJ 1980 Control ofNew Mexican locust and the effect on planted ponderosa pine in central Arizona US For Ser Res Note RM-386 Rocky Mountain For Range ExpStn Fort Collins CO
Gosling PG Samuel YK Jones SK 1995 A systematic examination of germination temperature chipping and water temperaturesoak duration pretreatments on the seeds ofLeucaena leucocephala Seed Sci amp Technol 23521-532
Hamly DH 1932 Softening of the seeds of Melilotus alba Bot Gazette 93 345shy375
Hanna PJ 1984 Anatomical features of the seed coat ofAcacia kempeana (Mueller) which relate to increased germination rate induced by heat treatment New Phytol 96 23-29
Hanks JP Dick-Peddie WA 1974 Vegetation patterns of the White Mountains New Mexico Southwest Naturalist 18(4) 371-382
Hine S Harrington JT Dreesen DR 1997 Seed scarification requirements for Robinia neomexicana In Harrington JTtech coord Proc Third SouthwestemContainer Growers Conf Feb 12-13 1997 Ft Collins CO
Hopkinson JM 1993 The strophiole of leguminous seeds Newsletter- IntI Herbage Seed Production Res Grp 18 7-8
---- 1997 Differences in hardseededness between Cunningham and Tarramba leucaena In Leucnet News The Newsletter of the International LeucaenaRes and Devlt Netwk No4
ISTA (International Seed Testing Association) 1999 International rules for seed testing rules Seed Sci amp Technol27 Supplement
Kelly KM van Staden J 1987 The lens as the site of permeability in the papilionoid seed Aspalathus linearis J Plant Physiol 128(45) 395-404
73
Keresztesi B 1988 Black locust the tree of agriculture Outlook on Agr 17(2) 77~85
Kigel 1 and Galili G 1995 Seed development and germination Marcel Dekker New York 853 p
Klemmedson 10 1994 New Mexico locust and parent material Influence on macronutrients of forest floor and soil Soil Sci Soc Am 1 58 974~980
Kuo WH Tam AW 1988 The pathway of water absorption of mung bean seeds Seed Sci amp Technol16(1) 139~144
Leadem CL 1997 Dormancy--unlocking seed secrets In Landis TD Thompson lR tech coords National Proceedings Forestand Conservation Nursery Assocs Gen Tech Rep PNW-G TR-419 Portland OR USDA For Ser PNW Res Sta 43-52
Lin Y Wagner MR Seweyestewa A Honahni MD DeGomez M McMillin JD 1996 Influence of cultural treatments on seed germination and seedling growth of Robinia neomexicana Forest Farm and Community Tree Res Reports Volume 1
Marunda CT 1990 Effects of seed pretreatment on the development ofAcacia auriculforinis and A holosericea seedlings ACIAR Proceedings Series No 28
Meginnis HG 1937 Sulphuric acid treatment to increase germination of black locust seed USDA Circ 453 34 p
Miklas PN Towensend CE Ladd SL 1987 Seedcoat anatomy and the scarification ofcicer milkvetch seed Crop Sci 27 766-772
Monsen SB 1984 Use of shrubs on mine spoils In The Challenge ofProducing Native Plants for the Intermountain Area Proceedings Intermountain Nurserymans Association 1983 conference Las Vegas NV compo PM Murphy 26-31 Ogden UT USDA For Ser Gen Tech Report INT-l68
Olson DF 1974 Robinia L Locust In Seeds of Woody Plants in the US tech coord CS Schopmeyer 728-731 USDA For Ser Agr Handbk No450
Rehman S Loescher RNl Harris P1C 1998 Dormancy breaking and germination ofAcacia salicina Lindl seeds Seed Sci amp Techno127 553-557
Rolston MP 1978 Water impermeable seed dormancy Bot Rev 44 365-396
74
SAS Institute Inc 1989 SASSTAT Users Guide Version 6 4th Edition Volume 1 Cary NC SAS Institute Inc 943 p
SerratoValenti G Conara L Ferrando M Modenesi P 1993 Structural and histochemical features ofStylosanthes scabra (Leguminosae Papilionoidae) seed coat as related to water entry Can J Bot 71 834-840
Simpson BJ 1988 A field guide to Texas trees Austin TX Texas Monthly Press 372p
Tran VN Cavanagh AK 1984 Structural aspects ofdormancy In Seed Physiology Vol 2 Murray DR (ed) p1-44 Academic Press Australia
USDA Forest Service 1948 Woody-plant seed manual USDA Misc Pub1654 416p
Vines RA 1960 Trees Shrubs and Woody Vines of the Southwest Austin Univ ofTexas Press 1104 p
Wagner MR Larson RE Craig TP 1992 Ecology and silvics ofNew Mexico locust (Robinia neomexicana) with an emphasis on the arboreal form p53-57 In JW Hanover K Miller and S Plesko (eds) Proc Intemat Confon Black Locust Biology Culture and Utilization Mich State Univ USA
Wilson JK 1944 Immersing seeds of species of Robinia in boiling water hastens germination J For 42 453-454
Zimmerman RW Carpenter SB 1980 First-year coppice production from a 5shyyear old black locust stand on surface mine spoil p309-314 Centr Hardwd For ConfProc University ofMissouri Columbia
1987 Dell 1980 Hanna 1984) but the effect ofmechanical scarification on the
strophiole has not been studied
Alternative Method for Breaking Dormancy Percussion
Strophiole degradation has been examined in depth using an impaction
treatment more commonly known as percussion Hamly (1932) first proposed that
shaking or impacting seeds could split the macrosc1ereid cells at the strophiole
Hamly successfully released tension ofbent strophiolar cells of impermeable
Melilotus alba Medik (White sweet clover) seeds by delivering physical blows
resulting in cells that had split apart and were only slightly bent Ninety-two percent
oftreated seeds became permeable as compared to 05 of control seeds Barton
(1947) showed that percussion significantly increases permeability in black locust
seed Ballard (1976) reasoned that the integrated nature of the strophiole within the
seed coat means a blow on any part of the seed weakens the strophiole There are no
published studies on the use of percussion on New Mexico locust
Percussion methods vary and often seem SUbjective or ill-defined Hamly
(1932) placed seeds in a glass bottle and shook them three times a second for 20
minutes resulting in 180 oscillations per minute Ballard (1973) describes shaking
seeds in a closed metal container with at least fifty seeds per container at 850-900
JS(~l11alIOIlS per minute for 1 minute Kelly and van Staden (1987) placed seeds into
sealed glass flasks Flasks were shaken by a wrist-shaker at a rate of 250
~UUVJ~ per minute Subsequent germination rates were 45 after 2 hours of
55 after 3 hours 60 after 6 hours and 100 after 12 hours
12
There are several potential advantages of percussion over acid hot water and
mechanical scarification Kelly and van Staden (1987) note that a percussion
treatment only alters macrosclereid cells of the strophiole by loosening separating
and raising them They emphasize that minimal damage is done to the overall
anatomical structure of the seed In acid scarification of the same seed lot radicle
and cotyledon damage occurred Based on photographic evidence the authors
believe that this morphological damage was due to uneven pressure during
imbibition with water entering haphazardly wherever the acid had sufficiently
dissolved the coat This ultimately led to uneven hydration of the embryo In
percussed seeds water only entered through the s~ophiole when the entire seed coat
had been saturated resulting in controlled and even embryo hydration (Kelly and van
Staden 1987)
Although all successful scarification treatments may focus at least in part on
the strophiole for papilionoid legumes (Baskin and Baskin 1998) percussion may be
less damaging to seed coat structures particularly when compared with acid Kelly
and van Staden did not compare hot water or mechanical scarification methods
against percussion However the fungal infection associated with hot water and
mechanical scarification as with acid scarificatio~ have been directly associated with
seed damage (Rehman et al 1998 USDA Forest Service 1948) Embryo damage in
has been observed in hot-water scarified legume seeds (Rehman et al 1998
13
By limiting embryo damage percussion scarification reduces seedling
mortality compared to other treatments Kelly and van Staden (1987) noted that
seedlings from the percussion treatment were morphologically sound and vigorous
when compared to acid treatment seedlings The authors attribute this improved
seedling vigor to the controlled water uptake that limited anatomical damage
Despite a 12-hour percussion treatment morphological damage was not substantially
greater than untreated seeds In addition seedling mass measured 10 days following
germination was not significantly different between percussed and untreated seeds
A final advantage ofpercussion may be the ability to adequately treat the most
dormant seeds while avoiding harm to seeds that have already been made permeable
by the treatment or that were permeable to begin with Instead ofcorroding scalding
or scratching the seed coat percussion appears to be just a loosening of the
strophiole spring (Ballard 1973) Kelly and van Staden (1987) were able to
increase germination with a lengthy treatment (12 hours) Figuring out whether a
substantial cushion oftreatment durations exist for percussion ofNew Mexico locust
seed would be a significant improvement to current scarification methods where seed
damage and seedling health is an issue
The greatest challenge for applying percussion to New Mexico locust seeds is
standardizing a treatment that is both effective and readily reproducible by nurseries
with mechanical scarification this may require the development of specific
~UllDment to properly quantify and execute the treatment
14
f
Objectives
The first objective of the study is to detennine which scarification treatment
results in optimal gennination speed and percentage ofNew Mexico locust The
second objective is to describe treatment interactions with nine seed sources collected
in New Mexico
Hypothesis
A percussion treatment acts exclusively on the main area ofwater uptake in
New Mexico locust seed loosening the strophiole so that imbibition can occur This
direct mechanism ofdonnancy release will result in improved gennination rate and
overall gennination percentage for New Mexico locust seed compared to standard
treatments Standard treatments including acid hot water and mechanical
scarification while eventually weakening the strophiolar cleft cause excessive
morphological damage through generalized seed coat degradation These treatments
are therefore prone to inconsistency due to over and under-treatment
15
MATERIALS AND METHODS
Seed Collection and Cleaning
Seeds were collected from August 25 through September 15 2000 from nine
locations along a latitudinal gradient in New Mexico Seed source locations and
descriptive information are given in Figure 1 and Tables I and 2 Stands were
separated by at least 45 km
Pods were hand picked as they began to ripen and dehisce (open) Collections
were made from ground level to approximately 3 m from several trees at each site
Pods were dried in open-ended brown paper bags for two to four weeks at room
temperature (21-230C) to facilitate cleaning Dried pods were placed in a closed cloth
sack which was pounded against the ground to separate seeds from pods For pods
still containing seeds hand maceration (crunching pods with hands) completed seed
separation Debris was removed by threshing with the aid of a greenhouse fan A
South Dakota Seed Blower (Seedbureau Chicago Illinois) was used to remove
remaining chaff and light seeds by air density separation Seeds were placed in dry
storage in sealed quart-size zip-lock bags within a sealed plastic container Storage
temperatures ranged from 2-4oC Seeds remained in storage for approximately 7
months before the experiment
Experimental Design and Treatment Structure
The experiment compared the impact ofhot water bath and percussion
l11aLl11 on germination properties ofNew Mexico locust seed A single
16
-l
Figure 1 Seed source collection map All sources were collected within New Mexico USA separated by at least 4S lan
IIIRZltItW lIJPIirmiddot rllll1lbullbull_~~~_II
Table 1 Latitude longitude location elevation and collection date ofNew Mexico locust seed sources
Lot title Latitude (N) Longitude (W) Location Elevation (m) Collection Date
Raton 36deg5846 104deg2872 Raton Pass NM 2295 82500
Holman 36deg0306 105deg2429 HolmanNM 2476 911500
Jemez 35deg4500 106deg4100 Jemez Springs NM 1976 9900 00
Sandia 35deg1083 106deg2341 Cedar Crest NM 2432 82600
Manzano 34deg3689 106deg2494 ManzanoNM 2465 9200
Magdalena 33deg5953 107deg0903 Magdalena NM 2544 82900
Capitan 33deg2728 105deg4325 Alto NM 2189 911200
Sacramento 32deg5798 105deg4496 Cloudcroft NM 2630 91400 Gila 32deg5400 107deg4600 Kingston NM 2432 9700
f
Table 2 Mean seed weight and standard error by source
a
Source Mean seed weight (g) for Standard error 100 seeds n = 5
Raton Holman Jemez Sandia Manzano Magdalena Capitan Sacramento Gila
2872 b c 2522 d 1826 e 2753 c 2413 d 2559 d 2443 d 3130 a 3029 ab
00103 00403 00276 00301 00370 00374 00458 00279 00348
a Mean seed weights followed by the same letter are not significantly
different
19
experiment was conducted utilizing a randomized complete block design blocked by
time with a factorial treatment structure Experimental factors were seed source and
scarification treatment Nine seed sources from throughout New Mexico were used
(Figure 1 Table 1) Scarification treatments included a control three levels ofhot
water and four levels of percussion Hot water treatments included immersion at
100degC and boiling for periods of 30 seconds and 60 seconds Percussion treatment
levels were 1 2 4 and 8 minutes One hundred seeds per block were used to test
each treatment combination Four blocks of seed were tested one at a time (all seed
sources included in each run) in close chronological sequence (within 6 weeks) A
total of four 100-seed samples were used to test each source by scarification treatment
combination
Treatments
Hot water treatment of seeds utilized 50 ml test tubes filled with 30 ml
distilled water Each test tube was used to treat one 100-seed sample at a time
resulting in a ratio of4 parts water to 1 part seed by volume Test tubes were placed
in a beaker with the beaker filled with tap water to the same level as the water within
test tubes Nine test tubes (one per seed source) were placed in the beaker
simultaneously so that one replication of each seed sourcescarification treatment
cotnblnatlon was conducted simultaneously Hot water treatments include immersion
boiling For immersion treatments seeds were added to test tubes with the water
(boiling) and the test tubes were immediately removed from the beaker to
For the boiling treatments test tubes remained in the beaker with water at
20
boiling temperature for a set duration after the addition of seeds Following all
treatments test tubes with seeds cooled 24 hours in a test tube rack
Percussion treatment utilized a pneumatic paint shaker (Central Pneumatic
model 00422 Camarillo CA) Compressed air was maintained at 530 kPa plusmn30 kPa
(80 psi plusmn 5 psi) resulting in approximately 350 oscillations per minute Seeds were
placed in 118 ml (4 oz) soil tin cans (US Can Lobard IL) and the lids were secured
with duct tape Each can was used to treat one 100-seed sample To take advantage
ofgreatest lateral movement of the paint shaker a spacer (paint can) was used to
position the soil tin at the end of the shaking arm This allowed shaking to occur at
the greatest distance from the pivot point (fulcrum) Tins were placed perpendicular
to the direction of shaking motion allowing seeds to impact at greatest force by
hitting flat surfaces of the soil tin
Germination Testing
Before gennination testing all seeds were soaked 24 hours in reverse
osmosis-treated water (this was included in the process ofhot water treatment
jU~SCIlOea above) Following soaking seeds were placed in 100 cm Petri dishes lined
90 cm Whatman qualitative 1 filter paper circles (MG Scientific Pleasant
WI) One Petri dish was used for each treatment by source combination
papers were initially moistened with 5 mL reverse osmosis-treated water then
~VU1CUlJl moistened as needed to ensure adequate conditions for gennination
were monitored daily (at 300 PM) for gennination for 14 days in accordance
~UIUlnJlVUa Seed Testing Association (ISTA) standards for Robinia species
21
(1STA 1999) Tests were carried out at room temperature (22degC plusmnOSoC)
Germination was classified as protrusion of the radicle from the embryo by at least
1mm as viewed with the naked eye
X-ray Radiography
On August 132001 three lOO-seed samples from the Sacramento seed source
were treated for 14 or 8-minute percussion durations The seeds were sent
immediately by overnight delivery to the Faxitron X-Ray Corporation in Wheeling
Illinois where Dr Bob Karrfalt ofthe USDA Forest Service oversaw their imaging
Radiographs were made at SX with a Faxitron Specimen Radiography System Model
MX-20
Scanning Electron Microscope Photography
On February 192002 a Hitachi S-3200N Variable Pressure Scanning
Electron Microscope (Mountain View CA) was used to examine strophiolar cells of
untreated seeds and seeds percussed for 4 minutes Seeds were cut longitudinally
igt their entire length using a razor blade splitting the strophiolar region roughly in
Cross sections of seed samples magnifying the strophiolar region were viewed
600X under the microscope
Analysis
Categorical Analysis of Variance was used to determine treatment source and
(Le time) main effects and interactions The response variable was total
~vlI This procedure is a Chi-square test of homogeneity using the natural log
of germinated to non-germinated seed for each treatment Maximumshy
22
likelihood analysis was used to calculate Chi-square test statistics with their observed
significance levels A single contrast was conducted to cempare treatment means
against the control Observed significance levels less than or equal to 005 were
considered significant Pairwise comparisons were conducted between non-control
treatment mean percents using a conservative alp~a value of 005 divided by the
number of comparisons (Bonferroni approach) and approximate pairwise z statistics
Categorical analyses were performed using Proc Catmod (SAS Institute 1989)
RESULTS
Scarification seed source block and all interactions between factors
influenced germination (Table 3) The main effects are reported in the presence of
these interactions because the overall robustness of treatments and seed sources are of
interest Background levels ofnaturally occurring variation in seed sources are
seen in control germination percentages in Figure 2 In particular time variability
is unlikely to be under the control of the producer Appendix B details experimental
data for source by treatment gennination percents for each of the four blocks
Averaged over all treatments germination by source ranged from 40 to
79 (Figure 2) Averaged over all sources (Figure 3) and for each source (Figure 4
5) percussion scarification durations of4 or 8 minutes yielded greatest germination
Averaged over all sources germination increased with increasing durations of
percussion then leveled off for the 4- and 8-minute treatments (Figure 3) Immersion
and 30-second boiling scarification maximized germination for the hot water
treatments with a decrease in germination when seeds were boiled for 60 seconds
For all but two sources there is a similar ipcreasing trend in germination from
1to 4 minutes ofpercussion (Figure 4) The Holman source increased in germination
1 to 2 minutes then leveled off at 4 minutes The Jemez source changed little
1 to 4 minutes of treatment As treatment duration increased from 4 to 8
mutes five sources decreased in germination (Figure 4a) whereas three sources
24
Table 3 Categorical Analysis of Variance table for germination response to seed treatment seed source block and the interaction of the factors
Source of DF Chi-Square Observed Variability Significance Level
Source 8 98370 lt00001 Trt 7 233876 lt00001
contrast 1 16976 lt00001 SourceTrt 56 97077 lt00001 Block 3 5538 lt00001
SourceBlock 24 10162 lt00001 TrtBlock 21 7564 lt00001
SourceTrtBlock 168 34283 lt00001
S 4)
8 ~
0
sectg ~ 40 ~
N 0 0
20
o f0ltgt ~ ~4 jybshy ((iltgto ~~ K1ltgt 6~b-~ ~~ 0b-~~ ~~ ~b-~ ~b-~b
80
60 -I-- - ~- middotmiddotmiddotmiddotmiddot1
r -] Northern NM Sources
Central NM Sources
Southern NM Sources
sect-o ~
~b-v(i
SOURCE
Figure 2 Effect ofNew Mexico locust seed source on germination for data averaged over all scarification treatments Control germination is shown by solid line within bar
80
~ cd ~ ltl) () 60 ~ ~
N
t 0shyrti tshySltl)
40
0
20
o lmmersion Boiling 30 sec Boiling 60 sec Control 1 min 2min 4 min 8 min
Hot Water Treatments Percussion Treatments
Figure 3 Effect of scarification on germination ofNew Mexico locust for data averaged over all seed sources Means labelled with the same letter are not significantly different
100~----------------------~--~----------------~
80
60
ltU
~
40 IJ
sectgsI
l100
80
60
40
a I middotmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot middotImiddotmiddotmiddotmiddotmiddotmiddot E
_~iii-bull _ _ bull f --~--
t-- -~ ---- -shy ---- shyI ~ ---- - shy
L
fiII
~
b
- shy ---r
- Holman
Sacramento Sandia Gila Jemez
c ~
ltgt ~
Tmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot-I
j~=~=-~~-1
I middotmiddot ~ bull r
t tr
Y -------M-a-gd-a-Ie-n-a--
1 _ _
fCapitan
---- Manzano ~y -_ Raton
2 4 8
Time interval ofpercussion scarification (min)
Figure 4 Effect of seed source by treatment interaction on New Mexico locust germination for percussion scarification pattern a and pattern b
28
tv 0
~ d
~ A s o
1 c3
80
60
40
20
o
TIT
t------=middotfmiddotmiddot-middotmiddot-middotmiddot-middotmiddot 1- --- - - --=- - -shy--cE= - _--shy --shy ----shy ~ ~ ---shy ~ -~- _ --shy-------r 1
I
Raton Holman
-_ Jemez
--- Sandia Manzano Magdalena Capitan Sacramento Gila
Immersion Boiling 30 sec Boiling 60 sec
Hot Water Scarification
Figure 5 Effect of seed source by treatment interaction on New Mexico locust germination for hot water scarification
continued to increase in germination (Figure 4b) There was no difference between
the two treatment levels for the Manzano source (Figure 4b)
The effect ofhot water scarification also varied among sources with few
overall differences between hot water treatments (Figure 5) However boiling for 60
seconds yielded highest germination for the Jemez source whereas increasing
durations ofboiling decreased germination for the Raton source
In addition to consistently high germination 4- and 8-minute percussionshy
treated seeds germinated as fast or faster than all other treatments (Figure 6 Table 4)
050 measured as the number of days needed to complete 50 ofeventual
germination averaged 2 days for both ofthese treatments
X-ray radiographs were taken of seeds collected from the Sacramento source
percussed for 1 4 or 8 minutes (Figure 7) Seeds show signs of damage at 8 minutes
ofpercussion (Figure 8) Compared to an undamaged seed percussed for only 4
minutes (Figure 8a) damage at the 8-minute level includes fractured radicles pitting
of the seed coat and chips in seed coat structure (Figure 8b-d)
Scanning electron microscope (SEM) images at 600X reveal the direct effect
of 4 minutes of percussion on cells of the strophiolar region (Figure 9) Whereas
untreated seeds retain cellular organization in the strophiolar region strophiolar cells
seeds treated for 4 minutes ofpercussion separate and raise resulting in a
~-~e~U appearance
30
Umiddot-~middot--l-~---middotymiddotmiddotmiddot-----middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot--middot--middotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middotmiddotmiddotmiddotmiddotmiddotmiddot- -
050= gt1
-e-- Percussion 8 min -0- Percussion 4 min ---T- Percussion 2 min -----ltil- Percussion 1 min -- Immersion --0- Boiling 30 sec -+- Boiling 60 sec -ltgt- Control
2 4 6 8 10 12
Day Figure 6 Timecourse ofNew Mexico locust gennination following scarification
treatments G50 is the number of days to 50 of eventual germination
31
14
Table 4 Effect of seed source and treatment on G50 (days to 50 germination)
Seed Source Control Immersion B30sec B60sec Plmin P2min P4min P8min mean Raton 3 3 5 7 4 3 2 2 4 Holman 2 2 2 2 2 2 2 2 2
Jemez 4 6 6 5 3 2 2 2 4 Sandia 2 2 2 2 2 2 2 2 2
w tv Manzano 5 2 2 3 3 2 2 2 3
Magdalena 4 2 2 3 4 3 2 1 3
Capitan 5 4 2 2 3 2 2 2 3
Sacramento 3 2 2 2 4 3 2 2 3 Gila 3 2 2 2 2 2 2 2 2 mean 3 3 3 3 3 2 2 2 3 standard 11 14 16 18 09 05 0 03 08 deviation
Note Columns and rows may not agree due to rounding
Figure 7 X-ray radiographs ofSacramento seed source percussed for 1 4 or 8 minutes
33
Figure 8a-d Seed damage close-ups Figure a shows undamaged seed percussed for 4 minutes Figures b-d show seeds percussed for 8 minutes Damage includes fracturing ofradicle in b complete removal ofseed coat in c and pitting ofseed coat in d
34
Untreated Treated
Figure 9 SEM images ofuntreated vs 4-minute percussion-treated strophiolar cells ofNew Mexico locust seeds Treated cells are loosened separated and raised
DISCUSSION
Determining the Optimal Treatment
Averaging results of scarification treatmems across seed sources is a useful
tool to predict which treatment is most likely to be successful for an unknown seed
source Averaged over all sources four- and eight-minute percussion treatments
resulted in the highest germination 815 and 790 respectively and produced
germination that was as fast or faster than all other treatments Although there was no
difference between these two treatments on average an argument for 4 minutes of
percussion as a standard protocol for treating New Mexico locust seeds can be made
Descriptively 4 minutes of percussion improved germination over 8 minutes
ofpercussion for a majority of sources (five out of nine) 8 minutes of percussion
improved germination for three ofnine sources with no difference for a fmal source
For individual sources 4 minutes ofpercussion was always the best or secondmiddot best
treatment and was always equal to or better than all hot water treatments
Germination at 8 minutes ofpercussion was lower than all other percussion durations
for the Jemez and Gila sources and was lower than at least one hot water treatment for
these sources as well
The x-ray radiographs of the Sacramento seed source illustrate the excessive
internal and external seed damage that can occur with 8 minutes of percussion
germination counts 8-minute percussed seeds from Holman and Capitan
ClUll signs of damage that were visible to the naked eye (see below)
r~evertheless some of these seeds still fit the criteria for germination Neither x-ray
36
photography nor visible observations during seed counts revealed seed damage at the
4-minute percussion level In operational trials seeds percussed for 4 minutes
developed into healthy seedlings (Khadduri and Harrington unpublished data)
Strophiole and Seed Coat Behavior
Four minutes ofpercussion successfully breaks seed dormancy in a large
percentage ofNew Mexico locust seeds without excessively damaging the
surrounding seed coat or internal seed coat structures Most papilionoid legume
seeds including seeds ofNew Mexico locust have a specific region of the seed coat
known as the strophiole or lens (Hamly 1932) The strophiole is located on the
cotyledonary lobe of a locust seed (Figure 10 a-c) If seeds are not indiscriminately
damaged the strophiole is the first point of water entry to the embryo (Hopkinson
1997) When a papilionoid seed is percussed for the appropriate amount of time
repeated hits on the integrated seed coat loosen the constrained cells of the strophiolar
region without excessively damaging the rest of the seed coat (Ballard 1976) SEM
photos illuStrate that 4 minutes of percussion loosened and separated strophiolar cells
ofNew Mexico locust seeds when compared with constrained organized strophiolar
cells of untreated seeds (Figure 9) Research-has shown that water passes to the
embryo through the treated strophiole in a controlled manner and regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues
(Kelly and van Staden 1987)
37
10 Strophiole ofunimbibed New Mexico locust seed (a) and following uptake (bc)
38
In contrast hot-water treatments can excessively degrade the seed coat
(Rebman et al 1998 Marunda 1990) Random cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rebman et aI 1998 Marunda 1990) Along with a
proportionately large amount ofun-imbibed (thus under-treated) seeds (Figure l1a)
damage was observed in hot-water treated seeds from six sources during germination
counts Damage included seeds where the radicle failed to elongate (Figure 11 b)
seeds where the radicle elongated but was delayed in freeing itself from the seed coat
(Figure 11c) and seeds where the radicle completely broke off from the embryo
(Figure lId) In addition some seeds germinated in reverse (the cotyledon emerged
fIrst) If the radicle emerged completely from the seed coat they were counted as
germinated
While nearly all seeds treated for 8 minutes ofpercussion imbibed water
damage was evident in x-ray evaluations (Sacramento seed source) and in
germination counts (Holman and Capitan) at this treatment level Seed damage
during germination counts was similar to levels found in hot-water treated seeds
suggesting that over-treatment by both hot water and longer durations of percussion
may result in similar damage to seed structure
The Sacramento and Holman seed sources were two of the fIve sources that
opcrplltlpn in germination from the 4- to 8-minute durations of percussion
the benefIts ofloosening the strophiole as evidenced by imbibition of
all 8-minute treated seeds were outweighed by external and internal seed coat
39
a
b c d
11 Damage to New Mexico locust seeds observed during germination cotmts following hot water scarification 9a Hot water replicate including ~t seedlings and numerous unimbibed seeds 9b Close-up of seed where raallCle failed to elongate 9c Close-up ofseed where radicle elongated but was
-gtIn in freeing itself from the seed coat 9d Close-up ofseed where radicle broke off from embryo
40
damage (which also may be contributing to high imbibition) For the three sources
for which 8 minutes of percussion resulted in maximum germination extended
percussion of seeds necessary to loosen the strophiole may not have excessively
damaged the seed anatomy The Manzano source did not significantly change in
germination from 4 to 8 minutes of percussion suggesting that an increase in
permeable seeds at the 8-minute treatment duration was balanced by the number of
seeds that failed to germinate due to seed damage To summarize the 4-minute
percussion treatment improved germination over 8 minutes ofpercussion for a
majority of sources (five ofnine) the 8-minute percussion treatment improved
germination over 4-minutes ofpercussion for three sources and there was no
difference with the Manzano source Notably for the Raton source 8 minutes of
percussion increased germination by 235 percentage points over 4-minutes of
percussion (7875 vs 5525 respectively) the next best treatment Raton was the
only source to result in germination less than 75 for 4 minutes ofpercussion so a
grower may wish to apply longer percussion durations to lots that do not respond well
to this treatment level
Finally there was an interesting trend in the Jemez and Raton sources seed
that significantly contributed to the source by treatment interaction (Figure 12) A
~u test showed that the Jemez source had a significantly lighter seed weight than
other sources while the Raton source had an average seed weight For the Jemez
the longest duration of boiling increased germination Conversely only short
U4UVll) of percussion were needed to achieve high germination and the longest
41
80 _ Jemez a Raton
iD
~ C iD e iD
Q c 0 ~
c ~ iD 0
20
0------shy
Immersion Boiling 30 sec Boiling 60 sec
Hot Water Scarification
10degTr========~--------~------------------------------
80
60
40
20
_ Jemez Raton
0------shy
1 min 2 min 4 min 8 min
Percussion Scarification
Figure 12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources
42
b
--~----~~~~========~---- duration ofpercussion reduced germination For the Raton source longer durations
ofboiling consistently decreased germination whereas longer percussion treatments
consistently increased germination
A possible explanation is that a thick seed coat is associated with a loose
strophiole structure and that a thin seed coat is associated with a constrained
strophiole This contrast merits further study and may suggest an ecological
adaptation Further investigation ofthis phenomenon may contribute to successful
pre-screening ofunknown seed sources to determine optimal treatment leveL
43
CONCLUSION
Based on results obtained in pilot study (see Appendix) and thesis
experiments 4 minutes ofpercussion is recommended as a standard protocol for
treating an unknown source ofNew Mexico locust seed In addition to laboratory
results the 4-minute percussion treatment has resulted in germination greater than
80 and healthy seedling grow-outs for production purposes (unpublished data) In
laboratory experiments only one hundred seeds per 118 ml (40z) soil tin were
percussed at a time whereas for production we have treated up to five hundred seeds
per tin For large-scale production ofNew Mexico locust seedlings growers may
wish to experiment with larger quantities of seed and larger-size soil tins
Seed storage trials following percussion treatments were not conducted
However germination greater than 80 and healthy seedlings were obtained when
seeds were percussion-treated and subsequently stored at room-temperature (22degC)
for one week (unpublished data) Seed storage trials merit further study as the
standard hot water treatment leaves seeds in a softened condition that is not
conducive to storage
Hydroseeding is an effective method for direct seeding in reclamation
situations Percussion treatment may be conducive to hydro seeding as seeds remain
a dry relatively undamaged yet permeable condition following treatment Sulfuric
seeds also remain dry following treatment but extensive damage to seed coat
poor seedling vigor have been reported with this treatment (Kelly and van Staden
A grow-out study using a hydro seeder on a reclamation site testing percussion
44
against other treatments such as sulfuric acid and mechanical scarification also
merits further study
Papilionoids are the largest subfamily oflegumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust these legumes often play an integral role in the revegetation of
disturbed lands The pilot study (Appendix) indicates that percussion scarification
successfully treats the widely used black locust as well Developing superior
scarification methods to the standard hot water mechanical scarification and acid
treatments may facilitate the use ofpapilionoid legumes in reclamation projects
45
APPENDICES
APPENDIX A
PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR
NEW MEXICO LOCUST AND BLACK LOCUST SEEDS
Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4
Hot water and sulfuric acid soaks are traditional treatments for seeds of many
temperate woody legwnes including locusts However these scarification techniques
often produce inconsistent germination Percussion scarification where seeds are
repeatedly propelled against a hard surface was compared with hot water
scarification to evaluate treatment efficacy for New Mexico locust (Robinia
neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment
seeds were placed in a 98degC water bath which was immediately removed from the
heat source For percussion scarification seeds were placed in a soil sample tin and
agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the
control were followed by 24-hour water soaks Hot water baths resulted in 56 and
41 germination for New Mexico locust and black locust respectively For both
species nearly all durations ofpercussion increased germination over the hot water
treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3
4 and 5 minutes for black locust resulted in at least 90 germination Traditional
scarification treatments randomly degrade the entire seed coat which can lead to
tissue damage during water uptake Percussion scarification specifically weakens the
strophiole the natural source ofwater entry to the seed in papilionoid legwnes
INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom
2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031
48
Following percussion imbibition is controlled through the strophiole and underlying
tissue is protected
Keywords New Mexico locust black locust scarification percussion germination
Introduction
Locust trees (Robinia spp) are aggressive pioneer species that quickly
colonize disturbed land fix atmospheric nitrogen in the soil and stabilize slopes that
are prone to erosion (Klemmedson 1994 Ashby et al 1985) (Figure A1ab)
Recognition of these attributes has contributed to increased demand for locusts in
reclamation projects Locust seeds however exhibit physical dormancy where a
thick seed coat prevents the movement of water and gasses to the embryo (Leadem
1997)
Sulfuric acid or hot water soaks have traditionally been used to break seed
dormancy in macro-propagation of locusts Many growers are moving away from the
use of su1furic acid in treating locust seeds (Dreesen and Harrington 1997) In
addition to being dangerous acid soak durations must be specifically correlated to
seed lot (Olson 1974) Collections of native seed can vary tremendously in seed size
seed weight and hard seededness etc This variability limits the utility of sulfuric
acid soaks as even within a given lot some seeds may be damaged whereas others
are under-treated and fail to imbibe (take up water) A survey of Southwestern
container growers indicates that hot water baths are currently the most common
scarification method for locust seeds (Hine et al 1997) However hot water baths
also produce inconsistent germination (Lin et al 1996)
49
b
Figure AI New Mexico locust a primary invader following disturbance Ala Crown-sprouting at edge ofburn near Los Alamos NM--one year after Cerro Grande fire Alb Colonizing road cut Gila National Forest NM
50
Percussion scarification where seeds are repeatedly propelled against a hard
surface is an alternative dormancy-breaking method that has proven successful in
legumes related to locusts (Hamly 1932 Barton 1947) We compared this alternative
treatment to hot water scarification to evaluate treatment efficacy for New Mexico
locust and black locust seeds
Materials and Methods
Commercial New Mexico locust seeds (Western Native Seed Coaldale CO)
were collected fall 2000 in Huerfano County CO Black locust seeds were collected
September 2000 from Taos County NM by harvesting ripe pods from several trees at
a distance of up to 3 m from ground level Black locust seeds were allowed to air-dry
for 3 weeks and were threshed and separated from large chaffusing a greenhouse
fan A South Dakota Seed Blower (Chicago Illinltois) was used to remove fine chaff
from both seed lots Seeds were storedmiddotat 2-4C until the germination study was
conducted in March 2001
This study compared hot water scarification with percussion scarification
using treatment and species as experimental factors Along with a control
scarification treatments included immersion of seed in boiling water as well as
percussion durations of 1 2 3 45 or 10 minutes Four 100-seed samples were used
to test each species by scarification treatment combination
For the hot water treatment test tubes were filled with 30 mL water and
placed in a water-filled beaker (Figure A2a) The bath was raised to boiling (-98C at
51
b
c
Figure A2 Implementation ofhot water and percussion scarification A2a Hot water bath A2b Paint-shaker set-up A2c Placing soil tin containing seeds at end ofpaint shaker ann
52
Las Cruces NM elevation of 1300 meters) Each 100-seed replicate was placed in a
test tube and each test tube was immediately removed to cool at room temperature
Percussion scarification was implemented using a pneumatic paint shaker
(Central Pneumatic model 00422 Camarillo CA) (Figure A2b) This allowed
standardization of treatment intensity Compressed air pressure was maintained at
530kPa plusmn 30kPa (80psi plusmn 5psi) resulting in approximately 350 oscillations per
minute Each lOO-seed sample was placed in a 118 ml (4 oz) soil tin for shaking To
maximize lateral movement of paint shaker a paint can was used as a spacer to place
soil tins at the end of the shaking arm (Figure A2c) This allowed shaking to occur at
the greatest distance from the pivot point (fulcrum) Following all treatments
including the control seeds were soaked for twenty-four hours in distilled water prior
to plating out
Gennination was evaluated on lab benches at room temperature Each
replicate of seeds was placed in a 100 cm petri dish on moistened filter paper with
humidity maintained by enclosing the petri dishes in ziplock bags Gennination was
monitored daily for 14 days in accordance with International Seed Testing
Association standards for Robinia species (ISTA 1999) Gennination was defined as
protrusion of the radicle from the embryo by at least 1mm as viewed by the naked
eye
Categorical Analysis of Variance using Prpc Catmod (SAS Institute 1989)
was used to detennine treatment differences in germination percentages for each
source This procedure is an extension of a Chi-square test of homogeneity using the
53
natural log of the ratio of germinated to non-germinated seeds for each treatment Pshy
values less than 005 were considered significant A limited set of pairwiseshy
comparisons was conducted to compare treatment mean percents using a
conservative alpha value of 005 divided by the number of comparisons
Results
Percussion maximized germination for both New Mexico locust and black
locust There were three germination response levels for New Mexico locust (Figure
A3) First there was a low response in the control indicating a dormant seed lot
The hot water treatment and I-minute duration of percussion share a second
intermediate response Germination was highest at percussion levels from 2 to 10
minutes
A selected percussion treatment intermediate in time (5 minutes) produced the
steepest germination curve indicating the fastest germination rate (Figure A4) The
hot water treatment produced an intermediate curve or relatively slower germination
rate The control curve was very flat indicating the slowest germination rate for New
Mexico locust
Control seeds germinated poorly for black locust as well (Figure A5) Hot
water scarification improved germination over the control but all levels of percussion
improved germination over hot water The optimal durations of percussion treatment
were bracketed with a significant increase from the 1 to 2 minute percussion level a
high germination response from 2 to 5 minutes of percussion and finally a significant
54
~ d 25 CL)
p t1 0 ~ t1
g Vl ~ Vl
100~---------------------------------------------------------------
80
60
40
20
o Control Hot Water Percussion
I min Percussion
2 min Percussion
3 min
Scarification
Percussion 4 min
Figure A3 Effect of scarification on germination response for New Mexico locust
Percussion 5 min
=significant difference between adjacent treatment bars ns = no significant difference
Percussion 10 min
~ ~ () u agt
Il-t d o ~
~ ~ c3
100middotlr~~~------------------------------------e- Percussion 5min middot 0middot Hot Water -- Control
80-J=middot======
6b-middot-lmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middot--middotmiddot-middot~-middotmiddot -middotmiddot middot middotmiddot-middot 1
40
20
-----~-----------
0 0 0 middotmiddotmiddotmiddotmiddot0middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot0
0 --~-----y-----~------_---~--- T-----~
o shy -TDay 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A4 Effect of selected scarification treatments on germination speed for New Mexico locust First seven days of fourteen-day experiment are shown
Day 7
80
~ cd
5 60 ()
[) Cl d 0 1t1 d
40sect dUl
-J
20
o
ns
Scarification
Figure A5 Effect of scarification on germination response for black locust = significant difference between adjacent treatment bars ns = no significant difference
decrease at the 10-minute level This drop-off suggests that 10 minutes of percussion
over-treats seeds from this lot of-black locust
A selected percussion treatment intermediate in time (5 minutes) also had the
fastest germination rate for black locust (Figure A6)_ The hot water curve is
somewhat flatter indicating a slower germination rate The control curve is very flat
indicating a slow germination rate
A qualitative difference between hot water and percussion-treated seeds was
also observed Along with healthy germinants the hot water treatment produced
over-treated seeds (Figure A 7a) Examples of damaged germinants include seeds
where the radicle did not elongate (Figure A 7b) seeds where the radicle elongated
but was delayed in freeing itself from the seed coat (Figure A7c) and seeds where the
radicle broke off completely from the embryo (Figure A7d) In addition to these
examples of over-treatment there were numerous small dark seeds that failed to
imbibe water For percussion treatment nearly all seeds took up water (Figure A8a)
Germination was rapid and uniform with little damage evident (Figure A8b)
To summarize both New Mexico locust and black locust seed lots were
dormant The standard hot water scarification treatment improved germination but
only to 56 and 41 for New Mexico locust and black locust respectively Nearly
all percussion durations improved total germination as well as germination rate over
the hot water treatment Germination was greater than 90 for percussion durations
of 4 5 and 10 minutes for New Mexico locust and 3 4 and 5 minutes for black locust
58
CI)
sgt t CI) 0
igt 0
sect ttl t E
Vl 0
c3
100r==~==~~=r------------------------------------~ ___ Percussion 5 min
middotmiddotmiddot0middotmiddotmiddot Hot Water -lIf- Control
80r=========~--~~----------------------------~
60
40
20 -~-~---~---------- ---~------------I
omiddot
O
_~_----------lIf-----~bull -----_ shy---shy I
Day 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A6 Effect of selected scarification treatments on germination speed for black locust First seven days of fourteen-day experiment are shown
Day 7
a
b c d
Figure A7 Damage to New Mexico locust seeds observed during germination counts following hot water scarification A7a Hot water replicate including damaged seedlings and numerous unimbibed seeds A7b Close-up ofseed where radicle failed to elongate A7c Close-up ofseed where radicle elongated but was delayed in freeing itself from the seed coat A7d Close-up ofseed where radicle completely broke offfrom embryo
60
a
b
Figure A8 New Mexico locust seeds following percussion treatment at time of germination counts A8a Percussion replicate with uniformly healthy seedlings and near complete imbibition of seeds A8b Close-up ofpercussion-treated seedling
61
Discussion
Papilionoid seeds have a specific anatomical feature known as the strophiole
or lens-the natural site of water entry to the seed (Hamly 1932) The strophiole is
located on the cotyledonary lobe of a locust seed (Figure A9) When a papilionoid
legume seed is percussed for the appropriate amount of time repeated hits on the
integrated seed coat loosen the constrained cells of the strophiolar region without
excessively damaging the rest of the seed coat (Ballard 1976)
When a percussed seed is soaked water enters exclusively through the
strophiole in a controlled manner (Kelly and Staden 1987) This regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues This
contrasts with seeds that have been hot water or acid scarified treatments that can
randomly degrade the seed coat Unlocalized cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rehman et al 1998 Marunda 1990)
Papilionoids are the largest subfamily of legumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust and black locust these legumes often play an integral role in the
revegetation ofdisturbed lands Developing superior scarification methods to the
standard hot water and acid treatments should fadlitate the use ofthese legumes in
restoration projects
62
Figure A9 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (bc)
63
APPENDIXB
GERMINATION PERCENTS AND STANDARD ERRORS FOR SOURCE
BLOCK AND TREATMENT FOR NEW MEXICO LOCUST
Table B 1 Gennination percents and standard errors for source block and treatment for New Mexico locust
Source Block Treatment Germination Percentage Standard Error
Capitan 1 Immersion 74 437 Capitan 2 Immersion 69 462 Capitan 3 Immersion 71 454 Capitan 4 Immersion 67 470 Capitan Boiling 30 sec 79 407 Capitan 2 Boiling 30 sec 76 427 Capitan 3 Boiling 30 sec 72 449 Capitan 4 Boiling 30 sec 76 427 Capitan Boiling 60 sec 71 454 Capitan 2 Boiling 60 sec 54 498 Capitan 3 Boiling 60 sec 67 470 Capitan 4 Boiling 60 sec 76 427 Capitan I Control 5 218 Capitan 2 Control 7 255 Capitan 3 Control 4 196 Capitan 4 Control 5 218 Capitan 1 Percussion I min 47 499 Capitan 2 Percussion I min
470
Capitan Percussion I min 39 488
Capitan 4 Percussion I min 38 485 Capitan I Percussion 2 min 55 497 Capitan Capitan
2
Percussion 2 min Percussion 2 min
71 65
454 477
Capitan 4 Percussion 2 min 56 496 Capitan I Percussion 4 min 86 347 Capitan 2 Percussion 4 min 79 407 Capitan 3 Percussion 4 min 81 392 Capitan 4 Percussion 4 min 82 384 Capitan I Percussion 8 min 90 300 Capitan 2 Percussion 8 min 88 325 Capitan 3 Percussion 8 min 90 300 Capitan 4 Percussion 8 min 90 300 Gila I Immersion 71 454 Gila 2 Immersion 77 421 Gila
Immersion 75 433
Gila 4 Immersion 72 449
Gila I Boiling 30 sec 68 466
Gila 2 Boiling 30 sec 81 392
Gila 3 Boiling 30 sec 54 498
Gila 4 Boiling 30 sec 63 483
Gila I Boiling 60 sec 55 497
Gila 2 Boiling 60 sec 68 466
Gila 3 Boiling 60 sec 57 495
Gila 4 Boiling 60 sec 70 458
65
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Gila 2 Control 13 336 Gila 3 Control 13 336 Gila 4 Control 20 400 Gila 1 Percussion 1 min 66 474 Gila 2 Percussion 1 min 57 495 Gila 3 Percussion 1 min 73 444 Gila 4 Percussion 1 min 82 384 Gila 1 Percussion 2 min 81 392 Gila 2 Percussion 2 min 86 347 Gila 3 Percussion 2 min 85 357 Gila 4 Percussion 2 min 54 498 Gila 1 Percussion 4 min 79 407 Gila 2 Percussion 4 min 83 376 Gila 3 Percussion 4 min 84 367 Gila 4 Percussion 4 min 79 407 Gila 1 Percussion 8 min 51 500 Gila 2 Percussion 8 min 76 427 Gila 3 Percussion 8 min 52 500 Gila 4 Percussion 8 min 74 439 Holman 1 Immersion 83 376 Holman 2 Immersion 86 347 Holman 3 Immersion 81 392 Holman 4 Immersion 90 300 Holman 1 Boiling 30 sec 90 300 Holman 2 Boiling 30 sec 85 357 Holman 3 Boiling 30 sec 87 336 Holman 4 Boiling 30 sec 83 376 Holman 1 Boiling 60 sec 79 407 Holman 2 Boiling 60 sec 80 400 Holman 3 Boiling 60 sec 86 347 Holman 4 Boiling 60 sec 93 255 Holman Control 25 433 Holman 2 Control 19 392 Holman Control 21 407
Holman 4 Control 24 427
Holman Percussion 1 min 77 421
Holman 2 Percussion 1 min 85 357
Holman 3 Percussion 1 min 80 400
Holman 4 Percussion 1 min 89 313
Holman 1 Percussion 2 min 88 325
Holman 2 Percussion 2 min 96 196
Holman 3 Percussion 2 min 93 255
Holman 4 Percussion 2 min 96 196
Holman 1 Percussion 4 min 92 271
Holman 2 Percussion 4 min 97 171
66
I
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Holman 3 Percussion 4 min 91 286 Holman 4 Percussion 4 min 96 196 Holman 1 Percussion 8 min 88 325 Holman 2 Percussion 8 min 85 357 Holman 3 Percussion 8 min 83 376 Holman 4 Percussion 8 min 82 384 Jemez 1 Immersion 53 499 Jemez 2 Immersion 44 496 Jemez 3 Immersion 52 500 Jemez 4 Immersion 70 458 Jemez 1 Boiling 30 sec 58 494 Jemez 2 Boiling 30 sec 55 497 Jemez 3 Boiling 30 sec 48 500 Jemez 4 Boiling 30 sec 65 477 Jemez 1 Boiling 60 sec 68 466 Jemez 2 Boiling 60 sec 63 483 Jemez 3 Boiling 60 sec 73 444 Jemez 4 Boiling 60 sec 89 313 Jemez 1 Control 2 140 Jemez 2 Control 9 286 Jemez 3 Control 6 237 Jemez 4 Control 8 271 Jemez 1 Percussion 1 min 58 494 Jemez 2 Percussion 1 min 63 483 Jemez 3 Percussion 1 min 77 421 Jemez 4 Percussion 1 min 89 313 Jemez 1 Percussion 2 min 68 466 Jemez 2 Percussion 2 min 64 480 Jemez 3 Percussion 2 min 80 400 Jemez 4 Percussion 2 min 83 376 Jemez Percussion 4 min 68 466 Jemez 2 Percussion 4 min 79 407 Jemez 3 Percussion 4 min 70 458 Jemez 4 Percussion 4 min 83 376 Jemez 1 Percussion 8 min 52 500 Jemez 2 Percussion 8 min 59 492 Jemez 3 Percussion 8 min 61 488 Jemez 4 Percussion 8 min 70 458 Magdalena 1 Immersion 69 462 Magdalena 2 Immersion 69 462 Magdalena J Immersion 70 458 Magdalena 4 Immersion 85 357 Magdalena 1 Boiling 30 sec 65 477 Magdalena 2 Boiling 30 sec 71 454 Magdalena 3 Boiling 30 sec 68 466
67
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Magdalena 4 Boiling 30 sec 83 376 Magdalena 1 Boiling 60 sec 63 483 Magdalena 2 Boiling 60 sec 68 466 Magdalena 3 Boiling 60 sec 63 483 Magdalena 4 Boiling 60 sec 82 384 Magdalena 1 Control 11 313 Magdalena 2 Control 6 237 Magdalena 3 Control 5 218 Magdalena 4 Control 4 196 Magdalena Percussion 1 min 32 466 Magdalena 2 Percussion 1 min 45 497 Magdalena 3 Percussion 1 min 46 498 Magdalena 4 Percussion I min 36 480 Magdalena 1 Percussion 2 min 67 470 Magdalena 2 Percussion 2 min 68 466 Magdalena 3 Percussion 2 min 65 477 Magdalena 4 Percussion 2 min 81 392 Magdalena 1 Percussion 4 min 87 336 Magdalena 2 Percussion 4 min 97 171 Magdalena 3 Percussion 4 min 83 376 Magdalena 4 Percussion 4 min 97 171 Magdalena 1 Percussion 8 min 98 lAO Magdalena 2 Percussion 8 min 100 000 Magdalena 3 Percussion 8 min 96 196 Magdalena 4 Percussion 8 min 98 lAO Manzano 1 Immersion 67 470 Manzano 2 Immersion 57 495 Manzano 3 Immersion 72 4049 Manzano 4 Immersion 73 4044 Manzano 1 Boiling 30 sec 70 458 Manzano 2 Boiling 30 sec 64 480 Manzano 3 Boiling 30 sec 71 454 Manzano 4 Boiling 30 sec 80 400 Manzano I Boiling 60 sec 50 500 Manzano 2 Boiling 60 sec 53 499 Manzano 3 Boiling 60 sec 59 492 Manzano 4 Boiling 60 sec 74 439 Manzano I Control 4 196 Manzano 2 Control 8 271 Manzano 3 Control 1 099 Manzano 4 Control 6 237 Manzano 1 Percussion 1 min 52 500 Manzano 2 Percussion 1 min 39 488 Manzano 3 Percussion 1 min 36 480 Manzano 4 Percussion 1 min 51 500
68
Table B 1 Continued
Source Block Treatment Gerrni~ation Percentage Standard Error
Manzano Percussion 2 min 53 499 Manzano 2 Percussion 2 min 58 494 Manzano 3 Percussion 2 min 61 488 Manzano 4 Percussion 2 min 62 485 Manzano 1 Percussion 4 min 76 427 Manzano 2 Percussion 4 min 85 357 Manzano 3 Percussion 4 min 79 407 Manzano 4 Percussion 4 min 83 376 Manzano 1 Percussion 8 min 87 336 Manzano 2 Percussion 8 min 83 376 Manzano 3 Percussion 8 min 83 376 Manzano 4 Percussion 8 min 75 433 Raton 1 Immersion 54 498 Raton 2 Immersion 37 483 Raton 3 Immersion 39 488 Raton 4 Immersion 69 462 Raton 1 Boiling 30 sec 36 480 Raton 2 Boiling 30 sec 20 400 Raton 3 Boiling 30 sec 28 449 Raton 4 Boiling 30 sec 60 490 Raton 1 Boiling 60 sec 15 357 Raton 2 Boiling 60 sec 171 Raton 3 Boiling 60 sec 4 196 Raton 4 Boiling 60 sec 35 477 Raton 1 Control 4 196 Raton 2 Control 12 325 Raton 3 Control 2 140 Raton 4 Control 6 237 Raton 1 Percussion 1 min 52 500 Raton 2 Percussion I min 29 454 Raton 3 Percussion I min 35 477 Raton 4 Percussion 1 min 31 462 Raton 1 Percussion 2 min 55 497 Raton 2 Percussion 2 min 35 477 Raton 3 Percussion 2 min 33 470 Raton 4 Percussion 2 min 42 494 Raton 1 Percussion 4 min 65 477 Raton 2 Percussion 4 min 52 500 Raton Percussion 4 min 51 500 Raton 4 Percussion 4 min 53 499 Raton 1 Percussion 8 min 88 325 Raton 2 Percussion 8 min 80 400 Raton 3 Percussion 8 min 73 444 Raton 4 Percussion 8 min 74 439 Sacramento 1 Immersion 43 495
69
Table B 1 Continued
-Source Block Treatment Germination Percentage Standard Error
Sacramento 2 Immersion 30 458 Sacramento 3 Immersion 38 485 Sacramento 4 Immersion 56 496 Sacramento 1 Boiling 30 sec 47 499 Sacramento 2 Boiling 30 sec 48 500 Sacramento 3 Boiling 30 sec 48 500 Sacramento 4 Boiling 30 sec 56 496 Sacramento 1 Boiling 60 sec 52 500 Sacramento 2 Boiling 60 sec 67 470 Sacramento 3 Boiling 60 sec 43 495 Sacramento 4 Boiling 60 sec 48 500 Sacramento 1 Control 0 000 Sacramento 2 Control 3 171 Sacramento 3 Control 3 171 Sacramento 4 Control 099 Sacramento 1 Percussion 1 min 57 495 Sacramento 2 Percussion 1 min 61 488 Sacramento 3 Percussion 1 min 62 485 Sacramento 4 Percussion 1 min 55 497 Sacramento 1 Percussion 2 min 84 367 Sacramento 2 Percussion 2 min 70 458 Sacramento 3 Percussion 2 min 81 392 Sacramento 4 Percussion 2 min 69 462 Sacramento 1 Percussion 4 min 91 286 Sacramento 2 Percussion 4 min 84 367 Sacramento 3 Percussion 4 min 84 367 Sacramento 4 Percussion 4 min 94 237 Sacramento 1 Percussion 8 min 82 384 Sacramento 2 Percussion 8 min 81 392 Sacramento 3 Percussion 8 min 82 384 Sacramento 4 Percussion 8 min 81 392 Sandia 1 Immersion 76 427 Sandia 2 Immersion 70 458 Sandia 3 Immersion 70 458 Sandia 4 Immersion 81 392 Sandia 1 Boiling 30 sec 78 414 Sandia 2 Boiling 30 sec 71 454 Sandia 3 Boiling 30 sec 63 483 Sandia 4 Boiling 30 sec 78 414 Sandia 1 Boiling 60 sec 67 470 Sandia 2 Boiling 60 sec 65 477 Sandia 3 Boiling 60 sec 74 439 Sandia 4 Boiling 60 sec 66 474 Sandia 1 Control 16 367 Sandia 2 Control 7 255
70
Table B 1 Continued
Source Block Treatment Gennination Percentage Standard Error
Sandia 3 Control 8 271 Sandia 4 Control 16 367 Sandia 1 Percussion 1 min 67 470 Sandia 2 Percussion 1 min 61 488 Sandia 3 Percussion 1 min 66 474 Sandia 4 Percussion 1 min 59 492 Sandia I Percussion 2 min 82 384 Sandia 2 Percussion 2 min 83 376 Sandia 3 Percussion 2 min 81 392 Sandia 4 Percussion 2 min 84 367 Sandia 1 Percussion 4 min 89 313 Sandia 2 Percussion 4 min 86 347 Sandia 3 Percussion 4 min 89 313 Sandia 4 Percussion 4 min 85 357 Sandia 1 Percussion 8 min 80 400 Sandia 2 Percussion 8 min 77 421 Sandia 3 Percussion 8 min 69 462 Sandia 4 Percussion 8 min 66 474
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Hanna PJ 1984 Anatomical features of the seed coat ofAcacia kempeana (Mueller) which relate to increased germination rate induced by heat treatment New Phytol 96 23-29
Hanks JP Dick-Peddie WA 1974 Vegetation patterns of the White Mountains New Mexico Southwest Naturalist 18(4) 371-382
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---- 1997 Differences in hardseededness between Cunningham and Tarramba leucaena In Leucnet News The Newsletter of the International LeucaenaRes and Devlt Netwk No4
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Kelly KM van Staden J 1987 The lens as the site of permeability in the papilionoid seed Aspalathus linearis J Plant Physiol 128(45) 395-404
73
Keresztesi B 1988 Black locust the tree of agriculture Outlook on Agr 17(2) 77~85
Kigel 1 and Galili G 1995 Seed development and germination Marcel Dekker New York 853 p
Klemmedson 10 1994 New Mexico locust and parent material Influence on macronutrients of forest floor and soil Soil Sci Soc Am 1 58 974~980
Kuo WH Tam AW 1988 The pathway of water absorption of mung bean seeds Seed Sci amp Technol16(1) 139~144
Leadem CL 1997 Dormancy--unlocking seed secrets In Landis TD Thompson lR tech coords National Proceedings Forestand Conservation Nursery Assocs Gen Tech Rep PNW-G TR-419 Portland OR USDA For Ser PNW Res Sta 43-52
Lin Y Wagner MR Seweyestewa A Honahni MD DeGomez M McMillin JD 1996 Influence of cultural treatments on seed germination and seedling growth of Robinia neomexicana Forest Farm and Community Tree Res Reports Volume 1
Marunda CT 1990 Effects of seed pretreatment on the development ofAcacia auriculforinis and A holosericea seedlings ACIAR Proceedings Series No 28
Meginnis HG 1937 Sulphuric acid treatment to increase germination of black locust seed USDA Circ 453 34 p
Miklas PN Towensend CE Ladd SL 1987 Seedcoat anatomy and the scarification ofcicer milkvetch seed Crop Sci 27 766-772
Monsen SB 1984 Use of shrubs on mine spoils In The Challenge ofProducing Native Plants for the Intermountain Area Proceedings Intermountain Nurserymans Association 1983 conference Las Vegas NV compo PM Murphy 26-31 Ogden UT USDA For Ser Gen Tech Report INT-l68
Olson DF 1974 Robinia L Locust In Seeds of Woody Plants in the US tech coord CS Schopmeyer 728-731 USDA For Ser Agr Handbk No450
Rehman S Loescher RNl Harris P1C 1998 Dormancy breaking and germination ofAcacia salicina Lindl seeds Seed Sci amp Techno127 553-557
Rolston MP 1978 Water impermeable seed dormancy Bot Rev 44 365-396
74
SAS Institute Inc 1989 SASSTAT Users Guide Version 6 4th Edition Volume 1 Cary NC SAS Institute Inc 943 p
SerratoValenti G Conara L Ferrando M Modenesi P 1993 Structural and histochemical features ofStylosanthes scabra (Leguminosae Papilionoidae) seed coat as related to water entry Can J Bot 71 834-840
Simpson BJ 1988 A field guide to Texas trees Austin TX Texas Monthly Press 372p
Tran VN Cavanagh AK 1984 Structural aspects ofdormancy In Seed Physiology Vol 2 Murray DR (ed) p1-44 Academic Press Australia
USDA Forest Service 1948 Woody-plant seed manual USDA Misc Pub1654 416p
Vines RA 1960 Trees Shrubs and Woody Vines of the Southwest Austin Univ ofTexas Press 1104 p
Wagner MR Larson RE Craig TP 1992 Ecology and silvics ofNew Mexico locust (Robinia neomexicana) with an emphasis on the arboreal form p53-57 In JW Hanover K Miller and S Plesko (eds) Proc Intemat Confon Black Locust Biology Culture and Utilization Mich State Univ USA
Wilson JK 1944 Immersing seeds of species of Robinia in boiling water hastens germination J For 42 453-454
Zimmerman RW Carpenter SB 1980 First-year coppice production from a 5shyyear old black locust stand on surface mine spoil p309-314 Centr Hardwd For ConfProc University ofMissouri Columbia
There are several potential advantages of percussion over acid hot water and
mechanical scarification Kelly and van Staden (1987) note that a percussion
treatment only alters macrosclereid cells of the strophiole by loosening separating
and raising them They emphasize that minimal damage is done to the overall
anatomical structure of the seed In acid scarification of the same seed lot radicle
and cotyledon damage occurred Based on photographic evidence the authors
believe that this morphological damage was due to uneven pressure during
imbibition with water entering haphazardly wherever the acid had sufficiently
dissolved the coat This ultimately led to uneven hydration of the embryo In
percussed seeds water only entered through the s~ophiole when the entire seed coat
had been saturated resulting in controlled and even embryo hydration (Kelly and van
Staden 1987)
Although all successful scarification treatments may focus at least in part on
the strophiole for papilionoid legumes (Baskin and Baskin 1998) percussion may be
less damaging to seed coat structures particularly when compared with acid Kelly
and van Staden did not compare hot water or mechanical scarification methods
against percussion However the fungal infection associated with hot water and
mechanical scarification as with acid scarificatio~ have been directly associated with
seed damage (Rehman et al 1998 USDA Forest Service 1948) Embryo damage in
has been observed in hot-water scarified legume seeds (Rehman et al 1998
13
By limiting embryo damage percussion scarification reduces seedling
mortality compared to other treatments Kelly and van Staden (1987) noted that
seedlings from the percussion treatment were morphologically sound and vigorous
when compared to acid treatment seedlings The authors attribute this improved
seedling vigor to the controlled water uptake that limited anatomical damage
Despite a 12-hour percussion treatment morphological damage was not substantially
greater than untreated seeds In addition seedling mass measured 10 days following
germination was not significantly different between percussed and untreated seeds
A final advantage ofpercussion may be the ability to adequately treat the most
dormant seeds while avoiding harm to seeds that have already been made permeable
by the treatment or that were permeable to begin with Instead ofcorroding scalding
or scratching the seed coat percussion appears to be just a loosening of the
strophiole spring (Ballard 1973) Kelly and van Staden (1987) were able to
increase germination with a lengthy treatment (12 hours) Figuring out whether a
substantial cushion oftreatment durations exist for percussion ofNew Mexico locust
seed would be a significant improvement to current scarification methods where seed
damage and seedling health is an issue
The greatest challenge for applying percussion to New Mexico locust seeds is
standardizing a treatment that is both effective and readily reproducible by nurseries
with mechanical scarification this may require the development of specific
~UllDment to properly quantify and execute the treatment
14
f
Objectives
The first objective of the study is to detennine which scarification treatment
results in optimal gennination speed and percentage ofNew Mexico locust The
second objective is to describe treatment interactions with nine seed sources collected
in New Mexico
Hypothesis
A percussion treatment acts exclusively on the main area ofwater uptake in
New Mexico locust seed loosening the strophiole so that imbibition can occur This
direct mechanism ofdonnancy release will result in improved gennination rate and
overall gennination percentage for New Mexico locust seed compared to standard
treatments Standard treatments including acid hot water and mechanical
scarification while eventually weakening the strophiolar cleft cause excessive
morphological damage through generalized seed coat degradation These treatments
are therefore prone to inconsistency due to over and under-treatment
15
MATERIALS AND METHODS
Seed Collection and Cleaning
Seeds were collected from August 25 through September 15 2000 from nine
locations along a latitudinal gradient in New Mexico Seed source locations and
descriptive information are given in Figure 1 and Tables I and 2 Stands were
separated by at least 45 km
Pods were hand picked as they began to ripen and dehisce (open) Collections
were made from ground level to approximately 3 m from several trees at each site
Pods were dried in open-ended brown paper bags for two to four weeks at room
temperature (21-230C) to facilitate cleaning Dried pods were placed in a closed cloth
sack which was pounded against the ground to separate seeds from pods For pods
still containing seeds hand maceration (crunching pods with hands) completed seed
separation Debris was removed by threshing with the aid of a greenhouse fan A
South Dakota Seed Blower (Seedbureau Chicago Illinois) was used to remove
remaining chaff and light seeds by air density separation Seeds were placed in dry
storage in sealed quart-size zip-lock bags within a sealed plastic container Storage
temperatures ranged from 2-4oC Seeds remained in storage for approximately 7
months before the experiment
Experimental Design and Treatment Structure
The experiment compared the impact ofhot water bath and percussion
l11aLl11 on germination properties ofNew Mexico locust seed A single
16
-l
Figure 1 Seed source collection map All sources were collected within New Mexico USA separated by at least 4S lan
IIIRZltItW lIJPIirmiddot rllll1lbullbull_~~~_II
Table 1 Latitude longitude location elevation and collection date ofNew Mexico locust seed sources
Lot title Latitude (N) Longitude (W) Location Elevation (m) Collection Date
Raton 36deg5846 104deg2872 Raton Pass NM 2295 82500
Holman 36deg0306 105deg2429 HolmanNM 2476 911500
Jemez 35deg4500 106deg4100 Jemez Springs NM 1976 9900 00
Sandia 35deg1083 106deg2341 Cedar Crest NM 2432 82600
Manzano 34deg3689 106deg2494 ManzanoNM 2465 9200
Magdalena 33deg5953 107deg0903 Magdalena NM 2544 82900
Capitan 33deg2728 105deg4325 Alto NM 2189 911200
Sacramento 32deg5798 105deg4496 Cloudcroft NM 2630 91400 Gila 32deg5400 107deg4600 Kingston NM 2432 9700
f
Table 2 Mean seed weight and standard error by source
a
Source Mean seed weight (g) for Standard error 100 seeds n = 5
Raton Holman Jemez Sandia Manzano Magdalena Capitan Sacramento Gila
2872 b c 2522 d 1826 e 2753 c 2413 d 2559 d 2443 d 3130 a 3029 ab
00103 00403 00276 00301 00370 00374 00458 00279 00348
a Mean seed weights followed by the same letter are not significantly
different
19
experiment was conducted utilizing a randomized complete block design blocked by
time with a factorial treatment structure Experimental factors were seed source and
scarification treatment Nine seed sources from throughout New Mexico were used
(Figure 1 Table 1) Scarification treatments included a control three levels ofhot
water and four levels of percussion Hot water treatments included immersion at
100degC and boiling for periods of 30 seconds and 60 seconds Percussion treatment
levels were 1 2 4 and 8 minutes One hundred seeds per block were used to test
each treatment combination Four blocks of seed were tested one at a time (all seed
sources included in each run) in close chronological sequence (within 6 weeks) A
total of four 100-seed samples were used to test each source by scarification treatment
combination
Treatments
Hot water treatment of seeds utilized 50 ml test tubes filled with 30 ml
distilled water Each test tube was used to treat one 100-seed sample at a time
resulting in a ratio of4 parts water to 1 part seed by volume Test tubes were placed
in a beaker with the beaker filled with tap water to the same level as the water within
test tubes Nine test tubes (one per seed source) were placed in the beaker
simultaneously so that one replication of each seed sourcescarification treatment
cotnblnatlon was conducted simultaneously Hot water treatments include immersion
boiling For immersion treatments seeds were added to test tubes with the water
(boiling) and the test tubes were immediately removed from the beaker to
For the boiling treatments test tubes remained in the beaker with water at
20
boiling temperature for a set duration after the addition of seeds Following all
treatments test tubes with seeds cooled 24 hours in a test tube rack
Percussion treatment utilized a pneumatic paint shaker (Central Pneumatic
model 00422 Camarillo CA) Compressed air was maintained at 530 kPa plusmn30 kPa
(80 psi plusmn 5 psi) resulting in approximately 350 oscillations per minute Seeds were
placed in 118 ml (4 oz) soil tin cans (US Can Lobard IL) and the lids were secured
with duct tape Each can was used to treat one 100-seed sample To take advantage
ofgreatest lateral movement of the paint shaker a spacer (paint can) was used to
position the soil tin at the end of the shaking arm This allowed shaking to occur at
the greatest distance from the pivot point (fulcrum) Tins were placed perpendicular
to the direction of shaking motion allowing seeds to impact at greatest force by
hitting flat surfaces of the soil tin
Germination Testing
Before gennination testing all seeds were soaked 24 hours in reverse
osmosis-treated water (this was included in the process ofhot water treatment
jU~SCIlOea above) Following soaking seeds were placed in 100 cm Petri dishes lined
90 cm Whatman qualitative 1 filter paper circles (MG Scientific Pleasant
WI) One Petri dish was used for each treatment by source combination
papers were initially moistened with 5 mL reverse osmosis-treated water then
~VU1CUlJl moistened as needed to ensure adequate conditions for gennination
were monitored daily (at 300 PM) for gennination for 14 days in accordance
~UIUlnJlVUa Seed Testing Association (ISTA) standards for Robinia species
21
(1STA 1999) Tests were carried out at room temperature (22degC plusmnOSoC)
Germination was classified as protrusion of the radicle from the embryo by at least
1mm as viewed with the naked eye
X-ray Radiography
On August 132001 three lOO-seed samples from the Sacramento seed source
were treated for 14 or 8-minute percussion durations The seeds were sent
immediately by overnight delivery to the Faxitron X-Ray Corporation in Wheeling
Illinois where Dr Bob Karrfalt ofthe USDA Forest Service oversaw their imaging
Radiographs were made at SX with a Faxitron Specimen Radiography System Model
MX-20
Scanning Electron Microscope Photography
On February 192002 a Hitachi S-3200N Variable Pressure Scanning
Electron Microscope (Mountain View CA) was used to examine strophiolar cells of
untreated seeds and seeds percussed for 4 minutes Seeds were cut longitudinally
igt their entire length using a razor blade splitting the strophiolar region roughly in
Cross sections of seed samples magnifying the strophiolar region were viewed
600X under the microscope
Analysis
Categorical Analysis of Variance was used to determine treatment source and
(Le time) main effects and interactions The response variable was total
~vlI This procedure is a Chi-square test of homogeneity using the natural log
of germinated to non-germinated seed for each treatment Maximumshy
22
likelihood analysis was used to calculate Chi-square test statistics with their observed
significance levels A single contrast was conducted to cempare treatment means
against the control Observed significance levels less than or equal to 005 were
considered significant Pairwise comparisons were conducted between non-control
treatment mean percents using a conservative alp~a value of 005 divided by the
number of comparisons (Bonferroni approach) and approximate pairwise z statistics
Categorical analyses were performed using Proc Catmod (SAS Institute 1989)
RESULTS
Scarification seed source block and all interactions between factors
influenced germination (Table 3) The main effects are reported in the presence of
these interactions because the overall robustness of treatments and seed sources are of
interest Background levels ofnaturally occurring variation in seed sources are
seen in control germination percentages in Figure 2 In particular time variability
is unlikely to be under the control of the producer Appendix B details experimental
data for source by treatment gennination percents for each of the four blocks
Averaged over all treatments germination by source ranged from 40 to
79 (Figure 2) Averaged over all sources (Figure 3) and for each source (Figure 4
5) percussion scarification durations of4 or 8 minutes yielded greatest germination
Averaged over all sources germination increased with increasing durations of
percussion then leveled off for the 4- and 8-minute treatments (Figure 3) Immersion
and 30-second boiling scarification maximized germination for the hot water
treatments with a decrease in germination when seeds were boiled for 60 seconds
For all but two sources there is a similar ipcreasing trend in germination from
1to 4 minutes ofpercussion (Figure 4) The Holman source increased in germination
1 to 2 minutes then leveled off at 4 minutes The Jemez source changed little
1 to 4 minutes of treatment As treatment duration increased from 4 to 8
mutes five sources decreased in germination (Figure 4a) whereas three sources
24
Table 3 Categorical Analysis of Variance table for germination response to seed treatment seed source block and the interaction of the factors
Source of DF Chi-Square Observed Variability Significance Level
Source 8 98370 lt00001 Trt 7 233876 lt00001
contrast 1 16976 lt00001 SourceTrt 56 97077 lt00001 Block 3 5538 lt00001
SourceBlock 24 10162 lt00001 TrtBlock 21 7564 lt00001
SourceTrtBlock 168 34283 lt00001
S 4)
8 ~
0
sectg ~ 40 ~
N 0 0
20
o f0ltgt ~ ~4 jybshy ((iltgto ~~ K1ltgt 6~b-~ ~~ 0b-~~ ~~ ~b-~ ~b-~b
80
60 -I-- - ~- middotmiddotmiddotmiddotmiddot1
r -] Northern NM Sources
Central NM Sources
Southern NM Sources
sect-o ~
~b-v(i
SOURCE
Figure 2 Effect ofNew Mexico locust seed source on germination for data averaged over all scarification treatments Control germination is shown by solid line within bar
80
~ cd ~ ltl) () 60 ~ ~
N
t 0shyrti tshySltl)
40
0
20
o lmmersion Boiling 30 sec Boiling 60 sec Control 1 min 2min 4 min 8 min
Hot Water Treatments Percussion Treatments
Figure 3 Effect of scarification on germination ofNew Mexico locust for data averaged over all seed sources Means labelled with the same letter are not significantly different
100~----------------------~--~----------------~
80
60
ltU
~
40 IJ
sectgsI
l100
80
60
40
a I middotmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot middotImiddotmiddotmiddotmiddotmiddotmiddot E
_~iii-bull _ _ bull f --~--
t-- -~ ---- -shy ---- shyI ~ ---- - shy
L
fiII
~
b
- shy ---r
- Holman
Sacramento Sandia Gila Jemez
c ~
ltgt ~
Tmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot-I
j~=~=-~~-1
I middotmiddot ~ bull r
t tr
Y -------M-a-gd-a-Ie-n-a--
1 _ _
fCapitan
---- Manzano ~y -_ Raton
2 4 8
Time interval ofpercussion scarification (min)
Figure 4 Effect of seed source by treatment interaction on New Mexico locust germination for percussion scarification pattern a and pattern b
28
tv 0
~ d
~ A s o
1 c3
80
60
40
20
o
TIT
t------=middotfmiddotmiddot-middotmiddot-middotmiddot-middotmiddot 1- --- - - --=- - -shy--cE= - _--shy --shy ----shy ~ ~ ---shy ~ -~- _ --shy-------r 1
I
Raton Holman
-_ Jemez
--- Sandia Manzano Magdalena Capitan Sacramento Gila
Immersion Boiling 30 sec Boiling 60 sec
Hot Water Scarification
Figure 5 Effect of seed source by treatment interaction on New Mexico locust germination for hot water scarification
continued to increase in germination (Figure 4b) There was no difference between
the two treatment levels for the Manzano source (Figure 4b)
The effect ofhot water scarification also varied among sources with few
overall differences between hot water treatments (Figure 5) However boiling for 60
seconds yielded highest germination for the Jemez source whereas increasing
durations ofboiling decreased germination for the Raton source
In addition to consistently high germination 4- and 8-minute percussionshy
treated seeds germinated as fast or faster than all other treatments (Figure 6 Table 4)
050 measured as the number of days needed to complete 50 ofeventual
germination averaged 2 days for both ofthese treatments
X-ray radiographs were taken of seeds collected from the Sacramento source
percussed for 1 4 or 8 minutes (Figure 7) Seeds show signs of damage at 8 minutes
ofpercussion (Figure 8) Compared to an undamaged seed percussed for only 4
minutes (Figure 8a) damage at the 8-minute level includes fractured radicles pitting
of the seed coat and chips in seed coat structure (Figure 8b-d)
Scanning electron microscope (SEM) images at 600X reveal the direct effect
of 4 minutes of percussion on cells of the strophiolar region (Figure 9) Whereas
untreated seeds retain cellular organization in the strophiolar region strophiolar cells
seeds treated for 4 minutes ofpercussion separate and raise resulting in a
~-~e~U appearance
30
Umiddot-~middot--l-~---middotymiddotmiddotmiddot-----middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot--middot--middotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middotmiddotmiddotmiddotmiddotmiddotmiddot- -
050= gt1
-e-- Percussion 8 min -0- Percussion 4 min ---T- Percussion 2 min -----ltil- Percussion 1 min -- Immersion --0- Boiling 30 sec -+- Boiling 60 sec -ltgt- Control
2 4 6 8 10 12
Day Figure 6 Timecourse ofNew Mexico locust gennination following scarification
treatments G50 is the number of days to 50 of eventual germination
31
14
Table 4 Effect of seed source and treatment on G50 (days to 50 germination)
Seed Source Control Immersion B30sec B60sec Plmin P2min P4min P8min mean Raton 3 3 5 7 4 3 2 2 4 Holman 2 2 2 2 2 2 2 2 2
Jemez 4 6 6 5 3 2 2 2 4 Sandia 2 2 2 2 2 2 2 2 2
w tv Manzano 5 2 2 3 3 2 2 2 3
Magdalena 4 2 2 3 4 3 2 1 3
Capitan 5 4 2 2 3 2 2 2 3
Sacramento 3 2 2 2 4 3 2 2 3 Gila 3 2 2 2 2 2 2 2 2 mean 3 3 3 3 3 2 2 2 3 standard 11 14 16 18 09 05 0 03 08 deviation
Note Columns and rows may not agree due to rounding
Figure 7 X-ray radiographs ofSacramento seed source percussed for 1 4 or 8 minutes
33
Figure 8a-d Seed damage close-ups Figure a shows undamaged seed percussed for 4 minutes Figures b-d show seeds percussed for 8 minutes Damage includes fracturing ofradicle in b complete removal ofseed coat in c and pitting ofseed coat in d
34
Untreated Treated
Figure 9 SEM images ofuntreated vs 4-minute percussion-treated strophiolar cells ofNew Mexico locust seeds Treated cells are loosened separated and raised
DISCUSSION
Determining the Optimal Treatment
Averaging results of scarification treatmems across seed sources is a useful
tool to predict which treatment is most likely to be successful for an unknown seed
source Averaged over all sources four- and eight-minute percussion treatments
resulted in the highest germination 815 and 790 respectively and produced
germination that was as fast or faster than all other treatments Although there was no
difference between these two treatments on average an argument for 4 minutes of
percussion as a standard protocol for treating New Mexico locust seeds can be made
Descriptively 4 minutes of percussion improved germination over 8 minutes
ofpercussion for a majority of sources (five out of nine) 8 minutes of percussion
improved germination for three ofnine sources with no difference for a fmal source
For individual sources 4 minutes ofpercussion was always the best or secondmiddot best
treatment and was always equal to or better than all hot water treatments
Germination at 8 minutes ofpercussion was lower than all other percussion durations
for the Jemez and Gila sources and was lower than at least one hot water treatment for
these sources as well
The x-ray radiographs of the Sacramento seed source illustrate the excessive
internal and external seed damage that can occur with 8 minutes of percussion
germination counts 8-minute percussed seeds from Holman and Capitan
ClUll signs of damage that were visible to the naked eye (see below)
r~evertheless some of these seeds still fit the criteria for germination Neither x-ray
36
photography nor visible observations during seed counts revealed seed damage at the
4-minute percussion level In operational trials seeds percussed for 4 minutes
developed into healthy seedlings (Khadduri and Harrington unpublished data)
Strophiole and Seed Coat Behavior
Four minutes ofpercussion successfully breaks seed dormancy in a large
percentage ofNew Mexico locust seeds without excessively damaging the
surrounding seed coat or internal seed coat structures Most papilionoid legume
seeds including seeds ofNew Mexico locust have a specific region of the seed coat
known as the strophiole or lens (Hamly 1932) The strophiole is located on the
cotyledonary lobe of a locust seed (Figure 10 a-c) If seeds are not indiscriminately
damaged the strophiole is the first point of water entry to the embryo (Hopkinson
1997) When a papilionoid seed is percussed for the appropriate amount of time
repeated hits on the integrated seed coat loosen the constrained cells of the strophiolar
region without excessively damaging the rest of the seed coat (Ballard 1976) SEM
photos illuStrate that 4 minutes of percussion loosened and separated strophiolar cells
ofNew Mexico locust seeds when compared with constrained organized strophiolar
cells of untreated seeds (Figure 9) Research-has shown that water passes to the
embryo through the treated strophiole in a controlled manner and regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues
(Kelly and van Staden 1987)
37
10 Strophiole ofunimbibed New Mexico locust seed (a) and following uptake (bc)
38
In contrast hot-water treatments can excessively degrade the seed coat
(Rebman et al 1998 Marunda 1990) Random cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rebman et aI 1998 Marunda 1990) Along with a
proportionately large amount ofun-imbibed (thus under-treated) seeds (Figure l1a)
damage was observed in hot-water treated seeds from six sources during germination
counts Damage included seeds where the radicle failed to elongate (Figure 11 b)
seeds where the radicle elongated but was delayed in freeing itself from the seed coat
(Figure 11c) and seeds where the radicle completely broke off from the embryo
(Figure lId) In addition some seeds germinated in reverse (the cotyledon emerged
fIrst) If the radicle emerged completely from the seed coat they were counted as
germinated
While nearly all seeds treated for 8 minutes ofpercussion imbibed water
damage was evident in x-ray evaluations (Sacramento seed source) and in
germination counts (Holman and Capitan) at this treatment level Seed damage
during germination counts was similar to levels found in hot-water treated seeds
suggesting that over-treatment by both hot water and longer durations of percussion
may result in similar damage to seed structure
The Sacramento and Holman seed sources were two of the fIve sources that
opcrplltlpn in germination from the 4- to 8-minute durations of percussion
the benefIts ofloosening the strophiole as evidenced by imbibition of
all 8-minute treated seeds were outweighed by external and internal seed coat
39
a
b c d
11 Damage to New Mexico locust seeds observed during germination cotmts following hot water scarification 9a Hot water replicate including ~t seedlings and numerous unimbibed seeds 9b Close-up of seed where raallCle failed to elongate 9c Close-up ofseed where radicle elongated but was
-gtIn in freeing itself from the seed coat 9d Close-up ofseed where radicle broke off from embryo
40
damage (which also may be contributing to high imbibition) For the three sources
for which 8 minutes of percussion resulted in maximum germination extended
percussion of seeds necessary to loosen the strophiole may not have excessively
damaged the seed anatomy The Manzano source did not significantly change in
germination from 4 to 8 minutes of percussion suggesting that an increase in
permeable seeds at the 8-minute treatment duration was balanced by the number of
seeds that failed to germinate due to seed damage To summarize the 4-minute
percussion treatment improved germination over 8 minutes ofpercussion for a
majority of sources (five ofnine) the 8-minute percussion treatment improved
germination over 4-minutes ofpercussion for three sources and there was no
difference with the Manzano source Notably for the Raton source 8 minutes of
percussion increased germination by 235 percentage points over 4-minutes of
percussion (7875 vs 5525 respectively) the next best treatment Raton was the
only source to result in germination less than 75 for 4 minutes ofpercussion so a
grower may wish to apply longer percussion durations to lots that do not respond well
to this treatment level
Finally there was an interesting trend in the Jemez and Raton sources seed
that significantly contributed to the source by treatment interaction (Figure 12) A
~u test showed that the Jemez source had a significantly lighter seed weight than
other sources while the Raton source had an average seed weight For the Jemez
the longest duration of boiling increased germination Conversely only short
U4UVll) of percussion were needed to achieve high germination and the longest
41
80 _ Jemez a Raton
iD
~ C iD e iD
Q c 0 ~
c ~ iD 0
20
0------shy
Immersion Boiling 30 sec Boiling 60 sec
Hot Water Scarification
10degTr========~--------~------------------------------
80
60
40
20
_ Jemez Raton
0------shy
1 min 2 min 4 min 8 min
Percussion Scarification
Figure 12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources
42
b
--~----~~~~========~---- duration ofpercussion reduced germination For the Raton source longer durations
ofboiling consistently decreased germination whereas longer percussion treatments
consistently increased germination
A possible explanation is that a thick seed coat is associated with a loose
strophiole structure and that a thin seed coat is associated with a constrained
strophiole This contrast merits further study and may suggest an ecological
adaptation Further investigation ofthis phenomenon may contribute to successful
pre-screening ofunknown seed sources to determine optimal treatment leveL
43
CONCLUSION
Based on results obtained in pilot study (see Appendix) and thesis
experiments 4 minutes ofpercussion is recommended as a standard protocol for
treating an unknown source ofNew Mexico locust seed In addition to laboratory
results the 4-minute percussion treatment has resulted in germination greater than
80 and healthy seedling grow-outs for production purposes (unpublished data) In
laboratory experiments only one hundred seeds per 118 ml (40z) soil tin were
percussed at a time whereas for production we have treated up to five hundred seeds
per tin For large-scale production ofNew Mexico locust seedlings growers may
wish to experiment with larger quantities of seed and larger-size soil tins
Seed storage trials following percussion treatments were not conducted
However germination greater than 80 and healthy seedlings were obtained when
seeds were percussion-treated and subsequently stored at room-temperature (22degC)
for one week (unpublished data) Seed storage trials merit further study as the
standard hot water treatment leaves seeds in a softened condition that is not
conducive to storage
Hydroseeding is an effective method for direct seeding in reclamation
situations Percussion treatment may be conducive to hydro seeding as seeds remain
a dry relatively undamaged yet permeable condition following treatment Sulfuric
seeds also remain dry following treatment but extensive damage to seed coat
poor seedling vigor have been reported with this treatment (Kelly and van Staden
A grow-out study using a hydro seeder on a reclamation site testing percussion
44
against other treatments such as sulfuric acid and mechanical scarification also
merits further study
Papilionoids are the largest subfamily oflegumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust these legumes often play an integral role in the revegetation of
disturbed lands The pilot study (Appendix) indicates that percussion scarification
successfully treats the widely used black locust as well Developing superior
scarification methods to the standard hot water mechanical scarification and acid
treatments may facilitate the use ofpapilionoid legumes in reclamation projects
45
APPENDICES
APPENDIX A
PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR
NEW MEXICO LOCUST AND BLACK LOCUST SEEDS
Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4
Hot water and sulfuric acid soaks are traditional treatments for seeds of many
temperate woody legwnes including locusts However these scarification techniques
often produce inconsistent germination Percussion scarification where seeds are
repeatedly propelled against a hard surface was compared with hot water
scarification to evaluate treatment efficacy for New Mexico locust (Robinia
neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment
seeds were placed in a 98degC water bath which was immediately removed from the
heat source For percussion scarification seeds were placed in a soil sample tin and
agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the
control were followed by 24-hour water soaks Hot water baths resulted in 56 and
41 germination for New Mexico locust and black locust respectively For both
species nearly all durations ofpercussion increased germination over the hot water
treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3
4 and 5 minutes for black locust resulted in at least 90 germination Traditional
scarification treatments randomly degrade the entire seed coat which can lead to
tissue damage during water uptake Percussion scarification specifically weakens the
strophiole the natural source ofwater entry to the seed in papilionoid legwnes
INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom
2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031
48
Following percussion imbibition is controlled through the strophiole and underlying
tissue is protected
Keywords New Mexico locust black locust scarification percussion germination
Introduction
Locust trees (Robinia spp) are aggressive pioneer species that quickly
colonize disturbed land fix atmospheric nitrogen in the soil and stabilize slopes that
are prone to erosion (Klemmedson 1994 Ashby et al 1985) (Figure A1ab)
Recognition of these attributes has contributed to increased demand for locusts in
reclamation projects Locust seeds however exhibit physical dormancy where a
thick seed coat prevents the movement of water and gasses to the embryo (Leadem
1997)
Sulfuric acid or hot water soaks have traditionally been used to break seed
dormancy in macro-propagation of locusts Many growers are moving away from the
use of su1furic acid in treating locust seeds (Dreesen and Harrington 1997) In
addition to being dangerous acid soak durations must be specifically correlated to
seed lot (Olson 1974) Collections of native seed can vary tremendously in seed size
seed weight and hard seededness etc This variability limits the utility of sulfuric
acid soaks as even within a given lot some seeds may be damaged whereas others
are under-treated and fail to imbibe (take up water) A survey of Southwestern
container growers indicates that hot water baths are currently the most common
scarification method for locust seeds (Hine et al 1997) However hot water baths
also produce inconsistent germination (Lin et al 1996)
49
b
Figure AI New Mexico locust a primary invader following disturbance Ala Crown-sprouting at edge ofburn near Los Alamos NM--one year after Cerro Grande fire Alb Colonizing road cut Gila National Forest NM
50
Percussion scarification where seeds are repeatedly propelled against a hard
surface is an alternative dormancy-breaking method that has proven successful in
legumes related to locusts (Hamly 1932 Barton 1947) We compared this alternative
treatment to hot water scarification to evaluate treatment efficacy for New Mexico
locust and black locust seeds
Materials and Methods
Commercial New Mexico locust seeds (Western Native Seed Coaldale CO)
were collected fall 2000 in Huerfano County CO Black locust seeds were collected
September 2000 from Taos County NM by harvesting ripe pods from several trees at
a distance of up to 3 m from ground level Black locust seeds were allowed to air-dry
for 3 weeks and were threshed and separated from large chaffusing a greenhouse
fan A South Dakota Seed Blower (Chicago Illinltois) was used to remove fine chaff
from both seed lots Seeds were storedmiddotat 2-4C until the germination study was
conducted in March 2001
This study compared hot water scarification with percussion scarification
using treatment and species as experimental factors Along with a control
scarification treatments included immersion of seed in boiling water as well as
percussion durations of 1 2 3 45 or 10 minutes Four 100-seed samples were used
to test each species by scarification treatment combination
For the hot water treatment test tubes were filled with 30 mL water and
placed in a water-filled beaker (Figure A2a) The bath was raised to boiling (-98C at
51
b
c
Figure A2 Implementation ofhot water and percussion scarification A2a Hot water bath A2b Paint-shaker set-up A2c Placing soil tin containing seeds at end ofpaint shaker ann
52
Las Cruces NM elevation of 1300 meters) Each 100-seed replicate was placed in a
test tube and each test tube was immediately removed to cool at room temperature
Percussion scarification was implemented using a pneumatic paint shaker
(Central Pneumatic model 00422 Camarillo CA) (Figure A2b) This allowed
standardization of treatment intensity Compressed air pressure was maintained at
530kPa plusmn 30kPa (80psi plusmn 5psi) resulting in approximately 350 oscillations per
minute Each lOO-seed sample was placed in a 118 ml (4 oz) soil tin for shaking To
maximize lateral movement of paint shaker a paint can was used as a spacer to place
soil tins at the end of the shaking arm (Figure A2c) This allowed shaking to occur at
the greatest distance from the pivot point (fulcrum) Following all treatments
including the control seeds were soaked for twenty-four hours in distilled water prior
to plating out
Gennination was evaluated on lab benches at room temperature Each
replicate of seeds was placed in a 100 cm petri dish on moistened filter paper with
humidity maintained by enclosing the petri dishes in ziplock bags Gennination was
monitored daily for 14 days in accordance with International Seed Testing
Association standards for Robinia species (ISTA 1999) Gennination was defined as
protrusion of the radicle from the embryo by at least 1mm as viewed by the naked
eye
Categorical Analysis of Variance using Prpc Catmod (SAS Institute 1989)
was used to detennine treatment differences in germination percentages for each
source This procedure is an extension of a Chi-square test of homogeneity using the
53
natural log of the ratio of germinated to non-germinated seeds for each treatment Pshy
values less than 005 were considered significant A limited set of pairwiseshy
comparisons was conducted to compare treatment mean percents using a
conservative alpha value of 005 divided by the number of comparisons
Results
Percussion maximized germination for both New Mexico locust and black
locust There were three germination response levels for New Mexico locust (Figure
A3) First there was a low response in the control indicating a dormant seed lot
The hot water treatment and I-minute duration of percussion share a second
intermediate response Germination was highest at percussion levels from 2 to 10
minutes
A selected percussion treatment intermediate in time (5 minutes) produced the
steepest germination curve indicating the fastest germination rate (Figure A4) The
hot water treatment produced an intermediate curve or relatively slower germination
rate The control curve was very flat indicating the slowest germination rate for New
Mexico locust
Control seeds germinated poorly for black locust as well (Figure A5) Hot
water scarification improved germination over the control but all levels of percussion
improved germination over hot water The optimal durations of percussion treatment
were bracketed with a significant increase from the 1 to 2 minute percussion level a
high germination response from 2 to 5 minutes of percussion and finally a significant
54
~ d 25 CL)
p t1 0 ~ t1
g Vl ~ Vl
100~---------------------------------------------------------------
80
60
40
20
o Control Hot Water Percussion
I min Percussion
2 min Percussion
3 min
Scarification
Percussion 4 min
Figure A3 Effect of scarification on germination response for New Mexico locust
Percussion 5 min
=significant difference between adjacent treatment bars ns = no significant difference
Percussion 10 min
~ ~ () u agt
Il-t d o ~
~ ~ c3
100middotlr~~~------------------------------------e- Percussion 5min middot 0middot Hot Water -- Control
80-J=middot======
6b-middot-lmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middot--middotmiddot-middot~-middotmiddot -middotmiddot middot middotmiddot-middot 1
40
20
-----~-----------
0 0 0 middotmiddotmiddotmiddotmiddot0middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot0
0 --~-----y-----~------_---~--- T-----~
o shy -TDay 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A4 Effect of selected scarification treatments on germination speed for New Mexico locust First seven days of fourteen-day experiment are shown
Day 7
80
~ cd
5 60 ()
[) Cl d 0 1t1 d
40sect dUl
-J
20
o
ns
Scarification
Figure A5 Effect of scarification on germination response for black locust = significant difference between adjacent treatment bars ns = no significant difference
decrease at the 10-minute level This drop-off suggests that 10 minutes of percussion
over-treats seeds from this lot of-black locust
A selected percussion treatment intermediate in time (5 minutes) also had the
fastest germination rate for black locust (Figure A6)_ The hot water curve is
somewhat flatter indicating a slower germination rate The control curve is very flat
indicating a slow germination rate
A qualitative difference between hot water and percussion-treated seeds was
also observed Along with healthy germinants the hot water treatment produced
over-treated seeds (Figure A 7a) Examples of damaged germinants include seeds
where the radicle did not elongate (Figure A 7b) seeds where the radicle elongated
but was delayed in freeing itself from the seed coat (Figure A7c) and seeds where the
radicle broke off completely from the embryo (Figure A7d) In addition to these
examples of over-treatment there were numerous small dark seeds that failed to
imbibe water For percussion treatment nearly all seeds took up water (Figure A8a)
Germination was rapid and uniform with little damage evident (Figure A8b)
To summarize both New Mexico locust and black locust seed lots were
dormant The standard hot water scarification treatment improved germination but
only to 56 and 41 for New Mexico locust and black locust respectively Nearly
all percussion durations improved total germination as well as germination rate over
the hot water treatment Germination was greater than 90 for percussion durations
of 4 5 and 10 minutes for New Mexico locust and 3 4 and 5 minutes for black locust
58
CI)
sgt t CI) 0
igt 0
sect ttl t E
Vl 0
c3
100r==~==~~=r------------------------------------~ ___ Percussion 5 min
middotmiddotmiddot0middotmiddotmiddot Hot Water -lIf- Control
80r=========~--~~----------------------------~
60
40
20 -~-~---~---------- ---~------------I
omiddot
O
_~_----------lIf-----~bull -----_ shy---shy I
Day 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A6 Effect of selected scarification treatments on germination speed for black locust First seven days of fourteen-day experiment are shown
Day 7
a
b c d
Figure A7 Damage to New Mexico locust seeds observed during germination counts following hot water scarification A7a Hot water replicate including damaged seedlings and numerous unimbibed seeds A7b Close-up ofseed where radicle failed to elongate A7c Close-up ofseed where radicle elongated but was delayed in freeing itself from the seed coat A7d Close-up ofseed where radicle completely broke offfrom embryo
60
a
b
Figure A8 New Mexico locust seeds following percussion treatment at time of germination counts A8a Percussion replicate with uniformly healthy seedlings and near complete imbibition of seeds A8b Close-up ofpercussion-treated seedling
61
Discussion
Papilionoid seeds have a specific anatomical feature known as the strophiole
or lens-the natural site of water entry to the seed (Hamly 1932) The strophiole is
located on the cotyledonary lobe of a locust seed (Figure A9) When a papilionoid
legume seed is percussed for the appropriate amount of time repeated hits on the
integrated seed coat loosen the constrained cells of the strophiolar region without
excessively damaging the rest of the seed coat (Ballard 1976)
When a percussed seed is soaked water enters exclusively through the
strophiole in a controlled manner (Kelly and Staden 1987) This regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues This
contrasts with seeds that have been hot water or acid scarified treatments that can
randomly degrade the seed coat Unlocalized cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rehman et al 1998 Marunda 1990)
Papilionoids are the largest subfamily of legumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust and black locust these legumes often play an integral role in the
revegetation ofdisturbed lands Developing superior scarification methods to the
standard hot water and acid treatments should fadlitate the use ofthese legumes in
restoration projects
62
Figure A9 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (bc)
63
APPENDIXB
GERMINATION PERCENTS AND STANDARD ERRORS FOR SOURCE
BLOCK AND TREATMENT FOR NEW MEXICO LOCUST
Table B 1 Gennination percents and standard errors for source block and treatment for New Mexico locust
Source Block Treatment Germination Percentage Standard Error
Capitan 1 Immersion 74 437 Capitan 2 Immersion 69 462 Capitan 3 Immersion 71 454 Capitan 4 Immersion 67 470 Capitan Boiling 30 sec 79 407 Capitan 2 Boiling 30 sec 76 427 Capitan 3 Boiling 30 sec 72 449 Capitan 4 Boiling 30 sec 76 427 Capitan Boiling 60 sec 71 454 Capitan 2 Boiling 60 sec 54 498 Capitan 3 Boiling 60 sec 67 470 Capitan 4 Boiling 60 sec 76 427 Capitan I Control 5 218 Capitan 2 Control 7 255 Capitan 3 Control 4 196 Capitan 4 Control 5 218 Capitan 1 Percussion I min 47 499 Capitan 2 Percussion I min
470
Capitan Percussion I min 39 488
Capitan 4 Percussion I min 38 485 Capitan I Percussion 2 min 55 497 Capitan Capitan
2
Percussion 2 min Percussion 2 min
71 65
454 477
Capitan 4 Percussion 2 min 56 496 Capitan I Percussion 4 min 86 347 Capitan 2 Percussion 4 min 79 407 Capitan 3 Percussion 4 min 81 392 Capitan 4 Percussion 4 min 82 384 Capitan I Percussion 8 min 90 300 Capitan 2 Percussion 8 min 88 325 Capitan 3 Percussion 8 min 90 300 Capitan 4 Percussion 8 min 90 300 Gila I Immersion 71 454 Gila 2 Immersion 77 421 Gila
Immersion 75 433
Gila 4 Immersion 72 449
Gila I Boiling 30 sec 68 466
Gila 2 Boiling 30 sec 81 392
Gila 3 Boiling 30 sec 54 498
Gila 4 Boiling 30 sec 63 483
Gila I Boiling 60 sec 55 497
Gila 2 Boiling 60 sec 68 466
Gila 3 Boiling 60 sec 57 495
Gila 4 Boiling 60 sec 70 458
65
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Gila 2 Control 13 336 Gila 3 Control 13 336 Gila 4 Control 20 400 Gila 1 Percussion 1 min 66 474 Gila 2 Percussion 1 min 57 495 Gila 3 Percussion 1 min 73 444 Gila 4 Percussion 1 min 82 384 Gila 1 Percussion 2 min 81 392 Gila 2 Percussion 2 min 86 347 Gila 3 Percussion 2 min 85 357 Gila 4 Percussion 2 min 54 498 Gila 1 Percussion 4 min 79 407 Gila 2 Percussion 4 min 83 376 Gila 3 Percussion 4 min 84 367 Gila 4 Percussion 4 min 79 407 Gila 1 Percussion 8 min 51 500 Gila 2 Percussion 8 min 76 427 Gila 3 Percussion 8 min 52 500 Gila 4 Percussion 8 min 74 439 Holman 1 Immersion 83 376 Holman 2 Immersion 86 347 Holman 3 Immersion 81 392 Holman 4 Immersion 90 300 Holman 1 Boiling 30 sec 90 300 Holman 2 Boiling 30 sec 85 357 Holman 3 Boiling 30 sec 87 336 Holman 4 Boiling 30 sec 83 376 Holman 1 Boiling 60 sec 79 407 Holman 2 Boiling 60 sec 80 400 Holman 3 Boiling 60 sec 86 347 Holman 4 Boiling 60 sec 93 255 Holman Control 25 433 Holman 2 Control 19 392 Holman Control 21 407
Holman 4 Control 24 427
Holman Percussion 1 min 77 421
Holman 2 Percussion 1 min 85 357
Holman 3 Percussion 1 min 80 400
Holman 4 Percussion 1 min 89 313
Holman 1 Percussion 2 min 88 325
Holman 2 Percussion 2 min 96 196
Holman 3 Percussion 2 min 93 255
Holman 4 Percussion 2 min 96 196
Holman 1 Percussion 4 min 92 271
Holman 2 Percussion 4 min 97 171
66
I
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Holman 3 Percussion 4 min 91 286 Holman 4 Percussion 4 min 96 196 Holman 1 Percussion 8 min 88 325 Holman 2 Percussion 8 min 85 357 Holman 3 Percussion 8 min 83 376 Holman 4 Percussion 8 min 82 384 Jemez 1 Immersion 53 499 Jemez 2 Immersion 44 496 Jemez 3 Immersion 52 500 Jemez 4 Immersion 70 458 Jemez 1 Boiling 30 sec 58 494 Jemez 2 Boiling 30 sec 55 497 Jemez 3 Boiling 30 sec 48 500 Jemez 4 Boiling 30 sec 65 477 Jemez 1 Boiling 60 sec 68 466 Jemez 2 Boiling 60 sec 63 483 Jemez 3 Boiling 60 sec 73 444 Jemez 4 Boiling 60 sec 89 313 Jemez 1 Control 2 140 Jemez 2 Control 9 286 Jemez 3 Control 6 237 Jemez 4 Control 8 271 Jemez 1 Percussion 1 min 58 494 Jemez 2 Percussion 1 min 63 483 Jemez 3 Percussion 1 min 77 421 Jemez 4 Percussion 1 min 89 313 Jemez 1 Percussion 2 min 68 466 Jemez 2 Percussion 2 min 64 480 Jemez 3 Percussion 2 min 80 400 Jemez 4 Percussion 2 min 83 376 Jemez Percussion 4 min 68 466 Jemez 2 Percussion 4 min 79 407 Jemez 3 Percussion 4 min 70 458 Jemez 4 Percussion 4 min 83 376 Jemez 1 Percussion 8 min 52 500 Jemez 2 Percussion 8 min 59 492 Jemez 3 Percussion 8 min 61 488 Jemez 4 Percussion 8 min 70 458 Magdalena 1 Immersion 69 462 Magdalena 2 Immersion 69 462 Magdalena J Immersion 70 458 Magdalena 4 Immersion 85 357 Magdalena 1 Boiling 30 sec 65 477 Magdalena 2 Boiling 30 sec 71 454 Magdalena 3 Boiling 30 sec 68 466
67
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Magdalena 4 Boiling 30 sec 83 376 Magdalena 1 Boiling 60 sec 63 483 Magdalena 2 Boiling 60 sec 68 466 Magdalena 3 Boiling 60 sec 63 483 Magdalena 4 Boiling 60 sec 82 384 Magdalena 1 Control 11 313 Magdalena 2 Control 6 237 Magdalena 3 Control 5 218 Magdalena 4 Control 4 196 Magdalena Percussion 1 min 32 466 Magdalena 2 Percussion 1 min 45 497 Magdalena 3 Percussion 1 min 46 498 Magdalena 4 Percussion I min 36 480 Magdalena 1 Percussion 2 min 67 470 Magdalena 2 Percussion 2 min 68 466 Magdalena 3 Percussion 2 min 65 477 Magdalena 4 Percussion 2 min 81 392 Magdalena 1 Percussion 4 min 87 336 Magdalena 2 Percussion 4 min 97 171 Magdalena 3 Percussion 4 min 83 376 Magdalena 4 Percussion 4 min 97 171 Magdalena 1 Percussion 8 min 98 lAO Magdalena 2 Percussion 8 min 100 000 Magdalena 3 Percussion 8 min 96 196 Magdalena 4 Percussion 8 min 98 lAO Manzano 1 Immersion 67 470 Manzano 2 Immersion 57 495 Manzano 3 Immersion 72 4049 Manzano 4 Immersion 73 4044 Manzano 1 Boiling 30 sec 70 458 Manzano 2 Boiling 30 sec 64 480 Manzano 3 Boiling 30 sec 71 454 Manzano 4 Boiling 30 sec 80 400 Manzano I Boiling 60 sec 50 500 Manzano 2 Boiling 60 sec 53 499 Manzano 3 Boiling 60 sec 59 492 Manzano 4 Boiling 60 sec 74 439 Manzano I Control 4 196 Manzano 2 Control 8 271 Manzano 3 Control 1 099 Manzano 4 Control 6 237 Manzano 1 Percussion 1 min 52 500 Manzano 2 Percussion 1 min 39 488 Manzano 3 Percussion 1 min 36 480 Manzano 4 Percussion 1 min 51 500
68
Table B 1 Continued
Source Block Treatment Gerrni~ation Percentage Standard Error
Manzano Percussion 2 min 53 499 Manzano 2 Percussion 2 min 58 494 Manzano 3 Percussion 2 min 61 488 Manzano 4 Percussion 2 min 62 485 Manzano 1 Percussion 4 min 76 427 Manzano 2 Percussion 4 min 85 357 Manzano 3 Percussion 4 min 79 407 Manzano 4 Percussion 4 min 83 376 Manzano 1 Percussion 8 min 87 336 Manzano 2 Percussion 8 min 83 376 Manzano 3 Percussion 8 min 83 376 Manzano 4 Percussion 8 min 75 433 Raton 1 Immersion 54 498 Raton 2 Immersion 37 483 Raton 3 Immersion 39 488 Raton 4 Immersion 69 462 Raton 1 Boiling 30 sec 36 480 Raton 2 Boiling 30 sec 20 400 Raton 3 Boiling 30 sec 28 449 Raton 4 Boiling 30 sec 60 490 Raton 1 Boiling 60 sec 15 357 Raton 2 Boiling 60 sec 171 Raton 3 Boiling 60 sec 4 196 Raton 4 Boiling 60 sec 35 477 Raton 1 Control 4 196 Raton 2 Control 12 325 Raton 3 Control 2 140 Raton 4 Control 6 237 Raton 1 Percussion 1 min 52 500 Raton 2 Percussion I min 29 454 Raton 3 Percussion I min 35 477 Raton 4 Percussion 1 min 31 462 Raton 1 Percussion 2 min 55 497 Raton 2 Percussion 2 min 35 477 Raton 3 Percussion 2 min 33 470 Raton 4 Percussion 2 min 42 494 Raton 1 Percussion 4 min 65 477 Raton 2 Percussion 4 min 52 500 Raton Percussion 4 min 51 500 Raton 4 Percussion 4 min 53 499 Raton 1 Percussion 8 min 88 325 Raton 2 Percussion 8 min 80 400 Raton 3 Percussion 8 min 73 444 Raton 4 Percussion 8 min 74 439 Sacramento 1 Immersion 43 495
69
Table B 1 Continued
-Source Block Treatment Germination Percentage Standard Error
Sacramento 2 Immersion 30 458 Sacramento 3 Immersion 38 485 Sacramento 4 Immersion 56 496 Sacramento 1 Boiling 30 sec 47 499 Sacramento 2 Boiling 30 sec 48 500 Sacramento 3 Boiling 30 sec 48 500 Sacramento 4 Boiling 30 sec 56 496 Sacramento 1 Boiling 60 sec 52 500 Sacramento 2 Boiling 60 sec 67 470 Sacramento 3 Boiling 60 sec 43 495 Sacramento 4 Boiling 60 sec 48 500 Sacramento 1 Control 0 000 Sacramento 2 Control 3 171 Sacramento 3 Control 3 171 Sacramento 4 Control 099 Sacramento 1 Percussion 1 min 57 495 Sacramento 2 Percussion 1 min 61 488 Sacramento 3 Percussion 1 min 62 485 Sacramento 4 Percussion 1 min 55 497 Sacramento 1 Percussion 2 min 84 367 Sacramento 2 Percussion 2 min 70 458 Sacramento 3 Percussion 2 min 81 392 Sacramento 4 Percussion 2 min 69 462 Sacramento 1 Percussion 4 min 91 286 Sacramento 2 Percussion 4 min 84 367 Sacramento 3 Percussion 4 min 84 367 Sacramento 4 Percussion 4 min 94 237 Sacramento 1 Percussion 8 min 82 384 Sacramento 2 Percussion 8 min 81 392 Sacramento 3 Percussion 8 min 82 384 Sacramento 4 Percussion 8 min 81 392 Sandia 1 Immersion 76 427 Sandia 2 Immersion 70 458 Sandia 3 Immersion 70 458 Sandia 4 Immersion 81 392 Sandia 1 Boiling 30 sec 78 414 Sandia 2 Boiling 30 sec 71 454 Sandia 3 Boiling 30 sec 63 483 Sandia 4 Boiling 30 sec 78 414 Sandia 1 Boiling 60 sec 67 470 Sandia 2 Boiling 60 sec 65 477 Sandia 3 Boiling 60 sec 74 439 Sandia 4 Boiling 60 sec 66 474 Sandia 1 Control 16 367 Sandia 2 Control 7 255
70
Table B 1 Continued
Source Block Treatment Gennination Percentage Standard Error
Sandia 3 Control 8 271 Sandia 4 Control 16 367 Sandia 1 Percussion 1 min 67 470 Sandia 2 Percussion 1 min 61 488 Sandia 3 Percussion 1 min 66 474 Sandia 4 Percussion 1 min 59 492 Sandia I Percussion 2 min 82 384 Sandia 2 Percussion 2 min 83 376 Sandia 3 Percussion 2 min 81 392 Sandia 4 Percussion 2 min 84 367 Sandia 1 Percussion 4 min 89 313 Sandia 2 Percussion 4 min 86 347 Sandia 3 Percussion 4 min 89 313 Sandia 4 Percussion 4 min 85 357 Sandia 1 Percussion 8 min 80 400 Sandia 2 Percussion 8 min 77 421 Sandia 3 Percussion 8 min 69 462 Sandia 4 Percussion 8 min 66 474
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USDA Forest Service 1948 Woody-plant seed manual USDA Misc Pub1654 416p
Vines RA 1960 Trees Shrubs and Woody Vines of the Southwest Austin Univ ofTexas Press 1104 p
Wagner MR Larson RE Craig TP 1992 Ecology and silvics ofNew Mexico locust (Robinia neomexicana) with an emphasis on the arboreal form p53-57 In JW Hanover K Miller and S Plesko (eds) Proc Intemat Confon Black Locust Biology Culture and Utilization Mich State Univ USA
Wilson JK 1944 Immersing seeds of species of Robinia in boiling water hastens germination J For 42 453-454
Zimmerman RW Carpenter SB 1980 First-year coppice production from a 5shyyear old black locust stand on surface mine spoil p309-314 Centr Hardwd For ConfProc University ofMissouri Columbia
By limiting embryo damage percussion scarification reduces seedling
mortality compared to other treatments Kelly and van Staden (1987) noted that
seedlings from the percussion treatment were morphologically sound and vigorous
when compared to acid treatment seedlings The authors attribute this improved
seedling vigor to the controlled water uptake that limited anatomical damage
Despite a 12-hour percussion treatment morphological damage was not substantially
greater than untreated seeds In addition seedling mass measured 10 days following
germination was not significantly different between percussed and untreated seeds
A final advantage ofpercussion may be the ability to adequately treat the most
dormant seeds while avoiding harm to seeds that have already been made permeable
by the treatment or that were permeable to begin with Instead ofcorroding scalding
or scratching the seed coat percussion appears to be just a loosening of the
strophiole spring (Ballard 1973) Kelly and van Staden (1987) were able to
increase germination with a lengthy treatment (12 hours) Figuring out whether a
substantial cushion oftreatment durations exist for percussion ofNew Mexico locust
seed would be a significant improvement to current scarification methods where seed
damage and seedling health is an issue
The greatest challenge for applying percussion to New Mexico locust seeds is
standardizing a treatment that is both effective and readily reproducible by nurseries
with mechanical scarification this may require the development of specific
~UllDment to properly quantify and execute the treatment
14
f
Objectives
The first objective of the study is to detennine which scarification treatment
results in optimal gennination speed and percentage ofNew Mexico locust The
second objective is to describe treatment interactions with nine seed sources collected
in New Mexico
Hypothesis
A percussion treatment acts exclusively on the main area ofwater uptake in
New Mexico locust seed loosening the strophiole so that imbibition can occur This
direct mechanism ofdonnancy release will result in improved gennination rate and
overall gennination percentage for New Mexico locust seed compared to standard
treatments Standard treatments including acid hot water and mechanical
scarification while eventually weakening the strophiolar cleft cause excessive
morphological damage through generalized seed coat degradation These treatments
are therefore prone to inconsistency due to over and under-treatment
15
MATERIALS AND METHODS
Seed Collection and Cleaning
Seeds were collected from August 25 through September 15 2000 from nine
locations along a latitudinal gradient in New Mexico Seed source locations and
descriptive information are given in Figure 1 and Tables I and 2 Stands were
separated by at least 45 km
Pods were hand picked as they began to ripen and dehisce (open) Collections
were made from ground level to approximately 3 m from several trees at each site
Pods were dried in open-ended brown paper bags for two to four weeks at room
temperature (21-230C) to facilitate cleaning Dried pods were placed in a closed cloth
sack which was pounded against the ground to separate seeds from pods For pods
still containing seeds hand maceration (crunching pods with hands) completed seed
separation Debris was removed by threshing with the aid of a greenhouse fan A
South Dakota Seed Blower (Seedbureau Chicago Illinois) was used to remove
remaining chaff and light seeds by air density separation Seeds were placed in dry
storage in sealed quart-size zip-lock bags within a sealed plastic container Storage
temperatures ranged from 2-4oC Seeds remained in storage for approximately 7
months before the experiment
Experimental Design and Treatment Structure
The experiment compared the impact ofhot water bath and percussion
l11aLl11 on germination properties ofNew Mexico locust seed A single
16
-l
Figure 1 Seed source collection map All sources were collected within New Mexico USA separated by at least 4S lan
IIIRZltItW lIJPIirmiddot rllll1lbullbull_~~~_II
Table 1 Latitude longitude location elevation and collection date ofNew Mexico locust seed sources
Lot title Latitude (N) Longitude (W) Location Elevation (m) Collection Date
Raton 36deg5846 104deg2872 Raton Pass NM 2295 82500
Holman 36deg0306 105deg2429 HolmanNM 2476 911500
Jemez 35deg4500 106deg4100 Jemez Springs NM 1976 9900 00
Sandia 35deg1083 106deg2341 Cedar Crest NM 2432 82600
Manzano 34deg3689 106deg2494 ManzanoNM 2465 9200
Magdalena 33deg5953 107deg0903 Magdalena NM 2544 82900
Capitan 33deg2728 105deg4325 Alto NM 2189 911200
Sacramento 32deg5798 105deg4496 Cloudcroft NM 2630 91400 Gila 32deg5400 107deg4600 Kingston NM 2432 9700
f
Table 2 Mean seed weight and standard error by source
a
Source Mean seed weight (g) for Standard error 100 seeds n = 5
Raton Holman Jemez Sandia Manzano Magdalena Capitan Sacramento Gila
2872 b c 2522 d 1826 e 2753 c 2413 d 2559 d 2443 d 3130 a 3029 ab
00103 00403 00276 00301 00370 00374 00458 00279 00348
a Mean seed weights followed by the same letter are not significantly
different
19
experiment was conducted utilizing a randomized complete block design blocked by
time with a factorial treatment structure Experimental factors were seed source and
scarification treatment Nine seed sources from throughout New Mexico were used
(Figure 1 Table 1) Scarification treatments included a control three levels ofhot
water and four levels of percussion Hot water treatments included immersion at
100degC and boiling for periods of 30 seconds and 60 seconds Percussion treatment
levels were 1 2 4 and 8 minutes One hundred seeds per block were used to test
each treatment combination Four blocks of seed were tested one at a time (all seed
sources included in each run) in close chronological sequence (within 6 weeks) A
total of four 100-seed samples were used to test each source by scarification treatment
combination
Treatments
Hot water treatment of seeds utilized 50 ml test tubes filled with 30 ml
distilled water Each test tube was used to treat one 100-seed sample at a time
resulting in a ratio of4 parts water to 1 part seed by volume Test tubes were placed
in a beaker with the beaker filled with tap water to the same level as the water within
test tubes Nine test tubes (one per seed source) were placed in the beaker
simultaneously so that one replication of each seed sourcescarification treatment
cotnblnatlon was conducted simultaneously Hot water treatments include immersion
boiling For immersion treatments seeds were added to test tubes with the water
(boiling) and the test tubes were immediately removed from the beaker to
For the boiling treatments test tubes remained in the beaker with water at
20
boiling temperature for a set duration after the addition of seeds Following all
treatments test tubes with seeds cooled 24 hours in a test tube rack
Percussion treatment utilized a pneumatic paint shaker (Central Pneumatic
model 00422 Camarillo CA) Compressed air was maintained at 530 kPa plusmn30 kPa
(80 psi plusmn 5 psi) resulting in approximately 350 oscillations per minute Seeds were
placed in 118 ml (4 oz) soil tin cans (US Can Lobard IL) and the lids were secured
with duct tape Each can was used to treat one 100-seed sample To take advantage
ofgreatest lateral movement of the paint shaker a spacer (paint can) was used to
position the soil tin at the end of the shaking arm This allowed shaking to occur at
the greatest distance from the pivot point (fulcrum) Tins were placed perpendicular
to the direction of shaking motion allowing seeds to impact at greatest force by
hitting flat surfaces of the soil tin
Germination Testing
Before gennination testing all seeds were soaked 24 hours in reverse
osmosis-treated water (this was included in the process ofhot water treatment
jU~SCIlOea above) Following soaking seeds were placed in 100 cm Petri dishes lined
90 cm Whatman qualitative 1 filter paper circles (MG Scientific Pleasant
WI) One Petri dish was used for each treatment by source combination
papers were initially moistened with 5 mL reverse osmosis-treated water then
~VU1CUlJl moistened as needed to ensure adequate conditions for gennination
were monitored daily (at 300 PM) for gennination for 14 days in accordance
~UIUlnJlVUa Seed Testing Association (ISTA) standards for Robinia species
21
(1STA 1999) Tests were carried out at room temperature (22degC plusmnOSoC)
Germination was classified as protrusion of the radicle from the embryo by at least
1mm as viewed with the naked eye
X-ray Radiography
On August 132001 three lOO-seed samples from the Sacramento seed source
were treated for 14 or 8-minute percussion durations The seeds were sent
immediately by overnight delivery to the Faxitron X-Ray Corporation in Wheeling
Illinois where Dr Bob Karrfalt ofthe USDA Forest Service oversaw their imaging
Radiographs were made at SX with a Faxitron Specimen Radiography System Model
MX-20
Scanning Electron Microscope Photography
On February 192002 a Hitachi S-3200N Variable Pressure Scanning
Electron Microscope (Mountain View CA) was used to examine strophiolar cells of
untreated seeds and seeds percussed for 4 minutes Seeds were cut longitudinally
igt their entire length using a razor blade splitting the strophiolar region roughly in
Cross sections of seed samples magnifying the strophiolar region were viewed
600X under the microscope
Analysis
Categorical Analysis of Variance was used to determine treatment source and
(Le time) main effects and interactions The response variable was total
~vlI This procedure is a Chi-square test of homogeneity using the natural log
of germinated to non-germinated seed for each treatment Maximumshy
22
likelihood analysis was used to calculate Chi-square test statistics with their observed
significance levels A single contrast was conducted to cempare treatment means
against the control Observed significance levels less than or equal to 005 were
considered significant Pairwise comparisons were conducted between non-control
treatment mean percents using a conservative alp~a value of 005 divided by the
number of comparisons (Bonferroni approach) and approximate pairwise z statistics
Categorical analyses were performed using Proc Catmod (SAS Institute 1989)
RESULTS
Scarification seed source block and all interactions between factors
influenced germination (Table 3) The main effects are reported in the presence of
these interactions because the overall robustness of treatments and seed sources are of
interest Background levels ofnaturally occurring variation in seed sources are
seen in control germination percentages in Figure 2 In particular time variability
is unlikely to be under the control of the producer Appendix B details experimental
data for source by treatment gennination percents for each of the four blocks
Averaged over all treatments germination by source ranged from 40 to
79 (Figure 2) Averaged over all sources (Figure 3) and for each source (Figure 4
5) percussion scarification durations of4 or 8 minutes yielded greatest germination
Averaged over all sources germination increased with increasing durations of
percussion then leveled off for the 4- and 8-minute treatments (Figure 3) Immersion
and 30-second boiling scarification maximized germination for the hot water
treatments with a decrease in germination when seeds were boiled for 60 seconds
For all but two sources there is a similar ipcreasing trend in germination from
1to 4 minutes ofpercussion (Figure 4) The Holman source increased in germination
1 to 2 minutes then leveled off at 4 minutes The Jemez source changed little
1 to 4 minutes of treatment As treatment duration increased from 4 to 8
mutes five sources decreased in germination (Figure 4a) whereas three sources
24
Table 3 Categorical Analysis of Variance table for germination response to seed treatment seed source block and the interaction of the factors
Source of DF Chi-Square Observed Variability Significance Level
Source 8 98370 lt00001 Trt 7 233876 lt00001
contrast 1 16976 lt00001 SourceTrt 56 97077 lt00001 Block 3 5538 lt00001
SourceBlock 24 10162 lt00001 TrtBlock 21 7564 lt00001
SourceTrtBlock 168 34283 lt00001
S 4)
8 ~
0
sectg ~ 40 ~
N 0 0
20
o f0ltgt ~ ~4 jybshy ((iltgto ~~ K1ltgt 6~b-~ ~~ 0b-~~ ~~ ~b-~ ~b-~b
80
60 -I-- - ~- middotmiddotmiddotmiddotmiddot1
r -] Northern NM Sources
Central NM Sources
Southern NM Sources
sect-o ~
~b-v(i
SOURCE
Figure 2 Effect ofNew Mexico locust seed source on germination for data averaged over all scarification treatments Control germination is shown by solid line within bar
80
~ cd ~ ltl) () 60 ~ ~
N
t 0shyrti tshySltl)
40
0
20
o lmmersion Boiling 30 sec Boiling 60 sec Control 1 min 2min 4 min 8 min
Hot Water Treatments Percussion Treatments
Figure 3 Effect of scarification on germination ofNew Mexico locust for data averaged over all seed sources Means labelled with the same letter are not significantly different
100~----------------------~--~----------------~
80
60
ltU
~
40 IJ
sectgsI
l100
80
60
40
a I middotmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot middotImiddotmiddotmiddotmiddotmiddotmiddot E
_~iii-bull _ _ bull f --~--
t-- -~ ---- -shy ---- shyI ~ ---- - shy
L
fiII
~
b
- shy ---r
- Holman
Sacramento Sandia Gila Jemez
c ~
ltgt ~
Tmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot-I
j~=~=-~~-1
I middotmiddot ~ bull r
t tr
Y -------M-a-gd-a-Ie-n-a--
1 _ _
fCapitan
---- Manzano ~y -_ Raton
2 4 8
Time interval ofpercussion scarification (min)
Figure 4 Effect of seed source by treatment interaction on New Mexico locust germination for percussion scarification pattern a and pattern b
28
tv 0
~ d
~ A s o
1 c3
80
60
40
20
o
TIT
t------=middotfmiddotmiddot-middotmiddot-middotmiddot-middotmiddot 1- --- - - --=- - -shy--cE= - _--shy --shy ----shy ~ ~ ---shy ~ -~- _ --shy-------r 1
I
Raton Holman
-_ Jemez
--- Sandia Manzano Magdalena Capitan Sacramento Gila
Immersion Boiling 30 sec Boiling 60 sec
Hot Water Scarification
Figure 5 Effect of seed source by treatment interaction on New Mexico locust germination for hot water scarification
continued to increase in germination (Figure 4b) There was no difference between
the two treatment levels for the Manzano source (Figure 4b)
The effect ofhot water scarification also varied among sources with few
overall differences between hot water treatments (Figure 5) However boiling for 60
seconds yielded highest germination for the Jemez source whereas increasing
durations ofboiling decreased germination for the Raton source
In addition to consistently high germination 4- and 8-minute percussionshy
treated seeds germinated as fast or faster than all other treatments (Figure 6 Table 4)
050 measured as the number of days needed to complete 50 ofeventual
germination averaged 2 days for both ofthese treatments
X-ray radiographs were taken of seeds collected from the Sacramento source
percussed for 1 4 or 8 minutes (Figure 7) Seeds show signs of damage at 8 minutes
ofpercussion (Figure 8) Compared to an undamaged seed percussed for only 4
minutes (Figure 8a) damage at the 8-minute level includes fractured radicles pitting
of the seed coat and chips in seed coat structure (Figure 8b-d)
Scanning electron microscope (SEM) images at 600X reveal the direct effect
of 4 minutes of percussion on cells of the strophiolar region (Figure 9) Whereas
untreated seeds retain cellular organization in the strophiolar region strophiolar cells
seeds treated for 4 minutes ofpercussion separate and raise resulting in a
~-~e~U appearance
30
Umiddot-~middot--l-~---middotymiddotmiddotmiddot-----middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot--middot--middotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middotmiddotmiddotmiddotmiddotmiddotmiddot- -
050= gt1
-e-- Percussion 8 min -0- Percussion 4 min ---T- Percussion 2 min -----ltil- Percussion 1 min -- Immersion --0- Boiling 30 sec -+- Boiling 60 sec -ltgt- Control
2 4 6 8 10 12
Day Figure 6 Timecourse ofNew Mexico locust gennination following scarification
treatments G50 is the number of days to 50 of eventual germination
31
14
Table 4 Effect of seed source and treatment on G50 (days to 50 germination)
Seed Source Control Immersion B30sec B60sec Plmin P2min P4min P8min mean Raton 3 3 5 7 4 3 2 2 4 Holman 2 2 2 2 2 2 2 2 2
Jemez 4 6 6 5 3 2 2 2 4 Sandia 2 2 2 2 2 2 2 2 2
w tv Manzano 5 2 2 3 3 2 2 2 3
Magdalena 4 2 2 3 4 3 2 1 3
Capitan 5 4 2 2 3 2 2 2 3
Sacramento 3 2 2 2 4 3 2 2 3 Gila 3 2 2 2 2 2 2 2 2 mean 3 3 3 3 3 2 2 2 3 standard 11 14 16 18 09 05 0 03 08 deviation
Note Columns and rows may not agree due to rounding
Figure 7 X-ray radiographs ofSacramento seed source percussed for 1 4 or 8 minutes
33
Figure 8a-d Seed damage close-ups Figure a shows undamaged seed percussed for 4 minutes Figures b-d show seeds percussed for 8 minutes Damage includes fracturing ofradicle in b complete removal ofseed coat in c and pitting ofseed coat in d
34
Untreated Treated
Figure 9 SEM images ofuntreated vs 4-minute percussion-treated strophiolar cells ofNew Mexico locust seeds Treated cells are loosened separated and raised
DISCUSSION
Determining the Optimal Treatment
Averaging results of scarification treatmems across seed sources is a useful
tool to predict which treatment is most likely to be successful for an unknown seed
source Averaged over all sources four- and eight-minute percussion treatments
resulted in the highest germination 815 and 790 respectively and produced
germination that was as fast or faster than all other treatments Although there was no
difference between these two treatments on average an argument for 4 minutes of
percussion as a standard protocol for treating New Mexico locust seeds can be made
Descriptively 4 minutes of percussion improved germination over 8 minutes
ofpercussion for a majority of sources (five out of nine) 8 minutes of percussion
improved germination for three ofnine sources with no difference for a fmal source
For individual sources 4 minutes ofpercussion was always the best or secondmiddot best
treatment and was always equal to or better than all hot water treatments
Germination at 8 minutes ofpercussion was lower than all other percussion durations
for the Jemez and Gila sources and was lower than at least one hot water treatment for
these sources as well
The x-ray radiographs of the Sacramento seed source illustrate the excessive
internal and external seed damage that can occur with 8 minutes of percussion
germination counts 8-minute percussed seeds from Holman and Capitan
ClUll signs of damage that were visible to the naked eye (see below)
r~evertheless some of these seeds still fit the criteria for germination Neither x-ray
36
photography nor visible observations during seed counts revealed seed damage at the
4-minute percussion level In operational trials seeds percussed for 4 minutes
developed into healthy seedlings (Khadduri and Harrington unpublished data)
Strophiole and Seed Coat Behavior
Four minutes ofpercussion successfully breaks seed dormancy in a large
percentage ofNew Mexico locust seeds without excessively damaging the
surrounding seed coat or internal seed coat structures Most papilionoid legume
seeds including seeds ofNew Mexico locust have a specific region of the seed coat
known as the strophiole or lens (Hamly 1932) The strophiole is located on the
cotyledonary lobe of a locust seed (Figure 10 a-c) If seeds are not indiscriminately
damaged the strophiole is the first point of water entry to the embryo (Hopkinson
1997) When a papilionoid seed is percussed for the appropriate amount of time
repeated hits on the integrated seed coat loosen the constrained cells of the strophiolar
region without excessively damaging the rest of the seed coat (Ballard 1976) SEM
photos illuStrate that 4 minutes of percussion loosened and separated strophiolar cells
ofNew Mexico locust seeds when compared with constrained organized strophiolar
cells of untreated seeds (Figure 9) Research-has shown that water passes to the
embryo through the treated strophiole in a controlled manner and regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues
(Kelly and van Staden 1987)
37
10 Strophiole ofunimbibed New Mexico locust seed (a) and following uptake (bc)
38
In contrast hot-water treatments can excessively degrade the seed coat
(Rebman et al 1998 Marunda 1990) Random cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rebman et aI 1998 Marunda 1990) Along with a
proportionately large amount ofun-imbibed (thus under-treated) seeds (Figure l1a)
damage was observed in hot-water treated seeds from six sources during germination
counts Damage included seeds where the radicle failed to elongate (Figure 11 b)
seeds where the radicle elongated but was delayed in freeing itself from the seed coat
(Figure 11c) and seeds where the radicle completely broke off from the embryo
(Figure lId) In addition some seeds germinated in reverse (the cotyledon emerged
fIrst) If the radicle emerged completely from the seed coat they were counted as
germinated
While nearly all seeds treated for 8 minutes ofpercussion imbibed water
damage was evident in x-ray evaluations (Sacramento seed source) and in
germination counts (Holman and Capitan) at this treatment level Seed damage
during germination counts was similar to levels found in hot-water treated seeds
suggesting that over-treatment by both hot water and longer durations of percussion
may result in similar damage to seed structure
The Sacramento and Holman seed sources were two of the fIve sources that
opcrplltlpn in germination from the 4- to 8-minute durations of percussion
the benefIts ofloosening the strophiole as evidenced by imbibition of
all 8-minute treated seeds were outweighed by external and internal seed coat
39
a
b c d
11 Damage to New Mexico locust seeds observed during germination cotmts following hot water scarification 9a Hot water replicate including ~t seedlings and numerous unimbibed seeds 9b Close-up of seed where raallCle failed to elongate 9c Close-up ofseed where radicle elongated but was
-gtIn in freeing itself from the seed coat 9d Close-up ofseed where radicle broke off from embryo
40
damage (which also may be contributing to high imbibition) For the three sources
for which 8 minutes of percussion resulted in maximum germination extended
percussion of seeds necessary to loosen the strophiole may not have excessively
damaged the seed anatomy The Manzano source did not significantly change in
germination from 4 to 8 minutes of percussion suggesting that an increase in
permeable seeds at the 8-minute treatment duration was balanced by the number of
seeds that failed to germinate due to seed damage To summarize the 4-minute
percussion treatment improved germination over 8 minutes ofpercussion for a
majority of sources (five ofnine) the 8-minute percussion treatment improved
germination over 4-minutes ofpercussion for three sources and there was no
difference with the Manzano source Notably for the Raton source 8 minutes of
percussion increased germination by 235 percentage points over 4-minutes of
percussion (7875 vs 5525 respectively) the next best treatment Raton was the
only source to result in germination less than 75 for 4 minutes ofpercussion so a
grower may wish to apply longer percussion durations to lots that do not respond well
to this treatment level
Finally there was an interesting trend in the Jemez and Raton sources seed
that significantly contributed to the source by treatment interaction (Figure 12) A
~u test showed that the Jemez source had a significantly lighter seed weight than
other sources while the Raton source had an average seed weight For the Jemez
the longest duration of boiling increased germination Conversely only short
U4UVll) of percussion were needed to achieve high germination and the longest
41
80 _ Jemez a Raton
iD
~ C iD e iD
Q c 0 ~
c ~ iD 0
20
0------shy
Immersion Boiling 30 sec Boiling 60 sec
Hot Water Scarification
10degTr========~--------~------------------------------
80
60
40
20
_ Jemez Raton
0------shy
1 min 2 min 4 min 8 min
Percussion Scarification
Figure 12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources
42
b
--~----~~~~========~---- duration ofpercussion reduced germination For the Raton source longer durations
ofboiling consistently decreased germination whereas longer percussion treatments
consistently increased germination
A possible explanation is that a thick seed coat is associated with a loose
strophiole structure and that a thin seed coat is associated with a constrained
strophiole This contrast merits further study and may suggest an ecological
adaptation Further investigation ofthis phenomenon may contribute to successful
pre-screening ofunknown seed sources to determine optimal treatment leveL
43
CONCLUSION
Based on results obtained in pilot study (see Appendix) and thesis
experiments 4 minutes ofpercussion is recommended as a standard protocol for
treating an unknown source ofNew Mexico locust seed In addition to laboratory
results the 4-minute percussion treatment has resulted in germination greater than
80 and healthy seedling grow-outs for production purposes (unpublished data) In
laboratory experiments only one hundred seeds per 118 ml (40z) soil tin were
percussed at a time whereas for production we have treated up to five hundred seeds
per tin For large-scale production ofNew Mexico locust seedlings growers may
wish to experiment with larger quantities of seed and larger-size soil tins
Seed storage trials following percussion treatments were not conducted
However germination greater than 80 and healthy seedlings were obtained when
seeds were percussion-treated and subsequently stored at room-temperature (22degC)
for one week (unpublished data) Seed storage trials merit further study as the
standard hot water treatment leaves seeds in a softened condition that is not
conducive to storage
Hydroseeding is an effective method for direct seeding in reclamation
situations Percussion treatment may be conducive to hydro seeding as seeds remain
a dry relatively undamaged yet permeable condition following treatment Sulfuric
seeds also remain dry following treatment but extensive damage to seed coat
poor seedling vigor have been reported with this treatment (Kelly and van Staden
A grow-out study using a hydro seeder on a reclamation site testing percussion
44
against other treatments such as sulfuric acid and mechanical scarification also
merits further study
Papilionoids are the largest subfamily oflegumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust these legumes often play an integral role in the revegetation of
disturbed lands The pilot study (Appendix) indicates that percussion scarification
successfully treats the widely used black locust as well Developing superior
scarification methods to the standard hot water mechanical scarification and acid
treatments may facilitate the use ofpapilionoid legumes in reclamation projects
45
APPENDICES
APPENDIX A
PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR
NEW MEXICO LOCUST AND BLACK LOCUST SEEDS
Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4
Hot water and sulfuric acid soaks are traditional treatments for seeds of many
temperate woody legwnes including locusts However these scarification techniques
often produce inconsistent germination Percussion scarification where seeds are
repeatedly propelled against a hard surface was compared with hot water
scarification to evaluate treatment efficacy for New Mexico locust (Robinia
neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment
seeds were placed in a 98degC water bath which was immediately removed from the
heat source For percussion scarification seeds were placed in a soil sample tin and
agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the
control were followed by 24-hour water soaks Hot water baths resulted in 56 and
41 germination for New Mexico locust and black locust respectively For both
species nearly all durations ofpercussion increased germination over the hot water
treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3
4 and 5 minutes for black locust resulted in at least 90 germination Traditional
scarification treatments randomly degrade the entire seed coat which can lead to
tissue damage during water uptake Percussion scarification specifically weakens the
strophiole the natural source ofwater entry to the seed in papilionoid legwnes
INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom
2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031
48
Following percussion imbibition is controlled through the strophiole and underlying
tissue is protected
Keywords New Mexico locust black locust scarification percussion germination
Introduction
Locust trees (Robinia spp) are aggressive pioneer species that quickly
colonize disturbed land fix atmospheric nitrogen in the soil and stabilize slopes that
are prone to erosion (Klemmedson 1994 Ashby et al 1985) (Figure A1ab)
Recognition of these attributes has contributed to increased demand for locusts in
reclamation projects Locust seeds however exhibit physical dormancy where a
thick seed coat prevents the movement of water and gasses to the embryo (Leadem
1997)
Sulfuric acid or hot water soaks have traditionally been used to break seed
dormancy in macro-propagation of locusts Many growers are moving away from the
use of su1furic acid in treating locust seeds (Dreesen and Harrington 1997) In
addition to being dangerous acid soak durations must be specifically correlated to
seed lot (Olson 1974) Collections of native seed can vary tremendously in seed size
seed weight and hard seededness etc This variability limits the utility of sulfuric
acid soaks as even within a given lot some seeds may be damaged whereas others
are under-treated and fail to imbibe (take up water) A survey of Southwestern
container growers indicates that hot water baths are currently the most common
scarification method for locust seeds (Hine et al 1997) However hot water baths
also produce inconsistent germination (Lin et al 1996)
49
b
Figure AI New Mexico locust a primary invader following disturbance Ala Crown-sprouting at edge ofburn near Los Alamos NM--one year after Cerro Grande fire Alb Colonizing road cut Gila National Forest NM
50
Percussion scarification where seeds are repeatedly propelled against a hard
surface is an alternative dormancy-breaking method that has proven successful in
legumes related to locusts (Hamly 1932 Barton 1947) We compared this alternative
treatment to hot water scarification to evaluate treatment efficacy for New Mexico
locust and black locust seeds
Materials and Methods
Commercial New Mexico locust seeds (Western Native Seed Coaldale CO)
were collected fall 2000 in Huerfano County CO Black locust seeds were collected
September 2000 from Taos County NM by harvesting ripe pods from several trees at
a distance of up to 3 m from ground level Black locust seeds were allowed to air-dry
for 3 weeks and were threshed and separated from large chaffusing a greenhouse
fan A South Dakota Seed Blower (Chicago Illinltois) was used to remove fine chaff
from both seed lots Seeds were storedmiddotat 2-4C until the germination study was
conducted in March 2001
This study compared hot water scarification with percussion scarification
using treatment and species as experimental factors Along with a control
scarification treatments included immersion of seed in boiling water as well as
percussion durations of 1 2 3 45 or 10 minutes Four 100-seed samples were used
to test each species by scarification treatment combination
For the hot water treatment test tubes were filled with 30 mL water and
placed in a water-filled beaker (Figure A2a) The bath was raised to boiling (-98C at
51
b
c
Figure A2 Implementation ofhot water and percussion scarification A2a Hot water bath A2b Paint-shaker set-up A2c Placing soil tin containing seeds at end ofpaint shaker ann
52
Las Cruces NM elevation of 1300 meters) Each 100-seed replicate was placed in a
test tube and each test tube was immediately removed to cool at room temperature
Percussion scarification was implemented using a pneumatic paint shaker
(Central Pneumatic model 00422 Camarillo CA) (Figure A2b) This allowed
standardization of treatment intensity Compressed air pressure was maintained at
530kPa plusmn 30kPa (80psi plusmn 5psi) resulting in approximately 350 oscillations per
minute Each lOO-seed sample was placed in a 118 ml (4 oz) soil tin for shaking To
maximize lateral movement of paint shaker a paint can was used as a spacer to place
soil tins at the end of the shaking arm (Figure A2c) This allowed shaking to occur at
the greatest distance from the pivot point (fulcrum) Following all treatments
including the control seeds were soaked for twenty-four hours in distilled water prior
to plating out
Gennination was evaluated on lab benches at room temperature Each
replicate of seeds was placed in a 100 cm petri dish on moistened filter paper with
humidity maintained by enclosing the petri dishes in ziplock bags Gennination was
monitored daily for 14 days in accordance with International Seed Testing
Association standards for Robinia species (ISTA 1999) Gennination was defined as
protrusion of the radicle from the embryo by at least 1mm as viewed by the naked
eye
Categorical Analysis of Variance using Prpc Catmod (SAS Institute 1989)
was used to detennine treatment differences in germination percentages for each
source This procedure is an extension of a Chi-square test of homogeneity using the
53
natural log of the ratio of germinated to non-germinated seeds for each treatment Pshy
values less than 005 were considered significant A limited set of pairwiseshy
comparisons was conducted to compare treatment mean percents using a
conservative alpha value of 005 divided by the number of comparisons
Results
Percussion maximized germination for both New Mexico locust and black
locust There were three germination response levels for New Mexico locust (Figure
A3) First there was a low response in the control indicating a dormant seed lot
The hot water treatment and I-minute duration of percussion share a second
intermediate response Germination was highest at percussion levels from 2 to 10
minutes
A selected percussion treatment intermediate in time (5 minutes) produced the
steepest germination curve indicating the fastest germination rate (Figure A4) The
hot water treatment produced an intermediate curve or relatively slower germination
rate The control curve was very flat indicating the slowest germination rate for New
Mexico locust
Control seeds germinated poorly for black locust as well (Figure A5) Hot
water scarification improved germination over the control but all levels of percussion
improved germination over hot water The optimal durations of percussion treatment
were bracketed with a significant increase from the 1 to 2 minute percussion level a
high germination response from 2 to 5 minutes of percussion and finally a significant
54
~ d 25 CL)
p t1 0 ~ t1
g Vl ~ Vl
100~---------------------------------------------------------------
80
60
40
20
o Control Hot Water Percussion
I min Percussion
2 min Percussion
3 min
Scarification
Percussion 4 min
Figure A3 Effect of scarification on germination response for New Mexico locust
Percussion 5 min
=significant difference between adjacent treatment bars ns = no significant difference
Percussion 10 min
~ ~ () u agt
Il-t d o ~
~ ~ c3
100middotlr~~~------------------------------------e- Percussion 5min middot 0middot Hot Water -- Control
80-J=middot======
6b-middot-lmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middot--middotmiddot-middot~-middotmiddot -middotmiddot middot middotmiddot-middot 1
40
20
-----~-----------
0 0 0 middotmiddotmiddotmiddotmiddot0middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot0
0 --~-----y-----~------_---~--- T-----~
o shy -TDay 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A4 Effect of selected scarification treatments on germination speed for New Mexico locust First seven days of fourteen-day experiment are shown
Day 7
80
~ cd
5 60 ()
[) Cl d 0 1t1 d
40sect dUl
-J
20
o
ns
Scarification
Figure A5 Effect of scarification on germination response for black locust = significant difference between adjacent treatment bars ns = no significant difference
decrease at the 10-minute level This drop-off suggests that 10 minutes of percussion
over-treats seeds from this lot of-black locust
A selected percussion treatment intermediate in time (5 minutes) also had the
fastest germination rate for black locust (Figure A6)_ The hot water curve is
somewhat flatter indicating a slower germination rate The control curve is very flat
indicating a slow germination rate
A qualitative difference between hot water and percussion-treated seeds was
also observed Along with healthy germinants the hot water treatment produced
over-treated seeds (Figure A 7a) Examples of damaged germinants include seeds
where the radicle did not elongate (Figure A 7b) seeds where the radicle elongated
but was delayed in freeing itself from the seed coat (Figure A7c) and seeds where the
radicle broke off completely from the embryo (Figure A7d) In addition to these
examples of over-treatment there were numerous small dark seeds that failed to
imbibe water For percussion treatment nearly all seeds took up water (Figure A8a)
Germination was rapid and uniform with little damage evident (Figure A8b)
To summarize both New Mexico locust and black locust seed lots were
dormant The standard hot water scarification treatment improved germination but
only to 56 and 41 for New Mexico locust and black locust respectively Nearly
all percussion durations improved total germination as well as germination rate over
the hot water treatment Germination was greater than 90 for percussion durations
of 4 5 and 10 minutes for New Mexico locust and 3 4 and 5 minutes for black locust
58
CI)
sgt t CI) 0
igt 0
sect ttl t E
Vl 0
c3
100r==~==~~=r------------------------------------~ ___ Percussion 5 min
middotmiddotmiddot0middotmiddotmiddot Hot Water -lIf- Control
80r=========~--~~----------------------------~
60
40
20 -~-~---~---------- ---~------------I
omiddot
O
_~_----------lIf-----~bull -----_ shy---shy I
Day 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A6 Effect of selected scarification treatments on germination speed for black locust First seven days of fourteen-day experiment are shown
Day 7
a
b c d
Figure A7 Damage to New Mexico locust seeds observed during germination counts following hot water scarification A7a Hot water replicate including damaged seedlings and numerous unimbibed seeds A7b Close-up ofseed where radicle failed to elongate A7c Close-up ofseed where radicle elongated but was delayed in freeing itself from the seed coat A7d Close-up ofseed where radicle completely broke offfrom embryo
60
a
b
Figure A8 New Mexico locust seeds following percussion treatment at time of germination counts A8a Percussion replicate with uniformly healthy seedlings and near complete imbibition of seeds A8b Close-up ofpercussion-treated seedling
61
Discussion
Papilionoid seeds have a specific anatomical feature known as the strophiole
or lens-the natural site of water entry to the seed (Hamly 1932) The strophiole is
located on the cotyledonary lobe of a locust seed (Figure A9) When a papilionoid
legume seed is percussed for the appropriate amount of time repeated hits on the
integrated seed coat loosen the constrained cells of the strophiolar region without
excessively damaging the rest of the seed coat (Ballard 1976)
When a percussed seed is soaked water enters exclusively through the
strophiole in a controlled manner (Kelly and Staden 1987) This regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues This
contrasts with seeds that have been hot water or acid scarified treatments that can
randomly degrade the seed coat Unlocalized cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rehman et al 1998 Marunda 1990)
Papilionoids are the largest subfamily of legumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust and black locust these legumes often play an integral role in the
revegetation ofdisturbed lands Developing superior scarification methods to the
standard hot water and acid treatments should fadlitate the use ofthese legumes in
restoration projects
62
Figure A9 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (bc)
63
APPENDIXB
GERMINATION PERCENTS AND STANDARD ERRORS FOR SOURCE
BLOCK AND TREATMENT FOR NEW MEXICO LOCUST
Table B 1 Gennination percents and standard errors for source block and treatment for New Mexico locust
Source Block Treatment Germination Percentage Standard Error
Capitan 1 Immersion 74 437 Capitan 2 Immersion 69 462 Capitan 3 Immersion 71 454 Capitan 4 Immersion 67 470 Capitan Boiling 30 sec 79 407 Capitan 2 Boiling 30 sec 76 427 Capitan 3 Boiling 30 sec 72 449 Capitan 4 Boiling 30 sec 76 427 Capitan Boiling 60 sec 71 454 Capitan 2 Boiling 60 sec 54 498 Capitan 3 Boiling 60 sec 67 470 Capitan 4 Boiling 60 sec 76 427 Capitan I Control 5 218 Capitan 2 Control 7 255 Capitan 3 Control 4 196 Capitan 4 Control 5 218 Capitan 1 Percussion I min 47 499 Capitan 2 Percussion I min
470
Capitan Percussion I min 39 488
Capitan 4 Percussion I min 38 485 Capitan I Percussion 2 min 55 497 Capitan Capitan
2
Percussion 2 min Percussion 2 min
71 65
454 477
Capitan 4 Percussion 2 min 56 496 Capitan I Percussion 4 min 86 347 Capitan 2 Percussion 4 min 79 407 Capitan 3 Percussion 4 min 81 392 Capitan 4 Percussion 4 min 82 384 Capitan I Percussion 8 min 90 300 Capitan 2 Percussion 8 min 88 325 Capitan 3 Percussion 8 min 90 300 Capitan 4 Percussion 8 min 90 300 Gila I Immersion 71 454 Gila 2 Immersion 77 421 Gila
Immersion 75 433
Gila 4 Immersion 72 449
Gila I Boiling 30 sec 68 466
Gila 2 Boiling 30 sec 81 392
Gila 3 Boiling 30 sec 54 498
Gila 4 Boiling 30 sec 63 483
Gila I Boiling 60 sec 55 497
Gila 2 Boiling 60 sec 68 466
Gila 3 Boiling 60 sec 57 495
Gila 4 Boiling 60 sec 70 458
65
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Gila 2 Control 13 336 Gila 3 Control 13 336 Gila 4 Control 20 400 Gila 1 Percussion 1 min 66 474 Gila 2 Percussion 1 min 57 495 Gila 3 Percussion 1 min 73 444 Gila 4 Percussion 1 min 82 384 Gila 1 Percussion 2 min 81 392 Gila 2 Percussion 2 min 86 347 Gila 3 Percussion 2 min 85 357 Gila 4 Percussion 2 min 54 498 Gila 1 Percussion 4 min 79 407 Gila 2 Percussion 4 min 83 376 Gila 3 Percussion 4 min 84 367 Gila 4 Percussion 4 min 79 407 Gila 1 Percussion 8 min 51 500 Gila 2 Percussion 8 min 76 427 Gila 3 Percussion 8 min 52 500 Gila 4 Percussion 8 min 74 439 Holman 1 Immersion 83 376 Holman 2 Immersion 86 347 Holman 3 Immersion 81 392 Holman 4 Immersion 90 300 Holman 1 Boiling 30 sec 90 300 Holman 2 Boiling 30 sec 85 357 Holman 3 Boiling 30 sec 87 336 Holman 4 Boiling 30 sec 83 376 Holman 1 Boiling 60 sec 79 407 Holman 2 Boiling 60 sec 80 400 Holman 3 Boiling 60 sec 86 347 Holman 4 Boiling 60 sec 93 255 Holman Control 25 433 Holman 2 Control 19 392 Holman Control 21 407
Holman 4 Control 24 427
Holman Percussion 1 min 77 421
Holman 2 Percussion 1 min 85 357
Holman 3 Percussion 1 min 80 400
Holman 4 Percussion 1 min 89 313
Holman 1 Percussion 2 min 88 325
Holman 2 Percussion 2 min 96 196
Holman 3 Percussion 2 min 93 255
Holman 4 Percussion 2 min 96 196
Holman 1 Percussion 4 min 92 271
Holman 2 Percussion 4 min 97 171
66
I
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Holman 3 Percussion 4 min 91 286 Holman 4 Percussion 4 min 96 196 Holman 1 Percussion 8 min 88 325 Holman 2 Percussion 8 min 85 357 Holman 3 Percussion 8 min 83 376 Holman 4 Percussion 8 min 82 384 Jemez 1 Immersion 53 499 Jemez 2 Immersion 44 496 Jemez 3 Immersion 52 500 Jemez 4 Immersion 70 458 Jemez 1 Boiling 30 sec 58 494 Jemez 2 Boiling 30 sec 55 497 Jemez 3 Boiling 30 sec 48 500 Jemez 4 Boiling 30 sec 65 477 Jemez 1 Boiling 60 sec 68 466 Jemez 2 Boiling 60 sec 63 483 Jemez 3 Boiling 60 sec 73 444 Jemez 4 Boiling 60 sec 89 313 Jemez 1 Control 2 140 Jemez 2 Control 9 286 Jemez 3 Control 6 237 Jemez 4 Control 8 271 Jemez 1 Percussion 1 min 58 494 Jemez 2 Percussion 1 min 63 483 Jemez 3 Percussion 1 min 77 421 Jemez 4 Percussion 1 min 89 313 Jemez 1 Percussion 2 min 68 466 Jemez 2 Percussion 2 min 64 480 Jemez 3 Percussion 2 min 80 400 Jemez 4 Percussion 2 min 83 376 Jemez Percussion 4 min 68 466 Jemez 2 Percussion 4 min 79 407 Jemez 3 Percussion 4 min 70 458 Jemez 4 Percussion 4 min 83 376 Jemez 1 Percussion 8 min 52 500 Jemez 2 Percussion 8 min 59 492 Jemez 3 Percussion 8 min 61 488 Jemez 4 Percussion 8 min 70 458 Magdalena 1 Immersion 69 462 Magdalena 2 Immersion 69 462 Magdalena J Immersion 70 458 Magdalena 4 Immersion 85 357 Magdalena 1 Boiling 30 sec 65 477 Magdalena 2 Boiling 30 sec 71 454 Magdalena 3 Boiling 30 sec 68 466
67
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Magdalena 4 Boiling 30 sec 83 376 Magdalena 1 Boiling 60 sec 63 483 Magdalena 2 Boiling 60 sec 68 466 Magdalena 3 Boiling 60 sec 63 483 Magdalena 4 Boiling 60 sec 82 384 Magdalena 1 Control 11 313 Magdalena 2 Control 6 237 Magdalena 3 Control 5 218 Magdalena 4 Control 4 196 Magdalena Percussion 1 min 32 466 Magdalena 2 Percussion 1 min 45 497 Magdalena 3 Percussion 1 min 46 498 Magdalena 4 Percussion I min 36 480 Magdalena 1 Percussion 2 min 67 470 Magdalena 2 Percussion 2 min 68 466 Magdalena 3 Percussion 2 min 65 477 Magdalena 4 Percussion 2 min 81 392 Magdalena 1 Percussion 4 min 87 336 Magdalena 2 Percussion 4 min 97 171 Magdalena 3 Percussion 4 min 83 376 Magdalena 4 Percussion 4 min 97 171 Magdalena 1 Percussion 8 min 98 lAO Magdalena 2 Percussion 8 min 100 000 Magdalena 3 Percussion 8 min 96 196 Magdalena 4 Percussion 8 min 98 lAO Manzano 1 Immersion 67 470 Manzano 2 Immersion 57 495 Manzano 3 Immersion 72 4049 Manzano 4 Immersion 73 4044 Manzano 1 Boiling 30 sec 70 458 Manzano 2 Boiling 30 sec 64 480 Manzano 3 Boiling 30 sec 71 454 Manzano 4 Boiling 30 sec 80 400 Manzano I Boiling 60 sec 50 500 Manzano 2 Boiling 60 sec 53 499 Manzano 3 Boiling 60 sec 59 492 Manzano 4 Boiling 60 sec 74 439 Manzano I Control 4 196 Manzano 2 Control 8 271 Manzano 3 Control 1 099 Manzano 4 Control 6 237 Manzano 1 Percussion 1 min 52 500 Manzano 2 Percussion 1 min 39 488 Manzano 3 Percussion 1 min 36 480 Manzano 4 Percussion 1 min 51 500
68
Table B 1 Continued
Source Block Treatment Gerrni~ation Percentage Standard Error
Manzano Percussion 2 min 53 499 Manzano 2 Percussion 2 min 58 494 Manzano 3 Percussion 2 min 61 488 Manzano 4 Percussion 2 min 62 485 Manzano 1 Percussion 4 min 76 427 Manzano 2 Percussion 4 min 85 357 Manzano 3 Percussion 4 min 79 407 Manzano 4 Percussion 4 min 83 376 Manzano 1 Percussion 8 min 87 336 Manzano 2 Percussion 8 min 83 376 Manzano 3 Percussion 8 min 83 376 Manzano 4 Percussion 8 min 75 433 Raton 1 Immersion 54 498 Raton 2 Immersion 37 483 Raton 3 Immersion 39 488 Raton 4 Immersion 69 462 Raton 1 Boiling 30 sec 36 480 Raton 2 Boiling 30 sec 20 400 Raton 3 Boiling 30 sec 28 449 Raton 4 Boiling 30 sec 60 490 Raton 1 Boiling 60 sec 15 357 Raton 2 Boiling 60 sec 171 Raton 3 Boiling 60 sec 4 196 Raton 4 Boiling 60 sec 35 477 Raton 1 Control 4 196 Raton 2 Control 12 325 Raton 3 Control 2 140 Raton 4 Control 6 237 Raton 1 Percussion 1 min 52 500 Raton 2 Percussion I min 29 454 Raton 3 Percussion I min 35 477 Raton 4 Percussion 1 min 31 462 Raton 1 Percussion 2 min 55 497 Raton 2 Percussion 2 min 35 477 Raton 3 Percussion 2 min 33 470 Raton 4 Percussion 2 min 42 494 Raton 1 Percussion 4 min 65 477 Raton 2 Percussion 4 min 52 500 Raton Percussion 4 min 51 500 Raton 4 Percussion 4 min 53 499 Raton 1 Percussion 8 min 88 325 Raton 2 Percussion 8 min 80 400 Raton 3 Percussion 8 min 73 444 Raton 4 Percussion 8 min 74 439 Sacramento 1 Immersion 43 495
69
Table B 1 Continued
-Source Block Treatment Germination Percentage Standard Error
Sacramento 2 Immersion 30 458 Sacramento 3 Immersion 38 485 Sacramento 4 Immersion 56 496 Sacramento 1 Boiling 30 sec 47 499 Sacramento 2 Boiling 30 sec 48 500 Sacramento 3 Boiling 30 sec 48 500 Sacramento 4 Boiling 30 sec 56 496 Sacramento 1 Boiling 60 sec 52 500 Sacramento 2 Boiling 60 sec 67 470 Sacramento 3 Boiling 60 sec 43 495 Sacramento 4 Boiling 60 sec 48 500 Sacramento 1 Control 0 000 Sacramento 2 Control 3 171 Sacramento 3 Control 3 171 Sacramento 4 Control 099 Sacramento 1 Percussion 1 min 57 495 Sacramento 2 Percussion 1 min 61 488 Sacramento 3 Percussion 1 min 62 485 Sacramento 4 Percussion 1 min 55 497 Sacramento 1 Percussion 2 min 84 367 Sacramento 2 Percussion 2 min 70 458 Sacramento 3 Percussion 2 min 81 392 Sacramento 4 Percussion 2 min 69 462 Sacramento 1 Percussion 4 min 91 286 Sacramento 2 Percussion 4 min 84 367 Sacramento 3 Percussion 4 min 84 367 Sacramento 4 Percussion 4 min 94 237 Sacramento 1 Percussion 8 min 82 384 Sacramento 2 Percussion 8 min 81 392 Sacramento 3 Percussion 8 min 82 384 Sacramento 4 Percussion 8 min 81 392 Sandia 1 Immersion 76 427 Sandia 2 Immersion 70 458 Sandia 3 Immersion 70 458 Sandia 4 Immersion 81 392 Sandia 1 Boiling 30 sec 78 414 Sandia 2 Boiling 30 sec 71 454 Sandia 3 Boiling 30 sec 63 483 Sandia 4 Boiling 30 sec 78 414 Sandia 1 Boiling 60 sec 67 470 Sandia 2 Boiling 60 sec 65 477 Sandia 3 Boiling 60 sec 74 439 Sandia 4 Boiling 60 sec 66 474 Sandia 1 Control 16 367 Sandia 2 Control 7 255
70
Table B 1 Continued
Source Block Treatment Gennination Percentage Standard Error
Sandia 3 Control 8 271 Sandia 4 Control 16 367 Sandia 1 Percussion 1 min 67 470 Sandia 2 Percussion 1 min 61 488 Sandia 3 Percussion 1 min 66 474 Sandia 4 Percussion 1 min 59 492 Sandia I Percussion 2 min 82 384 Sandia 2 Percussion 2 min 83 376 Sandia 3 Percussion 2 min 81 392 Sandia 4 Percussion 2 min 84 367 Sandia 1 Percussion 4 min 89 313 Sandia 2 Percussion 4 min 86 347 Sandia 3 Percussion 4 min 89 313 Sandia 4 Percussion 4 min 85 357 Sandia 1 Percussion 8 min 80 400 Sandia 2 Percussion 8 min 77 421 Sandia 3 Percussion 8 min 69 462 Sandia 4 Percussion 8 min 66 474
LITERATURE CITED
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Ballard LAT 1973 Physical barriers to gennination Seed Sci Technoll 285-303
___ 1976 Strophiolar water conduction in the seeds ofTrifolieae induced by action on the testa at nonstrophiolar sites Austr J Plant Physiol3 465-469
Barton LV 1947 Special studies on seed coat impenneability Contrib Boyce Thompson Inst 14 355-362
Baskin CC Baskin JM 1998 Seeds Ecology biogeography and evolution of donnancy and gennination Academic Press London UK 666 p
Bewley JD 1997 Seed germination and donnancy Plant Ce119 1055-1066
Bewley JD Black M 1994 Seeds physiology of development and germination 2nd ed Plenum Press New York NY 445 p
Briscoe CB 1996 Growing Nitrogen Fixing Trees in Acid Soils In MH Powell (ed) Nitrogen Fixing Trees for Acid Soils- A Field ManuaL Winrock International Morrilton AR 22 p
Copeland LO McDonald MB 1995 Principles of Seed Science and Technology 3rded Chapman amp Hall New York NY 409 p
Cox RE Klett JE 1984 Seed gennination requirements of native Colorado plants for use in the landscape Plant Propag 30(2) 6-10
Dell B 1980 Structure and function ofthe strophiolar plug in seeds ofAlbizia Lophantha Am J Bot 67 556-563
Dick-Peddie WA 1993 New Mexico Vegetation Past Present and Future University ofNew Mexico Press Albuquerque NM 244 p
Dreesen DR 2001 Propagation protocol for production ofcontainer Robinia neomexicana plants Los Lunas Plant Materials Center Los Lunas New Mexico In Native Plant Network URL httpwwwnativeplantnetworkorg (accessed 9 January 2002) Moscow (ID) University ofIdaho CoIl Natural Resources For Res Nursery
72
Dreesen DR Harrington JT 1997 Propagation of native plants for restoration projects in the Southwestern US- Preliminary Investigations In Landis TD Thompson JR tech coords National Proceedings Forest and Conservation Nursery Assocs Gen Tech Rep PNW-GTR-419 Portland OR USDA For Ser PNW Res Sta 77-88
Gottfried GJ 1980 Control ofNew Mexican locust and the effect on planted ponderosa pine in central Arizona US For Ser Res Note RM-386 Rocky Mountain For Range ExpStn Fort Collins CO
Gosling PG Samuel YK Jones SK 1995 A systematic examination of germination temperature chipping and water temperaturesoak duration pretreatments on the seeds ofLeucaena leucocephala Seed Sci amp Technol 23521-532
Hamly DH 1932 Softening of the seeds of Melilotus alba Bot Gazette 93 345shy375
Hanna PJ 1984 Anatomical features of the seed coat ofAcacia kempeana (Mueller) which relate to increased germination rate induced by heat treatment New Phytol 96 23-29
Hanks JP Dick-Peddie WA 1974 Vegetation patterns of the White Mountains New Mexico Southwest Naturalist 18(4) 371-382
Hine S Harrington JT Dreesen DR 1997 Seed scarification requirements for Robinia neomexicana In Harrington JTtech coord Proc Third SouthwestemContainer Growers Conf Feb 12-13 1997 Ft Collins CO
Hopkinson JM 1993 The strophiole of leguminous seeds Newsletter- IntI Herbage Seed Production Res Grp 18 7-8
---- 1997 Differences in hardseededness between Cunningham and Tarramba leucaena In Leucnet News The Newsletter of the International LeucaenaRes and Devlt Netwk No4
ISTA (International Seed Testing Association) 1999 International rules for seed testing rules Seed Sci amp Technol27 Supplement
Kelly KM van Staden J 1987 The lens as the site of permeability in the papilionoid seed Aspalathus linearis J Plant Physiol 128(45) 395-404
73
Keresztesi B 1988 Black locust the tree of agriculture Outlook on Agr 17(2) 77~85
Kigel 1 and Galili G 1995 Seed development and germination Marcel Dekker New York 853 p
Klemmedson 10 1994 New Mexico locust and parent material Influence on macronutrients of forest floor and soil Soil Sci Soc Am 1 58 974~980
Kuo WH Tam AW 1988 The pathway of water absorption of mung bean seeds Seed Sci amp Technol16(1) 139~144
Leadem CL 1997 Dormancy--unlocking seed secrets In Landis TD Thompson lR tech coords National Proceedings Forestand Conservation Nursery Assocs Gen Tech Rep PNW-G TR-419 Portland OR USDA For Ser PNW Res Sta 43-52
Lin Y Wagner MR Seweyestewa A Honahni MD DeGomez M McMillin JD 1996 Influence of cultural treatments on seed germination and seedling growth of Robinia neomexicana Forest Farm and Community Tree Res Reports Volume 1
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Meginnis HG 1937 Sulphuric acid treatment to increase germination of black locust seed USDA Circ 453 34 p
Miklas PN Towensend CE Ladd SL 1987 Seedcoat anatomy and the scarification ofcicer milkvetch seed Crop Sci 27 766-772
Monsen SB 1984 Use of shrubs on mine spoils In The Challenge ofProducing Native Plants for the Intermountain Area Proceedings Intermountain Nurserymans Association 1983 conference Las Vegas NV compo PM Murphy 26-31 Ogden UT USDA For Ser Gen Tech Report INT-l68
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Rehman S Loescher RNl Harris P1C 1998 Dormancy breaking and germination ofAcacia salicina Lindl seeds Seed Sci amp Techno127 553-557
Rolston MP 1978 Water impermeable seed dormancy Bot Rev 44 365-396
74
SAS Institute Inc 1989 SASSTAT Users Guide Version 6 4th Edition Volume 1 Cary NC SAS Institute Inc 943 p
SerratoValenti G Conara L Ferrando M Modenesi P 1993 Structural and histochemical features ofStylosanthes scabra (Leguminosae Papilionoidae) seed coat as related to water entry Can J Bot 71 834-840
Simpson BJ 1988 A field guide to Texas trees Austin TX Texas Monthly Press 372p
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USDA Forest Service 1948 Woody-plant seed manual USDA Misc Pub1654 416p
Vines RA 1960 Trees Shrubs and Woody Vines of the Southwest Austin Univ ofTexas Press 1104 p
Wagner MR Larson RE Craig TP 1992 Ecology and silvics ofNew Mexico locust (Robinia neomexicana) with an emphasis on the arboreal form p53-57 In JW Hanover K Miller and S Plesko (eds) Proc Intemat Confon Black Locust Biology Culture and Utilization Mich State Univ USA
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Zimmerman RW Carpenter SB 1980 First-year coppice production from a 5shyyear old black locust stand on surface mine spoil p309-314 Centr Hardwd For ConfProc University ofMissouri Columbia
f
Objectives
The first objective of the study is to detennine which scarification treatment
results in optimal gennination speed and percentage ofNew Mexico locust The
second objective is to describe treatment interactions with nine seed sources collected
in New Mexico
Hypothesis
A percussion treatment acts exclusively on the main area ofwater uptake in
New Mexico locust seed loosening the strophiole so that imbibition can occur This
direct mechanism ofdonnancy release will result in improved gennination rate and
overall gennination percentage for New Mexico locust seed compared to standard
treatments Standard treatments including acid hot water and mechanical
scarification while eventually weakening the strophiolar cleft cause excessive
morphological damage through generalized seed coat degradation These treatments
are therefore prone to inconsistency due to over and under-treatment
15
MATERIALS AND METHODS
Seed Collection and Cleaning
Seeds were collected from August 25 through September 15 2000 from nine
locations along a latitudinal gradient in New Mexico Seed source locations and
descriptive information are given in Figure 1 and Tables I and 2 Stands were
separated by at least 45 km
Pods were hand picked as they began to ripen and dehisce (open) Collections
were made from ground level to approximately 3 m from several trees at each site
Pods were dried in open-ended brown paper bags for two to four weeks at room
temperature (21-230C) to facilitate cleaning Dried pods were placed in a closed cloth
sack which was pounded against the ground to separate seeds from pods For pods
still containing seeds hand maceration (crunching pods with hands) completed seed
separation Debris was removed by threshing with the aid of a greenhouse fan A
South Dakota Seed Blower (Seedbureau Chicago Illinois) was used to remove
remaining chaff and light seeds by air density separation Seeds were placed in dry
storage in sealed quart-size zip-lock bags within a sealed plastic container Storage
temperatures ranged from 2-4oC Seeds remained in storage for approximately 7
months before the experiment
Experimental Design and Treatment Structure
The experiment compared the impact ofhot water bath and percussion
l11aLl11 on germination properties ofNew Mexico locust seed A single
16
-l
Figure 1 Seed source collection map All sources were collected within New Mexico USA separated by at least 4S lan
IIIRZltItW lIJPIirmiddot rllll1lbullbull_~~~_II
Table 1 Latitude longitude location elevation and collection date ofNew Mexico locust seed sources
Lot title Latitude (N) Longitude (W) Location Elevation (m) Collection Date
Raton 36deg5846 104deg2872 Raton Pass NM 2295 82500
Holman 36deg0306 105deg2429 HolmanNM 2476 911500
Jemez 35deg4500 106deg4100 Jemez Springs NM 1976 9900 00
Sandia 35deg1083 106deg2341 Cedar Crest NM 2432 82600
Manzano 34deg3689 106deg2494 ManzanoNM 2465 9200
Magdalena 33deg5953 107deg0903 Magdalena NM 2544 82900
Capitan 33deg2728 105deg4325 Alto NM 2189 911200
Sacramento 32deg5798 105deg4496 Cloudcroft NM 2630 91400 Gila 32deg5400 107deg4600 Kingston NM 2432 9700
f
Table 2 Mean seed weight and standard error by source
a
Source Mean seed weight (g) for Standard error 100 seeds n = 5
Raton Holman Jemez Sandia Manzano Magdalena Capitan Sacramento Gila
2872 b c 2522 d 1826 e 2753 c 2413 d 2559 d 2443 d 3130 a 3029 ab
00103 00403 00276 00301 00370 00374 00458 00279 00348
a Mean seed weights followed by the same letter are not significantly
different
19
experiment was conducted utilizing a randomized complete block design blocked by
time with a factorial treatment structure Experimental factors were seed source and
scarification treatment Nine seed sources from throughout New Mexico were used
(Figure 1 Table 1) Scarification treatments included a control three levels ofhot
water and four levels of percussion Hot water treatments included immersion at
100degC and boiling for periods of 30 seconds and 60 seconds Percussion treatment
levels were 1 2 4 and 8 minutes One hundred seeds per block were used to test
each treatment combination Four blocks of seed were tested one at a time (all seed
sources included in each run) in close chronological sequence (within 6 weeks) A
total of four 100-seed samples were used to test each source by scarification treatment
combination
Treatments
Hot water treatment of seeds utilized 50 ml test tubes filled with 30 ml
distilled water Each test tube was used to treat one 100-seed sample at a time
resulting in a ratio of4 parts water to 1 part seed by volume Test tubes were placed
in a beaker with the beaker filled with tap water to the same level as the water within
test tubes Nine test tubes (one per seed source) were placed in the beaker
simultaneously so that one replication of each seed sourcescarification treatment
cotnblnatlon was conducted simultaneously Hot water treatments include immersion
boiling For immersion treatments seeds were added to test tubes with the water
(boiling) and the test tubes were immediately removed from the beaker to
For the boiling treatments test tubes remained in the beaker with water at
20
boiling temperature for a set duration after the addition of seeds Following all
treatments test tubes with seeds cooled 24 hours in a test tube rack
Percussion treatment utilized a pneumatic paint shaker (Central Pneumatic
model 00422 Camarillo CA) Compressed air was maintained at 530 kPa plusmn30 kPa
(80 psi plusmn 5 psi) resulting in approximately 350 oscillations per minute Seeds were
placed in 118 ml (4 oz) soil tin cans (US Can Lobard IL) and the lids were secured
with duct tape Each can was used to treat one 100-seed sample To take advantage
ofgreatest lateral movement of the paint shaker a spacer (paint can) was used to
position the soil tin at the end of the shaking arm This allowed shaking to occur at
the greatest distance from the pivot point (fulcrum) Tins were placed perpendicular
to the direction of shaking motion allowing seeds to impact at greatest force by
hitting flat surfaces of the soil tin
Germination Testing
Before gennination testing all seeds were soaked 24 hours in reverse
osmosis-treated water (this was included in the process ofhot water treatment
jU~SCIlOea above) Following soaking seeds were placed in 100 cm Petri dishes lined
90 cm Whatman qualitative 1 filter paper circles (MG Scientific Pleasant
WI) One Petri dish was used for each treatment by source combination
papers were initially moistened with 5 mL reverse osmosis-treated water then
~VU1CUlJl moistened as needed to ensure adequate conditions for gennination
were monitored daily (at 300 PM) for gennination for 14 days in accordance
~UIUlnJlVUa Seed Testing Association (ISTA) standards for Robinia species
21
(1STA 1999) Tests were carried out at room temperature (22degC plusmnOSoC)
Germination was classified as protrusion of the radicle from the embryo by at least
1mm as viewed with the naked eye
X-ray Radiography
On August 132001 three lOO-seed samples from the Sacramento seed source
were treated for 14 or 8-minute percussion durations The seeds were sent
immediately by overnight delivery to the Faxitron X-Ray Corporation in Wheeling
Illinois where Dr Bob Karrfalt ofthe USDA Forest Service oversaw their imaging
Radiographs were made at SX with a Faxitron Specimen Radiography System Model
MX-20
Scanning Electron Microscope Photography
On February 192002 a Hitachi S-3200N Variable Pressure Scanning
Electron Microscope (Mountain View CA) was used to examine strophiolar cells of
untreated seeds and seeds percussed for 4 minutes Seeds were cut longitudinally
igt their entire length using a razor blade splitting the strophiolar region roughly in
Cross sections of seed samples magnifying the strophiolar region were viewed
600X under the microscope
Analysis
Categorical Analysis of Variance was used to determine treatment source and
(Le time) main effects and interactions The response variable was total
~vlI This procedure is a Chi-square test of homogeneity using the natural log
of germinated to non-germinated seed for each treatment Maximumshy
22
likelihood analysis was used to calculate Chi-square test statistics with their observed
significance levels A single contrast was conducted to cempare treatment means
against the control Observed significance levels less than or equal to 005 were
considered significant Pairwise comparisons were conducted between non-control
treatment mean percents using a conservative alp~a value of 005 divided by the
number of comparisons (Bonferroni approach) and approximate pairwise z statistics
Categorical analyses were performed using Proc Catmod (SAS Institute 1989)
RESULTS
Scarification seed source block and all interactions between factors
influenced germination (Table 3) The main effects are reported in the presence of
these interactions because the overall robustness of treatments and seed sources are of
interest Background levels ofnaturally occurring variation in seed sources are
seen in control germination percentages in Figure 2 In particular time variability
is unlikely to be under the control of the producer Appendix B details experimental
data for source by treatment gennination percents for each of the four blocks
Averaged over all treatments germination by source ranged from 40 to
79 (Figure 2) Averaged over all sources (Figure 3) and for each source (Figure 4
5) percussion scarification durations of4 or 8 minutes yielded greatest germination
Averaged over all sources germination increased with increasing durations of
percussion then leveled off for the 4- and 8-minute treatments (Figure 3) Immersion
and 30-second boiling scarification maximized germination for the hot water
treatments with a decrease in germination when seeds were boiled for 60 seconds
For all but two sources there is a similar ipcreasing trend in germination from
1to 4 minutes ofpercussion (Figure 4) The Holman source increased in germination
1 to 2 minutes then leveled off at 4 minutes The Jemez source changed little
1 to 4 minutes of treatment As treatment duration increased from 4 to 8
mutes five sources decreased in germination (Figure 4a) whereas three sources
24
Table 3 Categorical Analysis of Variance table for germination response to seed treatment seed source block and the interaction of the factors
Source of DF Chi-Square Observed Variability Significance Level
Source 8 98370 lt00001 Trt 7 233876 lt00001
contrast 1 16976 lt00001 SourceTrt 56 97077 lt00001 Block 3 5538 lt00001
SourceBlock 24 10162 lt00001 TrtBlock 21 7564 lt00001
SourceTrtBlock 168 34283 lt00001
S 4)
8 ~
0
sectg ~ 40 ~
N 0 0
20
o f0ltgt ~ ~4 jybshy ((iltgto ~~ K1ltgt 6~b-~ ~~ 0b-~~ ~~ ~b-~ ~b-~b
80
60 -I-- - ~- middotmiddotmiddotmiddotmiddot1
r -] Northern NM Sources
Central NM Sources
Southern NM Sources
sect-o ~
~b-v(i
SOURCE
Figure 2 Effect ofNew Mexico locust seed source on germination for data averaged over all scarification treatments Control germination is shown by solid line within bar
80
~ cd ~ ltl) () 60 ~ ~
N
t 0shyrti tshySltl)
40
0
20
o lmmersion Boiling 30 sec Boiling 60 sec Control 1 min 2min 4 min 8 min
Hot Water Treatments Percussion Treatments
Figure 3 Effect of scarification on germination ofNew Mexico locust for data averaged over all seed sources Means labelled with the same letter are not significantly different
100~----------------------~--~----------------~
80
60
ltU
~
40 IJ
sectgsI
l100
80
60
40
a I middotmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot middotImiddotmiddotmiddotmiddotmiddotmiddot E
_~iii-bull _ _ bull f --~--
t-- -~ ---- -shy ---- shyI ~ ---- - shy
L
fiII
~
b
- shy ---r
- Holman
Sacramento Sandia Gila Jemez
c ~
ltgt ~
Tmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot-I
j~=~=-~~-1
I middotmiddot ~ bull r
t tr
Y -------M-a-gd-a-Ie-n-a--
1 _ _
fCapitan
---- Manzano ~y -_ Raton
2 4 8
Time interval ofpercussion scarification (min)
Figure 4 Effect of seed source by treatment interaction on New Mexico locust germination for percussion scarification pattern a and pattern b
28
tv 0
~ d
~ A s o
1 c3
80
60
40
20
o
TIT
t------=middotfmiddotmiddot-middotmiddot-middotmiddot-middotmiddot 1- --- - - --=- - -shy--cE= - _--shy --shy ----shy ~ ~ ---shy ~ -~- _ --shy-------r 1
I
Raton Holman
-_ Jemez
--- Sandia Manzano Magdalena Capitan Sacramento Gila
Immersion Boiling 30 sec Boiling 60 sec
Hot Water Scarification
Figure 5 Effect of seed source by treatment interaction on New Mexico locust germination for hot water scarification
continued to increase in germination (Figure 4b) There was no difference between
the two treatment levels for the Manzano source (Figure 4b)
The effect ofhot water scarification also varied among sources with few
overall differences between hot water treatments (Figure 5) However boiling for 60
seconds yielded highest germination for the Jemez source whereas increasing
durations ofboiling decreased germination for the Raton source
In addition to consistently high germination 4- and 8-minute percussionshy
treated seeds germinated as fast or faster than all other treatments (Figure 6 Table 4)
050 measured as the number of days needed to complete 50 ofeventual
germination averaged 2 days for both ofthese treatments
X-ray radiographs were taken of seeds collected from the Sacramento source
percussed for 1 4 or 8 minutes (Figure 7) Seeds show signs of damage at 8 minutes
ofpercussion (Figure 8) Compared to an undamaged seed percussed for only 4
minutes (Figure 8a) damage at the 8-minute level includes fractured radicles pitting
of the seed coat and chips in seed coat structure (Figure 8b-d)
Scanning electron microscope (SEM) images at 600X reveal the direct effect
of 4 minutes of percussion on cells of the strophiolar region (Figure 9) Whereas
untreated seeds retain cellular organization in the strophiolar region strophiolar cells
seeds treated for 4 minutes ofpercussion separate and raise resulting in a
~-~e~U appearance
30
Umiddot-~middot--l-~---middotymiddotmiddotmiddot-----middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot--middot--middotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middotmiddotmiddotmiddotmiddotmiddotmiddot- -
050= gt1
-e-- Percussion 8 min -0- Percussion 4 min ---T- Percussion 2 min -----ltil- Percussion 1 min -- Immersion --0- Boiling 30 sec -+- Boiling 60 sec -ltgt- Control
2 4 6 8 10 12
Day Figure 6 Timecourse ofNew Mexico locust gennination following scarification
treatments G50 is the number of days to 50 of eventual germination
31
14
Table 4 Effect of seed source and treatment on G50 (days to 50 germination)
Seed Source Control Immersion B30sec B60sec Plmin P2min P4min P8min mean Raton 3 3 5 7 4 3 2 2 4 Holman 2 2 2 2 2 2 2 2 2
Jemez 4 6 6 5 3 2 2 2 4 Sandia 2 2 2 2 2 2 2 2 2
w tv Manzano 5 2 2 3 3 2 2 2 3
Magdalena 4 2 2 3 4 3 2 1 3
Capitan 5 4 2 2 3 2 2 2 3
Sacramento 3 2 2 2 4 3 2 2 3 Gila 3 2 2 2 2 2 2 2 2 mean 3 3 3 3 3 2 2 2 3 standard 11 14 16 18 09 05 0 03 08 deviation
Note Columns and rows may not agree due to rounding
Figure 7 X-ray radiographs ofSacramento seed source percussed for 1 4 or 8 minutes
33
Figure 8a-d Seed damage close-ups Figure a shows undamaged seed percussed for 4 minutes Figures b-d show seeds percussed for 8 minutes Damage includes fracturing ofradicle in b complete removal ofseed coat in c and pitting ofseed coat in d
34
Untreated Treated
Figure 9 SEM images ofuntreated vs 4-minute percussion-treated strophiolar cells ofNew Mexico locust seeds Treated cells are loosened separated and raised
DISCUSSION
Determining the Optimal Treatment
Averaging results of scarification treatmems across seed sources is a useful
tool to predict which treatment is most likely to be successful for an unknown seed
source Averaged over all sources four- and eight-minute percussion treatments
resulted in the highest germination 815 and 790 respectively and produced
germination that was as fast or faster than all other treatments Although there was no
difference between these two treatments on average an argument for 4 minutes of
percussion as a standard protocol for treating New Mexico locust seeds can be made
Descriptively 4 minutes of percussion improved germination over 8 minutes
ofpercussion for a majority of sources (five out of nine) 8 minutes of percussion
improved germination for three ofnine sources with no difference for a fmal source
For individual sources 4 minutes ofpercussion was always the best or secondmiddot best
treatment and was always equal to or better than all hot water treatments
Germination at 8 minutes ofpercussion was lower than all other percussion durations
for the Jemez and Gila sources and was lower than at least one hot water treatment for
these sources as well
The x-ray radiographs of the Sacramento seed source illustrate the excessive
internal and external seed damage that can occur with 8 minutes of percussion
germination counts 8-minute percussed seeds from Holman and Capitan
ClUll signs of damage that were visible to the naked eye (see below)
r~evertheless some of these seeds still fit the criteria for germination Neither x-ray
36
photography nor visible observations during seed counts revealed seed damage at the
4-minute percussion level In operational trials seeds percussed for 4 minutes
developed into healthy seedlings (Khadduri and Harrington unpublished data)
Strophiole and Seed Coat Behavior
Four minutes ofpercussion successfully breaks seed dormancy in a large
percentage ofNew Mexico locust seeds without excessively damaging the
surrounding seed coat or internal seed coat structures Most papilionoid legume
seeds including seeds ofNew Mexico locust have a specific region of the seed coat
known as the strophiole or lens (Hamly 1932) The strophiole is located on the
cotyledonary lobe of a locust seed (Figure 10 a-c) If seeds are not indiscriminately
damaged the strophiole is the first point of water entry to the embryo (Hopkinson
1997) When a papilionoid seed is percussed for the appropriate amount of time
repeated hits on the integrated seed coat loosen the constrained cells of the strophiolar
region without excessively damaging the rest of the seed coat (Ballard 1976) SEM
photos illuStrate that 4 minutes of percussion loosened and separated strophiolar cells
ofNew Mexico locust seeds when compared with constrained organized strophiolar
cells of untreated seeds (Figure 9) Research-has shown that water passes to the
embryo through the treated strophiole in a controlled manner and regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues
(Kelly and van Staden 1987)
37
10 Strophiole ofunimbibed New Mexico locust seed (a) and following uptake (bc)
38
In contrast hot-water treatments can excessively degrade the seed coat
(Rebman et al 1998 Marunda 1990) Random cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rebman et aI 1998 Marunda 1990) Along with a
proportionately large amount ofun-imbibed (thus under-treated) seeds (Figure l1a)
damage was observed in hot-water treated seeds from six sources during germination
counts Damage included seeds where the radicle failed to elongate (Figure 11 b)
seeds where the radicle elongated but was delayed in freeing itself from the seed coat
(Figure 11c) and seeds where the radicle completely broke off from the embryo
(Figure lId) In addition some seeds germinated in reverse (the cotyledon emerged
fIrst) If the radicle emerged completely from the seed coat they were counted as
germinated
While nearly all seeds treated for 8 minutes ofpercussion imbibed water
damage was evident in x-ray evaluations (Sacramento seed source) and in
germination counts (Holman and Capitan) at this treatment level Seed damage
during germination counts was similar to levels found in hot-water treated seeds
suggesting that over-treatment by both hot water and longer durations of percussion
may result in similar damage to seed structure
The Sacramento and Holman seed sources were two of the fIve sources that
opcrplltlpn in germination from the 4- to 8-minute durations of percussion
the benefIts ofloosening the strophiole as evidenced by imbibition of
all 8-minute treated seeds were outweighed by external and internal seed coat
39
a
b c d
11 Damage to New Mexico locust seeds observed during germination cotmts following hot water scarification 9a Hot water replicate including ~t seedlings and numerous unimbibed seeds 9b Close-up of seed where raallCle failed to elongate 9c Close-up ofseed where radicle elongated but was
-gtIn in freeing itself from the seed coat 9d Close-up ofseed where radicle broke off from embryo
40
damage (which also may be contributing to high imbibition) For the three sources
for which 8 minutes of percussion resulted in maximum germination extended
percussion of seeds necessary to loosen the strophiole may not have excessively
damaged the seed anatomy The Manzano source did not significantly change in
germination from 4 to 8 minutes of percussion suggesting that an increase in
permeable seeds at the 8-minute treatment duration was balanced by the number of
seeds that failed to germinate due to seed damage To summarize the 4-minute
percussion treatment improved germination over 8 minutes ofpercussion for a
majority of sources (five ofnine) the 8-minute percussion treatment improved
germination over 4-minutes ofpercussion for three sources and there was no
difference with the Manzano source Notably for the Raton source 8 minutes of
percussion increased germination by 235 percentage points over 4-minutes of
percussion (7875 vs 5525 respectively) the next best treatment Raton was the
only source to result in germination less than 75 for 4 minutes ofpercussion so a
grower may wish to apply longer percussion durations to lots that do not respond well
to this treatment level
Finally there was an interesting trend in the Jemez and Raton sources seed
that significantly contributed to the source by treatment interaction (Figure 12) A
~u test showed that the Jemez source had a significantly lighter seed weight than
other sources while the Raton source had an average seed weight For the Jemez
the longest duration of boiling increased germination Conversely only short
U4UVll) of percussion were needed to achieve high germination and the longest
41
80 _ Jemez a Raton
iD
~ C iD e iD
Q c 0 ~
c ~ iD 0
20
0------shy
Immersion Boiling 30 sec Boiling 60 sec
Hot Water Scarification
10degTr========~--------~------------------------------
80
60
40
20
_ Jemez Raton
0------shy
1 min 2 min 4 min 8 min
Percussion Scarification
Figure 12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources
42
b
--~----~~~~========~---- duration ofpercussion reduced germination For the Raton source longer durations
ofboiling consistently decreased germination whereas longer percussion treatments
consistently increased germination
A possible explanation is that a thick seed coat is associated with a loose
strophiole structure and that a thin seed coat is associated with a constrained
strophiole This contrast merits further study and may suggest an ecological
adaptation Further investigation ofthis phenomenon may contribute to successful
pre-screening ofunknown seed sources to determine optimal treatment leveL
43
CONCLUSION
Based on results obtained in pilot study (see Appendix) and thesis
experiments 4 minutes ofpercussion is recommended as a standard protocol for
treating an unknown source ofNew Mexico locust seed In addition to laboratory
results the 4-minute percussion treatment has resulted in germination greater than
80 and healthy seedling grow-outs for production purposes (unpublished data) In
laboratory experiments only one hundred seeds per 118 ml (40z) soil tin were
percussed at a time whereas for production we have treated up to five hundred seeds
per tin For large-scale production ofNew Mexico locust seedlings growers may
wish to experiment with larger quantities of seed and larger-size soil tins
Seed storage trials following percussion treatments were not conducted
However germination greater than 80 and healthy seedlings were obtained when
seeds were percussion-treated and subsequently stored at room-temperature (22degC)
for one week (unpublished data) Seed storage trials merit further study as the
standard hot water treatment leaves seeds in a softened condition that is not
conducive to storage
Hydroseeding is an effective method for direct seeding in reclamation
situations Percussion treatment may be conducive to hydro seeding as seeds remain
a dry relatively undamaged yet permeable condition following treatment Sulfuric
seeds also remain dry following treatment but extensive damage to seed coat
poor seedling vigor have been reported with this treatment (Kelly and van Staden
A grow-out study using a hydro seeder on a reclamation site testing percussion
44
against other treatments such as sulfuric acid and mechanical scarification also
merits further study
Papilionoids are the largest subfamily oflegumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust these legumes often play an integral role in the revegetation of
disturbed lands The pilot study (Appendix) indicates that percussion scarification
successfully treats the widely used black locust as well Developing superior
scarification methods to the standard hot water mechanical scarification and acid
treatments may facilitate the use ofpapilionoid legumes in reclamation projects
45
APPENDICES
APPENDIX A
PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR
NEW MEXICO LOCUST AND BLACK LOCUST SEEDS
Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4
Hot water and sulfuric acid soaks are traditional treatments for seeds of many
temperate woody legwnes including locusts However these scarification techniques
often produce inconsistent germination Percussion scarification where seeds are
repeatedly propelled against a hard surface was compared with hot water
scarification to evaluate treatment efficacy for New Mexico locust (Robinia
neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment
seeds were placed in a 98degC water bath which was immediately removed from the
heat source For percussion scarification seeds were placed in a soil sample tin and
agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the
control were followed by 24-hour water soaks Hot water baths resulted in 56 and
41 germination for New Mexico locust and black locust respectively For both
species nearly all durations ofpercussion increased germination over the hot water
treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3
4 and 5 minutes for black locust resulted in at least 90 germination Traditional
scarification treatments randomly degrade the entire seed coat which can lead to
tissue damage during water uptake Percussion scarification specifically weakens the
strophiole the natural source ofwater entry to the seed in papilionoid legwnes
INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom
2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031
48
Following percussion imbibition is controlled through the strophiole and underlying
tissue is protected
Keywords New Mexico locust black locust scarification percussion germination
Introduction
Locust trees (Robinia spp) are aggressive pioneer species that quickly
colonize disturbed land fix atmospheric nitrogen in the soil and stabilize slopes that
are prone to erosion (Klemmedson 1994 Ashby et al 1985) (Figure A1ab)
Recognition of these attributes has contributed to increased demand for locusts in
reclamation projects Locust seeds however exhibit physical dormancy where a
thick seed coat prevents the movement of water and gasses to the embryo (Leadem
1997)
Sulfuric acid or hot water soaks have traditionally been used to break seed
dormancy in macro-propagation of locusts Many growers are moving away from the
use of su1furic acid in treating locust seeds (Dreesen and Harrington 1997) In
addition to being dangerous acid soak durations must be specifically correlated to
seed lot (Olson 1974) Collections of native seed can vary tremendously in seed size
seed weight and hard seededness etc This variability limits the utility of sulfuric
acid soaks as even within a given lot some seeds may be damaged whereas others
are under-treated and fail to imbibe (take up water) A survey of Southwestern
container growers indicates that hot water baths are currently the most common
scarification method for locust seeds (Hine et al 1997) However hot water baths
also produce inconsistent germination (Lin et al 1996)
49
b
Figure AI New Mexico locust a primary invader following disturbance Ala Crown-sprouting at edge ofburn near Los Alamos NM--one year after Cerro Grande fire Alb Colonizing road cut Gila National Forest NM
50
Percussion scarification where seeds are repeatedly propelled against a hard
surface is an alternative dormancy-breaking method that has proven successful in
legumes related to locusts (Hamly 1932 Barton 1947) We compared this alternative
treatment to hot water scarification to evaluate treatment efficacy for New Mexico
locust and black locust seeds
Materials and Methods
Commercial New Mexico locust seeds (Western Native Seed Coaldale CO)
were collected fall 2000 in Huerfano County CO Black locust seeds were collected
September 2000 from Taos County NM by harvesting ripe pods from several trees at
a distance of up to 3 m from ground level Black locust seeds were allowed to air-dry
for 3 weeks and were threshed and separated from large chaffusing a greenhouse
fan A South Dakota Seed Blower (Chicago Illinltois) was used to remove fine chaff
from both seed lots Seeds were storedmiddotat 2-4C until the germination study was
conducted in March 2001
This study compared hot water scarification with percussion scarification
using treatment and species as experimental factors Along with a control
scarification treatments included immersion of seed in boiling water as well as
percussion durations of 1 2 3 45 or 10 minutes Four 100-seed samples were used
to test each species by scarification treatment combination
For the hot water treatment test tubes were filled with 30 mL water and
placed in a water-filled beaker (Figure A2a) The bath was raised to boiling (-98C at
51
b
c
Figure A2 Implementation ofhot water and percussion scarification A2a Hot water bath A2b Paint-shaker set-up A2c Placing soil tin containing seeds at end ofpaint shaker ann
52
Las Cruces NM elevation of 1300 meters) Each 100-seed replicate was placed in a
test tube and each test tube was immediately removed to cool at room temperature
Percussion scarification was implemented using a pneumatic paint shaker
(Central Pneumatic model 00422 Camarillo CA) (Figure A2b) This allowed
standardization of treatment intensity Compressed air pressure was maintained at
530kPa plusmn 30kPa (80psi plusmn 5psi) resulting in approximately 350 oscillations per
minute Each lOO-seed sample was placed in a 118 ml (4 oz) soil tin for shaking To
maximize lateral movement of paint shaker a paint can was used as a spacer to place
soil tins at the end of the shaking arm (Figure A2c) This allowed shaking to occur at
the greatest distance from the pivot point (fulcrum) Following all treatments
including the control seeds were soaked for twenty-four hours in distilled water prior
to plating out
Gennination was evaluated on lab benches at room temperature Each
replicate of seeds was placed in a 100 cm petri dish on moistened filter paper with
humidity maintained by enclosing the petri dishes in ziplock bags Gennination was
monitored daily for 14 days in accordance with International Seed Testing
Association standards for Robinia species (ISTA 1999) Gennination was defined as
protrusion of the radicle from the embryo by at least 1mm as viewed by the naked
eye
Categorical Analysis of Variance using Prpc Catmod (SAS Institute 1989)
was used to detennine treatment differences in germination percentages for each
source This procedure is an extension of a Chi-square test of homogeneity using the
53
natural log of the ratio of germinated to non-germinated seeds for each treatment Pshy
values less than 005 were considered significant A limited set of pairwiseshy
comparisons was conducted to compare treatment mean percents using a
conservative alpha value of 005 divided by the number of comparisons
Results
Percussion maximized germination for both New Mexico locust and black
locust There were three germination response levels for New Mexico locust (Figure
A3) First there was a low response in the control indicating a dormant seed lot
The hot water treatment and I-minute duration of percussion share a second
intermediate response Germination was highest at percussion levels from 2 to 10
minutes
A selected percussion treatment intermediate in time (5 minutes) produced the
steepest germination curve indicating the fastest germination rate (Figure A4) The
hot water treatment produced an intermediate curve or relatively slower germination
rate The control curve was very flat indicating the slowest germination rate for New
Mexico locust
Control seeds germinated poorly for black locust as well (Figure A5) Hot
water scarification improved germination over the control but all levels of percussion
improved germination over hot water The optimal durations of percussion treatment
were bracketed with a significant increase from the 1 to 2 minute percussion level a
high germination response from 2 to 5 minutes of percussion and finally a significant
54
~ d 25 CL)
p t1 0 ~ t1
g Vl ~ Vl
100~---------------------------------------------------------------
80
60
40
20
o Control Hot Water Percussion
I min Percussion
2 min Percussion
3 min
Scarification
Percussion 4 min
Figure A3 Effect of scarification on germination response for New Mexico locust
Percussion 5 min
=significant difference between adjacent treatment bars ns = no significant difference
Percussion 10 min
~ ~ () u agt
Il-t d o ~
~ ~ c3
100middotlr~~~------------------------------------e- Percussion 5min middot 0middot Hot Water -- Control
80-J=middot======
6b-middot-lmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middot--middotmiddot-middot~-middotmiddot -middotmiddot middot middotmiddot-middot 1
40
20
-----~-----------
0 0 0 middotmiddotmiddotmiddotmiddot0middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot0
0 --~-----y-----~------_---~--- T-----~
o shy -TDay 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A4 Effect of selected scarification treatments on germination speed for New Mexico locust First seven days of fourteen-day experiment are shown
Day 7
80
~ cd
5 60 ()
[) Cl d 0 1t1 d
40sect dUl
-J
20
o
ns
Scarification
Figure A5 Effect of scarification on germination response for black locust = significant difference between adjacent treatment bars ns = no significant difference
decrease at the 10-minute level This drop-off suggests that 10 minutes of percussion
over-treats seeds from this lot of-black locust
A selected percussion treatment intermediate in time (5 minutes) also had the
fastest germination rate for black locust (Figure A6)_ The hot water curve is
somewhat flatter indicating a slower germination rate The control curve is very flat
indicating a slow germination rate
A qualitative difference between hot water and percussion-treated seeds was
also observed Along with healthy germinants the hot water treatment produced
over-treated seeds (Figure A 7a) Examples of damaged germinants include seeds
where the radicle did not elongate (Figure A 7b) seeds where the radicle elongated
but was delayed in freeing itself from the seed coat (Figure A7c) and seeds where the
radicle broke off completely from the embryo (Figure A7d) In addition to these
examples of over-treatment there were numerous small dark seeds that failed to
imbibe water For percussion treatment nearly all seeds took up water (Figure A8a)
Germination was rapid and uniform with little damage evident (Figure A8b)
To summarize both New Mexico locust and black locust seed lots were
dormant The standard hot water scarification treatment improved germination but
only to 56 and 41 for New Mexico locust and black locust respectively Nearly
all percussion durations improved total germination as well as germination rate over
the hot water treatment Germination was greater than 90 for percussion durations
of 4 5 and 10 minutes for New Mexico locust and 3 4 and 5 minutes for black locust
58
CI)
sgt t CI) 0
igt 0
sect ttl t E
Vl 0
c3
100r==~==~~=r------------------------------------~ ___ Percussion 5 min
middotmiddotmiddot0middotmiddotmiddot Hot Water -lIf- Control
80r=========~--~~----------------------------~
60
40
20 -~-~---~---------- ---~------------I
omiddot
O
_~_----------lIf-----~bull -----_ shy---shy I
Day 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A6 Effect of selected scarification treatments on germination speed for black locust First seven days of fourteen-day experiment are shown
Day 7
a
b c d
Figure A7 Damage to New Mexico locust seeds observed during germination counts following hot water scarification A7a Hot water replicate including damaged seedlings and numerous unimbibed seeds A7b Close-up ofseed where radicle failed to elongate A7c Close-up ofseed where radicle elongated but was delayed in freeing itself from the seed coat A7d Close-up ofseed where radicle completely broke offfrom embryo
60
a
b
Figure A8 New Mexico locust seeds following percussion treatment at time of germination counts A8a Percussion replicate with uniformly healthy seedlings and near complete imbibition of seeds A8b Close-up ofpercussion-treated seedling
61
Discussion
Papilionoid seeds have a specific anatomical feature known as the strophiole
or lens-the natural site of water entry to the seed (Hamly 1932) The strophiole is
located on the cotyledonary lobe of a locust seed (Figure A9) When a papilionoid
legume seed is percussed for the appropriate amount of time repeated hits on the
integrated seed coat loosen the constrained cells of the strophiolar region without
excessively damaging the rest of the seed coat (Ballard 1976)
When a percussed seed is soaked water enters exclusively through the
strophiole in a controlled manner (Kelly and Staden 1987) This regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues This
contrasts with seeds that have been hot water or acid scarified treatments that can
randomly degrade the seed coat Unlocalized cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rehman et al 1998 Marunda 1990)
Papilionoids are the largest subfamily of legumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust and black locust these legumes often play an integral role in the
revegetation ofdisturbed lands Developing superior scarification methods to the
standard hot water and acid treatments should fadlitate the use ofthese legumes in
restoration projects
62
Figure A9 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (bc)
63
APPENDIXB
GERMINATION PERCENTS AND STANDARD ERRORS FOR SOURCE
BLOCK AND TREATMENT FOR NEW MEXICO LOCUST
Table B 1 Gennination percents and standard errors for source block and treatment for New Mexico locust
Source Block Treatment Germination Percentage Standard Error
Capitan 1 Immersion 74 437 Capitan 2 Immersion 69 462 Capitan 3 Immersion 71 454 Capitan 4 Immersion 67 470 Capitan Boiling 30 sec 79 407 Capitan 2 Boiling 30 sec 76 427 Capitan 3 Boiling 30 sec 72 449 Capitan 4 Boiling 30 sec 76 427 Capitan Boiling 60 sec 71 454 Capitan 2 Boiling 60 sec 54 498 Capitan 3 Boiling 60 sec 67 470 Capitan 4 Boiling 60 sec 76 427 Capitan I Control 5 218 Capitan 2 Control 7 255 Capitan 3 Control 4 196 Capitan 4 Control 5 218 Capitan 1 Percussion I min 47 499 Capitan 2 Percussion I min
470
Capitan Percussion I min 39 488
Capitan 4 Percussion I min 38 485 Capitan I Percussion 2 min 55 497 Capitan Capitan
2
Percussion 2 min Percussion 2 min
71 65
454 477
Capitan 4 Percussion 2 min 56 496 Capitan I Percussion 4 min 86 347 Capitan 2 Percussion 4 min 79 407 Capitan 3 Percussion 4 min 81 392 Capitan 4 Percussion 4 min 82 384 Capitan I Percussion 8 min 90 300 Capitan 2 Percussion 8 min 88 325 Capitan 3 Percussion 8 min 90 300 Capitan 4 Percussion 8 min 90 300 Gila I Immersion 71 454 Gila 2 Immersion 77 421 Gila
Immersion 75 433
Gila 4 Immersion 72 449
Gila I Boiling 30 sec 68 466
Gila 2 Boiling 30 sec 81 392
Gila 3 Boiling 30 sec 54 498
Gila 4 Boiling 30 sec 63 483
Gila I Boiling 60 sec 55 497
Gila 2 Boiling 60 sec 68 466
Gila 3 Boiling 60 sec 57 495
Gila 4 Boiling 60 sec 70 458
65
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Gila 2 Control 13 336 Gila 3 Control 13 336 Gila 4 Control 20 400 Gila 1 Percussion 1 min 66 474 Gila 2 Percussion 1 min 57 495 Gila 3 Percussion 1 min 73 444 Gila 4 Percussion 1 min 82 384 Gila 1 Percussion 2 min 81 392 Gila 2 Percussion 2 min 86 347 Gila 3 Percussion 2 min 85 357 Gila 4 Percussion 2 min 54 498 Gila 1 Percussion 4 min 79 407 Gila 2 Percussion 4 min 83 376 Gila 3 Percussion 4 min 84 367 Gila 4 Percussion 4 min 79 407 Gila 1 Percussion 8 min 51 500 Gila 2 Percussion 8 min 76 427 Gila 3 Percussion 8 min 52 500 Gila 4 Percussion 8 min 74 439 Holman 1 Immersion 83 376 Holman 2 Immersion 86 347 Holman 3 Immersion 81 392 Holman 4 Immersion 90 300 Holman 1 Boiling 30 sec 90 300 Holman 2 Boiling 30 sec 85 357 Holman 3 Boiling 30 sec 87 336 Holman 4 Boiling 30 sec 83 376 Holman 1 Boiling 60 sec 79 407 Holman 2 Boiling 60 sec 80 400 Holman 3 Boiling 60 sec 86 347 Holman 4 Boiling 60 sec 93 255 Holman Control 25 433 Holman 2 Control 19 392 Holman Control 21 407
Holman 4 Control 24 427
Holman Percussion 1 min 77 421
Holman 2 Percussion 1 min 85 357
Holman 3 Percussion 1 min 80 400
Holman 4 Percussion 1 min 89 313
Holman 1 Percussion 2 min 88 325
Holman 2 Percussion 2 min 96 196
Holman 3 Percussion 2 min 93 255
Holman 4 Percussion 2 min 96 196
Holman 1 Percussion 4 min 92 271
Holman 2 Percussion 4 min 97 171
66
I
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Holman 3 Percussion 4 min 91 286 Holman 4 Percussion 4 min 96 196 Holman 1 Percussion 8 min 88 325 Holman 2 Percussion 8 min 85 357 Holman 3 Percussion 8 min 83 376 Holman 4 Percussion 8 min 82 384 Jemez 1 Immersion 53 499 Jemez 2 Immersion 44 496 Jemez 3 Immersion 52 500 Jemez 4 Immersion 70 458 Jemez 1 Boiling 30 sec 58 494 Jemez 2 Boiling 30 sec 55 497 Jemez 3 Boiling 30 sec 48 500 Jemez 4 Boiling 30 sec 65 477 Jemez 1 Boiling 60 sec 68 466 Jemez 2 Boiling 60 sec 63 483 Jemez 3 Boiling 60 sec 73 444 Jemez 4 Boiling 60 sec 89 313 Jemez 1 Control 2 140 Jemez 2 Control 9 286 Jemez 3 Control 6 237 Jemez 4 Control 8 271 Jemez 1 Percussion 1 min 58 494 Jemez 2 Percussion 1 min 63 483 Jemez 3 Percussion 1 min 77 421 Jemez 4 Percussion 1 min 89 313 Jemez 1 Percussion 2 min 68 466 Jemez 2 Percussion 2 min 64 480 Jemez 3 Percussion 2 min 80 400 Jemez 4 Percussion 2 min 83 376 Jemez Percussion 4 min 68 466 Jemez 2 Percussion 4 min 79 407 Jemez 3 Percussion 4 min 70 458 Jemez 4 Percussion 4 min 83 376 Jemez 1 Percussion 8 min 52 500 Jemez 2 Percussion 8 min 59 492 Jemez 3 Percussion 8 min 61 488 Jemez 4 Percussion 8 min 70 458 Magdalena 1 Immersion 69 462 Magdalena 2 Immersion 69 462 Magdalena J Immersion 70 458 Magdalena 4 Immersion 85 357 Magdalena 1 Boiling 30 sec 65 477 Magdalena 2 Boiling 30 sec 71 454 Magdalena 3 Boiling 30 sec 68 466
67
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Magdalena 4 Boiling 30 sec 83 376 Magdalena 1 Boiling 60 sec 63 483 Magdalena 2 Boiling 60 sec 68 466 Magdalena 3 Boiling 60 sec 63 483 Magdalena 4 Boiling 60 sec 82 384 Magdalena 1 Control 11 313 Magdalena 2 Control 6 237 Magdalena 3 Control 5 218 Magdalena 4 Control 4 196 Magdalena Percussion 1 min 32 466 Magdalena 2 Percussion 1 min 45 497 Magdalena 3 Percussion 1 min 46 498 Magdalena 4 Percussion I min 36 480 Magdalena 1 Percussion 2 min 67 470 Magdalena 2 Percussion 2 min 68 466 Magdalena 3 Percussion 2 min 65 477 Magdalena 4 Percussion 2 min 81 392 Magdalena 1 Percussion 4 min 87 336 Magdalena 2 Percussion 4 min 97 171 Magdalena 3 Percussion 4 min 83 376 Magdalena 4 Percussion 4 min 97 171 Magdalena 1 Percussion 8 min 98 lAO Magdalena 2 Percussion 8 min 100 000 Magdalena 3 Percussion 8 min 96 196 Magdalena 4 Percussion 8 min 98 lAO Manzano 1 Immersion 67 470 Manzano 2 Immersion 57 495 Manzano 3 Immersion 72 4049 Manzano 4 Immersion 73 4044 Manzano 1 Boiling 30 sec 70 458 Manzano 2 Boiling 30 sec 64 480 Manzano 3 Boiling 30 sec 71 454 Manzano 4 Boiling 30 sec 80 400 Manzano I Boiling 60 sec 50 500 Manzano 2 Boiling 60 sec 53 499 Manzano 3 Boiling 60 sec 59 492 Manzano 4 Boiling 60 sec 74 439 Manzano I Control 4 196 Manzano 2 Control 8 271 Manzano 3 Control 1 099 Manzano 4 Control 6 237 Manzano 1 Percussion 1 min 52 500 Manzano 2 Percussion 1 min 39 488 Manzano 3 Percussion 1 min 36 480 Manzano 4 Percussion 1 min 51 500
68
Table B 1 Continued
Source Block Treatment Gerrni~ation Percentage Standard Error
Manzano Percussion 2 min 53 499 Manzano 2 Percussion 2 min 58 494 Manzano 3 Percussion 2 min 61 488 Manzano 4 Percussion 2 min 62 485 Manzano 1 Percussion 4 min 76 427 Manzano 2 Percussion 4 min 85 357 Manzano 3 Percussion 4 min 79 407 Manzano 4 Percussion 4 min 83 376 Manzano 1 Percussion 8 min 87 336 Manzano 2 Percussion 8 min 83 376 Manzano 3 Percussion 8 min 83 376 Manzano 4 Percussion 8 min 75 433 Raton 1 Immersion 54 498 Raton 2 Immersion 37 483 Raton 3 Immersion 39 488 Raton 4 Immersion 69 462 Raton 1 Boiling 30 sec 36 480 Raton 2 Boiling 30 sec 20 400 Raton 3 Boiling 30 sec 28 449 Raton 4 Boiling 30 sec 60 490 Raton 1 Boiling 60 sec 15 357 Raton 2 Boiling 60 sec 171 Raton 3 Boiling 60 sec 4 196 Raton 4 Boiling 60 sec 35 477 Raton 1 Control 4 196 Raton 2 Control 12 325 Raton 3 Control 2 140 Raton 4 Control 6 237 Raton 1 Percussion 1 min 52 500 Raton 2 Percussion I min 29 454 Raton 3 Percussion I min 35 477 Raton 4 Percussion 1 min 31 462 Raton 1 Percussion 2 min 55 497 Raton 2 Percussion 2 min 35 477 Raton 3 Percussion 2 min 33 470 Raton 4 Percussion 2 min 42 494 Raton 1 Percussion 4 min 65 477 Raton 2 Percussion 4 min 52 500 Raton Percussion 4 min 51 500 Raton 4 Percussion 4 min 53 499 Raton 1 Percussion 8 min 88 325 Raton 2 Percussion 8 min 80 400 Raton 3 Percussion 8 min 73 444 Raton 4 Percussion 8 min 74 439 Sacramento 1 Immersion 43 495
69
Table B 1 Continued
-Source Block Treatment Germination Percentage Standard Error
Sacramento 2 Immersion 30 458 Sacramento 3 Immersion 38 485 Sacramento 4 Immersion 56 496 Sacramento 1 Boiling 30 sec 47 499 Sacramento 2 Boiling 30 sec 48 500 Sacramento 3 Boiling 30 sec 48 500 Sacramento 4 Boiling 30 sec 56 496 Sacramento 1 Boiling 60 sec 52 500 Sacramento 2 Boiling 60 sec 67 470 Sacramento 3 Boiling 60 sec 43 495 Sacramento 4 Boiling 60 sec 48 500 Sacramento 1 Control 0 000 Sacramento 2 Control 3 171 Sacramento 3 Control 3 171 Sacramento 4 Control 099 Sacramento 1 Percussion 1 min 57 495 Sacramento 2 Percussion 1 min 61 488 Sacramento 3 Percussion 1 min 62 485 Sacramento 4 Percussion 1 min 55 497 Sacramento 1 Percussion 2 min 84 367 Sacramento 2 Percussion 2 min 70 458 Sacramento 3 Percussion 2 min 81 392 Sacramento 4 Percussion 2 min 69 462 Sacramento 1 Percussion 4 min 91 286 Sacramento 2 Percussion 4 min 84 367 Sacramento 3 Percussion 4 min 84 367 Sacramento 4 Percussion 4 min 94 237 Sacramento 1 Percussion 8 min 82 384 Sacramento 2 Percussion 8 min 81 392 Sacramento 3 Percussion 8 min 82 384 Sacramento 4 Percussion 8 min 81 392 Sandia 1 Immersion 76 427 Sandia 2 Immersion 70 458 Sandia 3 Immersion 70 458 Sandia 4 Immersion 81 392 Sandia 1 Boiling 30 sec 78 414 Sandia 2 Boiling 30 sec 71 454 Sandia 3 Boiling 30 sec 63 483 Sandia 4 Boiling 30 sec 78 414 Sandia 1 Boiling 60 sec 67 470 Sandia 2 Boiling 60 sec 65 477 Sandia 3 Boiling 60 sec 74 439 Sandia 4 Boiling 60 sec 66 474 Sandia 1 Control 16 367 Sandia 2 Control 7 255
70
Table B 1 Continued
Source Block Treatment Gennination Percentage Standard Error
Sandia 3 Control 8 271 Sandia 4 Control 16 367 Sandia 1 Percussion 1 min 67 470 Sandia 2 Percussion 1 min 61 488 Sandia 3 Percussion 1 min 66 474 Sandia 4 Percussion 1 min 59 492 Sandia I Percussion 2 min 82 384 Sandia 2 Percussion 2 min 83 376 Sandia 3 Percussion 2 min 81 392 Sandia 4 Percussion 2 min 84 367 Sandia 1 Percussion 4 min 89 313 Sandia 2 Percussion 4 min 86 347 Sandia 3 Percussion 4 min 89 313 Sandia 4 Percussion 4 min 85 357 Sandia 1 Percussion 8 min 80 400 Sandia 2 Percussion 8 min 77 421 Sandia 3 Percussion 8 min 69 462 Sandia 4 Percussion 8 min 66 474
LITERATURE CITED
Ashby WC Vogel WG Rogers NF 1985 Black locust in the reclamation equation General Technical Report Northeastern Forest Experiment Station USDA Forest Service NE-I05 12 p
Ballard LAT 1973 Physical barriers to gennination Seed Sci Technoll 285-303
___ 1976 Strophiolar water conduction in the seeds ofTrifolieae induced by action on the testa at nonstrophiolar sites Austr J Plant Physiol3 465-469
Barton LV 1947 Special studies on seed coat impenneability Contrib Boyce Thompson Inst 14 355-362
Baskin CC Baskin JM 1998 Seeds Ecology biogeography and evolution of donnancy and gennination Academic Press London UK 666 p
Bewley JD 1997 Seed germination and donnancy Plant Ce119 1055-1066
Bewley JD Black M 1994 Seeds physiology of development and germination 2nd ed Plenum Press New York NY 445 p
Briscoe CB 1996 Growing Nitrogen Fixing Trees in Acid Soils In MH Powell (ed) Nitrogen Fixing Trees for Acid Soils- A Field ManuaL Winrock International Morrilton AR 22 p
Copeland LO McDonald MB 1995 Principles of Seed Science and Technology 3rded Chapman amp Hall New York NY 409 p
Cox RE Klett JE 1984 Seed gennination requirements of native Colorado plants for use in the landscape Plant Propag 30(2) 6-10
Dell B 1980 Structure and function ofthe strophiolar plug in seeds ofAlbizia Lophantha Am J Bot 67 556-563
Dick-Peddie WA 1993 New Mexico Vegetation Past Present and Future University ofNew Mexico Press Albuquerque NM 244 p
Dreesen DR 2001 Propagation protocol for production ofcontainer Robinia neomexicana plants Los Lunas Plant Materials Center Los Lunas New Mexico In Native Plant Network URL httpwwwnativeplantnetworkorg (accessed 9 January 2002) Moscow (ID) University ofIdaho CoIl Natural Resources For Res Nursery
72
Dreesen DR Harrington JT 1997 Propagation of native plants for restoration projects in the Southwestern US- Preliminary Investigations In Landis TD Thompson JR tech coords National Proceedings Forest and Conservation Nursery Assocs Gen Tech Rep PNW-GTR-419 Portland OR USDA For Ser PNW Res Sta 77-88
Gottfried GJ 1980 Control ofNew Mexican locust and the effect on planted ponderosa pine in central Arizona US For Ser Res Note RM-386 Rocky Mountain For Range ExpStn Fort Collins CO
Gosling PG Samuel YK Jones SK 1995 A systematic examination of germination temperature chipping and water temperaturesoak duration pretreatments on the seeds ofLeucaena leucocephala Seed Sci amp Technol 23521-532
Hamly DH 1932 Softening of the seeds of Melilotus alba Bot Gazette 93 345shy375
Hanna PJ 1984 Anatomical features of the seed coat ofAcacia kempeana (Mueller) which relate to increased germination rate induced by heat treatment New Phytol 96 23-29
Hanks JP Dick-Peddie WA 1974 Vegetation patterns of the White Mountains New Mexico Southwest Naturalist 18(4) 371-382
Hine S Harrington JT Dreesen DR 1997 Seed scarification requirements for Robinia neomexicana In Harrington JTtech coord Proc Third SouthwestemContainer Growers Conf Feb 12-13 1997 Ft Collins CO
Hopkinson JM 1993 The strophiole of leguminous seeds Newsletter- IntI Herbage Seed Production Res Grp 18 7-8
---- 1997 Differences in hardseededness between Cunningham and Tarramba leucaena In Leucnet News The Newsletter of the International LeucaenaRes and Devlt Netwk No4
ISTA (International Seed Testing Association) 1999 International rules for seed testing rules Seed Sci amp Technol27 Supplement
Kelly KM van Staden J 1987 The lens as the site of permeability in the papilionoid seed Aspalathus linearis J Plant Physiol 128(45) 395-404
73
Keresztesi B 1988 Black locust the tree of agriculture Outlook on Agr 17(2) 77~85
Kigel 1 and Galili G 1995 Seed development and germination Marcel Dekker New York 853 p
Klemmedson 10 1994 New Mexico locust and parent material Influence on macronutrients of forest floor and soil Soil Sci Soc Am 1 58 974~980
Kuo WH Tam AW 1988 The pathway of water absorption of mung bean seeds Seed Sci amp Technol16(1) 139~144
Leadem CL 1997 Dormancy--unlocking seed secrets In Landis TD Thompson lR tech coords National Proceedings Forestand Conservation Nursery Assocs Gen Tech Rep PNW-G TR-419 Portland OR USDA For Ser PNW Res Sta 43-52
Lin Y Wagner MR Seweyestewa A Honahni MD DeGomez M McMillin JD 1996 Influence of cultural treatments on seed germination and seedling growth of Robinia neomexicana Forest Farm and Community Tree Res Reports Volume 1
Marunda CT 1990 Effects of seed pretreatment on the development ofAcacia auriculforinis and A holosericea seedlings ACIAR Proceedings Series No 28
Meginnis HG 1937 Sulphuric acid treatment to increase germination of black locust seed USDA Circ 453 34 p
Miklas PN Towensend CE Ladd SL 1987 Seedcoat anatomy and the scarification ofcicer milkvetch seed Crop Sci 27 766-772
Monsen SB 1984 Use of shrubs on mine spoils In The Challenge ofProducing Native Plants for the Intermountain Area Proceedings Intermountain Nurserymans Association 1983 conference Las Vegas NV compo PM Murphy 26-31 Ogden UT USDA For Ser Gen Tech Report INT-l68
Olson DF 1974 Robinia L Locust In Seeds of Woody Plants in the US tech coord CS Schopmeyer 728-731 USDA For Ser Agr Handbk No450
Rehman S Loescher RNl Harris P1C 1998 Dormancy breaking and germination ofAcacia salicina Lindl seeds Seed Sci amp Techno127 553-557
Rolston MP 1978 Water impermeable seed dormancy Bot Rev 44 365-396
74
SAS Institute Inc 1989 SASSTAT Users Guide Version 6 4th Edition Volume 1 Cary NC SAS Institute Inc 943 p
SerratoValenti G Conara L Ferrando M Modenesi P 1993 Structural and histochemical features ofStylosanthes scabra (Leguminosae Papilionoidae) seed coat as related to water entry Can J Bot 71 834-840
Simpson BJ 1988 A field guide to Texas trees Austin TX Texas Monthly Press 372p
Tran VN Cavanagh AK 1984 Structural aspects ofdormancy In Seed Physiology Vol 2 Murray DR (ed) p1-44 Academic Press Australia
USDA Forest Service 1948 Woody-plant seed manual USDA Misc Pub1654 416p
Vines RA 1960 Trees Shrubs and Woody Vines of the Southwest Austin Univ ofTexas Press 1104 p
Wagner MR Larson RE Craig TP 1992 Ecology and silvics ofNew Mexico locust (Robinia neomexicana) with an emphasis on the arboreal form p53-57 In JW Hanover K Miller and S Plesko (eds) Proc Intemat Confon Black Locust Biology Culture and Utilization Mich State Univ USA
Wilson JK 1944 Immersing seeds of species of Robinia in boiling water hastens germination J For 42 453-454
Zimmerman RW Carpenter SB 1980 First-year coppice production from a 5shyyear old black locust stand on surface mine spoil p309-314 Centr Hardwd For ConfProc University ofMissouri Columbia
MATERIALS AND METHODS
Seed Collection and Cleaning
Seeds were collected from August 25 through September 15 2000 from nine
locations along a latitudinal gradient in New Mexico Seed source locations and
descriptive information are given in Figure 1 and Tables I and 2 Stands were
separated by at least 45 km
Pods were hand picked as they began to ripen and dehisce (open) Collections
were made from ground level to approximately 3 m from several trees at each site
Pods were dried in open-ended brown paper bags for two to four weeks at room
temperature (21-230C) to facilitate cleaning Dried pods were placed in a closed cloth
sack which was pounded against the ground to separate seeds from pods For pods
still containing seeds hand maceration (crunching pods with hands) completed seed
separation Debris was removed by threshing with the aid of a greenhouse fan A
South Dakota Seed Blower (Seedbureau Chicago Illinois) was used to remove
remaining chaff and light seeds by air density separation Seeds were placed in dry
storage in sealed quart-size zip-lock bags within a sealed plastic container Storage
temperatures ranged from 2-4oC Seeds remained in storage for approximately 7
months before the experiment
Experimental Design and Treatment Structure
The experiment compared the impact ofhot water bath and percussion
l11aLl11 on germination properties ofNew Mexico locust seed A single
16
-l
Figure 1 Seed source collection map All sources were collected within New Mexico USA separated by at least 4S lan
IIIRZltItW lIJPIirmiddot rllll1lbullbull_~~~_II
Table 1 Latitude longitude location elevation and collection date ofNew Mexico locust seed sources
Lot title Latitude (N) Longitude (W) Location Elevation (m) Collection Date
Raton 36deg5846 104deg2872 Raton Pass NM 2295 82500
Holman 36deg0306 105deg2429 HolmanNM 2476 911500
Jemez 35deg4500 106deg4100 Jemez Springs NM 1976 9900 00
Sandia 35deg1083 106deg2341 Cedar Crest NM 2432 82600
Manzano 34deg3689 106deg2494 ManzanoNM 2465 9200
Magdalena 33deg5953 107deg0903 Magdalena NM 2544 82900
Capitan 33deg2728 105deg4325 Alto NM 2189 911200
Sacramento 32deg5798 105deg4496 Cloudcroft NM 2630 91400 Gila 32deg5400 107deg4600 Kingston NM 2432 9700
f
Table 2 Mean seed weight and standard error by source
a
Source Mean seed weight (g) for Standard error 100 seeds n = 5
Raton Holman Jemez Sandia Manzano Magdalena Capitan Sacramento Gila
2872 b c 2522 d 1826 e 2753 c 2413 d 2559 d 2443 d 3130 a 3029 ab
00103 00403 00276 00301 00370 00374 00458 00279 00348
a Mean seed weights followed by the same letter are not significantly
different
19
experiment was conducted utilizing a randomized complete block design blocked by
time with a factorial treatment structure Experimental factors were seed source and
scarification treatment Nine seed sources from throughout New Mexico were used
(Figure 1 Table 1) Scarification treatments included a control three levels ofhot
water and four levels of percussion Hot water treatments included immersion at
100degC and boiling for periods of 30 seconds and 60 seconds Percussion treatment
levels were 1 2 4 and 8 minutes One hundred seeds per block were used to test
each treatment combination Four blocks of seed were tested one at a time (all seed
sources included in each run) in close chronological sequence (within 6 weeks) A
total of four 100-seed samples were used to test each source by scarification treatment
combination
Treatments
Hot water treatment of seeds utilized 50 ml test tubes filled with 30 ml
distilled water Each test tube was used to treat one 100-seed sample at a time
resulting in a ratio of4 parts water to 1 part seed by volume Test tubes were placed
in a beaker with the beaker filled with tap water to the same level as the water within
test tubes Nine test tubes (one per seed source) were placed in the beaker
simultaneously so that one replication of each seed sourcescarification treatment
cotnblnatlon was conducted simultaneously Hot water treatments include immersion
boiling For immersion treatments seeds were added to test tubes with the water
(boiling) and the test tubes were immediately removed from the beaker to
For the boiling treatments test tubes remained in the beaker with water at
20
boiling temperature for a set duration after the addition of seeds Following all
treatments test tubes with seeds cooled 24 hours in a test tube rack
Percussion treatment utilized a pneumatic paint shaker (Central Pneumatic
model 00422 Camarillo CA) Compressed air was maintained at 530 kPa plusmn30 kPa
(80 psi plusmn 5 psi) resulting in approximately 350 oscillations per minute Seeds were
placed in 118 ml (4 oz) soil tin cans (US Can Lobard IL) and the lids were secured
with duct tape Each can was used to treat one 100-seed sample To take advantage
ofgreatest lateral movement of the paint shaker a spacer (paint can) was used to
position the soil tin at the end of the shaking arm This allowed shaking to occur at
the greatest distance from the pivot point (fulcrum) Tins were placed perpendicular
to the direction of shaking motion allowing seeds to impact at greatest force by
hitting flat surfaces of the soil tin
Germination Testing
Before gennination testing all seeds were soaked 24 hours in reverse
osmosis-treated water (this was included in the process ofhot water treatment
jU~SCIlOea above) Following soaking seeds were placed in 100 cm Petri dishes lined
90 cm Whatman qualitative 1 filter paper circles (MG Scientific Pleasant
WI) One Petri dish was used for each treatment by source combination
papers were initially moistened with 5 mL reverse osmosis-treated water then
~VU1CUlJl moistened as needed to ensure adequate conditions for gennination
were monitored daily (at 300 PM) for gennination for 14 days in accordance
~UIUlnJlVUa Seed Testing Association (ISTA) standards for Robinia species
21
(1STA 1999) Tests were carried out at room temperature (22degC plusmnOSoC)
Germination was classified as protrusion of the radicle from the embryo by at least
1mm as viewed with the naked eye
X-ray Radiography
On August 132001 three lOO-seed samples from the Sacramento seed source
were treated for 14 or 8-minute percussion durations The seeds were sent
immediately by overnight delivery to the Faxitron X-Ray Corporation in Wheeling
Illinois where Dr Bob Karrfalt ofthe USDA Forest Service oversaw their imaging
Radiographs were made at SX with a Faxitron Specimen Radiography System Model
MX-20
Scanning Electron Microscope Photography
On February 192002 a Hitachi S-3200N Variable Pressure Scanning
Electron Microscope (Mountain View CA) was used to examine strophiolar cells of
untreated seeds and seeds percussed for 4 minutes Seeds were cut longitudinally
igt their entire length using a razor blade splitting the strophiolar region roughly in
Cross sections of seed samples magnifying the strophiolar region were viewed
600X under the microscope
Analysis
Categorical Analysis of Variance was used to determine treatment source and
(Le time) main effects and interactions The response variable was total
~vlI This procedure is a Chi-square test of homogeneity using the natural log
of germinated to non-germinated seed for each treatment Maximumshy
22
likelihood analysis was used to calculate Chi-square test statistics with their observed
significance levels A single contrast was conducted to cempare treatment means
against the control Observed significance levels less than or equal to 005 were
considered significant Pairwise comparisons were conducted between non-control
treatment mean percents using a conservative alp~a value of 005 divided by the
number of comparisons (Bonferroni approach) and approximate pairwise z statistics
Categorical analyses were performed using Proc Catmod (SAS Institute 1989)
RESULTS
Scarification seed source block and all interactions between factors
influenced germination (Table 3) The main effects are reported in the presence of
these interactions because the overall robustness of treatments and seed sources are of
interest Background levels ofnaturally occurring variation in seed sources are
seen in control germination percentages in Figure 2 In particular time variability
is unlikely to be under the control of the producer Appendix B details experimental
data for source by treatment gennination percents for each of the four blocks
Averaged over all treatments germination by source ranged from 40 to
79 (Figure 2) Averaged over all sources (Figure 3) and for each source (Figure 4
5) percussion scarification durations of4 or 8 minutes yielded greatest germination
Averaged over all sources germination increased with increasing durations of
percussion then leveled off for the 4- and 8-minute treatments (Figure 3) Immersion
and 30-second boiling scarification maximized germination for the hot water
treatments with a decrease in germination when seeds were boiled for 60 seconds
For all but two sources there is a similar ipcreasing trend in germination from
1to 4 minutes ofpercussion (Figure 4) The Holman source increased in germination
1 to 2 minutes then leveled off at 4 minutes The Jemez source changed little
1 to 4 minutes of treatment As treatment duration increased from 4 to 8
mutes five sources decreased in germination (Figure 4a) whereas three sources
24
Table 3 Categorical Analysis of Variance table for germination response to seed treatment seed source block and the interaction of the factors
Source of DF Chi-Square Observed Variability Significance Level
Source 8 98370 lt00001 Trt 7 233876 lt00001
contrast 1 16976 lt00001 SourceTrt 56 97077 lt00001 Block 3 5538 lt00001
SourceBlock 24 10162 lt00001 TrtBlock 21 7564 lt00001
SourceTrtBlock 168 34283 lt00001
S 4)
8 ~
0
sectg ~ 40 ~
N 0 0
20
o f0ltgt ~ ~4 jybshy ((iltgto ~~ K1ltgt 6~b-~ ~~ 0b-~~ ~~ ~b-~ ~b-~b
80
60 -I-- - ~- middotmiddotmiddotmiddotmiddot1
r -] Northern NM Sources
Central NM Sources
Southern NM Sources
sect-o ~
~b-v(i
SOURCE
Figure 2 Effect ofNew Mexico locust seed source on germination for data averaged over all scarification treatments Control germination is shown by solid line within bar
80
~ cd ~ ltl) () 60 ~ ~
N
t 0shyrti tshySltl)
40
0
20
o lmmersion Boiling 30 sec Boiling 60 sec Control 1 min 2min 4 min 8 min
Hot Water Treatments Percussion Treatments
Figure 3 Effect of scarification on germination ofNew Mexico locust for data averaged over all seed sources Means labelled with the same letter are not significantly different
100~----------------------~--~----------------~
80
60
ltU
~
40 IJ
sectgsI
l100
80
60
40
a I middotmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot middotImiddotmiddotmiddotmiddotmiddotmiddot E
_~iii-bull _ _ bull f --~--
t-- -~ ---- -shy ---- shyI ~ ---- - shy
L
fiII
~
b
- shy ---r
- Holman
Sacramento Sandia Gila Jemez
c ~
ltgt ~
Tmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot-I
j~=~=-~~-1
I middotmiddot ~ bull r
t tr
Y -------M-a-gd-a-Ie-n-a--
1 _ _
fCapitan
---- Manzano ~y -_ Raton
2 4 8
Time interval ofpercussion scarification (min)
Figure 4 Effect of seed source by treatment interaction on New Mexico locust germination for percussion scarification pattern a and pattern b
28
tv 0
~ d
~ A s o
1 c3
80
60
40
20
o
TIT
t------=middotfmiddotmiddot-middotmiddot-middotmiddot-middotmiddot 1- --- - - --=- - -shy--cE= - _--shy --shy ----shy ~ ~ ---shy ~ -~- _ --shy-------r 1
I
Raton Holman
-_ Jemez
--- Sandia Manzano Magdalena Capitan Sacramento Gila
Immersion Boiling 30 sec Boiling 60 sec
Hot Water Scarification
Figure 5 Effect of seed source by treatment interaction on New Mexico locust germination for hot water scarification
continued to increase in germination (Figure 4b) There was no difference between
the two treatment levels for the Manzano source (Figure 4b)
The effect ofhot water scarification also varied among sources with few
overall differences between hot water treatments (Figure 5) However boiling for 60
seconds yielded highest germination for the Jemez source whereas increasing
durations ofboiling decreased germination for the Raton source
In addition to consistently high germination 4- and 8-minute percussionshy
treated seeds germinated as fast or faster than all other treatments (Figure 6 Table 4)
050 measured as the number of days needed to complete 50 ofeventual
germination averaged 2 days for both ofthese treatments
X-ray radiographs were taken of seeds collected from the Sacramento source
percussed for 1 4 or 8 minutes (Figure 7) Seeds show signs of damage at 8 minutes
ofpercussion (Figure 8) Compared to an undamaged seed percussed for only 4
minutes (Figure 8a) damage at the 8-minute level includes fractured radicles pitting
of the seed coat and chips in seed coat structure (Figure 8b-d)
Scanning electron microscope (SEM) images at 600X reveal the direct effect
of 4 minutes of percussion on cells of the strophiolar region (Figure 9) Whereas
untreated seeds retain cellular organization in the strophiolar region strophiolar cells
seeds treated for 4 minutes ofpercussion separate and raise resulting in a
~-~e~U appearance
30
Umiddot-~middot--l-~---middotymiddotmiddotmiddot-----middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot--middot--middotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middotmiddotmiddotmiddotmiddotmiddotmiddot- -
050= gt1
-e-- Percussion 8 min -0- Percussion 4 min ---T- Percussion 2 min -----ltil- Percussion 1 min -- Immersion --0- Boiling 30 sec -+- Boiling 60 sec -ltgt- Control
2 4 6 8 10 12
Day Figure 6 Timecourse ofNew Mexico locust gennination following scarification
treatments G50 is the number of days to 50 of eventual germination
31
14
Table 4 Effect of seed source and treatment on G50 (days to 50 germination)
Seed Source Control Immersion B30sec B60sec Plmin P2min P4min P8min mean Raton 3 3 5 7 4 3 2 2 4 Holman 2 2 2 2 2 2 2 2 2
Jemez 4 6 6 5 3 2 2 2 4 Sandia 2 2 2 2 2 2 2 2 2
w tv Manzano 5 2 2 3 3 2 2 2 3
Magdalena 4 2 2 3 4 3 2 1 3
Capitan 5 4 2 2 3 2 2 2 3
Sacramento 3 2 2 2 4 3 2 2 3 Gila 3 2 2 2 2 2 2 2 2 mean 3 3 3 3 3 2 2 2 3 standard 11 14 16 18 09 05 0 03 08 deviation
Note Columns and rows may not agree due to rounding
Figure 7 X-ray radiographs ofSacramento seed source percussed for 1 4 or 8 minutes
33
Figure 8a-d Seed damage close-ups Figure a shows undamaged seed percussed for 4 minutes Figures b-d show seeds percussed for 8 minutes Damage includes fracturing ofradicle in b complete removal ofseed coat in c and pitting ofseed coat in d
34
Untreated Treated
Figure 9 SEM images ofuntreated vs 4-minute percussion-treated strophiolar cells ofNew Mexico locust seeds Treated cells are loosened separated and raised
DISCUSSION
Determining the Optimal Treatment
Averaging results of scarification treatmems across seed sources is a useful
tool to predict which treatment is most likely to be successful for an unknown seed
source Averaged over all sources four- and eight-minute percussion treatments
resulted in the highest germination 815 and 790 respectively and produced
germination that was as fast or faster than all other treatments Although there was no
difference between these two treatments on average an argument for 4 minutes of
percussion as a standard protocol for treating New Mexico locust seeds can be made
Descriptively 4 minutes of percussion improved germination over 8 minutes
ofpercussion for a majority of sources (five out of nine) 8 minutes of percussion
improved germination for three ofnine sources with no difference for a fmal source
For individual sources 4 minutes ofpercussion was always the best or secondmiddot best
treatment and was always equal to or better than all hot water treatments
Germination at 8 minutes ofpercussion was lower than all other percussion durations
for the Jemez and Gila sources and was lower than at least one hot water treatment for
these sources as well
The x-ray radiographs of the Sacramento seed source illustrate the excessive
internal and external seed damage that can occur with 8 minutes of percussion
germination counts 8-minute percussed seeds from Holman and Capitan
ClUll signs of damage that were visible to the naked eye (see below)
r~evertheless some of these seeds still fit the criteria for germination Neither x-ray
36
photography nor visible observations during seed counts revealed seed damage at the
4-minute percussion level In operational trials seeds percussed for 4 minutes
developed into healthy seedlings (Khadduri and Harrington unpublished data)
Strophiole and Seed Coat Behavior
Four minutes ofpercussion successfully breaks seed dormancy in a large
percentage ofNew Mexico locust seeds without excessively damaging the
surrounding seed coat or internal seed coat structures Most papilionoid legume
seeds including seeds ofNew Mexico locust have a specific region of the seed coat
known as the strophiole or lens (Hamly 1932) The strophiole is located on the
cotyledonary lobe of a locust seed (Figure 10 a-c) If seeds are not indiscriminately
damaged the strophiole is the first point of water entry to the embryo (Hopkinson
1997) When a papilionoid seed is percussed for the appropriate amount of time
repeated hits on the integrated seed coat loosen the constrained cells of the strophiolar
region without excessively damaging the rest of the seed coat (Ballard 1976) SEM
photos illuStrate that 4 minutes of percussion loosened and separated strophiolar cells
ofNew Mexico locust seeds when compared with constrained organized strophiolar
cells of untreated seeds (Figure 9) Research-has shown that water passes to the
embryo through the treated strophiole in a controlled manner and regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues
(Kelly and van Staden 1987)
37
10 Strophiole ofunimbibed New Mexico locust seed (a) and following uptake (bc)
38
In contrast hot-water treatments can excessively degrade the seed coat
(Rebman et al 1998 Marunda 1990) Random cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rebman et aI 1998 Marunda 1990) Along with a
proportionately large amount ofun-imbibed (thus under-treated) seeds (Figure l1a)
damage was observed in hot-water treated seeds from six sources during germination
counts Damage included seeds where the radicle failed to elongate (Figure 11 b)
seeds where the radicle elongated but was delayed in freeing itself from the seed coat
(Figure 11c) and seeds where the radicle completely broke off from the embryo
(Figure lId) In addition some seeds germinated in reverse (the cotyledon emerged
fIrst) If the radicle emerged completely from the seed coat they were counted as
germinated
While nearly all seeds treated for 8 minutes ofpercussion imbibed water
damage was evident in x-ray evaluations (Sacramento seed source) and in
germination counts (Holman and Capitan) at this treatment level Seed damage
during germination counts was similar to levels found in hot-water treated seeds
suggesting that over-treatment by both hot water and longer durations of percussion
may result in similar damage to seed structure
The Sacramento and Holman seed sources were two of the fIve sources that
opcrplltlpn in germination from the 4- to 8-minute durations of percussion
the benefIts ofloosening the strophiole as evidenced by imbibition of
all 8-minute treated seeds were outweighed by external and internal seed coat
39
a
b c d
11 Damage to New Mexico locust seeds observed during germination cotmts following hot water scarification 9a Hot water replicate including ~t seedlings and numerous unimbibed seeds 9b Close-up of seed where raallCle failed to elongate 9c Close-up ofseed where radicle elongated but was
-gtIn in freeing itself from the seed coat 9d Close-up ofseed where radicle broke off from embryo
40
damage (which also may be contributing to high imbibition) For the three sources
for which 8 minutes of percussion resulted in maximum germination extended
percussion of seeds necessary to loosen the strophiole may not have excessively
damaged the seed anatomy The Manzano source did not significantly change in
germination from 4 to 8 minutes of percussion suggesting that an increase in
permeable seeds at the 8-minute treatment duration was balanced by the number of
seeds that failed to germinate due to seed damage To summarize the 4-minute
percussion treatment improved germination over 8 minutes ofpercussion for a
majority of sources (five ofnine) the 8-minute percussion treatment improved
germination over 4-minutes ofpercussion for three sources and there was no
difference with the Manzano source Notably for the Raton source 8 minutes of
percussion increased germination by 235 percentage points over 4-minutes of
percussion (7875 vs 5525 respectively) the next best treatment Raton was the
only source to result in germination less than 75 for 4 minutes ofpercussion so a
grower may wish to apply longer percussion durations to lots that do not respond well
to this treatment level
Finally there was an interesting trend in the Jemez and Raton sources seed
that significantly contributed to the source by treatment interaction (Figure 12) A
~u test showed that the Jemez source had a significantly lighter seed weight than
other sources while the Raton source had an average seed weight For the Jemez
the longest duration of boiling increased germination Conversely only short
U4UVll) of percussion were needed to achieve high germination and the longest
41
80 _ Jemez a Raton
iD
~ C iD e iD
Q c 0 ~
c ~ iD 0
20
0------shy
Immersion Boiling 30 sec Boiling 60 sec
Hot Water Scarification
10degTr========~--------~------------------------------
80
60
40
20
_ Jemez Raton
0------shy
1 min 2 min 4 min 8 min
Percussion Scarification
Figure 12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources
42
b
--~----~~~~========~---- duration ofpercussion reduced germination For the Raton source longer durations
ofboiling consistently decreased germination whereas longer percussion treatments
consistently increased germination
A possible explanation is that a thick seed coat is associated with a loose
strophiole structure and that a thin seed coat is associated with a constrained
strophiole This contrast merits further study and may suggest an ecological
adaptation Further investigation ofthis phenomenon may contribute to successful
pre-screening ofunknown seed sources to determine optimal treatment leveL
43
CONCLUSION
Based on results obtained in pilot study (see Appendix) and thesis
experiments 4 minutes ofpercussion is recommended as a standard protocol for
treating an unknown source ofNew Mexico locust seed In addition to laboratory
results the 4-minute percussion treatment has resulted in germination greater than
80 and healthy seedling grow-outs for production purposes (unpublished data) In
laboratory experiments only one hundred seeds per 118 ml (40z) soil tin were
percussed at a time whereas for production we have treated up to five hundred seeds
per tin For large-scale production ofNew Mexico locust seedlings growers may
wish to experiment with larger quantities of seed and larger-size soil tins
Seed storage trials following percussion treatments were not conducted
However germination greater than 80 and healthy seedlings were obtained when
seeds were percussion-treated and subsequently stored at room-temperature (22degC)
for one week (unpublished data) Seed storage trials merit further study as the
standard hot water treatment leaves seeds in a softened condition that is not
conducive to storage
Hydroseeding is an effective method for direct seeding in reclamation
situations Percussion treatment may be conducive to hydro seeding as seeds remain
a dry relatively undamaged yet permeable condition following treatment Sulfuric
seeds also remain dry following treatment but extensive damage to seed coat
poor seedling vigor have been reported with this treatment (Kelly and van Staden
A grow-out study using a hydro seeder on a reclamation site testing percussion
44
against other treatments such as sulfuric acid and mechanical scarification also
merits further study
Papilionoids are the largest subfamily oflegumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust these legumes often play an integral role in the revegetation of
disturbed lands The pilot study (Appendix) indicates that percussion scarification
successfully treats the widely used black locust as well Developing superior
scarification methods to the standard hot water mechanical scarification and acid
treatments may facilitate the use ofpapilionoid legumes in reclamation projects
45
APPENDICES
APPENDIX A
PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR
NEW MEXICO LOCUST AND BLACK LOCUST SEEDS
Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4
Hot water and sulfuric acid soaks are traditional treatments for seeds of many
temperate woody legwnes including locusts However these scarification techniques
often produce inconsistent germination Percussion scarification where seeds are
repeatedly propelled against a hard surface was compared with hot water
scarification to evaluate treatment efficacy for New Mexico locust (Robinia
neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment
seeds were placed in a 98degC water bath which was immediately removed from the
heat source For percussion scarification seeds were placed in a soil sample tin and
agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the
control were followed by 24-hour water soaks Hot water baths resulted in 56 and
41 germination for New Mexico locust and black locust respectively For both
species nearly all durations ofpercussion increased germination over the hot water
treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3
4 and 5 minutes for black locust resulted in at least 90 germination Traditional
scarification treatments randomly degrade the entire seed coat which can lead to
tissue damage during water uptake Percussion scarification specifically weakens the
strophiole the natural source ofwater entry to the seed in papilionoid legwnes
INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom
2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031
48
Following percussion imbibition is controlled through the strophiole and underlying
tissue is protected
Keywords New Mexico locust black locust scarification percussion germination
Introduction
Locust trees (Robinia spp) are aggressive pioneer species that quickly
colonize disturbed land fix atmospheric nitrogen in the soil and stabilize slopes that
are prone to erosion (Klemmedson 1994 Ashby et al 1985) (Figure A1ab)
Recognition of these attributes has contributed to increased demand for locusts in
reclamation projects Locust seeds however exhibit physical dormancy where a
thick seed coat prevents the movement of water and gasses to the embryo (Leadem
1997)
Sulfuric acid or hot water soaks have traditionally been used to break seed
dormancy in macro-propagation of locusts Many growers are moving away from the
use of su1furic acid in treating locust seeds (Dreesen and Harrington 1997) In
addition to being dangerous acid soak durations must be specifically correlated to
seed lot (Olson 1974) Collections of native seed can vary tremendously in seed size
seed weight and hard seededness etc This variability limits the utility of sulfuric
acid soaks as even within a given lot some seeds may be damaged whereas others
are under-treated and fail to imbibe (take up water) A survey of Southwestern
container growers indicates that hot water baths are currently the most common
scarification method for locust seeds (Hine et al 1997) However hot water baths
also produce inconsistent germination (Lin et al 1996)
49
b
Figure AI New Mexico locust a primary invader following disturbance Ala Crown-sprouting at edge ofburn near Los Alamos NM--one year after Cerro Grande fire Alb Colonizing road cut Gila National Forest NM
50
Percussion scarification where seeds are repeatedly propelled against a hard
surface is an alternative dormancy-breaking method that has proven successful in
legumes related to locusts (Hamly 1932 Barton 1947) We compared this alternative
treatment to hot water scarification to evaluate treatment efficacy for New Mexico
locust and black locust seeds
Materials and Methods
Commercial New Mexico locust seeds (Western Native Seed Coaldale CO)
were collected fall 2000 in Huerfano County CO Black locust seeds were collected
September 2000 from Taos County NM by harvesting ripe pods from several trees at
a distance of up to 3 m from ground level Black locust seeds were allowed to air-dry
for 3 weeks and were threshed and separated from large chaffusing a greenhouse
fan A South Dakota Seed Blower (Chicago Illinltois) was used to remove fine chaff
from both seed lots Seeds were storedmiddotat 2-4C until the germination study was
conducted in March 2001
This study compared hot water scarification with percussion scarification
using treatment and species as experimental factors Along with a control
scarification treatments included immersion of seed in boiling water as well as
percussion durations of 1 2 3 45 or 10 minutes Four 100-seed samples were used
to test each species by scarification treatment combination
For the hot water treatment test tubes were filled with 30 mL water and
placed in a water-filled beaker (Figure A2a) The bath was raised to boiling (-98C at
51
b
c
Figure A2 Implementation ofhot water and percussion scarification A2a Hot water bath A2b Paint-shaker set-up A2c Placing soil tin containing seeds at end ofpaint shaker ann
52
Las Cruces NM elevation of 1300 meters) Each 100-seed replicate was placed in a
test tube and each test tube was immediately removed to cool at room temperature
Percussion scarification was implemented using a pneumatic paint shaker
(Central Pneumatic model 00422 Camarillo CA) (Figure A2b) This allowed
standardization of treatment intensity Compressed air pressure was maintained at
530kPa plusmn 30kPa (80psi plusmn 5psi) resulting in approximately 350 oscillations per
minute Each lOO-seed sample was placed in a 118 ml (4 oz) soil tin for shaking To
maximize lateral movement of paint shaker a paint can was used as a spacer to place
soil tins at the end of the shaking arm (Figure A2c) This allowed shaking to occur at
the greatest distance from the pivot point (fulcrum) Following all treatments
including the control seeds were soaked for twenty-four hours in distilled water prior
to plating out
Gennination was evaluated on lab benches at room temperature Each
replicate of seeds was placed in a 100 cm petri dish on moistened filter paper with
humidity maintained by enclosing the petri dishes in ziplock bags Gennination was
monitored daily for 14 days in accordance with International Seed Testing
Association standards for Robinia species (ISTA 1999) Gennination was defined as
protrusion of the radicle from the embryo by at least 1mm as viewed by the naked
eye
Categorical Analysis of Variance using Prpc Catmod (SAS Institute 1989)
was used to detennine treatment differences in germination percentages for each
source This procedure is an extension of a Chi-square test of homogeneity using the
53
natural log of the ratio of germinated to non-germinated seeds for each treatment Pshy
values less than 005 were considered significant A limited set of pairwiseshy
comparisons was conducted to compare treatment mean percents using a
conservative alpha value of 005 divided by the number of comparisons
Results
Percussion maximized germination for both New Mexico locust and black
locust There were three germination response levels for New Mexico locust (Figure
A3) First there was a low response in the control indicating a dormant seed lot
The hot water treatment and I-minute duration of percussion share a second
intermediate response Germination was highest at percussion levels from 2 to 10
minutes
A selected percussion treatment intermediate in time (5 minutes) produced the
steepest germination curve indicating the fastest germination rate (Figure A4) The
hot water treatment produced an intermediate curve or relatively slower germination
rate The control curve was very flat indicating the slowest germination rate for New
Mexico locust
Control seeds germinated poorly for black locust as well (Figure A5) Hot
water scarification improved germination over the control but all levels of percussion
improved germination over hot water The optimal durations of percussion treatment
were bracketed with a significant increase from the 1 to 2 minute percussion level a
high germination response from 2 to 5 minutes of percussion and finally a significant
54
~ d 25 CL)
p t1 0 ~ t1
g Vl ~ Vl
100~---------------------------------------------------------------
80
60
40
20
o Control Hot Water Percussion
I min Percussion
2 min Percussion
3 min
Scarification
Percussion 4 min
Figure A3 Effect of scarification on germination response for New Mexico locust
Percussion 5 min
=significant difference between adjacent treatment bars ns = no significant difference
Percussion 10 min
~ ~ () u agt
Il-t d o ~
~ ~ c3
100middotlr~~~------------------------------------e- Percussion 5min middot 0middot Hot Water -- Control
80-J=middot======
6b-middot-lmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middot--middotmiddot-middot~-middotmiddot -middotmiddot middot middotmiddot-middot 1
40
20
-----~-----------
0 0 0 middotmiddotmiddotmiddotmiddot0middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot0
0 --~-----y-----~------_---~--- T-----~
o shy -TDay 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A4 Effect of selected scarification treatments on germination speed for New Mexico locust First seven days of fourteen-day experiment are shown
Day 7
80
~ cd
5 60 ()
[) Cl d 0 1t1 d
40sect dUl
-J
20
o
ns
Scarification
Figure A5 Effect of scarification on germination response for black locust = significant difference between adjacent treatment bars ns = no significant difference
decrease at the 10-minute level This drop-off suggests that 10 minutes of percussion
over-treats seeds from this lot of-black locust
A selected percussion treatment intermediate in time (5 minutes) also had the
fastest germination rate for black locust (Figure A6)_ The hot water curve is
somewhat flatter indicating a slower germination rate The control curve is very flat
indicating a slow germination rate
A qualitative difference between hot water and percussion-treated seeds was
also observed Along with healthy germinants the hot water treatment produced
over-treated seeds (Figure A 7a) Examples of damaged germinants include seeds
where the radicle did not elongate (Figure A 7b) seeds where the radicle elongated
but was delayed in freeing itself from the seed coat (Figure A7c) and seeds where the
radicle broke off completely from the embryo (Figure A7d) In addition to these
examples of over-treatment there were numerous small dark seeds that failed to
imbibe water For percussion treatment nearly all seeds took up water (Figure A8a)
Germination was rapid and uniform with little damage evident (Figure A8b)
To summarize both New Mexico locust and black locust seed lots were
dormant The standard hot water scarification treatment improved germination but
only to 56 and 41 for New Mexico locust and black locust respectively Nearly
all percussion durations improved total germination as well as germination rate over
the hot water treatment Germination was greater than 90 for percussion durations
of 4 5 and 10 minutes for New Mexico locust and 3 4 and 5 minutes for black locust
58
CI)
sgt t CI) 0
igt 0
sect ttl t E
Vl 0
c3
100r==~==~~=r------------------------------------~ ___ Percussion 5 min
middotmiddotmiddot0middotmiddotmiddot Hot Water -lIf- Control
80r=========~--~~----------------------------~
60
40
20 -~-~---~---------- ---~------------I
omiddot
O
_~_----------lIf-----~bull -----_ shy---shy I
Day 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A6 Effect of selected scarification treatments on germination speed for black locust First seven days of fourteen-day experiment are shown
Day 7
a
b c d
Figure A7 Damage to New Mexico locust seeds observed during germination counts following hot water scarification A7a Hot water replicate including damaged seedlings and numerous unimbibed seeds A7b Close-up ofseed where radicle failed to elongate A7c Close-up ofseed where radicle elongated but was delayed in freeing itself from the seed coat A7d Close-up ofseed where radicle completely broke offfrom embryo
60
a
b
Figure A8 New Mexico locust seeds following percussion treatment at time of germination counts A8a Percussion replicate with uniformly healthy seedlings and near complete imbibition of seeds A8b Close-up ofpercussion-treated seedling
61
Discussion
Papilionoid seeds have a specific anatomical feature known as the strophiole
or lens-the natural site of water entry to the seed (Hamly 1932) The strophiole is
located on the cotyledonary lobe of a locust seed (Figure A9) When a papilionoid
legume seed is percussed for the appropriate amount of time repeated hits on the
integrated seed coat loosen the constrained cells of the strophiolar region without
excessively damaging the rest of the seed coat (Ballard 1976)
When a percussed seed is soaked water enters exclusively through the
strophiole in a controlled manner (Kelly and Staden 1987) This regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues This
contrasts with seeds that have been hot water or acid scarified treatments that can
randomly degrade the seed coat Unlocalized cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rehman et al 1998 Marunda 1990)
Papilionoids are the largest subfamily of legumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust and black locust these legumes often play an integral role in the
revegetation ofdisturbed lands Developing superior scarification methods to the
standard hot water and acid treatments should fadlitate the use ofthese legumes in
restoration projects
62
Figure A9 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (bc)
63
APPENDIXB
GERMINATION PERCENTS AND STANDARD ERRORS FOR SOURCE
BLOCK AND TREATMENT FOR NEW MEXICO LOCUST
Table B 1 Gennination percents and standard errors for source block and treatment for New Mexico locust
Source Block Treatment Germination Percentage Standard Error
Capitan 1 Immersion 74 437 Capitan 2 Immersion 69 462 Capitan 3 Immersion 71 454 Capitan 4 Immersion 67 470 Capitan Boiling 30 sec 79 407 Capitan 2 Boiling 30 sec 76 427 Capitan 3 Boiling 30 sec 72 449 Capitan 4 Boiling 30 sec 76 427 Capitan Boiling 60 sec 71 454 Capitan 2 Boiling 60 sec 54 498 Capitan 3 Boiling 60 sec 67 470 Capitan 4 Boiling 60 sec 76 427 Capitan I Control 5 218 Capitan 2 Control 7 255 Capitan 3 Control 4 196 Capitan 4 Control 5 218 Capitan 1 Percussion I min 47 499 Capitan 2 Percussion I min
470
Capitan Percussion I min 39 488
Capitan 4 Percussion I min 38 485 Capitan I Percussion 2 min 55 497 Capitan Capitan
2
Percussion 2 min Percussion 2 min
71 65
454 477
Capitan 4 Percussion 2 min 56 496 Capitan I Percussion 4 min 86 347 Capitan 2 Percussion 4 min 79 407 Capitan 3 Percussion 4 min 81 392 Capitan 4 Percussion 4 min 82 384 Capitan I Percussion 8 min 90 300 Capitan 2 Percussion 8 min 88 325 Capitan 3 Percussion 8 min 90 300 Capitan 4 Percussion 8 min 90 300 Gila I Immersion 71 454 Gila 2 Immersion 77 421 Gila
Immersion 75 433
Gila 4 Immersion 72 449
Gila I Boiling 30 sec 68 466
Gila 2 Boiling 30 sec 81 392
Gila 3 Boiling 30 sec 54 498
Gila 4 Boiling 30 sec 63 483
Gila I Boiling 60 sec 55 497
Gila 2 Boiling 60 sec 68 466
Gila 3 Boiling 60 sec 57 495
Gila 4 Boiling 60 sec 70 458
65
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Gila 2 Control 13 336 Gila 3 Control 13 336 Gila 4 Control 20 400 Gila 1 Percussion 1 min 66 474 Gila 2 Percussion 1 min 57 495 Gila 3 Percussion 1 min 73 444 Gila 4 Percussion 1 min 82 384 Gila 1 Percussion 2 min 81 392 Gila 2 Percussion 2 min 86 347 Gila 3 Percussion 2 min 85 357 Gila 4 Percussion 2 min 54 498 Gila 1 Percussion 4 min 79 407 Gila 2 Percussion 4 min 83 376 Gila 3 Percussion 4 min 84 367 Gila 4 Percussion 4 min 79 407 Gila 1 Percussion 8 min 51 500 Gila 2 Percussion 8 min 76 427 Gila 3 Percussion 8 min 52 500 Gila 4 Percussion 8 min 74 439 Holman 1 Immersion 83 376 Holman 2 Immersion 86 347 Holman 3 Immersion 81 392 Holman 4 Immersion 90 300 Holman 1 Boiling 30 sec 90 300 Holman 2 Boiling 30 sec 85 357 Holman 3 Boiling 30 sec 87 336 Holman 4 Boiling 30 sec 83 376 Holman 1 Boiling 60 sec 79 407 Holman 2 Boiling 60 sec 80 400 Holman 3 Boiling 60 sec 86 347 Holman 4 Boiling 60 sec 93 255 Holman Control 25 433 Holman 2 Control 19 392 Holman Control 21 407
Holman 4 Control 24 427
Holman Percussion 1 min 77 421
Holman 2 Percussion 1 min 85 357
Holman 3 Percussion 1 min 80 400
Holman 4 Percussion 1 min 89 313
Holman 1 Percussion 2 min 88 325
Holman 2 Percussion 2 min 96 196
Holman 3 Percussion 2 min 93 255
Holman 4 Percussion 2 min 96 196
Holman 1 Percussion 4 min 92 271
Holman 2 Percussion 4 min 97 171
66
I
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Holman 3 Percussion 4 min 91 286 Holman 4 Percussion 4 min 96 196 Holman 1 Percussion 8 min 88 325 Holman 2 Percussion 8 min 85 357 Holman 3 Percussion 8 min 83 376 Holman 4 Percussion 8 min 82 384 Jemez 1 Immersion 53 499 Jemez 2 Immersion 44 496 Jemez 3 Immersion 52 500 Jemez 4 Immersion 70 458 Jemez 1 Boiling 30 sec 58 494 Jemez 2 Boiling 30 sec 55 497 Jemez 3 Boiling 30 sec 48 500 Jemez 4 Boiling 30 sec 65 477 Jemez 1 Boiling 60 sec 68 466 Jemez 2 Boiling 60 sec 63 483 Jemez 3 Boiling 60 sec 73 444 Jemez 4 Boiling 60 sec 89 313 Jemez 1 Control 2 140 Jemez 2 Control 9 286 Jemez 3 Control 6 237 Jemez 4 Control 8 271 Jemez 1 Percussion 1 min 58 494 Jemez 2 Percussion 1 min 63 483 Jemez 3 Percussion 1 min 77 421 Jemez 4 Percussion 1 min 89 313 Jemez 1 Percussion 2 min 68 466 Jemez 2 Percussion 2 min 64 480 Jemez 3 Percussion 2 min 80 400 Jemez 4 Percussion 2 min 83 376 Jemez Percussion 4 min 68 466 Jemez 2 Percussion 4 min 79 407 Jemez 3 Percussion 4 min 70 458 Jemez 4 Percussion 4 min 83 376 Jemez 1 Percussion 8 min 52 500 Jemez 2 Percussion 8 min 59 492 Jemez 3 Percussion 8 min 61 488 Jemez 4 Percussion 8 min 70 458 Magdalena 1 Immersion 69 462 Magdalena 2 Immersion 69 462 Magdalena J Immersion 70 458 Magdalena 4 Immersion 85 357 Magdalena 1 Boiling 30 sec 65 477 Magdalena 2 Boiling 30 sec 71 454 Magdalena 3 Boiling 30 sec 68 466
67
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Magdalena 4 Boiling 30 sec 83 376 Magdalena 1 Boiling 60 sec 63 483 Magdalena 2 Boiling 60 sec 68 466 Magdalena 3 Boiling 60 sec 63 483 Magdalena 4 Boiling 60 sec 82 384 Magdalena 1 Control 11 313 Magdalena 2 Control 6 237 Magdalena 3 Control 5 218 Magdalena 4 Control 4 196 Magdalena Percussion 1 min 32 466 Magdalena 2 Percussion 1 min 45 497 Magdalena 3 Percussion 1 min 46 498 Magdalena 4 Percussion I min 36 480 Magdalena 1 Percussion 2 min 67 470 Magdalena 2 Percussion 2 min 68 466 Magdalena 3 Percussion 2 min 65 477 Magdalena 4 Percussion 2 min 81 392 Magdalena 1 Percussion 4 min 87 336 Magdalena 2 Percussion 4 min 97 171 Magdalena 3 Percussion 4 min 83 376 Magdalena 4 Percussion 4 min 97 171 Magdalena 1 Percussion 8 min 98 lAO Magdalena 2 Percussion 8 min 100 000 Magdalena 3 Percussion 8 min 96 196 Magdalena 4 Percussion 8 min 98 lAO Manzano 1 Immersion 67 470 Manzano 2 Immersion 57 495 Manzano 3 Immersion 72 4049 Manzano 4 Immersion 73 4044 Manzano 1 Boiling 30 sec 70 458 Manzano 2 Boiling 30 sec 64 480 Manzano 3 Boiling 30 sec 71 454 Manzano 4 Boiling 30 sec 80 400 Manzano I Boiling 60 sec 50 500 Manzano 2 Boiling 60 sec 53 499 Manzano 3 Boiling 60 sec 59 492 Manzano 4 Boiling 60 sec 74 439 Manzano I Control 4 196 Manzano 2 Control 8 271 Manzano 3 Control 1 099 Manzano 4 Control 6 237 Manzano 1 Percussion 1 min 52 500 Manzano 2 Percussion 1 min 39 488 Manzano 3 Percussion 1 min 36 480 Manzano 4 Percussion 1 min 51 500
68
Table B 1 Continued
Source Block Treatment Gerrni~ation Percentage Standard Error
Manzano Percussion 2 min 53 499 Manzano 2 Percussion 2 min 58 494 Manzano 3 Percussion 2 min 61 488 Manzano 4 Percussion 2 min 62 485 Manzano 1 Percussion 4 min 76 427 Manzano 2 Percussion 4 min 85 357 Manzano 3 Percussion 4 min 79 407 Manzano 4 Percussion 4 min 83 376 Manzano 1 Percussion 8 min 87 336 Manzano 2 Percussion 8 min 83 376 Manzano 3 Percussion 8 min 83 376 Manzano 4 Percussion 8 min 75 433 Raton 1 Immersion 54 498 Raton 2 Immersion 37 483 Raton 3 Immersion 39 488 Raton 4 Immersion 69 462 Raton 1 Boiling 30 sec 36 480 Raton 2 Boiling 30 sec 20 400 Raton 3 Boiling 30 sec 28 449 Raton 4 Boiling 30 sec 60 490 Raton 1 Boiling 60 sec 15 357 Raton 2 Boiling 60 sec 171 Raton 3 Boiling 60 sec 4 196 Raton 4 Boiling 60 sec 35 477 Raton 1 Control 4 196 Raton 2 Control 12 325 Raton 3 Control 2 140 Raton 4 Control 6 237 Raton 1 Percussion 1 min 52 500 Raton 2 Percussion I min 29 454 Raton 3 Percussion I min 35 477 Raton 4 Percussion 1 min 31 462 Raton 1 Percussion 2 min 55 497 Raton 2 Percussion 2 min 35 477 Raton 3 Percussion 2 min 33 470 Raton 4 Percussion 2 min 42 494 Raton 1 Percussion 4 min 65 477 Raton 2 Percussion 4 min 52 500 Raton Percussion 4 min 51 500 Raton 4 Percussion 4 min 53 499 Raton 1 Percussion 8 min 88 325 Raton 2 Percussion 8 min 80 400 Raton 3 Percussion 8 min 73 444 Raton 4 Percussion 8 min 74 439 Sacramento 1 Immersion 43 495
69
Table B 1 Continued
-Source Block Treatment Germination Percentage Standard Error
Sacramento 2 Immersion 30 458 Sacramento 3 Immersion 38 485 Sacramento 4 Immersion 56 496 Sacramento 1 Boiling 30 sec 47 499 Sacramento 2 Boiling 30 sec 48 500 Sacramento 3 Boiling 30 sec 48 500 Sacramento 4 Boiling 30 sec 56 496 Sacramento 1 Boiling 60 sec 52 500 Sacramento 2 Boiling 60 sec 67 470 Sacramento 3 Boiling 60 sec 43 495 Sacramento 4 Boiling 60 sec 48 500 Sacramento 1 Control 0 000 Sacramento 2 Control 3 171 Sacramento 3 Control 3 171 Sacramento 4 Control 099 Sacramento 1 Percussion 1 min 57 495 Sacramento 2 Percussion 1 min 61 488 Sacramento 3 Percussion 1 min 62 485 Sacramento 4 Percussion 1 min 55 497 Sacramento 1 Percussion 2 min 84 367 Sacramento 2 Percussion 2 min 70 458 Sacramento 3 Percussion 2 min 81 392 Sacramento 4 Percussion 2 min 69 462 Sacramento 1 Percussion 4 min 91 286 Sacramento 2 Percussion 4 min 84 367 Sacramento 3 Percussion 4 min 84 367 Sacramento 4 Percussion 4 min 94 237 Sacramento 1 Percussion 8 min 82 384 Sacramento 2 Percussion 8 min 81 392 Sacramento 3 Percussion 8 min 82 384 Sacramento 4 Percussion 8 min 81 392 Sandia 1 Immersion 76 427 Sandia 2 Immersion 70 458 Sandia 3 Immersion 70 458 Sandia 4 Immersion 81 392 Sandia 1 Boiling 30 sec 78 414 Sandia 2 Boiling 30 sec 71 454 Sandia 3 Boiling 30 sec 63 483 Sandia 4 Boiling 30 sec 78 414 Sandia 1 Boiling 60 sec 67 470 Sandia 2 Boiling 60 sec 65 477 Sandia 3 Boiling 60 sec 74 439 Sandia 4 Boiling 60 sec 66 474 Sandia 1 Control 16 367 Sandia 2 Control 7 255
70
Table B 1 Continued
Source Block Treatment Gennination Percentage Standard Error
Sandia 3 Control 8 271 Sandia 4 Control 16 367 Sandia 1 Percussion 1 min 67 470 Sandia 2 Percussion 1 min 61 488 Sandia 3 Percussion 1 min 66 474 Sandia 4 Percussion 1 min 59 492 Sandia I Percussion 2 min 82 384 Sandia 2 Percussion 2 min 83 376 Sandia 3 Percussion 2 min 81 392 Sandia 4 Percussion 2 min 84 367 Sandia 1 Percussion 4 min 89 313 Sandia 2 Percussion 4 min 86 347 Sandia 3 Percussion 4 min 89 313 Sandia 4 Percussion 4 min 85 357 Sandia 1 Percussion 8 min 80 400 Sandia 2 Percussion 8 min 77 421 Sandia 3 Percussion 8 min 69 462 Sandia 4 Percussion 8 min 66 474
LITERATURE CITED
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Ballard LAT 1973 Physical barriers to gennination Seed Sci Technoll 285-303
___ 1976 Strophiolar water conduction in the seeds ofTrifolieae induced by action on the testa at nonstrophiolar sites Austr J Plant Physiol3 465-469
Barton LV 1947 Special studies on seed coat impenneability Contrib Boyce Thompson Inst 14 355-362
Baskin CC Baskin JM 1998 Seeds Ecology biogeography and evolution of donnancy and gennination Academic Press London UK 666 p
Bewley JD 1997 Seed germination and donnancy Plant Ce119 1055-1066
Bewley JD Black M 1994 Seeds physiology of development and germination 2nd ed Plenum Press New York NY 445 p
Briscoe CB 1996 Growing Nitrogen Fixing Trees in Acid Soils In MH Powell (ed) Nitrogen Fixing Trees for Acid Soils- A Field ManuaL Winrock International Morrilton AR 22 p
Copeland LO McDonald MB 1995 Principles of Seed Science and Technology 3rded Chapman amp Hall New York NY 409 p
Cox RE Klett JE 1984 Seed gennination requirements of native Colorado plants for use in the landscape Plant Propag 30(2) 6-10
Dell B 1980 Structure and function ofthe strophiolar plug in seeds ofAlbizia Lophantha Am J Bot 67 556-563
Dick-Peddie WA 1993 New Mexico Vegetation Past Present and Future University ofNew Mexico Press Albuquerque NM 244 p
Dreesen DR 2001 Propagation protocol for production ofcontainer Robinia neomexicana plants Los Lunas Plant Materials Center Los Lunas New Mexico In Native Plant Network URL httpwwwnativeplantnetworkorg (accessed 9 January 2002) Moscow (ID) University ofIdaho CoIl Natural Resources For Res Nursery
72
Dreesen DR Harrington JT 1997 Propagation of native plants for restoration projects in the Southwestern US- Preliminary Investigations In Landis TD Thompson JR tech coords National Proceedings Forest and Conservation Nursery Assocs Gen Tech Rep PNW-GTR-419 Portland OR USDA For Ser PNW Res Sta 77-88
Gottfried GJ 1980 Control ofNew Mexican locust and the effect on planted ponderosa pine in central Arizona US For Ser Res Note RM-386 Rocky Mountain For Range ExpStn Fort Collins CO
Gosling PG Samuel YK Jones SK 1995 A systematic examination of germination temperature chipping and water temperaturesoak duration pretreatments on the seeds ofLeucaena leucocephala Seed Sci amp Technol 23521-532
Hamly DH 1932 Softening of the seeds of Melilotus alba Bot Gazette 93 345shy375
Hanna PJ 1984 Anatomical features of the seed coat ofAcacia kempeana (Mueller) which relate to increased germination rate induced by heat treatment New Phytol 96 23-29
Hanks JP Dick-Peddie WA 1974 Vegetation patterns of the White Mountains New Mexico Southwest Naturalist 18(4) 371-382
Hine S Harrington JT Dreesen DR 1997 Seed scarification requirements for Robinia neomexicana In Harrington JTtech coord Proc Third SouthwestemContainer Growers Conf Feb 12-13 1997 Ft Collins CO
Hopkinson JM 1993 The strophiole of leguminous seeds Newsletter- IntI Herbage Seed Production Res Grp 18 7-8
---- 1997 Differences in hardseededness between Cunningham and Tarramba leucaena In Leucnet News The Newsletter of the International LeucaenaRes and Devlt Netwk No4
ISTA (International Seed Testing Association) 1999 International rules for seed testing rules Seed Sci amp Technol27 Supplement
Kelly KM van Staden J 1987 The lens as the site of permeability in the papilionoid seed Aspalathus linearis J Plant Physiol 128(45) 395-404
73
Keresztesi B 1988 Black locust the tree of agriculture Outlook on Agr 17(2) 77~85
Kigel 1 and Galili G 1995 Seed development and germination Marcel Dekker New York 853 p
Klemmedson 10 1994 New Mexico locust and parent material Influence on macronutrients of forest floor and soil Soil Sci Soc Am 1 58 974~980
Kuo WH Tam AW 1988 The pathway of water absorption of mung bean seeds Seed Sci amp Technol16(1) 139~144
Leadem CL 1997 Dormancy--unlocking seed secrets In Landis TD Thompson lR tech coords National Proceedings Forestand Conservation Nursery Assocs Gen Tech Rep PNW-G TR-419 Portland OR USDA For Ser PNW Res Sta 43-52
Lin Y Wagner MR Seweyestewa A Honahni MD DeGomez M McMillin JD 1996 Influence of cultural treatments on seed germination and seedling growth of Robinia neomexicana Forest Farm and Community Tree Res Reports Volume 1
Marunda CT 1990 Effects of seed pretreatment on the development ofAcacia auriculforinis and A holosericea seedlings ACIAR Proceedings Series No 28
Meginnis HG 1937 Sulphuric acid treatment to increase germination of black locust seed USDA Circ 453 34 p
Miklas PN Towensend CE Ladd SL 1987 Seedcoat anatomy and the scarification ofcicer milkvetch seed Crop Sci 27 766-772
Monsen SB 1984 Use of shrubs on mine spoils In The Challenge ofProducing Native Plants for the Intermountain Area Proceedings Intermountain Nurserymans Association 1983 conference Las Vegas NV compo PM Murphy 26-31 Ogden UT USDA For Ser Gen Tech Report INT-l68
Olson DF 1974 Robinia L Locust In Seeds of Woody Plants in the US tech coord CS Schopmeyer 728-731 USDA For Ser Agr Handbk No450
Rehman S Loescher RNl Harris P1C 1998 Dormancy breaking and germination ofAcacia salicina Lindl seeds Seed Sci amp Techno127 553-557
Rolston MP 1978 Water impermeable seed dormancy Bot Rev 44 365-396
74
SAS Institute Inc 1989 SASSTAT Users Guide Version 6 4th Edition Volume 1 Cary NC SAS Institute Inc 943 p
SerratoValenti G Conara L Ferrando M Modenesi P 1993 Structural and histochemical features ofStylosanthes scabra (Leguminosae Papilionoidae) seed coat as related to water entry Can J Bot 71 834-840
Simpson BJ 1988 A field guide to Texas trees Austin TX Texas Monthly Press 372p
Tran VN Cavanagh AK 1984 Structural aspects ofdormancy In Seed Physiology Vol 2 Murray DR (ed) p1-44 Academic Press Australia
USDA Forest Service 1948 Woody-plant seed manual USDA Misc Pub1654 416p
Vines RA 1960 Trees Shrubs and Woody Vines of the Southwest Austin Univ ofTexas Press 1104 p
Wagner MR Larson RE Craig TP 1992 Ecology and silvics ofNew Mexico locust (Robinia neomexicana) with an emphasis on the arboreal form p53-57 In JW Hanover K Miller and S Plesko (eds) Proc Intemat Confon Black Locust Biology Culture and Utilization Mich State Univ USA
Wilson JK 1944 Immersing seeds of species of Robinia in boiling water hastens germination J For 42 453-454
Zimmerman RW Carpenter SB 1980 First-year coppice production from a 5shyyear old black locust stand on surface mine spoil p309-314 Centr Hardwd For ConfProc University ofMissouri Columbia
-l
Figure 1 Seed source collection map All sources were collected within New Mexico USA separated by at least 4S lan
IIIRZltItW lIJPIirmiddot rllll1lbullbull_~~~_II
Table 1 Latitude longitude location elevation and collection date ofNew Mexico locust seed sources
Lot title Latitude (N) Longitude (W) Location Elevation (m) Collection Date
Raton 36deg5846 104deg2872 Raton Pass NM 2295 82500
Holman 36deg0306 105deg2429 HolmanNM 2476 911500
Jemez 35deg4500 106deg4100 Jemez Springs NM 1976 9900 00
Sandia 35deg1083 106deg2341 Cedar Crest NM 2432 82600
Manzano 34deg3689 106deg2494 ManzanoNM 2465 9200
Magdalena 33deg5953 107deg0903 Magdalena NM 2544 82900
Capitan 33deg2728 105deg4325 Alto NM 2189 911200
Sacramento 32deg5798 105deg4496 Cloudcroft NM 2630 91400 Gila 32deg5400 107deg4600 Kingston NM 2432 9700
f
Table 2 Mean seed weight and standard error by source
a
Source Mean seed weight (g) for Standard error 100 seeds n = 5
Raton Holman Jemez Sandia Manzano Magdalena Capitan Sacramento Gila
2872 b c 2522 d 1826 e 2753 c 2413 d 2559 d 2443 d 3130 a 3029 ab
00103 00403 00276 00301 00370 00374 00458 00279 00348
a Mean seed weights followed by the same letter are not significantly
different
19
experiment was conducted utilizing a randomized complete block design blocked by
time with a factorial treatment structure Experimental factors were seed source and
scarification treatment Nine seed sources from throughout New Mexico were used
(Figure 1 Table 1) Scarification treatments included a control three levels ofhot
water and four levels of percussion Hot water treatments included immersion at
100degC and boiling for periods of 30 seconds and 60 seconds Percussion treatment
levels were 1 2 4 and 8 minutes One hundred seeds per block were used to test
each treatment combination Four blocks of seed were tested one at a time (all seed
sources included in each run) in close chronological sequence (within 6 weeks) A
total of four 100-seed samples were used to test each source by scarification treatment
combination
Treatments
Hot water treatment of seeds utilized 50 ml test tubes filled with 30 ml
distilled water Each test tube was used to treat one 100-seed sample at a time
resulting in a ratio of4 parts water to 1 part seed by volume Test tubes were placed
in a beaker with the beaker filled with tap water to the same level as the water within
test tubes Nine test tubes (one per seed source) were placed in the beaker
simultaneously so that one replication of each seed sourcescarification treatment
cotnblnatlon was conducted simultaneously Hot water treatments include immersion
boiling For immersion treatments seeds were added to test tubes with the water
(boiling) and the test tubes were immediately removed from the beaker to
For the boiling treatments test tubes remained in the beaker with water at
20
boiling temperature for a set duration after the addition of seeds Following all
treatments test tubes with seeds cooled 24 hours in a test tube rack
Percussion treatment utilized a pneumatic paint shaker (Central Pneumatic
model 00422 Camarillo CA) Compressed air was maintained at 530 kPa plusmn30 kPa
(80 psi plusmn 5 psi) resulting in approximately 350 oscillations per minute Seeds were
placed in 118 ml (4 oz) soil tin cans (US Can Lobard IL) and the lids were secured
with duct tape Each can was used to treat one 100-seed sample To take advantage
ofgreatest lateral movement of the paint shaker a spacer (paint can) was used to
position the soil tin at the end of the shaking arm This allowed shaking to occur at
the greatest distance from the pivot point (fulcrum) Tins were placed perpendicular
to the direction of shaking motion allowing seeds to impact at greatest force by
hitting flat surfaces of the soil tin
Germination Testing
Before gennination testing all seeds were soaked 24 hours in reverse
osmosis-treated water (this was included in the process ofhot water treatment
jU~SCIlOea above) Following soaking seeds were placed in 100 cm Petri dishes lined
90 cm Whatman qualitative 1 filter paper circles (MG Scientific Pleasant
WI) One Petri dish was used for each treatment by source combination
papers were initially moistened with 5 mL reverse osmosis-treated water then
~VU1CUlJl moistened as needed to ensure adequate conditions for gennination
were monitored daily (at 300 PM) for gennination for 14 days in accordance
~UIUlnJlVUa Seed Testing Association (ISTA) standards for Robinia species
21
(1STA 1999) Tests were carried out at room temperature (22degC plusmnOSoC)
Germination was classified as protrusion of the radicle from the embryo by at least
1mm as viewed with the naked eye
X-ray Radiography
On August 132001 three lOO-seed samples from the Sacramento seed source
were treated for 14 or 8-minute percussion durations The seeds were sent
immediately by overnight delivery to the Faxitron X-Ray Corporation in Wheeling
Illinois where Dr Bob Karrfalt ofthe USDA Forest Service oversaw their imaging
Radiographs were made at SX with a Faxitron Specimen Radiography System Model
MX-20
Scanning Electron Microscope Photography
On February 192002 a Hitachi S-3200N Variable Pressure Scanning
Electron Microscope (Mountain View CA) was used to examine strophiolar cells of
untreated seeds and seeds percussed for 4 minutes Seeds were cut longitudinally
igt their entire length using a razor blade splitting the strophiolar region roughly in
Cross sections of seed samples magnifying the strophiolar region were viewed
600X under the microscope
Analysis
Categorical Analysis of Variance was used to determine treatment source and
(Le time) main effects and interactions The response variable was total
~vlI This procedure is a Chi-square test of homogeneity using the natural log
of germinated to non-germinated seed for each treatment Maximumshy
22
likelihood analysis was used to calculate Chi-square test statistics with their observed
significance levels A single contrast was conducted to cempare treatment means
against the control Observed significance levels less than or equal to 005 were
considered significant Pairwise comparisons were conducted between non-control
treatment mean percents using a conservative alp~a value of 005 divided by the
number of comparisons (Bonferroni approach) and approximate pairwise z statistics
Categorical analyses were performed using Proc Catmod (SAS Institute 1989)
RESULTS
Scarification seed source block and all interactions between factors
influenced germination (Table 3) The main effects are reported in the presence of
these interactions because the overall robustness of treatments and seed sources are of
interest Background levels ofnaturally occurring variation in seed sources are
seen in control germination percentages in Figure 2 In particular time variability
is unlikely to be under the control of the producer Appendix B details experimental
data for source by treatment gennination percents for each of the four blocks
Averaged over all treatments germination by source ranged from 40 to
79 (Figure 2) Averaged over all sources (Figure 3) and for each source (Figure 4
5) percussion scarification durations of4 or 8 minutes yielded greatest germination
Averaged over all sources germination increased with increasing durations of
percussion then leveled off for the 4- and 8-minute treatments (Figure 3) Immersion
and 30-second boiling scarification maximized germination for the hot water
treatments with a decrease in germination when seeds were boiled for 60 seconds
For all but two sources there is a similar ipcreasing trend in germination from
1to 4 minutes ofpercussion (Figure 4) The Holman source increased in germination
1 to 2 minutes then leveled off at 4 minutes The Jemez source changed little
1 to 4 minutes of treatment As treatment duration increased from 4 to 8
mutes five sources decreased in germination (Figure 4a) whereas three sources
24
Table 3 Categorical Analysis of Variance table for germination response to seed treatment seed source block and the interaction of the factors
Source of DF Chi-Square Observed Variability Significance Level
Source 8 98370 lt00001 Trt 7 233876 lt00001
contrast 1 16976 lt00001 SourceTrt 56 97077 lt00001 Block 3 5538 lt00001
SourceBlock 24 10162 lt00001 TrtBlock 21 7564 lt00001
SourceTrtBlock 168 34283 lt00001
S 4)
8 ~
0
sectg ~ 40 ~
N 0 0
20
o f0ltgt ~ ~4 jybshy ((iltgto ~~ K1ltgt 6~b-~ ~~ 0b-~~ ~~ ~b-~ ~b-~b
80
60 -I-- - ~- middotmiddotmiddotmiddotmiddot1
r -] Northern NM Sources
Central NM Sources
Southern NM Sources
sect-o ~
~b-v(i
SOURCE
Figure 2 Effect ofNew Mexico locust seed source on germination for data averaged over all scarification treatments Control germination is shown by solid line within bar
80
~ cd ~ ltl) () 60 ~ ~
N
t 0shyrti tshySltl)
40
0
20
o lmmersion Boiling 30 sec Boiling 60 sec Control 1 min 2min 4 min 8 min
Hot Water Treatments Percussion Treatments
Figure 3 Effect of scarification on germination ofNew Mexico locust for data averaged over all seed sources Means labelled with the same letter are not significantly different
100~----------------------~--~----------------~
80
60
ltU
~
40 IJ
sectgsI
l100
80
60
40
a I middotmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot middotImiddotmiddotmiddotmiddotmiddotmiddot E
_~iii-bull _ _ bull f --~--
t-- -~ ---- -shy ---- shyI ~ ---- - shy
L
fiII
~
b
- shy ---r
- Holman
Sacramento Sandia Gila Jemez
c ~
ltgt ~
Tmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot-I
j~=~=-~~-1
I middotmiddot ~ bull r
t tr
Y -------M-a-gd-a-Ie-n-a--
1 _ _
fCapitan
---- Manzano ~y -_ Raton
2 4 8
Time interval ofpercussion scarification (min)
Figure 4 Effect of seed source by treatment interaction on New Mexico locust germination for percussion scarification pattern a and pattern b
28
tv 0
~ d
~ A s o
1 c3
80
60
40
20
o
TIT
t------=middotfmiddotmiddot-middotmiddot-middotmiddot-middotmiddot 1- --- - - --=- - -shy--cE= - _--shy --shy ----shy ~ ~ ---shy ~ -~- _ --shy-------r 1
I
Raton Holman
-_ Jemez
--- Sandia Manzano Magdalena Capitan Sacramento Gila
Immersion Boiling 30 sec Boiling 60 sec
Hot Water Scarification
Figure 5 Effect of seed source by treatment interaction on New Mexico locust germination for hot water scarification
continued to increase in germination (Figure 4b) There was no difference between
the two treatment levels for the Manzano source (Figure 4b)
The effect ofhot water scarification also varied among sources with few
overall differences between hot water treatments (Figure 5) However boiling for 60
seconds yielded highest germination for the Jemez source whereas increasing
durations ofboiling decreased germination for the Raton source
In addition to consistently high germination 4- and 8-minute percussionshy
treated seeds germinated as fast or faster than all other treatments (Figure 6 Table 4)
050 measured as the number of days needed to complete 50 ofeventual
germination averaged 2 days for both ofthese treatments
X-ray radiographs were taken of seeds collected from the Sacramento source
percussed for 1 4 or 8 minutes (Figure 7) Seeds show signs of damage at 8 minutes
ofpercussion (Figure 8) Compared to an undamaged seed percussed for only 4
minutes (Figure 8a) damage at the 8-minute level includes fractured radicles pitting
of the seed coat and chips in seed coat structure (Figure 8b-d)
Scanning electron microscope (SEM) images at 600X reveal the direct effect
of 4 minutes of percussion on cells of the strophiolar region (Figure 9) Whereas
untreated seeds retain cellular organization in the strophiolar region strophiolar cells
seeds treated for 4 minutes ofpercussion separate and raise resulting in a
~-~e~U appearance
30
Umiddot-~middot--l-~---middotymiddotmiddotmiddot-----middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot--middot--middotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middotmiddotmiddotmiddotmiddotmiddotmiddot- -
050= gt1
-e-- Percussion 8 min -0- Percussion 4 min ---T- Percussion 2 min -----ltil- Percussion 1 min -- Immersion --0- Boiling 30 sec -+- Boiling 60 sec -ltgt- Control
2 4 6 8 10 12
Day Figure 6 Timecourse ofNew Mexico locust gennination following scarification
treatments G50 is the number of days to 50 of eventual germination
31
14
Table 4 Effect of seed source and treatment on G50 (days to 50 germination)
Seed Source Control Immersion B30sec B60sec Plmin P2min P4min P8min mean Raton 3 3 5 7 4 3 2 2 4 Holman 2 2 2 2 2 2 2 2 2
Jemez 4 6 6 5 3 2 2 2 4 Sandia 2 2 2 2 2 2 2 2 2
w tv Manzano 5 2 2 3 3 2 2 2 3
Magdalena 4 2 2 3 4 3 2 1 3
Capitan 5 4 2 2 3 2 2 2 3
Sacramento 3 2 2 2 4 3 2 2 3 Gila 3 2 2 2 2 2 2 2 2 mean 3 3 3 3 3 2 2 2 3 standard 11 14 16 18 09 05 0 03 08 deviation
Note Columns and rows may not agree due to rounding
Figure 7 X-ray radiographs ofSacramento seed source percussed for 1 4 or 8 minutes
33
Figure 8a-d Seed damage close-ups Figure a shows undamaged seed percussed for 4 minutes Figures b-d show seeds percussed for 8 minutes Damage includes fracturing ofradicle in b complete removal ofseed coat in c and pitting ofseed coat in d
34
Untreated Treated
Figure 9 SEM images ofuntreated vs 4-minute percussion-treated strophiolar cells ofNew Mexico locust seeds Treated cells are loosened separated and raised
DISCUSSION
Determining the Optimal Treatment
Averaging results of scarification treatmems across seed sources is a useful
tool to predict which treatment is most likely to be successful for an unknown seed
source Averaged over all sources four- and eight-minute percussion treatments
resulted in the highest germination 815 and 790 respectively and produced
germination that was as fast or faster than all other treatments Although there was no
difference between these two treatments on average an argument for 4 minutes of
percussion as a standard protocol for treating New Mexico locust seeds can be made
Descriptively 4 minutes of percussion improved germination over 8 minutes
ofpercussion for a majority of sources (five out of nine) 8 minutes of percussion
improved germination for three ofnine sources with no difference for a fmal source
For individual sources 4 minutes ofpercussion was always the best or secondmiddot best
treatment and was always equal to or better than all hot water treatments
Germination at 8 minutes ofpercussion was lower than all other percussion durations
for the Jemez and Gila sources and was lower than at least one hot water treatment for
these sources as well
The x-ray radiographs of the Sacramento seed source illustrate the excessive
internal and external seed damage that can occur with 8 minutes of percussion
germination counts 8-minute percussed seeds from Holman and Capitan
ClUll signs of damage that were visible to the naked eye (see below)
r~evertheless some of these seeds still fit the criteria for germination Neither x-ray
36
photography nor visible observations during seed counts revealed seed damage at the
4-minute percussion level In operational trials seeds percussed for 4 minutes
developed into healthy seedlings (Khadduri and Harrington unpublished data)
Strophiole and Seed Coat Behavior
Four minutes ofpercussion successfully breaks seed dormancy in a large
percentage ofNew Mexico locust seeds without excessively damaging the
surrounding seed coat or internal seed coat structures Most papilionoid legume
seeds including seeds ofNew Mexico locust have a specific region of the seed coat
known as the strophiole or lens (Hamly 1932) The strophiole is located on the
cotyledonary lobe of a locust seed (Figure 10 a-c) If seeds are not indiscriminately
damaged the strophiole is the first point of water entry to the embryo (Hopkinson
1997) When a papilionoid seed is percussed for the appropriate amount of time
repeated hits on the integrated seed coat loosen the constrained cells of the strophiolar
region without excessively damaging the rest of the seed coat (Ballard 1976) SEM
photos illuStrate that 4 minutes of percussion loosened and separated strophiolar cells
ofNew Mexico locust seeds when compared with constrained organized strophiolar
cells of untreated seeds (Figure 9) Research-has shown that water passes to the
embryo through the treated strophiole in a controlled manner and regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues
(Kelly and van Staden 1987)
37
10 Strophiole ofunimbibed New Mexico locust seed (a) and following uptake (bc)
38
In contrast hot-water treatments can excessively degrade the seed coat
(Rebman et al 1998 Marunda 1990) Random cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rebman et aI 1998 Marunda 1990) Along with a
proportionately large amount ofun-imbibed (thus under-treated) seeds (Figure l1a)
damage was observed in hot-water treated seeds from six sources during germination
counts Damage included seeds where the radicle failed to elongate (Figure 11 b)
seeds where the radicle elongated but was delayed in freeing itself from the seed coat
(Figure 11c) and seeds where the radicle completely broke off from the embryo
(Figure lId) In addition some seeds germinated in reverse (the cotyledon emerged
fIrst) If the radicle emerged completely from the seed coat they were counted as
germinated
While nearly all seeds treated for 8 minutes ofpercussion imbibed water
damage was evident in x-ray evaluations (Sacramento seed source) and in
germination counts (Holman and Capitan) at this treatment level Seed damage
during germination counts was similar to levels found in hot-water treated seeds
suggesting that over-treatment by both hot water and longer durations of percussion
may result in similar damage to seed structure
The Sacramento and Holman seed sources were two of the fIve sources that
opcrplltlpn in germination from the 4- to 8-minute durations of percussion
the benefIts ofloosening the strophiole as evidenced by imbibition of
all 8-minute treated seeds were outweighed by external and internal seed coat
39
a
b c d
11 Damage to New Mexico locust seeds observed during germination cotmts following hot water scarification 9a Hot water replicate including ~t seedlings and numerous unimbibed seeds 9b Close-up of seed where raallCle failed to elongate 9c Close-up ofseed where radicle elongated but was
-gtIn in freeing itself from the seed coat 9d Close-up ofseed where radicle broke off from embryo
40
damage (which also may be contributing to high imbibition) For the three sources
for which 8 minutes of percussion resulted in maximum germination extended
percussion of seeds necessary to loosen the strophiole may not have excessively
damaged the seed anatomy The Manzano source did not significantly change in
germination from 4 to 8 minutes of percussion suggesting that an increase in
permeable seeds at the 8-minute treatment duration was balanced by the number of
seeds that failed to germinate due to seed damage To summarize the 4-minute
percussion treatment improved germination over 8 minutes ofpercussion for a
majority of sources (five ofnine) the 8-minute percussion treatment improved
germination over 4-minutes ofpercussion for three sources and there was no
difference with the Manzano source Notably for the Raton source 8 minutes of
percussion increased germination by 235 percentage points over 4-minutes of
percussion (7875 vs 5525 respectively) the next best treatment Raton was the
only source to result in germination less than 75 for 4 minutes ofpercussion so a
grower may wish to apply longer percussion durations to lots that do not respond well
to this treatment level
Finally there was an interesting trend in the Jemez and Raton sources seed
that significantly contributed to the source by treatment interaction (Figure 12) A
~u test showed that the Jemez source had a significantly lighter seed weight than
other sources while the Raton source had an average seed weight For the Jemez
the longest duration of boiling increased germination Conversely only short
U4UVll) of percussion were needed to achieve high germination and the longest
41
80 _ Jemez a Raton
iD
~ C iD e iD
Q c 0 ~
c ~ iD 0
20
0------shy
Immersion Boiling 30 sec Boiling 60 sec
Hot Water Scarification
10degTr========~--------~------------------------------
80
60
40
20
_ Jemez Raton
0------shy
1 min 2 min 4 min 8 min
Percussion Scarification
Figure 12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources
42
b
--~----~~~~========~---- duration ofpercussion reduced germination For the Raton source longer durations
ofboiling consistently decreased germination whereas longer percussion treatments
consistently increased germination
A possible explanation is that a thick seed coat is associated with a loose
strophiole structure and that a thin seed coat is associated with a constrained
strophiole This contrast merits further study and may suggest an ecological
adaptation Further investigation ofthis phenomenon may contribute to successful
pre-screening ofunknown seed sources to determine optimal treatment leveL
43
CONCLUSION
Based on results obtained in pilot study (see Appendix) and thesis
experiments 4 minutes ofpercussion is recommended as a standard protocol for
treating an unknown source ofNew Mexico locust seed In addition to laboratory
results the 4-minute percussion treatment has resulted in germination greater than
80 and healthy seedling grow-outs for production purposes (unpublished data) In
laboratory experiments only one hundred seeds per 118 ml (40z) soil tin were
percussed at a time whereas for production we have treated up to five hundred seeds
per tin For large-scale production ofNew Mexico locust seedlings growers may
wish to experiment with larger quantities of seed and larger-size soil tins
Seed storage trials following percussion treatments were not conducted
However germination greater than 80 and healthy seedlings were obtained when
seeds were percussion-treated and subsequently stored at room-temperature (22degC)
for one week (unpublished data) Seed storage trials merit further study as the
standard hot water treatment leaves seeds in a softened condition that is not
conducive to storage
Hydroseeding is an effective method for direct seeding in reclamation
situations Percussion treatment may be conducive to hydro seeding as seeds remain
a dry relatively undamaged yet permeable condition following treatment Sulfuric
seeds also remain dry following treatment but extensive damage to seed coat
poor seedling vigor have been reported with this treatment (Kelly and van Staden
A grow-out study using a hydro seeder on a reclamation site testing percussion
44
against other treatments such as sulfuric acid and mechanical scarification also
merits further study
Papilionoids are the largest subfamily oflegumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust these legumes often play an integral role in the revegetation of
disturbed lands The pilot study (Appendix) indicates that percussion scarification
successfully treats the widely used black locust as well Developing superior
scarification methods to the standard hot water mechanical scarification and acid
treatments may facilitate the use ofpapilionoid legumes in reclamation projects
45
APPENDICES
APPENDIX A
PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR
NEW MEXICO LOCUST AND BLACK LOCUST SEEDS
Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4
Hot water and sulfuric acid soaks are traditional treatments for seeds of many
temperate woody legwnes including locusts However these scarification techniques
often produce inconsistent germination Percussion scarification where seeds are
repeatedly propelled against a hard surface was compared with hot water
scarification to evaluate treatment efficacy for New Mexico locust (Robinia
neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment
seeds were placed in a 98degC water bath which was immediately removed from the
heat source For percussion scarification seeds were placed in a soil sample tin and
agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the
control were followed by 24-hour water soaks Hot water baths resulted in 56 and
41 germination for New Mexico locust and black locust respectively For both
species nearly all durations ofpercussion increased germination over the hot water
treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3
4 and 5 minutes for black locust resulted in at least 90 germination Traditional
scarification treatments randomly degrade the entire seed coat which can lead to
tissue damage during water uptake Percussion scarification specifically weakens the
strophiole the natural source ofwater entry to the seed in papilionoid legwnes
INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom
2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031
48
Following percussion imbibition is controlled through the strophiole and underlying
tissue is protected
Keywords New Mexico locust black locust scarification percussion germination
Introduction
Locust trees (Robinia spp) are aggressive pioneer species that quickly
colonize disturbed land fix atmospheric nitrogen in the soil and stabilize slopes that
are prone to erosion (Klemmedson 1994 Ashby et al 1985) (Figure A1ab)
Recognition of these attributes has contributed to increased demand for locusts in
reclamation projects Locust seeds however exhibit physical dormancy where a
thick seed coat prevents the movement of water and gasses to the embryo (Leadem
1997)
Sulfuric acid or hot water soaks have traditionally been used to break seed
dormancy in macro-propagation of locusts Many growers are moving away from the
use of su1furic acid in treating locust seeds (Dreesen and Harrington 1997) In
addition to being dangerous acid soak durations must be specifically correlated to
seed lot (Olson 1974) Collections of native seed can vary tremendously in seed size
seed weight and hard seededness etc This variability limits the utility of sulfuric
acid soaks as even within a given lot some seeds may be damaged whereas others
are under-treated and fail to imbibe (take up water) A survey of Southwestern
container growers indicates that hot water baths are currently the most common
scarification method for locust seeds (Hine et al 1997) However hot water baths
also produce inconsistent germination (Lin et al 1996)
49
b
Figure AI New Mexico locust a primary invader following disturbance Ala Crown-sprouting at edge ofburn near Los Alamos NM--one year after Cerro Grande fire Alb Colonizing road cut Gila National Forest NM
50
Percussion scarification where seeds are repeatedly propelled against a hard
surface is an alternative dormancy-breaking method that has proven successful in
legumes related to locusts (Hamly 1932 Barton 1947) We compared this alternative
treatment to hot water scarification to evaluate treatment efficacy for New Mexico
locust and black locust seeds
Materials and Methods
Commercial New Mexico locust seeds (Western Native Seed Coaldale CO)
were collected fall 2000 in Huerfano County CO Black locust seeds were collected
September 2000 from Taos County NM by harvesting ripe pods from several trees at
a distance of up to 3 m from ground level Black locust seeds were allowed to air-dry
for 3 weeks and were threshed and separated from large chaffusing a greenhouse
fan A South Dakota Seed Blower (Chicago Illinltois) was used to remove fine chaff
from both seed lots Seeds were storedmiddotat 2-4C until the germination study was
conducted in March 2001
This study compared hot water scarification with percussion scarification
using treatment and species as experimental factors Along with a control
scarification treatments included immersion of seed in boiling water as well as
percussion durations of 1 2 3 45 or 10 minutes Four 100-seed samples were used
to test each species by scarification treatment combination
For the hot water treatment test tubes were filled with 30 mL water and
placed in a water-filled beaker (Figure A2a) The bath was raised to boiling (-98C at
51
b
c
Figure A2 Implementation ofhot water and percussion scarification A2a Hot water bath A2b Paint-shaker set-up A2c Placing soil tin containing seeds at end ofpaint shaker ann
52
Las Cruces NM elevation of 1300 meters) Each 100-seed replicate was placed in a
test tube and each test tube was immediately removed to cool at room temperature
Percussion scarification was implemented using a pneumatic paint shaker
(Central Pneumatic model 00422 Camarillo CA) (Figure A2b) This allowed
standardization of treatment intensity Compressed air pressure was maintained at
530kPa plusmn 30kPa (80psi plusmn 5psi) resulting in approximately 350 oscillations per
minute Each lOO-seed sample was placed in a 118 ml (4 oz) soil tin for shaking To
maximize lateral movement of paint shaker a paint can was used as a spacer to place
soil tins at the end of the shaking arm (Figure A2c) This allowed shaking to occur at
the greatest distance from the pivot point (fulcrum) Following all treatments
including the control seeds were soaked for twenty-four hours in distilled water prior
to plating out
Gennination was evaluated on lab benches at room temperature Each
replicate of seeds was placed in a 100 cm petri dish on moistened filter paper with
humidity maintained by enclosing the petri dishes in ziplock bags Gennination was
monitored daily for 14 days in accordance with International Seed Testing
Association standards for Robinia species (ISTA 1999) Gennination was defined as
protrusion of the radicle from the embryo by at least 1mm as viewed by the naked
eye
Categorical Analysis of Variance using Prpc Catmod (SAS Institute 1989)
was used to detennine treatment differences in germination percentages for each
source This procedure is an extension of a Chi-square test of homogeneity using the
53
natural log of the ratio of germinated to non-germinated seeds for each treatment Pshy
values less than 005 were considered significant A limited set of pairwiseshy
comparisons was conducted to compare treatment mean percents using a
conservative alpha value of 005 divided by the number of comparisons
Results
Percussion maximized germination for both New Mexico locust and black
locust There were three germination response levels for New Mexico locust (Figure
A3) First there was a low response in the control indicating a dormant seed lot
The hot water treatment and I-minute duration of percussion share a second
intermediate response Germination was highest at percussion levels from 2 to 10
minutes
A selected percussion treatment intermediate in time (5 minutes) produced the
steepest germination curve indicating the fastest germination rate (Figure A4) The
hot water treatment produced an intermediate curve or relatively slower germination
rate The control curve was very flat indicating the slowest germination rate for New
Mexico locust
Control seeds germinated poorly for black locust as well (Figure A5) Hot
water scarification improved germination over the control but all levels of percussion
improved germination over hot water The optimal durations of percussion treatment
were bracketed with a significant increase from the 1 to 2 minute percussion level a
high germination response from 2 to 5 minutes of percussion and finally a significant
54
~ d 25 CL)
p t1 0 ~ t1
g Vl ~ Vl
100~---------------------------------------------------------------
80
60
40
20
o Control Hot Water Percussion
I min Percussion
2 min Percussion
3 min
Scarification
Percussion 4 min
Figure A3 Effect of scarification on germination response for New Mexico locust
Percussion 5 min
=significant difference between adjacent treatment bars ns = no significant difference
Percussion 10 min
~ ~ () u agt
Il-t d o ~
~ ~ c3
100middotlr~~~------------------------------------e- Percussion 5min middot 0middot Hot Water -- Control
80-J=middot======
6b-middot-lmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middot--middotmiddot-middot~-middotmiddot -middotmiddot middot middotmiddot-middot 1
40
20
-----~-----------
0 0 0 middotmiddotmiddotmiddotmiddot0middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot0
0 --~-----y-----~------_---~--- T-----~
o shy -TDay 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A4 Effect of selected scarification treatments on germination speed for New Mexico locust First seven days of fourteen-day experiment are shown
Day 7
80
~ cd
5 60 ()
[) Cl d 0 1t1 d
40sect dUl
-J
20
o
ns
Scarification
Figure A5 Effect of scarification on germination response for black locust = significant difference between adjacent treatment bars ns = no significant difference
decrease at the 10-minute level This drop-off suggests that 10 minutes of percussion
over-treats seeds from this lot of-black locust
A selected percussion treatment intermediate in time (5 minutes) also had the
fastest germination rate for black locust (Figure A6)_ The hot water curve is
somewhat flatter indicating a slower germination rate The control curve is very flat
indicating a slow germination rate
A qualitative difference between hot water and percussion-treated seeds was
also observed Along with healthy germinants the hot water treatment produced
over-treated seeds (Figure A 7a) Examples of damaged germinants include seeds
where the radicle did not elongate (Figure A 7b) seeds where the radicle elongated
but was delayed in freeing itself from the seed coat (Figure A7c) and seeds where the
radicle broke off completely from the embryo (Figure A7d) In addition to these
examples of over-treatment there were numerous small dark seeds that failed to
imbibe water For percussion treatment nearly all seeds took up water (Figure A8a)
Germination was rapid and uniform with little damage evident (Figure A8b)
To summarize both New Mexico locust and black locust seed lots were
dormant The standard hot water scarification treatment improved germination but
only to 56 and 41 for New Mexico locust and black locust respectively Nearly
all percussion durations improved total germination as well as germination rate over
the hot water treatment Germination was greater than 90 for percussion durations
of 4 5 and 10 minutes for New Mexico locust and 3 4 and 5 minutes for black locust
58
CI)
sgt t CI) 0
igt 0
sect ttl t E
Vl 0
c3
100r==~==~~=r------------------------------------~ ___ Percussion 5 min
middotmiddotmiddot0middotmiddotmiddot Hot Water -lIf- Control
80r=========~--~~----------------------------~
60
40
20 -~-~---~---------- ---~------------I
omiddot
O
_~_----------lIf-----~bull -----_ shy---shy I
Day 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A6 Effect of selected scarification treatments on germination speed for black locust First seven days of fourteen-day experiment are shown
Day 7
a
b c d
Figure A7 Damage to New Mexico locust seeds observed during germination counts following hot water scarification A7a Hot water replicate including damaged seedlings and numerous unimbibed seeds A7b Close-up ofseed where radicle failed to elongate A7c Close-up ofseed where radicle elongated but was delayed in freeing itself from the seed coat A7d Close-up ofseed where radicle completely broke offfrom embryo
60
a
b
Figure A8 New Mexico locust seeds following percussion treatment at time of germination counts A8a Percussion replicate with uniformly healthy seedlings and near complete imbibition of seeds A8b Close-up ofpercussion-treated seedling
61
Discussion
Papilionoid seeds have a specific anatomical feature known as the strophiole
or lens-the natural site of water entry to the seed (Hamly 1932) The strophiole is
located on the cotyledonary lobe of a locust seed (Figure A9) When a papilionoid
legume seed is percussed for the appropriate amount of time repeated hits on the
integrated seed coat loosen the constrained cells of the strophiolar region without
excessively damaging the rest of the seed coat (Ballard 1976)
When a percussed seed is soaked water enters exclusively through the
strophiole in a controlled manner (Kelly and Staden 1987) This regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues This
contrasts with seeds that have been hot water or acid scarified treatments that can
randomly degrade the seed coat Unlocalized cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rehman et al 1998 Marunda 1990)
Papilionoids are the largest subfamily of legumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust and black locust these legumes often play an integral role in the
revegetation ofdisturbed lands Developing superior scarification methods to the
standard hot water and acid treatments should fadlitate the use ofthese legumes in
restoration projects
62
Figure A9 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (bc)
63
APPENDIXB
GERMINATION PERCENTS AND STANDARD ERRORS FOR SOURCE
BLOCK AND TREATMENT FOR NEW MEXICO LOCUST
Table B 1 Gennination percents and standard errors for source block and treatment for New Mexico locust
Source Block Treatment Germination Percentage Standard Error
Capitan 1 Immersion 74 437 Capitan 2 Immersion 69 462 Capitan 3 Immersion 71 454 Capitan 4 Immersion 67 470 Capitan Boiling 30 sec 79 407 Capitan 2 Boiling 30 sec 76 427 Capitan 3 Boiling 30 sec 72 449 Capitan 4 Boiling 30 sec 76 427 Capitan Boiling 60 sec 71 454 Capitan 2 Boiling 60 sec 54 498 Capitan 3 Boiling 60 sec 67 470 Capitan 4 Boiling 60 sec 76 427 Capitan I Control 5 218 Capitan 2 Control 7 255 Capitan 3 Control 4 196 Capitan 4 Control 5 218 Capitan 1 Percussion I min 47 499 Capitan 2 Percussion I min
470
Capitan Percussion I min 39 488
Capitan 4 Percussion I min 38 485 Capitan I Percussion 2 min 55 497 Capitan Capitan
2
Percussion 2 min Percussion 2 min
71 65
454 477
Capitan 4 Percussion 2 min 56 496 Capitan I Percussion 4 min 86 347 Capitan 2 Percussion 4 min 79 407 Capitan 3 Percussion 4 min 81 392 Capitan 4 Percussion 4 min 82 384 Capitan I Percussion 8 min 90 300 Capitan 2 Percussion 8 min 88 325 Capitan 3 Percussion 8 min 90 300 Capitan 4 Percussion 8 min 90 300 Gila I Immersion 71 454 Gila 2 Immersion 77 421 Gila
Immersion 75 433
Gila 4 Immersion 72 449
Gila I Boiling 30 sec 68 466
Gila 2 Boiling 30 sec 81 392
Gila 3 Boiling 30 sec 54 498
Gila 4 Boiling 30 sec 63 483
Gila I Boiling 60 sec 55 497
Gila 2 Boiling 60 sec 68 466
Gila 3 Boiling 60 sec 57 495
Gila 4 Boiling 60 sec 70 458
65
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Gila 2 Control 13 336 Gila 3 Control 13 336 Gila 4 Control 20 400 Gila 1 Percussion 1 min 66 474 Gila 2 Percussion 1 min 57 495 Gila 3 Percussion 1 min 73 444 Gila 4 Percussion 1 min 82 384 Gila 1 Percussion 2 min 81 392 Gila 2 Percussion 2 min 86 347 Gila 3 Percussion 2 min 85 357 Gila 4 Percussion 2 min 54 498 Gila 1 Percussion 4 min 79 407 Gila 2 Percussion 4 min 83 376 Gila 3 Percussion 4 min 84 367 Gila 4 Percussion 4 min 79 407 Gila 1 Percussion 8 min 51 500 Gila 2 Percussion 8 min 76 427 Gila 3 Percussion 8 min 52 500 Gila 4 Percussion 8 min 74 439 Holman 1 Immersion 83 376 Holman 2 Immersion 86 347 Holman 3 Immersion 81 392 Holman 4 Immersion 90 300 Holman 1 Boiling 30 sec 90 300 Holman 2 Boiling 30 sec 85 357 Holman 3 Boiling 30 sec 87 336 Holman 4 Boiling 30 sec 83 376 Holman 1 Boiling 60 sec 79 407 Holman 2 Boiling 60 sec 80 400 Holman 3 Boiling 60 sec 86 347 Holman 4 Boiling 60 sec 93 255 Holman Control 25 433 Holman 2 Control 19 392 Holman Control 21 407
Holman 4 Control 24 427
Holman Percussion 1 min 77 421
Holman 2 Percussion 1 min 85 357
Holman 3 Percussion 1 min 80 400
Holman 4 Percussion 1 min 89 313
Holman 1 Percussion 2 min 88 325
Holman 2 Percussion 2 min 96 196
Holman 3 Percussion 2 min 93 255
Holman 4 Percussion 2 min 96 196
Holman 1 Percussion 4 min 92 271
Holman 2 Percussion 4 min 97 171
66
I
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Holman 3 Percussion 4 min 91 286 Holman 4 Percussion 4 min 96 196 Holman 1 Percussion 8 min 88 325 Holman 2 Percussion 8 min 85 357 Holman 3 Percussion 8 min 83 376 Holman 4 Percussion 8 min 82 384 Jemez 1 Immersion 53 499 Jemez 2 Immersion 44 496 Jemez 3 Immersion 52 500 Jemez 4 Immersion 70 458 Jemez 1 Boiling 30 sec 58 494 Jemez 2 Boiling 30 sec 55 497 Jemez 3 Boiling 30 sec 48 500 Jemez 4 Boiling 30 sec 65 477 Jemez 1 Boiling 60 sec 68 466 Jemez 2 Boiling 60 sec 63 483 Jemez 3 Boiling 60 sec 73 444 Jemez 4 Boiling 60 sec 89 313 Jemez 1 Control 2 140 Jemez 2 Control 9 286 Jemez 3 Control 6 237 Jemez 4 Control 8 271 Jemez 1 Percussion 1 min 58 494 Jemez 2 Percussion 1 min 63 483 Jemez 3 Percussion 1 min 77 421 Jemez 4 Percussion 1 min 89 313 Jemez 1 Percussion 2 min 68 466 Jemez 2 Percussion 2 min 64 480 Jemez 3 Percussion 2 min 80 400 Jemez 4 Percussion 2 min 83 376 Jemez Percussion 4 min 68 466 Jemez 2 Percussion 4 min 79 407 Jemez 3 Percussion 4 min 70 458 Jemez 4 Percussion 4 min 83 376 Jemez 1 Percussion 8 min 52 500 Jemez 2 Percussion 8 min 59 492 Jemez 3 Percussion 8 min 61 488 Jemez 4 Percussion 8 min 70 458 Magdalena 1 Immersion 69 462 Magdalena 2 Immersion 69 462 Magdalena J Immersion 70 458 Magdalena 4 Immersion 85 357 Magdalena 1 Boiling 30 sec 65 477 Magdalena 2 Boiling 30 sec 71 454 Magdalena 3 Boiling 30 sec 68 466
67
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Magdalena 4 Boiling 30 sec 83 376 Magdalena 1 Boiling 60 sec 63 483 Magdalena 2 Boiling 60 sec 68 466 Magdalena 3 Boiling 60 sec 63 483 Magdalena 4 Boiling 60 sec 82 384 Magdalena 1 Control 11 313 Magdalena 2 Control 6 237 Magdalena 3 Control 5 218 Magdalena 4 Control 4 196 Magdalena Percussion 1 min 32 466 Magdalena 2 Percussion 1 min 45 497 Magdalena 3 Percussion 1 min 46 498 Magdalena 4 Percussion I min 36 480 Magdalena 1 Percussion 2 min 67 470 Magdalena 2 Percussion 2 min 68 466 Magdalena 3 Percussion 2 min 65 477 Magdalena 4 Percussion 2 min 81 392 Magdalena 1 Percussion 4 min 87 336 Magdalena 2 Percussion 4 min 97 171 Magdalena 3 Percussion 4 min 83 376 Magdalena 4 Percussion 4 min 97 171 Magdalena 1 Percussion 8 min 98 lAO Magdalena 2 Percussion 8 min 100 000 Magdalena 3 Percussion 8 min 96 196 Magdalena 4 Percussion 8 min 98 lAO Manzano 1 Immersion 67 470 Manzano 2 Immersion 57 495 Manzano 3 Immersion 72 4049 Manzano 4 Immersion 73 4044 Manzano 1 Boiling 30 sec 70 458 Manzano 2 Boiling 30 sec 64 480 Manzano 3 Boiling 30 sec 71 454 Manzano 4 Boiling 30 sec 80 400 Manzano I Boiling 60 sec 50 500 Manzano 2 Boiling 60 sec 53 499 Manzano 3 Boiling 60 sec 59 492 Manzano 4 Boiling 60 sec 74 439 Manzano I Control 4 196 Manzano 2 Control 8 271 Manzano 3 Control 1 099 Manzano 4 Control 6 237 Manzano 1 Percussion 1 min 52 500 Manzano 2 Percussion 1 min 39 488 Manzano 3 Percussion 1 min 36 480 Manzano 4 Percussion 1 min 51 500
68
Table B 1 Continued
Source Block Treatment Gerrni~ation Percentage Standard Error
Manzano Percussion 2 min 53 499 Manzano 2 Percussion 2 min 58 494 Manzano 3 Percussion 2 min 61 488 Manzano 4 Percussion 2 min 62 485 Manzano 1 Percussion 4 min 76 427 Manzano 2 Percussion 4 min 85 357 Manzano 3 Percussion 4 min 79 407 Manzano 4 Percussion 4 min 83 376 Manzano 1 Percussion 8 min 87 336 Manzano 2 Percussion 8 min 83 376 Manzano 3 Percussion 8 min 83 376 Manzano 4 Percussion 8 min 75 433 Raton 1 Immersion 54 498 Raton 2 Immersion 37 483 Raton 3 Immersion 39 488 Raton 4 Immersion 69 462 Raton 1 Boiling 30 sec 36 480 Raton 2 Boiling 30 sec 20 400 Raton 3 Boiling 30 sec 28 449 Raton 4 Boiling 30 sec 60 490 Raton 1 Boiling 60 sec 15 357 Raton 2 Boiling 60 sec 171 Raton 3 Boiling 60 sec 4 196 Raton 4 Boiling 60 sec 35 477 Raton 1 Control 4 196 Raton 2 Control 12 325 Raton 3 Control 2 140 Raton 4 Control 6 237 Raton 1 Percussion 1 min 52 500 Raton 2 Percussion I min 29 454 Raton 3 Percussion I min 35 477 Raton 4 Percussion 1 min 31 462 Raton 1 Percussion 2 min 55 497 Raton 2 Percussion 2 min 35 477 Raton 3 Percussion 2 min 33 470 Raton 4 Percussion 2 min 42 494 Raton 1 Percussion 4 min 65 477 Raton 2 Percussion 4 min 52 500 Raton Percussion 4 min 51 500 Raton 4 Percussion 4 min 53 499 Raton 1 Percussion 8 min 88 325 Raton 2 Percussion 8 min 80 400 Raton 3 Percussion 8 min 73 444 Raton 4 Percussion 8 min 74 439 Sacramento 1 Immersion 43 495
69
Table B 1 Continued
-Source Block Treatment Germination Percentage Standard Error
Sacramento 2 Immersion 30 458 Sacramento 3 Immersion 38 485 Sacramento 4 Immersion 56 496 Sacramento 1 Boiling 30 sec 47 499 Sacramento 2 Boiling 30 sec 48 500 Sacramento 3 Boiling 30 sec 48 500 Sacramento 4 Boiling 30 sec 56 496 Sacramento 1 Boiling 60 sec 52 500 Sacramento 2 Boiling 60 sec 67 470 Sacramento 3 Boiling 60 sec 43 495 Sacramento 4 Boiling 60 sec 48 500 Sacramento 1 Control 0 000 Sacramento 2 Control 3 171 Sacramento 3 Control 3 171 Sacramento 4 Control 099 Sacramento 1 Percussion 1 min 57 495 Sacramento 2 Percussion 1 min 61 488 Sacramento 3 Percussion 1 min 62 485 Sacramento 4 Percussion 1 min 55 497 Sacramento 1 Percussion 2 min 84 367 Sacramento 2 Percussion 2 min 70 458 Sacramento 3 Percussion 2 min 81 392 Sacramento 4 Percussion 2 min 69 462 Sacramento 1 Percussion 4 min 91 286 Sacramento 2 Percussion 4 min 84 367 Sacramento 3 Percussion 4 min 84 367 Sacramento 4 Percussion 4 min 94 237 Sacramento 1 Percussion 8 min 82 384 Sacramento 2 Percussion 8 min 81 392 Sacramento 3 Percussion 8 min 82 384 Sacramento 4 Percussion 8 min 81 392 Sandia 1 Immersion 76 427 Sandia 2 Immersion 70 458 Sandia 3 Immersion 70 458 Sandia 4 Immersion 81 392 Sandia 1 Boiling 30 sec 78 414 Sandia 2 Boiling 30 sec 71 454 Sandia 3 Boiling 30 sec 63 483 Sandia 4 Boiling 30 sec 78 414 Sandia 1 Boiling 60 sec 67 470 Sandia 2 Boiling 60 sec 65 477 Sandia 3 Boiling 60 sec 74 439 Sandia 4 Boiling 60 sec 66 474 Sandia 1 Control 16 367 Sandia 2 Control 7 255
70
Table B 1 Continued
Source Block Treatment Gennination Percentage Standard Error
Sandia 3 Control 8 271 Sandia 4 Control 16 367 Sandia 1 Percussion 1 min 67 470 Sandia 2 Percussion 1 min 61 488 Sandia 3 Percussion 1 min 66 474 Sandia 4 Percussion 1 min 59 492 Sandia I Percussion 2 min 82 384 Sandia 2 Percussion 2 min 83 376 Sandia 3 Percussion 2 min 81 392 Sandia 4 Percussion 2 min 84 367 Sandia 1 Percussion 4 min 89 313 Sandia 2 Percussion 4 min 86 347 Sandia 3 Percussion 4 min 89 313 Sandia 4 Percussion 4 min 85 357 Sandia 1 Percussion 8 min 80 400 Sandia 2 Percussion 8 min 77 421 Sandia 3 Percussion 8 min 69 462 Sandia 4 Percussion 8 min 66 474
LITERATURE CITED
Ashby WC Vogel WG Rogers NF 1985 Black locust in the reclamation equation General Technical Report Northeastern Forest Experiment Station USDA Forest Service NE-I05 12 p
Ballard LAT 1973 Physical barriers to gennination Seed Sci Technoll 285-303
___ 1976 Strophiolar water conduction in the seeds ofTrifolieae induced by action on the testa at nonstrophiolar sites Austr J Plant Physiol3 465-469
Barton LV 1947 Special studies on seed coat impenneability Contrib Boyce Thompson Inst 14 355-362
Baskin CC Baskin JM 1998 Seeds Ecology biogeography and evolution of donnancy and gennination Academic Press London UK 666 p
Bewley JD 1997 Seed germination and donnancy Plant Ce119 1055-1066
Bewley JD Black M 1994 Seeds physiology of development and germination 2nd ed Plenum Press New York NY 445 p
Briscoe CB 1996 Growing Nitrogen Fixing Trees in Acid Soils In MH Powell (ed) Nitrogen Fixing Trees for Acid Soils- A Field ManuaL Winrock International Morrilton AR 22 p
Copeland LO McDonald MB 1995 Principles of Seed Science and Technology 3rded Chapman amp Hall New York NY 409 p
Cox RE Klett JE 1984 Seed gennination requirements of native Colorado plants for use in the landscape Plant Propag 30(2) 6-10
Dell B 1980 Structure and function ofthe strophiolar plug in seeds ofAlbizia Lophantha Am J Bot 67 556-563
Dick-Peddie WA 1993 New Mexico Vegetation Past Present and Future University ofNew Mexico Press Albuquerque NM 244 p
Dreesen DR 2001 Propagation protocol for production ofcontainer Robinia neomexicana plants Los Lunas Plant Materials Center Los Lunas New Mexico In Native Plant Network URL httpwwwnativeplantnetworkorg (accessed 9 January 2002) Moscow (ID) University ofIdaho CoIl Natural Resources For Res Nursery
72
Dreesen DR Harrington JT 1997 Propagation of native plants for restoration projects in the Southwestern US- Preliminary Investigations In Landis TD Thompson JR tech coords National Proceedings Forest and Conservation Nursery Assocs Gen Tech Rep PNW-GTR-419 Portland OR USDA For Ser PNW Res Sta 77-88
Gottfried GJ 1980 Control ofNew Mexican locust and the effect on planted ponderosa pine in central Arizona US For Ser Res Note RM-386 Rocky Mountain For Range ExpStn Fort Collins CO
Gosling PG Samuel YK Jones SK 1995 A systematic examination of germination temperature chipping and water temperaturesoak duration pretreatments on the seeds ofLeucaena leucocephala Seed Sci amp Technol 23521-532
Hamly DH 1932 Softening of the seeds of Melilotus alba Bot Gazette 93 345shy375
Hanna PJ 1984 Anatomical features of the seed coat ofAcacia kempeana (Mueller) which relate to increased germination rate induced by heat treatment New Phytol 96 23-29
Hanks JP Dick-Peddie WA 1974 Vegetation patterns of the White Mountains New Mexico Southwest Naturalist 18(4) 371-382
Hine S Harrington JT Dreesen DR 1997 Seed scarification requirements for Robinia neomexicana In Harrington JTtech coord Proc Third SouthwestemContainer Growers Conf Feb 12-13 1997 Ft Collins CO
Hopkinson JM 1993 The strophiole of leguminous seeds Newsletter- IntI Herbage Seed Production Res Grp 18 7-8
---- 1997 Differences in hardseededness between Cunningham and Tarramba leucaena In Leucnet News The Newsletter of the International LeucaenaRes and Devlt Netwk No4
ISTA (International Seed Testing Association) 1999 International rules for seed testing rules Seed Sci amp Technol27 Supplement
Kelly KM van Staden J 1987 The lens as the site of permeability in the papilionoid seed Aspalathus linearis J Plant Physiol 128(45) 395-404
73
Keresztesi B 1988 Black locust the tree of agriculture Outlook on Agr 17(2) 77~85
Kigel 1 and Galili G 1995 Seed development and germination Marcel Dekker New York 853 p
Klemmedson 10 1994 New Mexico locust and parent material Influence on macronutrients of forest floor and soil Soil Sci Soc Am 1 58 974~980
Kuo WH Tam AW 1988 The pathway of water absorption of mung bean seeds Seed Sci amp Technol16(1) 139~144
Leadem CL 1997 Dormancy--unlocking seed secrets In Landis TD Thompson lR tech coords National Proceedings Forestand Conservation Nursery Assocs Gen Tech Rep PNW-G TR-419 Portland OR USDA For Ser PNW Res Sta 43-52
Lin Y Wagner MR Seweyestewa A Honahni MD DeGomez M McMillin JD 1996 Influence of cultural treatments on seed germination and seedling growth of Robinia neomexicana Forest Farm and Community Tree Res Reports Volume 1
Marunda CT 1990 Effects of seed pretreatment on the development ofAcacia auriculforinis and A holosericea seedlings ACIAR Proceedings Series No 28
Meginnis HG 1937 Sulphuric acid treatment to increase germination of black locust seed USDA Circ 453 34 p
Miklas PN Towensend CE Ladd SL 1987 Seedcoat anatomy and the scarification ofcicer milkvetch seed Crop Sci 27 766-772
Monsen SB 1984 Use of shrubs on mine spoils In The Challenge ofProducing Native Plants for the Intermountain Area Proceedings Intermountain Nurserymans Association 1983 conference Las Vegas NV compo PM Murphy 26-31 Ogden UT USDA For Ser Gen Tech Report INT-l68
Olson DF 1974 Robinia L Locust In Seeds of Woody Plants in the US tech coord CS Schopmeyer 728-731 USDA For Ser Agr Handbk No450
Rehman S Loescher RNl Harris P1C 1998 Dormancy breaking and germination ofAcacia salicina Lindl seeds Seed Sci amp Techno127 553-557
Rolston MP 1978 Water impermeable seed dormancy Bot Rev 44 365-396
74
SAS Institute Inc 1989 SASSTAT Users Guide Version 6 4th Edition Volume 1 Cary NC SAS Institute Inc 943 p
SerratoValenti G Conara L Ferrando M Modenesi P 1993 Structural and histochemical features ofStylosanthes scabra (Leguminosae Papilionoidae) seed coat as related to water entry Can J Bot 71 834-840
Simpson BJ 1988 A field guide to Texas trees Austin TX Texas Monthly Press 372p
Tran VN Cavanagh AK 1984 Structural aspects ofdormancy In Seed Physiology Vol 2 Murray DR (ed) p1-44 Academic Press Australia
USDA Forest Service 1948 Woody-plant seed manual USDA Misc Pub1654 416p
Vines RA 1960 Trees Shrubs and Woody Vines of the Southwest Austin Univ ofTexas Press 1104 p
Wagner MR Larson RE Craig TP 1992 Ecology and silvics ofNew Mexico locust (Robinia neomexicana) with an emphasis on the arboreal form p53-57 In JW Hanover K Miller and S Plesko (eds) Proc Intemat Confon Black Locust Biology Culture and Utilization Mich State Univ USA
Wilson JK 1944 Immersing seeds of species of Robinia in boiling water hastens germination J For 42 453-454
Zimmerman RW Carpenter SB 1980 First-year coppice production from a 5shyyear old black locust stand on surface mine spoil p309-314 Centr Hardwd For ConfProc University ofMissouri Columbia
IIIRZltItW lIJPIirmiddot rllll1lbullbull_~~~_II
Table 1 Latitude longitude location elevation and collection date ofNew Mexico locust seed sources
Lot title Latitude (N) Longitude (W) Location Elevation (m) Collection Date
Raton 36deg5846 104deg2872 Raton Pass NM 2295 82500
Holman 36deg0306 105deg2429 HolmanNM 2476 911500
Jemez 35deg4500 106deg4100 Jemez Springs NM 1976 9900 00
Sandia 35deg1083 106deg2341 Cedar Crest NM 2432 82600
Manzano 34deg3689 106deg2494 ManzanoNM 2465 9200
Magdalena 33deg5953 107deg0903 Magdalena NM 2544 82900
Capitan 33deg2728 105deg4325 Alto NM 2189 911200
Sacramento 32deg5798 105deg4496 Cloudcroft NM 2630 91400 Gila 32deg5400 107deg4600 Kingston NM 2432 9700
f
Table 2 Mean seed weight and standard error by source
a
Source Mean seed weight (g) for Standard error 100 seeds n = 5
Raton Holman Jemez Sandia Manzano Magdalena Capitan Sacramento Gila
2872 b c 2522 d 1826 e 2753 c 2413 d 2559 d 2443 d 3130 a 3029 ab
00103 00403 00276 00301 00370 00374 00458 00279 00348
a Mean seed weights followed by the same letter are not significantly
different
19
experiment was conducted utilizing a randomized complete block design blocked by
time with a factorial treatment structure Experimental factors were seed source and
scarification treatment Nine seed sources from throughout New Mexico were used
(Figure 1 Table 1) Scarification treatments included a control three levels ofhot
water and four levels of percussion Hot water treatments included immersion at
100degC and boiling for periods of 30 seconds and 60 seconds Percussion treatment
levels were 1 2 4 and 8 minutes One hundred seeds per block were used to test
each treatment combination Four blocks of seed were tested one at a time (all seed
sources included in each run) in close chronological sequence (within 6 weeks) A
total of four 100-seed samples were used to test each source by scarification treatment
combination
Treatments
Hot water treatment of seeds utilized 50 ml test tubes filled with 30 ml
distilled water Each test tube was used to treat one 100-seed sample at a time
resulting in a ratio of4 parts water to 1 part seed by volume Test tubes were placed
in a beaker with the beaker filled with tap water to the same level as the water within
test tubes Nine test tubes (one per seed source) were placed in the beaker
simultaneously so that one replication of each seed sourcescarification treatment
cotnblnatlon was conducted simultaneously Hot water treatments include immersion
boiling For immersion treatments seeds were added to test tubes with the water
(boiling) and the test tubes were immediately removed from the beaker to
For the boiling treatments test tubes remained in the beaker with water at
20
boiling temperature for a set duration after the addition of seeds Following all
treatments test tubes with seeds cooled 24 hours in a test tube rack
Percussion treatment utilized a pneumatic paint shaker (Central Pneumatic
model 00422 Camarillo CA) Compressed air was maintained at 530 kPa plusmn30 kPa
(80 psi plusmn 5 psi) resulting in approximately 350 oscillations per minute Seeds were
placed in 118 ml (4 oz) soil tin cans (US Can Lobard IL) and the lids were secured
with duct tape Each can was used to treat one 100-seed sample To take advantage
ofgreatest lateral movement of the paint shaker a spacer (paint can) was used to
position the soil tin at the end of the shaking arm This allowed shaking to occur at
the greatest distance from the pivot point (fulcrum) Tins were placed perpendicular
to the direction of shaking motion allowing seeds to impact at greatest force by
hitting flat surfaces of the soil tin
Germination Testing
Before gennination testing all seeds were soaked 24 hours in reverse
osmosis-treated water (this was included in the process ofhot water treatment
jU~SCIlOea above) Following soaking seeds were placed in 100 cm Petri dishes lined
90 cm Whatman qualitative 1 filter paper circles (MG Scientific Pleasant
WI) One Petri dish was used for each treatment by source combination
papers were initially moistened with 5 mL reverse osmosis-treated water then
~VU1CUlJl moistened as needed to ensure adequate conditions for gennination
were monitored daily (at 300 PM) for gennination for 14 days in accordance
~UIUlnJlVUa Seed Testing Association (ISTA) standards for Robinia species
21
(1STA 1999) Tests were carried out at room temperature (22degC plusmnOSoC)
Germination was classified as protrusion of the radicle from the embryo by at least
1mm as viewed with the naked eye
X-ray Radiography
On August 132001 three lOO-seed samples from the Sacramento seed source
were treated for 14 or 8-minute percussion durations The seeds were sent
immediately by overnight delivery to the Faxitron X-Ray Corporation in Wheeling
Illinois where Dr Bob Karrfalt ofthe USDA Forest Service oversaw their imaging
Radiographs were made at SX with a Faxitron Specimen Radiography System Model
MX-20
Scanning Electron Microscope Photography
On February 192002 a Hitachi S-3200N Variable Pressure Scanning
Electron Microscope (Mountain View CA) was used to examine strophiolar cells of
untreated seeds and seeds percussed for 4 minutes Seeds were cut longitudinally
igt their entire length using a razor blade splitting the strophiolar region roughly in
Cross sections of seed samples magnifying the strophiolar region were viewed
600X under the microscope
Analysis
Categorical Analysis of Variance was used to determine treatment source and
(Le time) main effects and interactions The response variable was total
~vlI This procedure is a Chi-square test of homogeneity using the natural log
of germinated to non-germinated seed for each treatment Maximumshy
22
likelihood analysis was used to calculate Chi-square test statistics with their observed
significance levels A single contrast was conducted to cempare treatment means
against the control Observed significance levels less than or equal to 005 were
considered significant Pairwise comparisons were conducted between non-control
treatment mean percents using a conservative alp~a value of 005 divided by the
number of comparisons (Bonferroni approach) and approximate pairwise z statistics
Categorical analyses were performed using Proc Catmod (SAS Institute 1989)
RESULTS
Scarification seed source block and all interactions between factors
influenced germination (Table 3) The main effects are reported in the presence of
these interactions because the overall robustness of treatments and seed sources are of
interest Background levels ofnaturally occurring variation in seed sources are
seen in control germination percentages in Figure 2 In particular time variability
is unlikely to be under the control of the producer Appendix B details experimental
data for source by treatment gennination percents for each of the four blocks
Averaged over all treatments germination by source ranged from 40 to
79 (Figure 2) Averaged over all sources (Figure 3) and for each source (Figure 4
5) percussion scarification durations of4 or 8 minutes yielded greatest germination
Averaged over all sources germination increased with increasing durations of
percussion then leveled off for the 4- and 8-minute treatments (Figure 3) Immersion
and 30-second boiling scarification maximized germination for the hot water
treatments with a decrease in germination when seeds were boiled for 60 seconds
For all but two sources there is a similar ipcreasing trend in germination from
1to 4 minutes ofpercussion (Figure 4) The Holman source increased in germination
1 to 2 minutes then leveled off at 4 minutes The Jemez source changed little
1 to 4 minutes of treatment As treatment duration increased from 4 to 8
mutes five sources decreased in germination (Figure 4a) whereas three sources
24
Table 3 Categorical Analysis of Variance table for germination response to seed treatment seed source block and the interaction of the factors
Source of DF Chi-Square Observed Variability Significance Level
Source 8 98370 lt00001 Trt 7 233876 lt00001
contrast 1 16976 lt00001 SourceTrt 56 97077 lt00001 Block 3 5538 lt00001
SourceBlock 24 10162 lt00001 TrtBlock 21 7564 lt00001
SourceTrtBlock 168 34283 lt00001
S 4)
8 ~
0
sectg ~ 40 ~
N 0 0
20
o f0ltgt ~ ~4 jybshy ((iltgto ~~ K1ltgt 6~b-~ ~~ 0b-~~ ~~ ~b-~ ~b-~b
80
60 -I-- - ~- middotmiddotmiddotmiddotmiddot1
r -] Northern NM Sources
Central NM Sources
Southern NM Sources
sect-o ~
~b-v(i
SOURCE
Figure 2 Effect ofNew Mexico locust seed source on germination for data averaged over all scarification treatments Control germination is shown by solid line within bar
80
~ cd ~ ltl) () 60 ~ ~
N
t 0shyrti tshySltl)
40
0
20
o lmmersion Boiling 30 sec Boiling 60 sec Control 1 min 2min 4 min 8 min
Hot Water Treatments Percussion Treatments
Figure 3 Effect of scarification on germination ofNew Mexico locust for data averaged over all seed sources Means labelled with the same letter are not significantly different
100~----------------------~--~----------------~
80
60
ltU
~
40 IJ
sectgsI
l100
80
60
40
a I middotmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot middotImiddotmiddotmiddotmiddotmiddotmiddot E
_~iii-bull _ _ bull f --~--
t-- -~ ---- -shy ---- shyI ~ ---- - shy
L
fiII
~
b
- shy ---r
- Holman
Sacramento Sandia Gila Jemez
c ~
ltgt ~
Tmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot-I
j~=~=-~~-1
I middotmiddot ~ bull r
t tr
Y -------M-a-gd-a-Ie-n-a--
1 _ _
fCapitan
---- Manzano ~y -_ Raton
2 4 8
Time interval ofpercussion scarification (min)
Figure 4 Effect of seed source by treatment interaction on New Mexico locust germination for percussion scarification pattern a and pattern b
28
tv 0
~ d
~ A s o
1 c3
80
60
40
20
o
TIT
t------=middotfmiddotmiddot-middotmiddot-middotmiddot-middotmiddot 1- --- - - --=- - -shy--cE= - _--shy --shy ----shy ~ ~ ---shy ~ -~- _ --shy-------r 1
I
Raton Holman
-_ Jemez
--- Sandia Manzano Magdalena Capitan Sacramento Gila
Immersion Boiling 30 sec Boiling 60 sec
Hot Water Scarification
Figure 5 Effect of seed source by treatment interaction on New Mexico locust germination for hot water scarification
continued to increase in germination (Figure 4b) There was no difference between
the two treatment levels for the Manzano source (Figure 4b)
The effect ofhot water scarification also varied among sources with few
overall differences between hot water treatments (Figure 5) However boiling for 60
seconds yielded highest germination for the Jemez source whereas increasing
durations ofboiling decreased germination for the Raton source
In addition to consistently high germination 4- and 8-minute percussionshy
treated seeds germinated as fast or faster than all other treatments (Figure 6 Table 4)
050 measured as the number of days needed to complete 50 ofeventual
germination averaged 2 days for both ofthese treatments
X-ray radiographs were taken of seeds collected from the Sacramento source
percussed for 1 4 or 8 minutes (Figure 7) Seeds show signs of damage at 8 minutes
ofpercussion (Figure 8) Compared to an undamaged seed percussed for only 4
minutes (Figure 8a) damage at the 8-minute level includes fractured radicles pitting
of the seed coat and chips in seed coat structure (Figure 8b-d)
Scanning electron microscope (SEM) images at 600X reveal the direct effect
of 4 minutes of percussion on cells of the strophiolar region (Figure 9) Whereas
untreated seeds retain cellular organization in the strophiolar region strophiolar cells
seeds treated for 4 minutes ofpercussion separate and raise resulting in a
~-~e~U appearance
30
Umiddot-~middot--l-~---middotymiddotmiddotmiddot-----middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot--middot--middotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middotmiddotmiddotmiddotmiddotmiddotmiddot- -
050= gt1
-e-- Percussion 8 min -0- Percussion 4 min ---T- Percussion 2 min -----ltil- Percussion 1 min -- Immersion --0- Boiling 30 sec -+- Boiling 60 sec -ltgt- Control
2 4 6 8 10 12
Day Figure 6 Timecourse ofNew Mexico locust gennination following scarification
treatments G50 is the number of days to 50 of eventual germination
31
14
Table 4 Effect of seed source and treatment on G50 (days to 50 germination)
Seed Source Control Immersion B30sec B60sec Plmin P2min P4min P8min mean Raton 3 3 5 7 4 3 2 2 4 Holman 2 2 2 2 2 2 2 2 2
Jemez 4 6 6 5 3 2 2 2 4 Sandia 2 2 2 2 2 2 2 2 2
w tv Manzano 5 2 2 3 3 2 2 2 3
Magdalena 4 2 2 3 4 3 2 1 3
Capitan 5 4 2 2 3 2 2 2 3
Sacramento 3 2 2 2 4 3 2 2 3 Gila 3 2 2 2 2 2 2 2 2 mean 3 3 3 3 3 2 2 2 3 standard 11 14 16 18 09 05 0 03 08 deviation
Note Columns and rows may not agree due to rounding
Figure 7 X-ray radiographs ofSacramento seed source percussed for 1 4 or 8 minutes
33
Figure 8a-d Seed damage close-ups Figure a shows undamaged seed percussed for 4 minutes Figures b-d show seeds percussed for 8 minutes Damage includes fracturing ofradicle in b complete removal ofseed coat in c and pitting ofseed coat in d
34
Untreated Treated
Figure 9 SEM images ofuntreated vs 4-minute percussion-treated strophiolar cells ofNew Mexico locust seeds Treated cells are loosened separated and raised
DISCUSSION
Determining the Optimal Treatment
Averaging results of scarification treatmems across seed sources is a useful
tool to predict which treatment is most likely to be successful for an unknown seed
source Averaged over all sources four- and eight-minute percussion treatments
resulted in the highest germination 815 and 790 respectively and produced
germination that was as fast or faster than all other treatments Although there was no
difference between these two treatments on average an argument for 4 minutes of
percussion as a standard protocol for treating New Mexico locust seeds can be made
Descriptively 4 minutes of percussion improved germination over 8 minutes
ofpercussion for a majority of sources (five out of nine) 8 minutes of percussion
improved germination for three ofnine sources with no difference for a fmal source
For individual sources 4 minutes ofpercussion was always the best or secondmiddot best
treatment and was always equal to or better than all hot water treatments
Germination at 8 minutes ofpercussion was lower than all other percussion durations
for the Jemez and Gila sources and was lower than at least one hot water treatment for
these sources as well
The x-ray radiographs of the Sacramento seed source illustrate the excessive
internal and external seed damage that can occur with 8 minutes of percussion
germination counts 8-minute percussed seeds from Holman and Capitan
ClUll signs of damage that were visible to the naked eye (see below)
r~evertheless some of these seeds still fit the criteria for germination Neither x-ray
36
photography nor visible observations during seed counts revealed seed damage at the
4-minute percussion level In operational trials seeds percussed for 4 minutes
developed into healthy seedlings (Khadduri and Harrington unpublished data)
Strophiole and Seed Coat Behavior
Four minutes ofpercussion successfully breaks seed dormancy in a large
percentage ofNew Mexico locust seeds without excessively damaging the
surrounding seed coat or internal seed coat structures Most papilionoid legume
seeds including seeds ofNew Mexico locust have a specific region of the seed coat
known as the strophiole or lens (Hamly 1932) The strophiole is located on the
cotyledonary lobe of a locust seed (Figure 10 a-c) If seeds are not indiscriminately
damaged the strophiole is the first point of water entry to the embryo (Hopkinson
1997) When a papilionoid seed is percussed for the appropriate amount of time
repeated hits on the integrated seed coat loosen the constrained cells of the strophiolar
region without excessively damaging the rest of the seed coat (Ballard 1976) SEM
photos illuStrate that 4 minutes of percussion loosened and separated strophiolar cells
ofNew Mexico locust seeds when compared with constrained organized strophiolar
cells of untreated seeds (Figure 9) Research-has shown that water passes to the
embryo through the treated strophiole in a controlled manner and regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues
(Kelly and van Staden 1987)
37
10 Strophiole ofunimbibed New Mexico locust seed (a) and following uptake (bc)
38
In contrast hot-water treatments can excessively degrade the seed coat
(Rebman et al 1998 Marunda 1990) Random cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rebman et aI 1998 Marunda 1990) Along with a
proportionately large amount ofun-imbibed (thus under-treated) seeds (Figure l1a)
damage was observed in hot-water treated seeds from six sources during germination
counts Damage included seeds where the radicle failed to elongate (Figure 11 b)
seeds where the radicle elongated but was delayed in freeing itself from the seed coat
(Figure 11c) and seeds where the radicle completely broke off from the embryo
(Figure lId) In addition some seeds germinated in reverse (the cotyledon emerged
fIrst) If the radicle emerged completely from the seed coat they were counted as
germinated
While nearly all seeds treated for 8 minutes ofpercussion imbibed water
damage was evident in x-ray evaluations (Sacramento seed source) and in
germination counts (Holman and Capitan) at this treatment level Seed damage
during germination counts was similar to levels found in hot-water treated seeds
suggesting that over-treatment by both hot water and longer durations of percussion
may result in similar damage to seed structure
The Sacramento and Holman seed sources were two of the fIve sources that
opcrplltlpn in germination from the 4- to 8-minute durations of percussion
the benefIts ofloosening the strophiole as evidenced by imbibition of
all 8-minute treated seeds were outweighed by external and internal seed coat
39
a
b c d
11 Damage to New Mexico locust seeds observed during germination cotmts following hot water scarification 9a Hot water replicate including ~t seedlings and numerous unimbibed seeds 9b Close-up of seed where raallCle failed to elongate 9c Close-up ofseed where radicle elongated but was
-gtIn in freeing itself from the seed coat 9d Close-up ofseed where radicle broke off from embryo
40
damage (which also may be contributing to high imbibition) For the three sources
for which 8 minutes of percussion resulted in maximum germination extended
percussion of seeds necessary to loosen the strophiole may not have excessively
damaged the seed anatomy The Manzano source did not significantly change in
germination from 4 to 8 minutes of percussion suggesting that an increase in
permeable seeds at the 8-minute treatment duration was balanced by the number of
seeds that failed to germinate due to seed damage To summarize the 4-minute
percussion treatment improved germination over 8 minutes ofpercussion for a
majority of sources (five ofnine) the 8-minute percussion treatment improved
germination over 4-minutes ofpercussion for three sources and there was no
difference with the Manzano source Notably for the Raton source 8 minutes of
percussion increased germination by 235 percentage points over 4-minutes of
percussion (7875 vs 5525 respectively) the next best treatment Raton was the
only source to result in germination less than 75 for 4 minutes ofpercussion so a
grower may wish to apply longer percussion durations to lots that do not respond well
to this treatment level
Finally there was an interesting trend in the Jemez and Raton sources seed
that significantly contributed to the source by treatment interaction (Figure 12) A
~u test showed that the Jemez source had a significantly lighter seed weight than
other sources while the Raton source had an average seed weight For the Jemez
the longest duration of boiling increased germination Conversely only short
U4UVll) of percussion were needed to achieve high germination and the longest
41
80 _ Jemez a Raton
iD
~ C iD e iD
Q c 0 ~
c ~ iD 0
20
0------shy
Immersion Boiling 30 sec Boiling 60 sec
Hot Water Scarification
10degTr========~--------~------------------------------
80
60
40
20
_ Jemez Raton
0------shy
1 min 2 min 4 min 8 min
Percussion Scarification
Figure 12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources
42
b
--~----~~~~========~---- duration ofpercussion reduced germination For the Raton source longer durations
ofboiling consistently decreased germination whereas longer percussion treatments
consistently increased germination
A possible explanation is that a thick seed coat is associated with a loose
strophiole structure and that a thin seed coat is associated with a constrained
strophiole This contrast merits further study and may suggest an ecological
adaptation Further investigation ofthis phenomenon may contribute to successful
pre-screening ofunknown seed sources to determine optimal treatment leveL
43
CONCLUSION
Based on results obtained in pilot study (see Appendix) and thesis
experiments 4 minutes ofpercussion is recommended as a standard protocol for
treating an unknown source ofNew Mexico locust seed In addition to laboratory
results the 4-minute percussion treatment has resulted in germination greater than
80 and healthy seedling grow-outs for production purposes (unpublished data) In
laboratory experiments only one hundred seeds per 118 ml (40z) soil tin were
percussed at a time whereas for production we have treated up to five hundred seeds
per tin For large-scale production ofNew Mexico locust seedlings growers may
wish to experiment with larger quantities of seed and larger-size soil tins
Seed storage trials following percussion treatments were not conducted
However germination greater than 80 and healthy seedlings were obtained when
seeds were percussion-treated and subsequently stored at room-temperature (22degC)
for one week (unpublished data) Seed storage trials merit further study as the
standard hot water treatment leaves seeds in a softened condition that is not
conducive to storage
Hydroseeding is an effective method for direct seeding in reclamation
situations Percussion treatment may be conducive to hydro seeding as seeds remain
a dry relatively undamaged yet permeable condition following treatment Sulfuric
seeds also remain dry following treatment but extensive damage to seed coat
poor seedling vigor have been reported with this treatment (Kelly and van Staden
A grow-out study using a hydro seeder on a reclamation site testing percussion
44
against other treatments such as sulfuric acid and mechanical scarification also
merits further study
Papilionoids are the largest subfamily oflegumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust these legumes often play an integral role in the revegetation of
disturbed lands The pilot study (Appendix) indicates that percussion scarification
successfully treats the widely used black locust as well Developing superior
scarification methods to the standard hot water mechanical scarification and acid
treatments may facilitate the use ofpapilionoid legumes in reclamation projects
45
APPENDICES
APPENDIX A
PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR
NEW MEXICO LOCUST AND BLACK LOCUST SEEDS
Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4
Hot water and sulfuric acid soaks are traditional treatments for seeds of many
temperate woody legwnes including locusts However these scarification techniques
often produce inconsistent germination Percussion scarification where seeds are
repeatedly propelled against a hard surface was compared with hot water
scarification to evaluate treatment efficacy for New Mexico locust (Robinia
neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment
seeds were placed in a 98degC water bath which was immediately removed from the
heat source For percussion scarification seeds were placed in a soil sample tin and
agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the
control were followed by 24-hour water soaks Hot water baths resulted in 56 and
41 germination for New Mexico locust and black locust respectively For both
species nearly all durations ofpercussion increased germination over the hot water
treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3
4 and 5 minutes for black locust resulted in at least 90 germination Traditional
scarification treatments randomly degrade the entire seed coat which can lead to
tissue damage during water uptake Percussion scarification specifically weakens the
strophiole the natural source ofwater entry to the seed in papilionoid legwnes
INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom
2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031
48
Following percussion imbibition is controlled through the strophiole and underlying
tissue is protected
Keywords New Mexico locust black locust scarification percussion germination
Introduction
Locust trees (Robinia spp) are aggressive pioneer species that quickly
colonize disturbed land fix atmospheric nitrogen in the soil and stabilize slopes that
are prone to erosion (Klemmedson 1994 Ashby et al 1985) (Figure A1ab)
Recognition of these attributes has contributed to increased demand for locusts in
reclamation projects Locust seeds however exhibit physical dormancy where a
thick seed coat prevents the movement of water and gasses to the embryo (Leadem
1997)
Sulfuric acid or hot water soaks have traditionally been used to break seed
dormancy in macro-propagation of locusts Many growers are moving away from the
use of su1furic acid in treating locust seeds (Dreesen and Harrington 1997) In
addition to being dangerous acid soak durations must be specifically correlated to
seed lot (Olson 1974) Collections of native seed can vary tremendously in seed size
seed weight and hard seededness etc This variability limits the utility of sulfuric
acid soaks as even within a given lot some seeds may be damaged whereas others
are under-treated and fail to imbibe (take up water) A survey of Southwestern
container growers indicates that hot water baths are currently the most common
scarification method for locust seeds (Hine et al 1997) However hot water baths
also produce inconsistent germination (Lin et al 1996)
49
b
Figure AI New Mexico locust a primary invader following disturbance Ala Crown-sprouting at edge ofburn near Los Alamos NM--one year after Cerro Grande fire Alb Colonizing road cut Gila National Forest NM
50
Percussion scarification where seeds are repeatedly propelled against a hard
surface is an alternative dormancy-breaking method that has proven successful in
legumes related to locusts (Hamly 1932 Barton 1947) We compared this alternative
treatment to hot water scarification to evaluate treatment efficacy for New Mexico
locust and black locust seeds
Materials and Methods
Commercial New Mexico locust seeds (Western Native Seed Coaldale CO)
were collected fall 2000 in Huerfano County CO Black locust seeds were collected
September 2000 from Taos County NM by harvesting ripe pods from several trees at
a distance of up to 3 m from ground level Black locust seeds were allowed to air-dry
for 3 weeks and were threshed and separated from large chaffusing a greenhouse
fan A South Dakota Seed Blower (Chicago Illinltois) was used to remove fine chaff
from both seed lots Seeds were storedmiddotat 2-4C until the germination study was
conducted in March 2001
This study compared hot water scarification with percussion scarification
using treatment and species as experimental factors Along with a control
scarification treatments included immersion of seed in boiling water as well as
percussion durations of 1 2 3 45 or 10 minutes Four 100-seed samples were used
to test each species by scarification treatment combination
For the hot water treatment test tubes were filled with 30 mL water and
placed in a water-filled beaker (Figure A2a) The bath was raised to boiling (-98C at
51
b
c
Figure A2 Implementation ofhot water and percussion scarification A2a Hot water bath A2b Paint-shaker set-up A2c Placing soil tin containing seeds at end ofpaint shaker ann
52
Las Cruces NM elevation of 1300 meters) Each 100-seed replicate was placed in a
test tube and each test tube was immediately removed to cool at room temperature
Percussion scarification was implemented using a pneumatic paint shaker
(Central Pneumatic model 00422 Camarillo CA) (Figure A2b) This allowed
standardization of treatment intensity Compressed air pressure was maintained at
530kPa plusmn 30kPa (80psi plusmn 5psi) resulting in approximately 350 oscillations per
minute Each lOO-seed sample was placed in a 118 ml (4 oz) soil tin for shaking To
maximize lateral movement of paint shaker a paint can was used as a spacer to place
soil tins at the end of the shaking arm (Figure A2c) This allowed shaking to occur at
the greatest distance from the pivot point (fulcrum) Following all treatments
including the control seeds were soaked for twenty-four hours in distilled water prior
to plating out
Gennination was evaluated on lab benches at room temperature Each
replicate of seeds was placed in a 100 cm petri dish on moistened filter paper with
humidity maintained by enclosing the petri dishes in ziplock bags Gennination was
monitored daily for 14 days in accordance with International Seed Testing
Association standards for Robinia species (ISTA 1999) Gennination was defined as
protrusion of the radicle from the embryo by at least 1mm as viewed by the naked
eye
Categorical Analysis of Variance using Prpc Catmod (SAS Institute 1989)
was used to detennine treatment differences in germination percentages for each
source This procedure is an extension of a Chi-square test of homogeneity using the
53
natural log of the ratio of germinated to non-germinated seeds for each treatment Pshy
values less than 005 were considered significant A limited set of pairwiseshy
comparisons was conducted to compare treatment mean percents using a
conservative alpha value of 005 divided by the number of comparisons
Results
Percussion maximized germination for both New Mexico locust and black
locust There were three germination response levels for New Mexico locust (Figure
A3) First there was a low response in the control indicating a dormant seed lot
The hot water treatment and I-minute duration of percussion share a second
intermediate response Germination was highest at percussion levels from 2 to 10
minutes
A selected percussion treatment intermediate in time (5 minutes) produced the
steepest germination curve indicating the fastest germination rate (Figure A4) The
hot water treatment produced an intermediate curve or relatively slower germination
rate The control curve was very flat indicating the slowest germination rate for New
Mexico locust
Control seeds germinated poorly for black locust as well (Figure A5) Hot
water scarification improved germination over the control but all levels of percussion
improved germination over hot water The optimal durations of percussion treatment
were bracketed with a significant increase from the 1 to 2 minute percussion level a
high germination response from 2 to 5 minutes of percussion and finally a significant
54
~ d 25 CL)
p t1 0 ~ t1
g Vl ~ Vl
100~---------------------------------------------------------------
80
60
40
20
o Control Hot Water Percussion
I min Percussion
2 min Percussion
3 min
Scarification
Percussion 4 min
Figure A3 Effect of scarification on germination response for New Mexico locust
Percussion 5 min
=significant difference between adjacent treatment bars ns = no significant difference
Percussion 10 min
~ ~ () u agt
Il-t d o ~
~ ~ c3
100middotlr~~~------------------------------------e- Percussion 5min middot 0middot Hot Water -- Control
80-J=middot======
6b-middot-lmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middot--middotmiddot-middot~-middotmiddot -middotmiddot middot middotmiddot-middot 1
40
20
-----~-----------
0 0 0 middotmiddotmiddotmiddotmiddot0middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot0
0 --~-----y-----~------_---~--- T-----~
o shy -TDay 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A4 Effect of selected scarification treatments on germination speed for New Mexico locust First seven days of fourteen-day experiment are shown
Day 7
80
~ cd
5 60 ()
[) Cl d 0 1t1 d
40sect dUl
-J
20
o
ns
Scarification
Figure A5 Effect of scarification on germination response for black locust = significant difference between adjacent treatment bars ns = no significant difference
decrease at the 10-minute level This drop-off suggests that 10 minutes of percussion
over-treats seeds from this lot of-black locust
A selected percussion treatment intermediate in time (5 minutes) also had the
fastest germination rate for black locust (Figure A6)_ The hot water curve is
somewhat flatter indicating a slower germination rate The control curve is very flat
indicating a slow germination rate
A qualitative difference between hot water and percussion-treated seeds was
also observed Along with healthy germinants the hot water treatment produced
over-treated seeds (Figure A 7a) Examples of damaged germinants include seeds
where the radicle did not elongate (Figure A 7b) seeds where the radicle elongated
but was delayed in freeing itself from the seed coat (Figure A7c) and seeds where the
radicle broke off completely from the embryo (Figure A7d) In addition to these
examples of over-treatment there were numerous small dark seeds that failed to
imbibe water For percussion treatment nearly all seeds took up water (Figure A8a)
Germination was rapid and uniform with little damage evident (Figure A8b)
To summarize both New Mexico locust and black locust seed lots were
dormant The standard hot water scarification treatment improved germination but
only to 56 and 41 for New Mexico locust and black locust respectively Nearly
all percussion durations improved total germination as well as germination rate over
the hot water treatment Germination was greater than 90 for percussion durations
of 4 5 and 10 minutes for New Mexico locust and 3 4 and 5 minutes for black locust
58
CI)
sgt t CI) 0
igt 0
sect ttl t E
Vl 0
c3
100r==~==~~=r------------------------------------~ ___ Percussion 5 min
middotmiddotmiddot0middotmiddotmiddot Hot Water -lIf- Control
80r=========~--~~----------------------------~
60
40
20 -~-~---~---------- ---~------------I
omiddot
O
_~_----------lIf-----~bull -----_ shy---shy I
Day 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A6 Effect of selected scarification treatments on germination speed for black locust First seven days of fourteen-day experiment are shown
Day 7
a
b c d
Figure A7 Damage to New Mexico locust seeds observed during germination counts following hot water scarification A7a Hot water replicate including damaged seedlings and numerous unimbibed seeds A7b Close-up ofseed where radicle failed to elongate A7c Close-up ofseed where radicle elongated but was delayed in freeing itself from the seed coat A7d Close-up ofseed where radicle completely broke offfrom embryo
60
a
b
Figure A8 New Mexico locust seeds following percussion treatment at time of germination counts A8a Percussion replicate with uniformly healthy seedlings and near complete imbibition of seeds A8b Close-up ofpercussion-treated seedling
61
Discussion
Papilionoid seeds have a specific anatomical feature known as the strophiole
or lens-the natural site of water entry to the seed (Hamly 1932) The strophiole is
located on the cotyledonary lobe of a locust seed (Figure A9) When a papilionoid
legume seed is percussed for the appropriate amount of time repeated hits on the
integrated seed coat loosen the constrained cells of the strophiolar region without
excessively damaging the rest of the seed coat (Ballard 1976)
When a percussed seed is soaked water enters exclusively through the
strophiole in a controlled manner (Kelly and Staden 1987) This regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues This
contrasts with seeds that have been hot water or acid scarified treatments that can
randomly degrade the seed coat Unlocalized cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rehman et al 1998 Marunda 1990)
Papilionoids are the largest subfamily of legumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust and black locust these legumes often play an integral role in the
revegetation ofdisturbed lands Developing superior scarification methods to the
standard hot water and acid treatments should fadlitate the use ofthese legumes in
restoration projects
62
Figure A9 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (bc)
63
APPENDIXB
GERMINATION PERCENTS AND STANDARD ERRORS FOR SOURCE
BLOCK AND TREATMENT FOR NEW MEXICO LOCUST
Table B 1 Gennination percents and standard errors for source block and treatment for New Mexico locust
Source Block Treatment Germination Percentage Standard Error
Capitan 1 Immersion 74 437 Capitan 2 Immersion 69 462 Capitan 3 Immersion 71 454 Capitan 4 Immersion 67 470 Capitan Boiling 30 sec 79 407 Capitan 2 Boiling 30 sec 76 427 Capitan 3 Boiling 30 sec 72 449 Capitan 4 Boiling 30 sec 76 427 Capitan Boiling 60 sec 71 454 Capitan 2 Boiling 60 sec 54 498 Capitan 3 Boiling 60 sec 67 470 Capitan 4 Boiling 60 sec 76 427 Capitan I Control 5 218 Capitan 2 Control 7 255 Capitan 3 Control 4 196 Capitan 4 Control 5 218 Capitan 1 Percussion I min 47 499 Capitan 2 Percussion I min
470
Capitan Percussion I min 39 488
Capitan 4 Percussion I min 38 485 Capitan I Percussion 2 min 55 497 Capitan Capitan
2
Percussion 2 min Percussion 2 min
71 65
454 477
Capitan 4 Percussion 2 min 56 496 Capitan I Percussion 4 min 86 347 Capitan 2 Percussion 4 min 79 407 Capitan 3 Percussion 4 min 81 392 Capitan 4 Percussion 4 min 82 384 Capitan I Percussion 8 min 90 300 Capitan 2 Percussion 8 min 88 325 Capitan 3 Percussion 8 min 90 300 Capitan 4 Percussion 8 min 90 300 Gila I Immersion 71 454 Gila 2 Immersion 77 421 Gila
Immersion 75 433
Gila 4 Immersion 72 449
Gila I Boiling 30 sec 68 466
Gila 2 Boiling 30 sec 81 392
Gila 3 Boiling 30 sec 54 498
Gila 4 Boiling 30 sec 63 483
Gila I Boiling 60 sec 55 497
Gila 2 Boiling 60 sec 68 466
Gila 3 Boiling 60 sec 57 495
Gila 4 Boiling 60 sec 70 458
65
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Gila 2 Control 13 336 Gila 3 Control 13 336 Gila 4 Control 20 400 Gila 1 Percussion 1 min 66 474 Gila 2 Percussion 1 min 57 495 Gila 3 Percussion 1 min 73 444 Gila 4 Percussion 1 min 82 384 Gila 1 Percussion 2 min 81 392 Gila 2 Percussion 2 min 86 347 Gila 3 Percussion 2 min 85 357 Gila 4 Percussion 2 min 54 498 Gila 1 Percussion 4 min 79 407 Gila 2 Percussion 4 min 83 376 Gila 3 Percussion 4 min 84 367 Gila 4 Percussion 4 min 79 407 Gila 1 Percussion 8 min 51 500 Gila 2 Percussion 8 min 76 427 Gila 3 Percussion 8 min 52 500 Gila 4 Percussion 8 min 74 439 Holman 1 Immersion 83 376 Holman 2 Immersion 86 347 Holman 3 Immersion 81 392 Holman 4 Immersion 90 300 Holman 1 Boiling 30 sec 90 300 Holman 2 Boiling 30 sec 85 357 Holman 3 Boiling 30 sec 87 336 Holman 4 Boiling 30 sec 83 376 Holman 1 Boiling 60 sec 79 407 Holman 2 Boiling 60 sec 80 400 Holman 3 Boiling 60 sec 86 347 Holman 4 Boiling 60 sec 93 255 Holman Control 25 433 Holman 2 Control 19 392 Holman Control 21 407
Holman 4 Control 24 427
Holman Percussion 1 min 77 421
Holman 2 Percussion 1 min 85 357
Holman 3 Percussion 1 min 80 400
Holman 4 Percussion 1 min 89 313
Holman 1 Percussion 2 min 88 325
Holman 2 Percussion 2 min 96 196
Holman 3 Percussion 2 min 93 255
Holman 4 Percussion 2 min 96 196
Holman 1 Percussion 4 min 92 271
Holman 2 Percussion 4 min 97 171
66
I
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Holman 3 Percussion 4 min 91 286 Holman 4 Percussion 4 min 96 196 Holman 1 Percussion 8 min 88 325 Holman 2 Percussion 8 min 85 357 Holman 3 Percussion 8 min 83 376 Holman 4 Percussion 8 min 82 384 Jemez 1 Immersion 53 499 Jemez 2 Immersion 44 496 Jemez 3 Immersion 52 500 Jemez 4 Immersion 70 458 Jemez 1 Boiling 30 sec 58 494 Jemez 2 Boiling 30 sec 55 497 Jemez 3 Boiling 30 sec 48 500 Jemez 4 Boiling 30 sec 65 477 Jemez 1 Boiling 60 sec 68 466 Jemez 2 Boiling 60 sec 63 483 Jemez 3 Boiling 60 sec 73 444 Jemez 4 Boiling 60 sec 89 313 Jemez 1 Control 2 140 Jemez 2 Control 9 286 Jemez 3 Control 6 237 Jemez 4 Control 8 271 Jemez 1 Percussion 1 min 58 494 Jemez 2 Percussion 1 min 63 483 Jemez 3 Percussion 1 min 77 421 Jemez 4 Percussion 1 min 89 313 Jemez 1 Percussion 2 min 68 466 Jemez 2 Percussion 2 min 64 480 Jemez 3 Percussion 2 min 80 400 Jemez 4 Percussion 2 min 83 376 Jemez Percussion 4 min 68 466 Jemez 2 Percussion 4 min 79 407 Jemez 3 Percussion 4 min 70 458 Jemez 4 Percussion 4 min 83 376 Jemez 1 Percussion 8 min 52 500 Jemez 2 Percussion 8 min 59 492 Jemez 3 Percussion 8 min 61 488 Jemez 4 Percussion 8 min 70 458 Magdalena 1 Immersion 69 462 Magdalena 2 Immersion 69 462 Magdalena J Immersion 70 458 Magdalena 4 Immersion 85 357 Magdalena 1 Boiling 30 sec 65 477 Magdalena 2 Boiling 30 sec 71 454 Magdalena 3 Boiling 30 sec 68 466
67
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Magdalena 4 Boiling 30 sec 83 376 Magdalena 1 Boiling 60 sec 63 483 Magdalena 2 Boiling 60 sec 68 466 Magdalena 3 Boiling 60 sec 63 483 Magdalena 4 Boiling 60 sec 82 384 Magdalena 1 Control 11 313 Magdalena 2 Control 6 237 Magdalena 3 Control 5 218 Magdalena 4 Control 4 196 Magdalena Percussion 1 min 32 466 Magdalena 2 Percussion 1 min 45 497 Magdalena 3 Percussion 1 min 46 498 Magdalena 4 Percussion I min 36 480 Magdalena 1 Percussion 2 min 67 470 Magdalena 2 Percussion 2 min 68 466 Magdalena 3 Percussion 2 min 65 477 Magdalena 4 Percussion 2 min 81 392 Magdalena 1 Percussion 4 min 87 336 Magdalena 2 Percussion 4 min 97 171 Magdalena 3 Percussion 4 min 83 376 Magdalena 4 Percussion 4 min 97 171 Magdalena 1 Percussion 8 min 98 lAO Magdalena 2 Percussion 8 min 100 000 Magdalena 3 Percussion 8 min 96 196 Magdalena 4 Percussion 8 min 98 lAO Manzano 1 Immersion 67 470 Manzano 2 Immersion 57 495 Manzano 3 Immersion 72 4049 Manzano 4 Immersion 73 4044 Manzano 1 Boiling 30 sec 70 458 Manzano 2 Boiling 30 sec 64 480 Manzano 3 Boiling 30 sec 71 454 Manzano 4 Boiling 30 sec 80 400 Manzano I Boiling 60 sec 50 500 Manzano 2 Boiling 60 sec 53 499 Manzano 3 Boiling 60 sec 59 492 Manzano 4 Boiling 60 sec 74 439 Manzano I Control 4 196 Manzano 2 Control 8 271 Manzano 3 Control 1 099 Manzano 4 Control 6 237 Manzano 1 Percussion 1 min 52 500 Manzano 2 Percussion 1 min 39 488 Manzano 3 Percussion 1 min 36 480 Manzano 4 Percussion 1 min 51 500
68
Table B 1 Continued
Source Block Treatment Gerrni~ation Percentage Standard Error
Manzano Percussion 2 min 53 499 Manzano 2 Percussion 2 min 58 494 Manzano 3 Percussion 2 min 61 488 Manzano 4 Percussion 2 min 62 485 Manzano 1 Percussion 4 min 76 427 Manzano 2 Percussion 4 min 85 357 Manzano 3 Percussion 4 min 79 407 Manzano 4 Percussion 4 min 83 376 Manzano 1 Percussion 8 min 87 336 Manzano 2 Percussion 8 min 83 376 Manzano 3 Percussion 8 min 83 376 Manzano 4 Percussion 8 min 75 433 Raton 1 Immersion 54 498 Raton 2 Immersion 37 483 Raton 3 Immersion 39 488 Raton 4 Immersion 69 462 Raton 1 Boiling 30 sec 36 480 Raton 2 Boiling 30 sec 20 400 Raton 3 Boiling 30 sec 28 449 Raton 4 Boiling 30 sec 60 490 Raton 1 Boiling 60 sec 15 357 Raton 2 Boiling 60 sec 171 Raton 3 Boiling 60 sec 4 196 Raton 4 Boiling 60 sec 35 477 Raton 1 Control 4 196 Raton 2 Control 12 325 Raton 3 Control 2 140 Raton 4 Control 6 237 Raton 1 Percussion 1 min 52 500 Raton 2 Percussion I min 29 454 Raton 3 Percussion I min 35 477 Raton 4 Percussion 1 min 31 462 Raton 1 Percussion 2 min 55 497 Raton 2 Percussion 2 min 35 477 Raton 3 Percussion 2 min 33 470 Raton 4 Percussion 2 min 42 494 Raton 1 Percussion 4 min 65 477 Raton 2 Percussion 4 min 52 500 Raton Percussion 4 min 51 500 Raton 4 Percussion 4 min 53 499 Raton 1 Percussion 8 min 88 325 Raton 2 Percussion 8 min 80 400 Raton 3 Percussion 8 min 73 444 Raton 4 Percussion 8 min 74 439 Sacramento 1 Immersion 43 495
69
Table B 1 Continued
-Source Block Treatment Germination Percentage Standard Error
Sacramento 2 Immersion 30 458 Sacramento 3 Immersion 38 485 Sacramento 4 Immersion 56 496 Sacramento 1 Boiling 30 sec 47 499 Sacramento 2 Boiling 30 sec 48 500 Sacramento 3 Boiling 30 sec 48 500 Sacramento 4 Boiling 30 sec 56 496 Sacramento 1 Boiling 60 sec 52 500 Sacramento 2 Boiling 60 sec 67 470 Sacramento 3 Boiling 60 sec 43 495 Sacramento 4 Boiling 60 sec 48 500 Sacramento 1 Control 0 000 Sacramento 2 Control 3 171 Sacramento 3 Control 3 171 Sacramento 4 Control 099 Sacramento 1 Percussion 1 min 57 495 Sacramento 2 Percussion 1 min 61 488 Sacramento 3 Percussion 1 min 62 485 Sacramento 4 Percussion 1 min 55 497 Sacramento 1 Percussion 2 min 84 367 Sacramento 2 Percussion 2 min 70 458 Sacramento 3 Percussion 2 min 81 392 Sacramento 4 Percussion 2 min 69 462 Sacramento 1 Percussion 4 min 91 286 Sacramento 2 Percussion 4 min 84 367 Sacramento 3 Percussion 4 min 84 367 Sacramento 4 Percussion 4 min 94 237 Sacramento 1 Percussion 8 min 82 384 Sacramento 2 Percussion 8 min 81 392 Sacramento 3 Percussion 8 min 82 384 Sacramento 4 Percussion 8 min 81 392 Sandia 1 Immersion 76 427 Sandia 2 Immersion 70 458 Sandia 3 Immersion 70 458 Sandia 4 Immersion 81 392 Sandia 1 Boiling 30 sec 78 414 Sandia 2 Boiling 30 sec 71 454 Sandia 3 Boiling 30 sec 63 483 Sandia 4 Boiling 30 sec 78 414 Sandia 1 Boiling 60 sec 67 470 Sandia 2 Boiling 60 sec 65 477 Sandia 3 Boiling 60 sec 74 439 Sandia 4 Boiling 60 sec 66 474 Sandia 1 Control 16 367 Sandia 2 Control 7 255
70
Table B 1 Continued
Source Block Treatment Gennination Percentage Standard Error
Sandia 3 Control 8 271 Sandia 4 Control 16 367 Sandia 1 Percussion 1 min 67 470 Sandia 2 Percussion 1 min 61 488 Sandia 3 Percussion 1 min 66 474 Sandia 4 Percussion 1 min 59 492 Sandia I Percussion 2 min 82 384 Sandia 2 Percussion 2 min 83 376 Sandia 3 Percussion 2 min 81 392 Sandia 4 Percussion 2 min 84 367 Sandia 1 Percussion 4 min 89 313 Sandia 2 Percussion 4 min 86 347 Sandia 3 Percussion 4 min 89 313 Sandia 4 Percussion 4 min 85 357 Sandia 1 Percussion 8 min 80 400 Sandia 2 Percussion 8 min 77 421 Sandia 3 Percussion 8 min 69 462 Sandia 4 Percussion 8 min 66 474
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Kigel 1 and Galili G 1995 Seed development and germination Marcel Dekker New York 853 p
Klemmedson 10 1994 New Mexico locust and parent material Influence on macronutrients of forest floor and soil Soil Sci Soc Am 1 58 974~980
Kuo WH Tam AW 1988 The pathway of water absorption of mung bean seeds Seed Sci amp Technol16(1) 139~144
Leadem CL 1997 Dormancy--unlocking seed secrets In Landis TD Thompson lR tech coords National Proceedings Forestand Conservation Nursery Assocs Gen Tech Rep PNW-G TR-419 Portland OR USDA For Ser PNW Res Sta 43-52
Lin Y Wagner MR Seweyestewa A Honahni MD DeGomez M McMillin JD 1996 Influence of cultural treatments on seed germination and seedling growth of Robinia neomexicana Forest Farm and Community Tree Res Reports Volume 1
Marunda CT 1990 Effects of seed pretreatment on the development ofAcacia auriculforinis and A holosericea seedlings ACIAR Proceedings Series No 28
Meginnis HG 1937 Sulphuric acid treatment to increase germination of black locust seed USDA Circ 453 34 p
Miklas PN Towensend CE Ladd SL 1987 Seedcoat anatomy and the scarification ofcicer milkvetch seed Crop Sci 27 766-772
Monsen SB 1984 Use of shrubs on mine spoils In The Challenge ofProducing Native Plants for the Intermountain Area Proceedings Intermountain Nurserymans Association 1983 conference Las Vegas NV compo PM Murphy 26-31 Ogden UT USDA For Ser Gen Tech Report INT-l68
Olson DF 1974 Robinia L Locust In Seeds of Woody Plants in the US tech coord CS Schopmeyer 728-731 USDA For Ser Agr Handbk No450
Rehman S Loescher RNl Harris P1C 1998 Dormancy breaking and germination ofAcacia salicina Lindl seeds Seed Sci amp Techno127 553-557
Rolston MP 1978 Water impermeable seed dormancy Bot Rev 44 365-396
74
SAS Institute Inc 1989 SASSTAT Users Guide Version 6 4th Edition Volume 1 Cary NC SAS Institute Inc 943 p
SerratoValenti G Conara L Ferrando M Modenesi P 1993 Structural and histochemical features ofStylosanthes scabra (Leguminosae Papilionoidae) seed coat as related to water entry Can J Bot 71 834-840
Simpson BJ 1988 A field guide to Texas trees Austin TX Texas Monthly Press 372p
Tran VN Cavanagh AK 1984 Structural aspects ofdormancy In Seed Physiology Vol 2 Murray DR (ed) p1-44 Academic Press Australia
USDA Forest Service 1948 Woody-plant seed manual USDA Misc Pub1654 416p
Vines RA 1960 Trees Shrubs and Woody Vines of the Southwest Austin Univ ofTexas Press 1104 p
Wagner MR Larson RE Craig TP 1992 Ecology and silvics ofNew Mexico locust (Robinia neomexicana) with an emphasis on the arboreal form p53-57 In JW Hanover K Miller and S Plesko (eds) Proc Intemat Confon Black Locust Biology Culture and Utilization Mich State Univ USA
Wilson JK 1944 Immersing seeds of species of Robinia in boiling water hastens germination J For 42 453-454
Zimmerman RW Carpenter SB 1980 First-year coppice production from a 5shyyear old black locust stand on surface mine spoil p309-314 Centr Hardwd For ConfProc University ofMissouri Columbia
f
Table 2 Mean seed weight and standard error by source
a
Source Mean seed weight (g) for Standard error 100 seeds n = 5
Raton Holman Jemez Sandia Manzano Magdalena Capitan Sacramento Gila
2872 b c 2522 d 1826 e 2753 c 2413 d 2559 d 2443 d 3130 a 3029 ab
00103 00403 00276 00301 00370 00374 00458 00279 00348
a Mean seed weights followed by the same letter are not significantly
different
19
experiment was conducted utilizing a randomized complete block design blocked by
time with a factorial treatment structure Experimental factors were seed source and
scarification treatment Nine seed sources from throughout New Mexico were used
(Figure 1 Table 1) Scarification treatments included a control three levels ofhot
water and four levels of percussion Hot water treatments included immersion at
100degC and boiling for periods of 30 seconds and 60 seconds Percussion treatment
levels were 1 2 4 and 8 minutes One hundred seeds per block were used to test
each treatment combination Four blocks of seed were tested one at a time (all seed
sources included in each run) in close chronological sequence (within 6 weeks) A
total of four 100-seed samples were used to test each source by scarification treatment
combination
Treatments
Hot water treatment of seeds utilized 50 ml test tubes filled with 30 ml
distilled water Each test tube was used to treat one 100-seed sample at a time
resulting in a ratio of4 parts water to 1 part seed by volume Test tubes were placed
in a beaker with the beaker filled with tap water to the same level as the water within
test tubes Nine test tubes (one per seed source) were placed in the beaker
simultaneously so that one replication of each seed sourcescarification treatment
cotnblnatlon was conducted simultaneously Hot water treatments include immersion
boiling For immersion treatments seeds were added to test tubes with the water
(boiling) and the test tubes were immediately removed from the beaker to
For the boiling treatments test tubes remained in the beaker with water at
20
boiling temperature for a set duration after the addition of seeds Following all
treatments test tubes with seeds cooled 24 hours in a test tube rack
Percussion treatment utilized a pneumatic paint shaker (Central Pneumatic
model 00422 Camarillo CA) Compressed air was maintained at 530 kPa plusmn30 kPa
(80 psi plusmn 5 psi) resulting in approximately 350 oscillations per minute Seeds were
placed in 118 ml (4 oz) soil tin cans (US Can Lobard IL) and the lids were secured
with duct tape Each can was used to treat one 100-seed sample To take advantage
ofgreatest lateral movement of the paint shaker a spacer (paint can) was used to
position the soil tin at the end of the shaking arm This allowed shaking to occur at
the greatest distance from the pivot point (fulcrum) Tins were placed perpendicular
to the direction of shaking motion allowing seeds to impact at greatest force by
hitting flat surfaces of the soil tin
Germination Testing
Before gennination testing all seeds were soaked 24 hours in reverse
osmosis-treated water (this was included in the process ofhot water treatment
jU~SCIlOea above) Following soaking seeds were placed in 100 cm Petri dishes lined
90 cm Whatman qualitative 1 filter paper circles (MG Scientific Pleasant
WI) One Petri dish was used for each treatment by source combination
papers were initially moistened with 5 mL reverse osmosis-treated water then
~VU1CUlJl moistened as needed to ensure adequate conditions for gennination
were monitored daily (at 300 PM) for gennination for 14 days in accordance
~UIUlnJlVUa Seed Testing Association (ISTA) standards for Robinia species
21
(1STA 1999) Tests were carried out at room temperature (22degC plusmnOSoC)
Germination was classified as protrusion of the radicle from the embryo by at least
1mm as viewed with the naked eye
X-ray Radiography
On August 132001 three lOO-seed samples from the Sacramento seed source
were treated for 14 or 8-minute percussion durations The seeds were sent
immediately by overnight delivery to the Faxitron X-Ray Corporation in Wheeling
Illinois where Dr Bob Karrfalt ofthe USDA Forest Service oversaw their imaging
Radiographs were made at SX with a Faxitron Specimen Radiography System Model
MX-20
Scanning Electron Microscope Photography
On February 192002 a Hitachi S-3200N Variable Pressure Scanning
Electron Microscope (Mountain View CA) was used to examine strophiolar cells of
untreated seeds and seeds percussed for 4 minutes Seeds were cut longitudinally
igt their entire length using a razor blade splitting the strophiolar region roughly in
Cross sections of seed samples magnifying the strophiolar region were viewed
600X under the microscope
Analysis
Categorical Analysis of Variance was used to determine treatment source and
(Le time) main effects and interactions The response variable was total
~vlI This procedure is a Chi-square test of homogeneity using the natural log
of germinated to non-germinated seed for each treatment Maximumshy
22
likelihood analysis was used to calculate Chi-square test statistics with their observed
significance levels A single contrast was conducted to cempare treatment means
against the control Observed significance levels less than or equal to 005 were
considered significant Pairwise comparisons were conducted between non-control
treatment mean percents using a conservative alp~a value of 005 divided by the
number of comparisons (Bonferroni approach) and approximate pairwise z statistics
Categorical analyses were performed using Proc Catmod (SAS Institute 1989)
RESULTS
Scarification seed source block and all interactions between factors
influenced germination (Table 3) The main effects are reported in the presence of
these interactions because the overall robustness of treatments and seed sources are of
interest Background levels ofnaturally occurring variation in seed sources are
seen in control germination percentages in Figure 2 In particular time variability
is unlikely to be under the control of the producer Appendix B details experimental
data for source by treatment gennination percents for each of the four blocks
Averaged over all treatments germination by source ranged from 40 to
79 (Figure 2) Averaged over all sources (Figure 3) and for each source (Figure 4
5) percussion scarification durations of4 or 8 minutes yielded greatest germination
Averaged over all sources germination increased with increasing durations of
percussion then leveled off for the 4- and 8-minute treatments (Figure 3) Immersion
and 30-second boiling scarification maximized germination for the hot water
treatments with a decrease in germination when seeds were boiled for 60 seconds
For all but two sources there is a similar ipcreasing trend in germination from
1to 4 minutes ofpercussion (Figure 4) The Holman source increased in germination
1 to 2 minutes then leveled off at 4 minutes The Jemez source changed little
1 to 4 minutes of treatment As treatment duration increased from 4 to 8
mutes five sources decreased in germination (Figure 4a) whereas three sources
24
Table 3 Categorical Analysis of Variance table for germination response to seed treatment seed source block and the interaction of the factors
Source of DF Chi-Square Observed Variability Significance Level
Source 8 98370 lt00001 Trt 7 233876 lt00001
contrast 1 16976 lt00001 SourceTrt 56 97077 lt00001 Block 3 5538 lt00001
SourceBlock 24 10162 lt00001 TrtBlock 21 7564 lt00001
SourceTrtBlock 168 34283 lt00001
S 4)
8 ~
0
sectg ~ 40 ~
N 0 0
20
o f0ltgt ~ ~4 jybshy ((iltgto ~~ K1ltgt 6~b-~ ~~ 0b-~~ ~~ ~b-~ ~b-~b
80
60 -I-- - ~- middotmiddotmiddotmiddotmiddot1
r -] Northern NM Sources
Central NM Sources
Southern NM Sources
sect-o ~
~b-v(i
SOURCE
Figure 2 Effect ofNew Mexico locust seed source on germination for data averaged over all scarification treatments Control germination is shown by solid line within bar
80
~ cd ~ ltl) () 60 ~ ~
N
t 0shyrti tshySltl)
40
0
20
o lmmersion Boiling 30 sec Boiling 60 sec Control 1 min 2min 4 min 8 min
Hot Water Treatments Percussion Treatments
Figure 3 Effect of scarification on germination ofNew Mexico locust for data averaged over all seed sources Means labelled with the same letter are not significantly different
100~----------------------~--~----------------~
80
60
ltU
~
40 IJ
sectgsI
l100
80
60
40
a I middotmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot middotImiddotmiddotmiddotmiddotmiddotmiddot E
_~iii-bull _ _ bull f --~--
t-- -~ ---- -shy ---- shyI ~ ---- - shy
L
fiII
~
b
- shy ---r
- Holman
Sacramento Sandia Gila Jemez
c ~
ltgt ~
Tmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot-I
j~=~=-~~-1
I middotmiddot ~ bull r
t tr
Y -------M-a-gd-a-Ie-n-a--
1 _ _
fCapitan
---- Manzano ~y -_ Raton
2 4 8
Time interval ofpercussion scarification (min)
Figure 4 Effect of seed source by treatment interaction on New Mexico locust germination for percussion scarification pattern a and pattern b
28
tv 0
~ d
~ A s o
1 c3
80
60
40
20
o
TIT
t------=middotfmiddotmiddot-middotmiddot-middotmiddot-middotmiddot 1- --- - - --=- - -shy--cE= - _--shy --shy ----shy ~ ~ ---shy ~ -~- _ --shy-------r 1
I
Raton Holman
-_ Jemez
--- Sandia Manzano Magdalena Capitan Sacramento Gila
Immersion Boiling 30 sec Boiling 60 sec
Hot Water Scarification
Figure 5 Effect of seed source by treatment interaction on New Mexico locust germination for hot water scarification
continued to increase in germination (Figure 4b) There was no difference between
the two treatment levels for the Manzano source (Figure 4b)
The effect ofhot water scarification also varied among sources with few
overall differences between hot water treatments (Figure 5) However boiling for 60
seconds yielded highest germination for the Jemez source whereas increasing
durations ofboiling decreased germination for the Raton source
In addition to consistently high germination 4- and 8-minute percussionshy
treated seeds germinated as fast or faster than all other treatments (Figure 6 Table 4)
050 measured as the number of days needed to complete 50 ofeventual
germination averaged 2 days for both ofthese treatments
X-ray radiographs were taken of seeds collected from the Sacramento source
percussed for 1 4 or 8 minutes (Figure 7) Seeds show signs of damage at 8 minutes
ofpercussion (Figure 8) Compared to an undamaged seed percussed for only 4
minutes (Figure 8a) damage at the 8-minute level includes fractured radicles pitting
of the seed coat and chips in seed coat structure (Figure 8b-d)
Scanning electron microscope (SEM) images at 600X reveal the direct effect
of 4 minutes of percussion on cells of the strophiolar region (Figure 9) Whereas
untreated seeds retain cellular organization in the strophiolar region strophiolar cells
seeds treated for 4 minutes ofpercussion separate and raise resulting in a
~-~e~U appearance
30
Umiddot-~middot--l-~---middotymiddotmiddotmiddot-----middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot--middot--middotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middotmiddotmiddotmiddotmiddotmiddotmiddot- -
050= gt1
-e-- Percussion 8 min -0- Percussion 4 min ---T- Percussion 2 min -----ltil- Percussion 1 min -- Immersion --0- Boiling 30 sec -+- Boiling 60 sec -ltgt- Control
2 4 6 8 10 12
Day Figure 6 Timecourse ofNew Mexico locust gennination following scarification
treatments G50 is the number of days to 50 of eventual germination
31
14
Table 4 Effect of seed source and treatment on G50 (days to 50 germination)
Seed Source Control Immersion B30sec B60sec Plmin P2min P4min P8min mean Raton 3 3 5 7 4 3 2 2 4 Holman 2 2 2 2 2 2 2 2 2
Jemez 4 6 6 5 3 2 2 2 4 Sandia 2 2 2 2 2 2 2 2 2
w tv Manzano 5 2 2 3 3 2 2 2 3
Magdalena 4 2 2 3 4 3 2 1 3
Capitan 5 4 2 2 3 2 2 2 3
Sacramento 3 2 2 2 4 3 2 2 3 Gila 3 2 2 2 2 2 2 2 2 mean 3 3 3 3 3 2 2 2 3 standard 11 14 16 18 09 05 0 03 08 deviation
Note Columns and rows may not agree due to rounding
Figure 7 X-ray radiographs ofSacramento seed source percussed for 1 4 or 8 minutes
33
Figure 8a-d Seed damage close-ups Figure a shows undamaged seed percussed for 4 minutes Figures b-d show seeds percussed for 8 minutes Damage includes fracturing ofradicle in b complete removal ofseed coat in c and pitting ofseed coat in d
34
Untreated Treated
Figure 9 SEM images ofuntreated vs 4-minute percussion-treated strophiolar cells ofNew Mexico locust seeds Treated cells are loosened separated and raised
DISCUSSION
Determining the Optimal Treatment
Averaging results of scarification treatmems across seed sources is a useful
tool to predict which treatment is most likely to be successful for an unknown seed
source Averaged over all sources four- and eight-minute percussion treatments
resulted in the highest germination 815 and 790 respectively and produced
germination that was as fast or faster than all other treatments Although there was no
difference between these two treatments on average an argument for 4 minutes of
percussion as a standard protocol for treating New Mexico locust seeds can be made
Descriptively 4 minutes of percussion improved germination over 8 minutes
ofpercussion for a majority of sources (five out of nine) 8 minutes of percussion
improved germination for three ofnine sources with no difference for a fmal source
For individual sources 4 minutes ofpercussion was always the best or secondmiddot best
treatment and was always equal to or better than all hot water treatments
Germination at 8 minutes ofpercussion was lower than all other percussion durations
for the Jemez and Gila sources and was lower than at least one hot water treatment for
these sources as well
The x-ray radiographs of the Sacramento seed source illustrate the excessive
internal and external seed damage that can occur with 8 minutes of percussion
germination counts 8-minute percussed seeds from Holman and Capitan
ClUll signs of damage that were visible to the naked eye (see below)
r~evertheless some of these seeds still fit the criteria for germination Neither x-ray
36
photography nor visible observations during seed counts revealed seed damage at the
4-minute percussion level In operational trials seeds percussed for 4 minutes
developed into healthy seedlings (Khadduri and Harrington unpublished data)
Strophiole and Seed Coat Behavior
Four minutes ofpercussion successfully breaks seed dormancy in a large
percentage ofNew Mexico locust seeds without excessively damaging the
surrounding seed coat or internal seed coat structures Most papilionoid legume
seeds including seeds ofNew Mexico locust have a specific region of the seed coat
known as the strophiole or lens (Hamly 1932) The strophiole is located on the
cotyledonary lobe of a locust seed (Figure 10 a-c) If seeds are not indiscriminately
damaged the strophiole is the first point of water entry to the embryo (Hopkinson
1997) When a papilionoid seed is percussed for the appropriate amount of time
repeated hits on the integrated seed coat loosen the constrained cells of the strophiolar
region without excessively damaging the rest of the seed coat (Ballard 1976) SEM
photos illuStrate that 4 minutes of percussion loosened and separated strophiolar cells
ofNew Mexico locust seeds when compared with constrained organized strophiolar
cells of untreated seeds (Figure 9) Research-has shown that water passes to the
embryo through the treated strophiole in a controlled manner and regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues
(Kelly and van Staden 1987)
37
10 Strophiole ofunimbibed New Mexico locust seed (a) and following uptake (bc)
38
In contrast hot-water treatments can excessively degrade the seed coat
(Rebman et al 1998 Marunda 1990) Random cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rebman et aI 1998 Marunda 1990) Along with a
proportionately large amount ofun-imbibed (thus under-treated) seeds (Figure l1a)
damage was observed in hot-water treated seeds from six sources during germination
counts Damage included seeds where the radicle failed to elongate (Figure 11 b)
seeds where the radicle elongated but was delayed in freeing itself from the seed coat
(Figure 11c) and seeds where the radicle completely broke off from the embryo
(Figure lId) In addition some seeds germinated in reverse (the cotyledon emerged
fIrst) If the radicle emerged completely from the seed coat they were counted as
germinated
While nearly all seeds treated for 8 minutes ofpercussion imbibed water
damage was evident in x-ray evaluations (Sacramento seed source) and in
germination counts (Holman and Capitan) at this treatment level Seed damage
during germination counts was similar to levels found in hot-water treated seeds
suggesting that over-treatment by both hot water and longer durations of percussion
may result in similar damage to seed structure
The Sacramento and Holman seed sources were two of the fIve sources that
opcrplltlpn in germination from the 4- to 8-minute durations of percussion
the benefIts ofloosening the strophiole as evidenced by imbibition of
all 8-minute treated seeds were outweighed by external and internal seed coat
39
a
b c d
11 Damage to New Mexico locust seeds observed during germination cotmts following hot water scarification 9a Hot water replicate including ~t seedlings and numerous unimbibed seeds 9b Close-up of seed where raallCle failed to elongate 9c Close-up ofseed where radicle elongated but was
-gtIn in freeing itself from the seed coat 9d Close-up ofseed where radicle broke off from embryo
40
damage (which also may be contributing to high imbibition) For the three sources
for which 8 minutes of percussion resulted in maximum germination extended
percussion of seeds necessary to loosen the strophiole may not have excessively
damaged the seed anatomy The Manzano source did not significantly change in
germination from 4 to 8 minutes of percussion suggesting that an increase in
permeable seeds at the 8-minute treatment duration was balanced by the number of
seeds that failed to germinate due to seed damage To summarize the 4-minute
percussion treatment improved germination over 8 minutes ofpercussion for a
majority of sources (five ofnine) the 8-minute percussion treatment improved
germination over 4-minutes ofpercussion for three sources and there was no
difference with the Manzano source Notably for the Raton source 8 minutes of
percussion increased germination by 235 percentage points over 4-minutes of
percussion (7875 vs 5525 respectively) the next best treatment Raton was the
only source to result in germination less than 75 for 4 minutes ofpercussion so a
grower may wish to apply longer percussion durations to lots that do not respond well
to this treatment level
Finally there was an interesting trend in the Jemez and Raton sources seed
that significantly contributed to the source by treatment interaction (Figure 12) A
~u test showed that the Jemez source had a significantly lighter seed weight than
other sources while the Raton source had an average seed weight For the Jemez
the longest duration of boiling increased germination Conversely only short
U4UVll) of percussion were needed to achieve high germination and the longest
41
80 _ Jemez a Raton
iD
~ C iD e iD
Q c 0 ~
c ~ iD 0
20
0------shy
Immersion Boiling 30 sec Boiling 60 sec
Hot Water Scarification
10degTr========~--------~------------------------------
80
60
40
20
_ Jemez Raton
0------shy
1 min 2 min 4 min 8 min
Percussion Scarification
Figure 12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources
42
b
--~----~~~~========~---- duration ofpercussion reduced germination For the Raton source longer durations
ofboiling consistently decreased germination whereas longer percussion treatments
consistently increased germination
A possible explanation is that a thick seed coat is associated with a loose
strophiole structure and that a thin seed coat is associated with a constrained
strophiole This contrast merits further study and may suggest an ecological
adaptation Further investigation ofthis phenomenon may contribute to successful
pre-screening ofunknown seed sources to determine optimal treatment leveL
43
CONCLUSION
Based on results obtained in pilot study (see Appendix) and thesis
experiments 4 minutes ofpercussion is recommended as a standard protocol for
treating an unknown source ofNew Mexico locust seed In addition to laboratory
results the 4-minute percussion treatment has resulted in germination greater than
80 and healthy seedling grow-outs for production purposes (unpublished data) In
laboratory experiments only one hundred seeds per 118 ml (40z) soil tin were
percussed at a time whereas for production we have treated up to five hundred seeds
per tin For large-scale production ofNew Mexico locust seedlings growers may
wish to experiment with larger quantities of seed and larger-size soil tins
Seed storage trials following percussion treatments were not conducted
However germination greater than 80 and healthy seedlings were obtained when
seeds were percussion-treated and subsequently stored at room-temperature (22degC)
for one week (unpublished data) Seed storage trials merit further study as the
standard hot water treatment leaves seeds in a softened condition that is not
conducive to storage
Hydroseeding is an effective method for direct seeding in reclamation
situations Percussion treatment may be conducive to hydro seeding as seeds remain
a dry relatively undamaged yet permeable condition following treatment Sulfuric
seeds also remain dry following treatment but extensive damage to seed coat
poor seedling vigor have been reported with this treatment (Kelly and van Staden
A grow-out study using a hydro seeder on a reclamation site testing percussion
44
against other treatments such as sulfuric acid and mechanical scarification also
merits further study
Papilionoids are the largest subfamily oflegumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust these legumes often play an integral role in the revegetation of
disturbed lands The pilot study (Appendix) indicates that percussion scarification
successfully treats the widely used black locust as well Developing superior
scarification methods to the standard hot water mechanical scarification and acid
treatments may facilitate the use ofpapilionoid legumes in reclamation projects
45
APPENDICES
APPENDIX A
PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR
NEW MEXICO LOCUST AND BLACK LOCUST SEEDS
Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4
Hot water and sulfuric acid soaks are traditional treatments for seeds of many
temperate woody legwnes including locusts However these scarification techniques
often produce inconsistent germination Percussion scarification where seeds are
repeatedly propelled against a hard surface was compared with hot water
scarification to evaluate treatment efficacy for New Mexico locust (Robinia
neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment
seeds were placed in a 98degC water bath which was immediately removed from the
heat source For percussion scarification seeds were placed in a soil sample tin and
agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the
control were followed by 24-hour water soaks Hot water baths resulted in 56 and
41 germination for New Mexico locust and black locust respectively For both
species nearly all durations ofpercussion increased germination over the hot water
treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3
4 and 5 minutes for black locust resulted in at least 90 germination Traditional
scarification treatments randomly degrade the entire seed coat which can lead to
tissue damage during water uptake Percussion scarification specifically weakens the
strophiole the natural source ofwater entry to the seed in papilionoid legwnes
INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom
2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031
48
Following percussion imbibition is controlled through the strophiole and underlying
tissue is protected
Keywords New Mexico locust black locust scarification percussion germination
Introduction
Locust trees (Robinia spp) are aggressive pioneer species that quickly
colonize disturbed land fix atmospheric nitrogen in the soil and stabilize slopes that
are prone to erosion (Klemmedson 1994 Ashby et al 1985) (Figure A1ab)
Recognition of these attributes has contributed to increased demand for locusts in
reclamation projects Locust seeds however exhibit physical dormancy where a
thick seed coat prevents the movement of water and gasses to the embryo (Leadem
1997)
Sulfuric acid or hot water soaks have traditionally been used to break seed
dormancy in macro-propagation of locusts Many growers are moving away from the
use of su1furic acid in treating locust seeds (Dreesen and Harrington 1997) In
addition to being dangerous acid soak durations must be specifically correlated to
seed lot (Olson 1974) Collections of native seed can vary tremendously in seed size
seed weight and hard seededness etc This variability limits the utility of sulfuric
acid soaks as even within a given lot some seeds may be damaged whereas others
are under-treated and fail to imbibe (take up water) A survey of Southwestern
container growers indicates that hot water baths are currently the most common
scarification method for locust seeds (Hine et al 1997) However hot water baths
also produce inconsistent germination (Lin et al 1996)
49
b
Figure AI New Mexico locust a primary invader following disturbance Ala Crown-sprouting at edge ofburn near Los Alamos NM--one year after Cerro Grande fire Alb Colonizing road cut Gila National Forest NM
50
Percussion scarification where seeds are repeatedly propelled against a hard
surface is an alternative dormancy-breaking method that has proven successful in
legumes related to locusts (Hamly 1932 Barton 1947) We compared this alternative
treatment to hot water scarification to evaluate treatment efficacy for New Mexico
locust and black locust seeds
Materials and Methods
Commercial New Mexico locust seeds (Western Native Seed Coaldale CO)
were collected fall 2000 in Huerfano County CO Black locust seeds were collected
September 2000 from Taos County NM by harvesting ripe pods from several trees at
a distance of up to 3 m from ground level Black locust seeds were allowed to air-dry
for 3 weeks and were threshed and separated from large chaffusing a greenhouse
fan A South Dakota Seed Blower (Chicago Illinltois) was used to remove fine chaff
from both seed lots Seeds were storedmiddotat 2-4C until the germination study was
conducted in March 2001
This study compared hot water scarification with percussion scarification
using treatment and species as experimental factors Along with a control
scarification treatments included immersion of seed in boiling water as well as
percussion durations of 1 2 3 45 or 10 minutes Four 100-seed samples were used
to test each species by scarification treatment combination
For the hot water treatment test tubes were filled with 30 mL water and
placed in a water-filled beaker (Figure A2a) The bath was raised to boiling (-98C at
51
b
c
Figure A2 Implementation ofhot water and percussion scarification A2a Hot water bath A2b Paint-shaker set-up A2c Placing soil tin containing seeds at end ofpaint shaker ann
52
Las Cruces NM elevation of 1300 meters) Each 100-seed replicate was placed in a
test tube and each test tube was immediately removed to cool at room temperature
Percussion scarification was implemented using a pneumatic paint shaker
(Central Pneumatic model 00422 Camarillo CA) (Figure A2b) This allowed
standardization of treatment intensity Compressed air pressure was maintained at
530kPa plusmn 30kPa (80psi plusmn 5psi) resulting in approximately 350 oscillations per
minute Each lOO-seed sample was placed in a 118 ml (4 oz) soil tin for shaking To
maximize lateral movement of paint shaker a paint can was used as a spacer to place
soil tins at the end of the shaking arm (Figure A2c) This allowed shaking to occur at
the greatest distance from the pivot point (fulcrum) Following all treatments
including the control seeds were soaked for twenty-four hours in distilled water prior
to plating out
Gennination was evaluated on lab benches at room temperature Each
replicate of seeds was placed in a 100 cm petri dish on moistened filter paper with
humidity maintained by enclosing the petri dishes in ziplock bags Gennination was
monitored daily for 14 days in accordance with International Seed Testing
Association standards for Robinia species (ISTA 1999) Gennination was defined as
protrusion of the radicle from the embryo by at least 1mm as viewed by the naked
eye
Categorical Analysis of Variance using Prpc Catmod (SAS Institute 1989)
was used to detennine treatment differences in germination percentages for each
source This procedure is an extension of a Chi-square test of homogeneity using the
53
natural log of the ratio of germinated to non-germinated seeds for each treatment Pshy
values less than 005 were considered significant A limited set of pairwiseshy
comparisons was conducted to compare treatment mean percents using a
conservative alpha value of 005 divided by the number of comparisons
Results
Percussion maximized germination for both New Mexico locust and black
locust There were three germination response levels for New Mexico locust (Figure
A3) First there was a low response in the control indicating a dormant seed lot
The hot water treatment and I-minute duration of percussion share a second
intermediate response Germination was highest at percussion levels from 2 to 10
minutes
A selected percussion treatment intermediate in time (5 minutes) produced the
steepest germination curve indicating the fastest germination rate (Figure A4) The
hot water treatment produced an intermediate curve or relatively slower germination
rate The control curve was very flat indicating the slowest germination rate for New
Mexico locust
Control seeds germinated poorly for black locust as well (Figure A5) Hot
water scarification improved germination over the control but all levels of percussion
improved germination over hot water The optimal durations of percussion treatment
were bracketed with a significant increase from the 1 to 2 minute percussion level a
high germination response from 2 to 5 minutes of percussion and finally a significant
54
~ d 25 CL)
p t1 0 ~ t1
g Vl ~ Vl
100~---------------------------------------------------------------
80
60
40
20
o Control Hot Water Percussion
I min Percussion
2 min Percussion
3 min
Scarification
Percussion 4 min
Figure A3 Effect of scarification on germination response for New Mexico locust
Percussion 5 min
=significant difference between adjacent treatment bars ns = no significant difference
Percussion 10 min
~ ~ () u agt
Il-t d o ~
~ ~ c3
100middotlr~~~------------------------------------e- Percussion 5min middot 0middot Hot Water -- Control
80-J=middot======
6b-middot-lmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middot--middotmiddot-middot~-middotmiddot -middotmiddot middot middotmiddot-middot 1
40
20
-----~-----------
0 0 0 middotmiddotmiddotmiddotmiddot0middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot0
0 --~-----y-----~------_---~--- T-----~
o shy -TDay 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A4 Effect of selected scarification treatments on germination speed for New Mexico locust First seven days of fourteen-day experiment are shown
Day 7
80
~ cd
5 60 ()
[) Cl d 0 1t1 d
40sect dUl
-J
20
o
ns
Scarification
Figure A5 Effect of scarification on germination response for black locust = significant difference between adjacent treatment bars ns = no significant difference
decrease at the 10-minute level This drop-off suggests that 10 minutes of percussion
over-treats seeds from this lot of-black locust
A selected percussion treatment intermediate in time (5 minutes) also had the
fastest germination rate for black locust (Figure A6)_ The hot water curve is
somewhat flatter indicating a slower germination rate The control curve is very flat
indicating a slow germination rate
A qualitative difference between hot water and percussion-treated seeds was
also observed Along with healthy germinants the hot water treatment produced
over-treated seeds (Figure A 7a) Examples of damaged germinants include seeds
where the radicle did not elongate (Figure A 7b) seeds where the radicle elongated
but was delayed in freeing itself from the seed coat (Figure A7c) and seeds where the
radicle broke off completely from the embryo (Figure A7d) In addition to these
examples of over-treatment there were numerous small dark seeds that failed to
imbibe water For percussion treatment nearly all seeds took up water (Figure A8a)
Germination was rapid and uniform with little damage evident (Figure A8b)
To summarize both New Mexico locust and black locust seed lots were
dormant The standard hot water scarification treatment improved germination but
only to 56 and 41 for New Mexico locust and black locust respectively Nearly
all percussion durations improved total germination as well as germination rate over
the hot water treatment Germination was greater than 90 for percussion durations
of 4 5 and 10 minutes for New Mexico locust and 3 4 and 5 minutes for black locust
58
CI)
sgt t CI) 0
igt 0
sect ttl t E
Vl 0
c3
100r==~==~~=r------------------------------------~ ___ Percussion 5 min
middotmiddotmiddot0middotmiddotmiddot Hot Water -lIf- Control
80r=========~--~~----------------------------~
60
40
20 -~-~---~---------- ---~------------I
omiddot
O
_~_----------lIf-----~bull -----_ shy---shy I
Day 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A6 Effect of selected scarification treatments on germination speed for black locust First seven days of fourteen-day experiment are shown
Day 7
a
b c d
Figure A7 Damage to New Mexico locust seeds observed during germination counts following hot water scarification A7a Hot water replicate including damaged seedlings and numerous unimbibed seeds A7b Close-up ofseed where radicle failed to elongate A7c Close-up ofseed where radicle elongated but was delayed in freeing itself from the seed coat A7d Close-up ofseed where radicle completely broke offfrom embryo
60
a
b
Figure A8 New Mexico locust seeds following percussion treatment at time of germination counts A8a Percussion replicate with uniformly healthy seedlings and near complete imbibition of seeds A8b Close-up ofpercussion-treated seedling
61
Discussion
Papilionoid seeds have a specific anatomical feature known as the strophiole
or lens-the natural site of water entry to the seed (Hamly 1932) The strophiole is
located on the cotyledonary lobe of a locust seed (Figure A9) When a papilionoid
legume seed is percussed for the appropriate amount of time repeated hits on the
integrated seed coat loosen the constrained cells of the strophiolar region without
excessively damaging the rest of the seed coat (Ballard 1976)
When a percussed seed is soaked water enters exclusively through the
strophiole in a controlled manner (Kelly and Staden 1987) This regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues This
contrasts with seeds that have been hot water or acid scarified treatments that can
randomly degrade the seed coat Unlocalized cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rehman et al 1998 Marunda 1990)
Papilionoids are the largest subfamily of legumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust and black locust these legumes often play an integral role in the
revegetation ofdisturbed lands Developing superior scarification methods to the
standard hot water and acid treatments should fadlitate the use ofthese legumes in
restoration projects
62
Figure A9 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (bc)
63
APPENDIXB
GERMINATION PERCENTS AND STANDARD ERRORS FOR SOURCE
BLOCK AND TREATMENT FOR NEW MEXICO LOCUST
Table B 1 Gennination percents and standard errors for source block and treatment for New Mexico locust
Source Block Treatment Germination Percentage Standard Error
Capitan 1 Immersion 74 437 Capitan 2 Immersion 69 462 Capitan 3 Immersion 71 454 Capitan 4 Immersion 67 470 Capitan Boiling 30 sec 79 407 Capitan 2 Boiling 30 sec 76 427 Capitan 3 Boiling 30 sec 72 449 Capitan 4 Boiling 30 sec 76 427 Capitan Boiling 60 sec 71 454 Capitan 2 Boiling 60 sec 54 498 Capitan 3 Boiling 60 sec 67 470 Capitan 4 Boiling 60 sec 76 427 Capitan I Control 5 218 Capitan 2 Control 7 255 Capitan 3 Control 4 196 Capitan 4 Control 5 218 Capitan 1 Percussion I min 47 499 Capitan 2 Percussion I min
470
Capitan Percussion I min 39 488
Capitan 4 Percussion I min 38 485 Capitan I Percussion 2 min 55 497 Capitan Capitan
2
Percussion 2 min Percussion 2 min
71 65
454 477
Capitan 4 Percussion 2 min 56 496 Capitan I Percussion 4 min 86 347 Capitan 2 Percussion 4 min 79 407 Capitan 3 Percussion 4 min 81 392 Capitan 4 Percussion 4 min 82 384 Capitan I Percussion 8 min 90 300 Capitan 2 Percussion 8 min 88 325 Capitan 3 Percussion 8 min 90 300 Capitan 4 Percussion 8 min 90 300 Gila I Immersion 71 454 Gila 2 Immersion 77 421 Gila
Immersion 75 433
Gila 4 Immersion 72 449
Gila I Boiling 30 sec 68 466
Gila 2 Boiling 30 sec 81 392
Gila 3 Boiling 30 sec 54 498
Gila 4 Boiling 30 sec 63 483
Gila I Boiling 60 sec 55 497
Gila 2 Boiling 60 sec 68 466
Gila 3 Boiling 60 sec 57 495
Gila 4 Boiling 60 sec 70 458
65
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Gila 2 Control 13 336 Gila 3 Control 13 336 Gila 4 Control 20 400 Gila 1 Percussion 1 min 66 474 Gila 2 Percussion 1 min 57 495 Gila 3 Percussion 1 min 73 444 Gila 4 Percussion 1 min 82 384 Gila 1 Percussion 2 min 81 392 Gila 2 Percussion 2 min 86 347 Gila 3 Percussion 2 min 85 357 Gila 4 Percussion 2 min 54 498 Gila 1 Percussion 4 min 79 407 Gila 2 Percussion 4 min 83 376 Gila 3 Percussion 4 min 84 367 Gila 4 Percussion 4 min 79 407 Gila 1 Percussion 8 min 51 500 Gila 2 Percussion 8 min 76 427 Gila 3 Percussion 8 min 52 500 Gila 4 Percussion 8 min 74 439 Holman 1 Immersion 83 376 Holman 2 Immersion 86 347 Holman 3 Immersion 81 392 Holman 4 Immersion 90 300 Holman 1 Boiling 30 sec 90 300 Holman 2 Boiling 30 sec 85 357 Holman 3 Boiling 30 sec 87 336 Holman 4 Boiling 30 sec 83 376 Holman 1 Boiling 60 sec 79 407 Holman 2 Boiling 60 sec 80 400 Holman 3 Boiling 60 sec 86 347 Holman 4 Boiling 60 sec 93 255 Holman Control 25 433 Holman 2 Control 19 392 Holman Control 21 407
Holman 4 Control 24 427
Holman Percussion 1 min 77 421
Holman 2 Percussion 1 min 85 357
Holman 3 Percussion 1 min 80 400
Holman 4 Percussion 1 min 89 313
Holman 1 Percussion 2 min 88 325
Holman 2 Percussion 2 min 96 196
Holman 3 Percussion 2 min 93 255
Holman 4 Percussion 2 min 96 196
Holman 1 Percussion 4 min 92 271
Holman 2 Percussion 4 min 97 171
66
I
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Holman 3 Percussion 4 min 91 286 Holman 4 Percussion 4 min 96 196 Holman 1 Percussion 8 min 88 325 Holman 2 Percussion 8 min 85 357 Holman 3 Percussion 8 min 83 376 Holman 4 Percussion 8 min 82 384 Jemez 1 Immersion 53 499 Jemez 2 Immersion 44 496 Jemez 3 Immersion 52 500 Jemez 4 Immersion 70 458 Jemez 1 Boiling 30 sec 58 494 Jemez 2 Boiling 30 sec 55 497 Jemez 3 Boiling 30 sec 48 500 Jemez 4 Boiling 30 sec 65 477 Jemez 1 Boiling 60 sec 68 466 Jemez 2 Boiling 60 sec 63 483 Jemez 3 Boiling 60 sec 73 444 Jemez 4 Boiling 60 sec 89 313 Jemez 1 Control 2 140 Jemez 2 Control 9 286 Jemez 3 Control 6 237 Jemez 4 Control 8 271 Jemez 1 Percussion 1 min 58 494 Jemez 2 Percussion 1 min 63 483 Jemez 3 Percussion 1 min 77 421 Jemez 4 Percussion 1 min 89 313 Jemez 1 Percussion 2 min 68 466 Jemez 2 Percussion 2 min 64 480 Jemez 3 Percussion 2 min 80 400 Jemez 4 Percussion 2 min 83 376 Jemez Percussion 4 min 68 466 Jemez 2 Percussion 4 min 79 407 Jemez 3 Percussion 4 min 70 458 Jemez 4 Percussion 4 min 83 376 Jemez 1 Percussion 8 min 52 500 Jemez 2 Percussion 8 min 59 492 Jemez 3 Percussion 8 min 61 488 Jemez 4 Percussion 8 min 70 458 Magdalena 1 Immersion 69 462 Magdalena 2 Immersion 69 462 Magdalena J Immersion 70 458 Magdalena 4 Immersion 85 357 Magdalena 1 Boiling 30 sec 65 477 Magdalena 2 Boiling 30 sec 71 454 Magdalena 3 Boiling 30 sec 68 466
67
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Magdalena 4 Boiling 30 sec 83 376 Magdalena 1 Boiling 60 sec 63 483 Magdalena 2 Boiling 60 sec 68 466 Magdalena 3 Boiling 60 sec 63 483 Magdalena 4 Boiling 60 sec 82 384 Magdalena 1 Control 11 313 Magdalena 2 Control 6 237 Magdalena 3 Control 5 218 Magdalena 4 Control 4 196 Magdalena Percussion 1 min 32 466 Magdalena 2 Percussion 1 min 45 497 Magdalena 3 Percussion 1 min 46 498 Magdalena 4 Percussion I min 36 480 Magdalena 1 Percussion 2 min 67 470 Magdalena 2 Percussion 2 min 68 466 Magdalena 3 Percussion 2 min 65 477 Magdalena 4 Percussion 2 min 81 392 Magdalena 1 Percussion 4 min 87 336 Magdalena 2 Percussion 4 min 97 171 Magdalena 3 Percussion 4 min 83 376 Magdalena 4 Percussion 4 min 97 171 Magdalena 1 Percussion 8 min 98 lAO Magdalena 2 Percussion 8 min 100 000 Magdalena 3 Percussion 8 min 96 196 Magdalena 4 Percussion 8 min 98 lAO Manzano 1 Immersion 67 470 Manzano 2 Immersion 57 495 Manzano 3 Immersion 72 4049 Manzano 4 Immersion 73 4044 Manzano 1 Boiling 30 sec 70 458 Manzano 2 Boiling 30 sec 64 480 Manzano 3 Boiling 30 sec 71 454 Manzano 4 Boiling 30 sec 80 400 Manzano I Boiling 60 sec 50 500 Manzano 2 Boiling 60 sec 53 499 Manzano 3 Boiling 60 sec 59 492 Manzano 4 Boiling 60 sec 74 439 Manzano I Control 4 196 Manzano 2 Control 8 271 Manzano 3 Control 1 099 Manzano 4 Control 6 237 Manzano 1 Percussion 1 min 52 500 Manzano 2 Percussion 1 min 39 488 Manzano 3 Percussion 1 min 36 480 Manzano 4 Percussion 1 min 51 500
68
Table B 1 Continued
Source Block Treatment Gerrni~ation Percentage Standard Error
Manzano Percussion 2 min 53 499 Manzano 2 Percussion 2 min 58 494 Manzano 3 Percussion 2 min 61 488 Manzano 4 Percussion 2 min 62 485 Manzano 1 Percussion 4 min 76 427 Manzano 2 Percussion 4 min 85 357 Manzano 3 Percussion 4 min 79 407 Manzano 4 Percussion 4 min 83 376 Manzano 1 Percussion 8 min 87 336 Manzano 2 Percussion 8 min 83 376 Manzano 3 Percussion 8 min 83 376 Manzano 4 Percussion 8 min 75 433 Raton 1 Immersion 54 498 Raton 2 Immersion 37 483 Raton 3 Immersion 39 488 Raton 4 Immersion 69 462 Raton 1 Boiling 30 sec 36 480 Raton 2 Boiling 30 sec 20 400 Raton 3 Boiling 30 sec 28 449 Raton 4 Boiling 30 sec 60 490 Raton 1 Boiling 60 sec 15 357 Raton 2 Boiling 60 sec 171 Raton 3 Boiling 60 sec 4 196 Raton 4 Boiling 60 sec 35 477 Raton 1 Control 4 196 Raton 2 Control 12 325 Raton 3 Control 2 140 Raton 4 Control 6 237 Raton 1 Percussion 1 min 52 500 Raton 2 Percussion I min 29 454 Raton 3 Percussion I min 35 477 Raton 4 Percussion 1 min 31 462 Raton 1 Percussion 2 min 55 497 Raton 2 Percussion 2 min 35 477 Raton 3 Percussion 2 min 33 470 Raton 4 Percussion 2 min 42 494 Raton 1 Percussion 4 min 65 477 Raton 2 Percussion 4 min 52 500 Raton Percussion 4 min 51 500 Raton 4 Percussion 4 min 53 499 Raton 1 Percussion 8 min 88 325 Raton 2 Percussion 8 min 80 400 Raton 3 Percussion 8 min 73 444 Raton 4 Percussion 8 min 74 439 Sacramento 1 Immersion 43 495
69
Table B 1 Continued
-Source Block Treatment Germination Percentage Standard Error
Sacramento 2 Immersion 30 458 Sacramento 3 Immersion 38 485 Sacramento 4 Immersion 56 496 Sacramento 1 Boiling 30 sec 47 499 Sacramento 2 Boiling 30 sec 48 500 Sacramento 3 Boiling 30 sec 48 500 Sacramento 4 Boiling 30 sec 56 496 Sacramento 1 Boiling 60 sec 52 500 Sacramento 2 Boiling 60 sec 67 470 Sacramento 3 Boiling 60 sec 43 495 Sacramento 4 Boiling 60 sec 48 500 Sacramento 1 Control 0 000 Sacramento 2 Control 3 171 Sacramento 3 Control 3 171 Sacramento 4 Control 099 Sacramento 1 Percussion 1 min 57 495 Sacramento 2 Percussion 1 min 61 488 Sacramento 3 Percussion 1 min 62 485 Sacramento 4 Percussion 1 min 55 497 Sacramento 1 Percussion 2 min 84 367 Sacramento 2 Percussion 2 min 70 458 Sacramento 3 Percussion 2 min 81 392 Sacramento 4 Percussion 2 min 69 462 Sacramento 1 Percussion 4 min 91 286 Sacramento 2 Percussion 4 min 84 367 Sacramento 3 Percussion 4 min 84 367 Sacramento 4 Percussion 4 min 94 237 Sacramento 1 Percussion 8 min 82 384 Sacramento 2 Percussion 8 min 81 392 Sacramento 3 Percussion 8 min 82 384 Sacramento 4 Percussion 8 min 81 392 Sandia 1 Immersion 76 427 Sandia 2 Immersion 70 458 Sandia 3 Immersion 70 458 Sandia 4 Immersion 81 392 Sandia 1 Boiling 30 sec 78 414 Sandia 2 Boiling 30 sec 71 454 Sandia 3 Boiling 30 sec 63 483 Sandia 4 Boiling 30 sec 78 414 Sandia 1 Boiling 60 sec 67 470 Sandia 2 Boiling 60 sec 65 477 Sandia 3 Boiling 60 sec 74 439 Sandia 4 Boiling 60 sec 66 474 Sandia 1 Control 16 367 Sandia 2 Control 7 255
70
Table B 1 Continued
Source Block Treatment Gennination Percentage Standard Error
Sandia 3 Control 8 271 Sandia 4 Control 16 367 Sandia 1 Percussion 1 min 67 470 Sandia 2 Percussion 1 min 61 488 Sandia 3 Percussion 1 min 66 474 Sandia 4 Percussion 1 min 59 492 Sandia I Percussion 2 min 82 384 Sandia 2 Percussion 2 min 83 376 Sandia 3 Percussion 2 min 81 392 Sandia 4 Percussion 2 min 84 367 Sandia 1 Percussion 4 min 89 313 Sandia 2 Percussion 4 min 86 347 Sandia 3 Percussion 4 min 89 313 Sandia 4 Percussion 4 min 85 357 Sandia 1 Percussion 8 min 80 400 Sandia 2 Percussion 8 min 77 421 Sandia 3 Percussion 8 min 69 462 Sandia 4 Percussion 8 min 66 474
LITERATURE CITED
Ashby WC Vogel WG Rogers NF 1985 Black locust in the reclamation equation General Technical Report Northeastern Forest Experiment Station USDA Forest Service NE-I05 12 p
Ballard LAT 1973 Physical barriers to gennination Seed Sci Technoll 285-303
___ 1976 Strophiolar water conduction in the seeds ofTrifolieae induced by action on the testa at nonstrophiolar sites Austr J Plant Physiol3 465-469
Barton LV 1947 Special studies on seed coat impenneability Contrib Boyce Thompson Inst 14 355-362
Baskin CC Baskin JM 1998 Seeds Ecology biogeography and evolution of donnancy and gennination Academic Press London UK 666 p
Bewley JD 1997 Seed germination and donnancy Plant Ce119 1055-1066
Bewley JD Black M 1994 Seeds physiology of development and germination 2nd ed Plenum Press New York NY 445 p
Briscoe CB 1996 Growing Nitrogen Fixing Trees in Acid Soils In MH Powell (ed) Nitrogen Fixing Trees for Acid Soils- A Field ManuaL Winrock International Morrilton AR 22 p
Copeland LO McDonald MB 1995 Principles of Seed Science and Technology 3rded Chapman amp Hall New York NY 409 p
Cox RE Klett JE 1984 Seed gennination requirements of native Colorado plants for use in the landscape Plant Propag 30(2) 6-10
Dell B 1980 Structure and function ofthe strophiolar plug in seeds ofAlbizia Lophantha Am J Bot 67 556-563
Dick-Peddie WA 1993 New Mexico Vegetation Past Present and Future University ofNew Mexico Press Albuquerque NM 244 p
Dreesen DR 2001 Propagation protocol for production ofcontainer Robinia neomexicana plants Los Lunas Plant Materials Center Los Lunas New Mexico In Native Plant Network URL httpwwwnativeplantnetworkorg (accessed 9 January 2002) Moscow (ID) University ofIdaho CoIl Natural Resources For Res Nursery
72
Dreesen DR Harrington JT 1997 Propagation of native plants for restoration projects in the Southwestern US- Preliminary Investigations In Landis TD Thompson JR tech coords National Proceedings Forest and Conservation Nursery Assocs Gen Tech Rep PNW-GTR-419 Portland OR USDA For Ser PNW Res Sta 77-88
Gottfried GJ 1980 Control ofNew Mexican locust and the effect on planted ponderosa pine in central Arizona US For Ser Res Note RM-386 Rocky Mountain For Range ExpStn Fort Collins CO
Gosling PG Samuel YK Jones SK 1995 A systematic examination of germination temperature chipping and water temperaturesoak duration pretreatments on the seeds ofLeucaena leucocephala Seed Sci amp Technol 23521-532
Hamly DH 1932 Softening of the seeds of Melilotus alba Bot Gazette 93 345shy375
Hanna PJ 1984 Anatomical features of the seed coat ofAcacia kempeana (Mueller) which relate to increased germination rate induced by heat treatment New Phytol 96 23-29
Hanks JP Dick-Peddie WA 1974 Vegetation patterns of the White Mountains New Mexico Southwest Naturalist 18(4) 371-382
Hine S Harrington JT Dreesen DR 1997 Seed scarification requirements for Robinia neomexicana In Harrington JTtech coord Proc Third SouthwestemContainer Growers Conf Feb 12-13 1997 Ft Collins CO
Hopkinson JM 1993 The strophiole of leguminous seeds Newsletter- IntI Herbage Seed Production Res Grp 18 7-8
---- 1997 Differences in hardseededness between Cunningham and Tarramba leucaena In Leucnet News The Newsletter of the International LeucaenaRes and Devlt Netwk No4
ISTA (International Seed Testing Association) 1999 International rules for seed testing rules Seed Sci amp Technol27 Supplement
Kelly KM van Staden J 1987 The lens as the site of permeability in the papilionoid seed Aspalathus linearis J Plant Physiol 128(45) 395-404
73
Keresztesi B 1988 Black locust the tree of agriculture Outlook on Agr 17(2) 77~85
Kigel 1 and Galili G 1995 Seed development and germination Marcel Dekker New York 853 p
Klemmedson 10 1994 New Mexico locust and parent material Influence on macronutrients of forest floor and soil Soil Sci Soc Am 1 58 974~980
Kuo WH Tam AW 1988 The pathway of water absorption of mung bean seeds Seed Sci amp Technol16(1) 139~144
Leadem CL 1997 Dormancy--unlocking seed secrets In Landis TD Thompson lR tech coords National Proceedings Forestand Conservation Nursery Assocs Gen Tech Rep PNW-G TR-419 Portland OR USDA For Ser PNW Res Sta 43-52
Lin Y Wagner MR Seweyestewa A Honahni MD DeGomez M McMillin JD 1996 Influence of cultural treatments on seed germination and seedling growth of Robinia neomexicana Forest Farm and Community Tree Res Reports Volume 1
Marunda CT 1990 Effects of seed pretreatment on the development ofAcacia auriculforinis and A holosericea seedlings ACIAR Proceedings Series No 28
Meginnis HG 1937 Sulphuric acid treatment to increase germination of black locust seed USDA Circ 453 34 p
Miklas PN Towensend CE Ladd SL 1987 Seedcoat anatomy and the scarification ofcicer milkvetch seed Crop Sci 27 766-772
Monsen SB 1984 Use of shrubs on mine spoils In The Challenge ofProducing Native Plants for the Intermountain Area Proceedings Intermountain Nurserymans Association 1983 conference Las Vegas NV compo PM Murphy 26-31 Ogden UT USDA For Ser Gen Tech Report INT-l68
Olson DF 1974 Robinia L Locust In Seeds of Woody Plants in the US tech coord CS Schopmeyer 728-731 USDA For Ser Agr Handbk No450
Rehman S Loescher RNl Harris P1C 1998 Dormancy breaking and germination ofAcacia salicina Lindl seeds Seed Sci amp Techno127 553-557
Rolston MP 1978 Water impermeable seed dormancy Bot Rev 44 365-396
74
SAS Institute Inc 1989 SASSTAT Users Guide Version 6 4th Edition Volume 1 Cary NC SAS Institute Inc 943 p
SerratoValenti G Conara L Ferrando M Modenesi P 1993 Structural and histochemical features ofStylosanthes scabra (Leguminosae Papilionoidae) seed coat as related to water entry Can J Bot 71 834-840
Simpson BJ 1988 A field guide to Texas trees Austin TX Texas Monthly Press 372p
Tran VN Cavanagh AK 1984 Structural aspects ofdormancy In Seed Physiology Vol 2 Murray DR (ed) p1-44 Academic Press Australia
USDA Forest Service 1948 Woody-plant seed manual USDA Misc Pub1654 416p
Vines RA 1960 Trees Shrubs and Woody Vines of the Southwest Austin Univ ofTexas Press 1104 p
Wagner MR Larson RE Craig TP 1992 Ecology and silvics ofNew Mexico locust (Robinia neomexicana) with an emphasis on the arboreal form p53-57 In JW Hanover K Miller and S Plesko (eds) Proc Intemat Confon Black Locust Biology Culture and Utilization Mich State Univ USA
Wilson JK 1944 Immersing seeds of species of Robinia in boiling water hastens germination J For 42 453-454
Zimmerman RW Carpenter SB 1980 First-year coppice production from a 5shyyear old black locust stand on surface mine spoil p309-314 Centr Hardwd For ConfProc University ofMissouri Columbia
experiment was conducted utilizing a randomized complete block design blocked by
time with a factorial treatment structure Experimental factors were seed source and
scarification treatment Nine seed sources from throughout New Mexico were used
(Figure 1 Table 1) Scarification treatments included a control three levels ofhot
water and four levels of percussion Hot water treatments included immersion at
100degC and boiling for periods of 30 seconds and 60 seconds Percussion treatment
levels were 1 2 4 and 8 minutes One hundred seeds per block were used to test
each treatment combination Four blocks of seed were tested one at a time (all seed
sources included in each run) in close chronological sequence (within 6 weeks) A
total of four 100-seed samples were used to test each source by scarification treatment
combination
Treatments
Hot water treatment of seeds utilized 50 ml test tubes filled with 30 ml
distilled water Each test tube was used to treat one 100-seed sample at a time
resulting in a ratio of4 parts water to 1 part seed by volume Test tubes were placed
in a beaker with the beaker filled with tap water to the same level as the water within
test tubes Nine test tubes (one per seed source) were placed in the beaker
simultaneously so that one replication of each seed sourcescarification treatment
cotnblnatlon was conducted simultaneously Hot water treatments include immersion
boiling For immersion treatments seeds were added to test tubes with the water
(boiling) and the test tubes were immediately removed from the beaker to
For the boiling treatments test tubes remained in the beaker with water at
20
boiling temperature for a set duration after the addition of seeds Following all
treatments test tubes with seeds cooled 24 hours in a test tube rack
Percussion treatment utilized a pneumatic paint shaker (Central Pneumatic
model 00422 Camarillo CA) Compressed air was maintained at 530 kPa plusmn30 kPa
(80 psi plusmn 5 psi) resulting in approximately 350 oscillations per minute Seeds were
placed in 118 ml (4 oz) soil tin cans (US Can Lobard IL) and the lids were secured
with duct tape Each can was used to treat one 100-seed sample To take advantage
ofgreatest lateral movement of the paint shaker a spacer (paint can) was used to
position the soil tin at the end of the shaking arm This allowed shaking to occur at
the greatest distance from the pivot point (fulcrum) Tins were placed perpendicular
to the direction of shaking motion allowing seeds to impact at greatest force by
hitting flat surfaces of the soil tin
Germination Testing
Before gennination testing all seeds were soaked 24 hours in reverse
osmosis-treated water (this was included in the process ofhot water treatment
jU~SCIlOea above) Following soaking seeds were placed in 100 cm Petri dishes lined
90 cm Whatman qualitative 1 filter paper circles (MG Scientific Pleasant
WI) One Petri dish was used for each treatment by source combination
papers were initially moistened with 5 mL reverse osmosis-treated water then
~VU1CUlJl moistened as needed to ensure adequate conditions for gennination
were monitored daily (at 300 PM) for gennination for 14 days in accordance
~UIUlnJlVUa Seed Testing Association (ISTA) standards for Robinia species
21
(1STA 1999) Tests were carried out at room temperature (22degC plusmnOSoC)
Germination was classified as protrusion of the radicle from the embryo by at least
1mm as viewed with the naked eye
X-ray Radiography
On August 132001 three lOO-seed samples from the Sacramento seed source
were treated for 14 or 8-minute percussion durations The seeds were sent
immediately by overnight delivery to the Faxitron X-Ray Corporation in Wheeling
Illinois where Dr Bob Karrfalt ofthe USDA Forest Service oversaw their imaging
Radiographs were made at SX with a Faxitron Specimen Radiography System Model
MX-20
Scanning Electron Microscope Photography
On February 192002 a Hitachi S-3200N Variable Pressure Scanning
Electron Microscope (Mountain View CA) was used to examine strophiolar cells of
untreated seeds and seeds percussed for 4 minutes Seeds were cut longitudinally
igt their entire length using a razor blade splitting the strophiolar region roughly in
Cross sections of seed samples magnifying the strophiolar region were viewed
600X under the microscope
Analysis
Categorical Analysis of Variance was used to determine treatment source and
(Le time) main effects and interactions The response variable was total
~vlI This procedure is a Chi-square test of homogeneity using the natural log
of germinated to non-germinated seed for each treatment Maximumshy
22
likelihood analysis was used to calculate Chi-square test statistics with their observed
significance levels A single contrast was conducted to cempare treatment means
against the control Observed significance levels less than or equal to 005 were
considered significant Pairwise comparisons were conducted between non-control
treatment mean percents using a conservative alp~a value of 005 divided by the
number of comparisons (Bonferroni approach) and approximate pairwise z statistics
Categorical analyses were performed using Proc Catmod (SAS Institute 1989)
RESULTS
Scarification seed source block and all interactions between factors
influenced germination (Table 3) The main effects are reported in the presence of
these interactions because the overall robustness of treatments and seed sources are of
interest Background levels ofnaturally occurring variation in seed sources are
seen in control germination percentages in Figure 2 In particular time variability
is unlikely to be under the control of the producer Appendix B details experimental
data for source by treatment gennination percents for each of the four blocks
Averaged over all treatments germination by source ranged from 40 to
79 (Figure 2) Averaged over all sources (Figure 3) and for each source (Figure 4
5) percussion scarification durations of4 or 8 minutes yielded greatest germination
Averaged over all sources germination increased with increasing durations of
percussion then leveled off for the 4- and 8-minute treatments (Figure 3) Immersion
and 30-second boiling scarification maximized germination for the hot water
treatments with a decrease in germination when seeds were boiled for 60 seconds
For all but two sources there is a similar ipcreasing trend in germination from
1to 4 minutes ofpercussion (Figure 4) The Holman source increased in germination
1 to 2 minutes then leveled off at 4 minutes The Jemez source changed little
1 to 4 minutes of treatment As treatment duration increased from 4 to 8
mutes five sources decreased in germination (Figure 4a) whereas three sources
24
Table 3 Categorical Analysis of Variance table for germination response to seed treatment seed source block and the interaction of the factors
Source of DF Chi-Square Observed Variability Significance Level
Source 8 98370 lt00001 Trt 7 233876 lt00001
contrast 1 16976 lt00001 SourceTrt 56 97077 lt00001 Block 3 5538 lt00001
SourceBlock 24 10162 lt00001 TrtBlock 21 7564 lt00001
SourceTrtBlock 168 34283 lt00001
S 4)
8 ~
0
sectg ~ 40 ~
N 0 0
20
o f0ltgt ~ ~4 jybshy ((iltgto ~~ K1ltgt 6~b-~ ~~ 0b-~~ ~~ ~b-~ ~b-~b
80
60 -I-- - ~- middotmiddotmiddotmiddotmiddot1
r -] Northern NM Sources
Central NM Sources
Southern NM Sources
sect-o ~
~b-v(i
SOURCE
Figure 2 Effect ofNew Mexico locust seed source on germination for data averaged over all scarification treatments Control germination is shown by solid line within bar
80
~ cd ~ ltl) () 60 ~ ~
N
t 0shyrti tshySltl)
40
0
20
o lmmersion Boiling 30 sec Boiling 60 sec Control 1 min 2min 4 min 8 min
Hot Water Treatments Percussion Treatments
Figure 3 Effect of scarification on germination ofNew Mexico locust for data averaged over all seed sources Means labelled with the same letter are not significantly different
100~----------------------~--~----------------~
80
60
ltU
~
40 IJ
sectgsI
l100
80
60
40
a I middotmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot middotImiddotmiddotmiddotmiddotmiddotmiddot E
_~iii-bull _ _ bull f --~--
t-- -~ ---- -shy ---- shyI ~ ---- - shy
L
fiII
~
b
- shy ---r
- Holman
Sacramento Sandia Gila Jemez
c ~
ltgt ~
Tmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot-I
j~=~=-~~-1
I middotmiddot ~ bull r
t tr
Y -------M-a-gd-a-Ie-n-a--
1 _ _
fCapitan
---- Manzano ~y -_ Raton
2 4 8
Time interval ofpercussion scarification (min)
Figure 4 Effect of seed source by treatment interaction on New Mexico locust germination for percussion scarification pattern a and pattern b
28
tv 0
~ d
~ A s o
1 c3
80
60
40
20
o
TIT
t------=middotfmiddotmiddot-middotmiddot-middotmiddot-middotmiddot 1- --- - - --=- - -shy--cE= - _--shy --shy ----shy ~ ~ ---shy ~ -~- _ --shy-------r 1
I
Raton Holman
-_ Jemez
--- Sandia Manzano Magdalena Capitan Sacramento Gila
Immersion Boiling 30 sec Boiling 60 sec
Hot Water Scarification
Figure 5 Effect of seed source by treatment interaction on New Mexico locust germination for hot water scarification
continued to increase in germination (Figure 4b) There was no difference between
the two treatment levels for the Manzano source (Figure 4b)
The effect ofhot water scarification also varied among sources with few
overall differences between hot water treatments (Figure 5) However boiling for 60
seconds yielded highest germination for the Jemez source whereas increasing
durations ofboiling decreased germination for the Raton source
In addition to consistently high germination 4- and 8-minute percussionshy
treated seeds germinated as fast or faster than all other treatments (Figure 6 Table 4)
050 measured as the number of days needed to complete 50 ofeventual
germination averaged 2 days for both ofthese treatments
X-ray radiographs were taken of seeds collected from the Sacramento source
percussed for 1 4 or 8 minutes (Figure 7) Seeds show signs of damage at 8 minutes
ofpercussion (Figure 8) Compared to an undamaged seed percussed for only 4
minutes (Figure 8a) damage at the 8-minute level includes fractured radicles pitting
of the seed coat and chips in seed coat structure (Figure 8b-d)
Scanning electron microscope (SEM) images at 600X reveal the direct effect
of 4 minutes of percussion on cells of the strophiolar region (Figure 9) Whereas
untreated seeds retain cellular organization in the strophiolar region strophiolar cells
seeds treated for 4 minutes ofpercussion separate and raise resulting in a
~-~e~U appearance
30
Umiddot-~middot--l-~---middotymiddotmiddotmiddot-----middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot--middot--middotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middotmiddotmiddotmiddotmiddotmiddotmiddot- -
050= gt1
-e-- Percussion 8 min -0- Percussion 4 min ---T- Percussion 2 min -----ltil- Percussion 1 min -- Immersion --0- Boiling 30 sec -+- Boiling 60 sec -ltgt- Control
2 4 6 8 10 12
Day Figure 6 Timecourse ofNew Mexico locust gennination following scarification
treatments G50 is the number of days to 50 of eventual germination
31
14
Table 4 Effect of seed source and treatment on G50 (days to 50 germination)
Seed Source Control Immersion B30sec B60sec Plmin P2min P4min P8min mean Raton 3 3 5 7 4 3 2 2 4 Holman 2 2 2 2 2 2 2 2 2
Jemez 4 6 6 5 3 2 2 2 4 Sandia 2 2 2 2 2 2 2 2 2
w tv Manzano 5 2 2 3 3 2 2 2 3
Magdalena 4 2 2 3 4 3 2 1 3
Capitan 5 4 2 2 3 2 2 2 3
Sacramento 3 2 2 2 4 3 2 2 3 Gila 3 2 2 2 2 2 2 2 2 mean 3 3 3 3 3 2 2 2 3 standard 11 14 16 18 09 05 0 03 08 deviation
Note Columns and rows may not agree due to rounding
Figure 7 X-ray radiographs ofSacramento seed source percussed for 1 4 or 8 minutes
33
Figure 8a-d Seed damage close-ups Figure a shows undamaged seed percussed for 4 minutes Figures b-d show seeds percussed for 8 minutes Damage includes fracturing ofradicle in b complete removal ofseed coat in c and pitting ofseed coat in d
34
Untreated Treated
Figure 9 SEM images ofuntreated vs 4-minute percussion-treated strophiolar cells ofNew Mexico locust seeds Treated cells are loosened separated and raised
DISCUSSION
Determining the Optimal Treatment
Averaging results of scarification treatmems across seed sources is a useful
tool to predict which treatment is most likely to be successful for an unknown seed
source Averaged over all sources four- and eight-minute percussion treatments
resulted in the highest germination 815 and 790 respectively and produced
germination that was as fast or faster than all other treatments Although there was no
difference between these two treatments on average an argument for 4 minutes of
percussion as a standard protocol for treating New Mexico locust seeds can be made
Descriptively 4 minutes of percussion improved germination over 8 minutes
ofpercussion for a majority of sources (five out of nine) 8 minutes of percussion
improved germination for three ofnine sources with no difference for a fmal source
For individual sources 4 minutes ofpercussion was always the best or secondmiddot best
treatment and was always equal to or better than all hot water treatments
Germination at 8 minutes ofpercussion was lower than all other percussion durations
for the Jemez and Gila sources and was lower than at least one hot water treatment for
these sources as well
The x-ray radiographs of the Sacramento seed source illustrate the excessive
internal and external seed damage that can occur with 8 minutes of percussion
germination counts 8-minute percussed seeds from Holman and Capitan
ClUll signs of damage that were visible to the naked eye (see below)
r~evertheless some of these seeds still fit the criteria for germination Neither x-ray
36
photography nor visible observations during seed counts revealed seed damage at the
4-minute percussion level In operational trials seeds percussed for 4 minutes
developed into healthy seedlings (Khadduri and Harrington unpublished data)
Strophiole and Seed Coat Behavior
Four minutes ofpercussion successfully breaks seed dormancy in a large
percentage ofNew Mexico locust seeds without excessively damaging the
surrounding seed coat or internal seed coat structures Most papilionoid legume
seeds including seeds ofNew Mexico locust have a specific region of the seed coat
known as the strophiole or lens (Hamly 1932) The strophiole is located on the
cotyledonary lobe of a locust seed (Figure 10 a-c) If seeds are not indiscriminately
damaged the strophiole is the first point of water entry to the embryo (Hopkinson
1997) When a papilionoid seed is percussed for the appropriate amount of time
repeated hits on the integrated seed coat loosen the constrained cells of the strophiolar
region without excessively damaging the rest of the seed coat (Ballard 1976) SEM
photos illuStrate that 4 minutes of percussion loosened and separated strophiolar cells
ofNew Mexico locust seeds when compared with constrained organized strophiolar
cells of untreated seeds (Figure 9) Research-has shown that water passes to the
embryo through the treated strophiole in a controlled manner and regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues
(Kelly and van Staden 1987)
37
10 Strophiole ofunimbibed New Mexico locust seed (a) and following uptake (bc)
38
In contrast hot-water treatments can excessively degrade the seed coat
(Rebman et al 1998 Marunda 1990) Random cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rebman et aI 1998 Marunda 1990) Along with a
proportionately large amount ofun-imbibed (thus under-treated) seeds (Figure l1a)
damage was observed in hot-water treated seeds from six sources during germination
counts Damage included seeds where the radicle failed to elongate (Figure 11 b)
seeds where the radicle elongated but was delayed in freeing itself from the seed coat
(Figure 11c) and seeds where the radicle completely broke off from the embryo
(Figure lId) In addition some seeds germinated in reverse (the cotyledon emerged
fIrst) If the radicle emerged completely from the seed coat they were counted as
germinated
While nearly all seeds treated for 8 minutes ofpercussion imbibed water
damage was evident in x-ray evaluations (Sacramento seed source) and in
germination counts (Holman and Capitan) at this treatment level Seed damage
during germination counts was similar to levels found in hot-water treated seeds
suggesting that over-treatment by both hot water and longer durations of percussion
may result in similar damage to seed structure
The Sacramento and Holman seed sources were two of the fIve sources that
opcrplltlpn in germination from the 4- to 8-minute durations of percussion
the benefIts ofloosening the strophiole as evidenced by imbibition of
all 8-minute treated seeds were outweighed by external and internal seed coat
39
a
b c d
11 Damage to New Mexico locust seeds observed during germination cotmts following hot water scarification 9a Hot water replicate including ~t seedlings and numerous unimbibed seeds 9b Close-up of seed where raallCle failed to elongate 9c Close-up ofseed where radicle elongated but was
-gtIn in freeing itself from the seed coat 9d Close-up ofseed where radicle broke off from embryo
40
damage (which also may be contributing to high imbibition) For the three sources
for which 8 minutes of percussion resulted in maximum germination extended
percussion of seeds necessary to loosen the strophiole may not have excessively
damaged the seed anatomy The Manzano source did not significantly change in
germination from 4 to 8 minutes of percussion suggesting that an increase in
permeable seeds at the 8-minute treatment duration was balanced by the number of
seeds that failed to germinate due to seed damage To summarize the 4-minute
percussion treatment improved germination over 8 minutes ofpercussion for a
majority of sources (five ofnine) the 8-minute percussion treatment improved
germination over 4-minutes ofpercussion for three sources and there was no
difference with the Manzano source Notably for the Raton source 8 minutes of
percussion increased germination by 235 percentage points over 4-minutes of
percussion (7875 vs 5525 respectively) the next best treatment Raton was the
only source to result in germination less than 75 for 4 minutes ofpercussion so a
grower may wish to apply longer percussion durations to lots that do not respond well
to this treatment level
Finally there was an interesting trend in the Jemez and Raton sources seed
that significantly contributed to the source by treatment interaction (Figure 12) A
~u test showed that the Jemez source had a significantly lighter seed weight than
other sources while the Raton source had an average seed weight For the Jemez
the longest duration of boiling increased germination Conversely only short
U4UVll) of percussion were needed to achieve high germination and the longest
41
80 _ Jemez a Raton
iD
~ C iD e iD
Q c 0 ~
c ~ iD 0
20
0------shy
Immersion Boiling 30 sec Boiling 60 sec
Hot Water Scarification
10degTr========~--------~------------------------------
80
60
40
20
_ Jemez Raton
0------shy
1 min 2 min 4 min 8 min
Percussion Scarification
Figure 12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources
42
b
--~----~~~~========~---- duration ofpercussion reduced germination For the Raton source longer durations
ofboiling consistently decreased germination whereas longer percussion treatments
consistently increased germination
A possible explanation is that a thick seed coat is associated with a loose
strophiole structure and that a thin seed coat is associated with a constrained
strophiole This contrast merits further study and may suggest an ecological
adaptation Further investigation ofthis phenomenon may contribute to successful
pre-screening ofunknown seed sources to determine optimal treatment leveL
43
CONCLUSION
Based on results obtained in pilot study (see Appendix) and thesis
experiments 4 minutes ofpercussion is recommended as a standard protocol for
treating an unknown source ofNew Mexico locust seed In addition to laboratory
results the 4-minute percussion treatment has resulted in germination greater than
80 and healthy seedling grow-outs for production purposes (unpublished data) In
laboratory experiments only one hundred seeds per 118 ml (40z) soil tin were
percussed at a time whereas for production we have treated up to five hundred seeds
per tin For large-scale production ofNew Mexico locust seedlings growers may
wish to experiment with larger quantities of seed and larger-size soil tins
Seed storage trials following percussion treatments were not conducted
However germination greater than 80 and healthy seedlings were obtained when
seeds were percussion-treated and subsequently stored at room-temperature (22degC)
for one week (unpublished data) Seed storage trials merit further study as the
standard hot water treatment leaves seeds in a softened condition that is not
conducive to storage
Hydroseeding is an effective method for direct seeding in reclamation
situations Percussion treatment may be conducive to hydro seeding as seeds remain
a dry relatively undamaged yet permeable condition following treatment Sulfuric
seeds also remain dry following treatment but extensive damage to seed coat
poor seedling vigor have been reported with this treatment (Kelly and van Staden
A grow-out study using a hydro seeder on a reclamation site testing percussion
44
against other treatments such as sulfuric acid and mechanical scarification also
merits further study
Papilionoids are the largest subfamily oflegumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust these legumes often play an integral role in the revegetation of
disturbed lands The pilot study (Appendix) indicates that percussion scarification
successfully treats the widely used black locust as well Developing superior
scarification methods to the standard hot water mechanical scarification and acid
treatments may facilitate the use ofpapilionoid legumes in reclamation projects
45
APPENDICES
APPENDIX A
PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR
NEW MEXICO LOCUST AND BLACK LOCUST SEEDS
Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4
Hot water and sulfuric acid soaks are traditional treatments for seeds of many
temperate woody legwnes including locusts However these scarification techniques
often produce inconsistent germination Percussion scarification where seeds are
repeatedly propelled against a hard surface was compared with hot water
scarification to evaluate treatment efficacy for New Mexico locust (Robinia
neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment
seeds were placed in a 98degC water bath which was immediately removed from the
heat source For percussion scarification seeds were placed in a soil sample tin and
agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the
control were followed by 24-hour water soaks Hot water baths resulted in 56 and
41 germination for New Mexico locust and black locust respectively For both
species nearly all durations ofpercussion increased germination over the hot water
treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3
4 and 5 minutes for black locust resulted in at least 90 germination Traditional
scarification treatments randomly degrade the entire seed coat which can lead to
tissue damage during water uptake Percussion scarification specifically weakens the
strophiole the natural source ofwater entry to the seed in papilionoid legwnes
INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom
2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031
48
Following percussion imbibition is controlled through the strophiole and underlying
tissue is protected
Keywords New Mexico locust black locust scarification percussion germination
Introduction
Locust trees (Robinia spp) are aggressive pioneer species that quickly
colonize disturbed land fix atmospheric nitrogen in the soil and stabilize slopes that
are prone to erosion (Klemmedson 1994 Ashby et al 1985) (Figure A1ab)
Recognition of these attributes has contributed to increased demand for locusts in
reclamation projects Locust seeds however exhibit physical dormancy where a
thick seed coat prevents the movement of water and gasses to the embryo (Leadem
1997)
Sulfuric acid or hot water soaks have traditionally been used to break seed
dormancy in macro-propagation of locusts Many growers are moving away from the
use of su1furic acid in treating locust seeds (Dreesen and Harrington 1997) In
addition to being dangerous acid soak durations must be specifically correlated to
seed lot (Olson 1974) Collections of native seed can vary tremendously in seed size
seed weight and hard seededness etc This variability limits the utility of sulfuric
acid soaks as even within a given lot some seeds may be damaged whereas others
are under-treated and fail to imbibe (take up water) A survey of Southwestern
container growers indicates that hot water baths are currently the most common
scarification method for locust seeds (Hine et al 1997) However hot water baths
also produce inconsistent germination (Lin et al 1996)
49
b
Figure AI New Mexico locust a primary invader following disturbance Ala Crown-sprouting at edge ofburn near Los Alamos NM--one year after Cerro Grande fire Alb Colonizing road cut Gila National Forest NM
50
Percussion scarification where seeds are repeatedly propelled against a hard
surface is an alternative dormancy-breaking method that has proven successful in
legumes related to locusts (Hamly 1932 Barton 1947) We compared this alternative
treatment to hot water scarification to evaluate treatment efficacy for New Mexico
locust and black locust seeds
Materials and Methods
Commercial New Mexico locust seeds (Western Native Seed Coaldale CO)
were collected fall 2000 in Huerfano County CO Black locust seeds were collected
September 2000 from Taos County NM by harvesting ripe pods from several trees at
a distance of up to 3 m from ground level Black locust seeds were allowed to air-dry
for 3 weeks and were threshed and separated from large chaffusing a greenhouse
fan A South Dakota Seed Blower (Chicago Illinltois) was used to remove fine chaff
from both seed lots Seeds were storedmiddotat 2-4C until the germination study was
conducted in March 2001
This study compared hot water scarification with percussion scarification
using treatment and species as experimental factors Along with a control
scarification treatments included immersion of seed in boiling water as well as
percussion durations of 1 2 3 45 or 10 minutes Four 100-seed samples were used
to test each species by scarification treatment combination
For the hot water treatment test tubes were filled with 30 mL water and
placed in a water-filled beaker (Figure A2a) The bath was raised to boiling (-98C at
51
b
c
Figure A2 Implementation ofhot water and percussion scarification A2a Hot water bath A2b Paint-shaker set-up A2c Placing soil tin containing seeds at end ofpaint shaker ann
52
Las Cruces NM elevation of 1300 meters) Each 100-seed replicate was placed in a
test tube and each test tube was immediately removed to cool at room temperature
Percussion scarification was implemented using a pneumatic paint shaker
(Central Pneumatic model 00422 Camarillo CA) (Figure A2b) This allowed
standardization of treatment intensity Compressed air pressure was maintained at
530kPa plusmn 30kPa (80psi plusmn 5psi) resulting in approximately 350 oscillations per
minute Each lOO-seed sample was placed in a 118 ml (4 oz) soil tin for shaking To
maximize lateral movement of paint shaker a paint can was used as a spacer to place
soil tins at the end of the shaking arm (Figure A2c) This allowed shaking to occur at
the greatest distance from the pivot point (fulcrum) Following all treatments
including the control seeds were soaked for twenty-four hours in distilled water prior
to plating out
Gennination was evaluated on lab benches at room temperature Each
replicate of seeds was placed in a 100 cm petri dish on moistened filter paper with
humidity maintained by enclosing the petri dishes in ziplock bags Gennination was
monitored daily for 14 days in accordance with International Seed Testing
Association standards for Robinia species (ISTA 1999) Gennination was defined as
protrusion of the radicle from the embryo by at least 1mm as viewed by the naked
eye
Categorical Analysis of Variance using Prpc Catmod (SAS Institute 1989)
was used to detennine treatment differences in germination percentages for each
source This procedure is an extension of a Chi-square test of homogeneity using the
53
natural log of the ratio of germinated to non-germinated seeds for each treatment Pshy
values less than 005 were considered significant A limited set of pairwiseshy
comparisons was conducted to compare treatment mean percents using a
conservative alpha value of 005 divided by the number of comparisons
Results
Percussion maximized germination for both New Mexico locust and black
locust There were three germination response levels for New Mexico locust (Figure
A3) First there was a low response in the control indicating a dormant seed lot
The hot water treatment and I-minute duration of percussion share a second
intermediate response Germination was highest at percussion levels from 2 to 10
minutes
A selected percussion treatment intermediate in time (5 minutes) produced the
steepest germination curve indicating the fastest germination rate (Figure A4) The
hot water treatment produced an intermediate curve or relatively slower germination
rate The control curve was very flat indicating the slowest germination rate for New
Mexico locust
Control seeds germinated poorly for black locust as well (Figure A5) Hot
water scarification improved germination over the control but all levels of percussion
improved germination over hot water The optimal durations of percussion treatment
were bracketed with a significant increase from the 1 to 2 minute percussion level a
high germination response from 2 to 5 minutes of percussion and finally a significant
54
~ d 25 CL)
p t1 0 ~ t1
g Vl ~ Vl
100~---------------------------------------------------------------
80
60
40
20
o Control Hot Water Percussion
I min Percussion
2 min Percussion
3 min
Scarification
Percussion 4 min
Figure A3 Effect of scarification on germination response for New Mexico locust
Percussion 5 min
=significant difference between adjacent treatment bars ns = no significant difference
Percussion 10 min
~ ~ () u agt
Il-t d o ~
~ ~ c3
100middotlr~~~------------------------------------e- Percussion 5min middot 0middot Hot Water -- Control
80-J=middot======
6b-middot-lmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middot--middotmiddot-middot~-middotmiddot -middotmiddot middot middotmiddot-middot 1
40
20
-----~-----------
0 0 0 middotmiddotmiddotmiddotmiddot0middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot0
0 --~-----y-----~------_---~--- T-----~
o shy -TDay 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A4 Effect of selected scarification treatments on germination speed for New Mexico locust First seven days of fourteen-day experiment are shown
Day 7
80
~ cd
5 60 ()
[) Cl d 0 1t1 d
40sect dUl
-J
20
o
ns
Scarification
Figure A5 Effect of scarification on germination response for black locust = significant difference between adjacent treatment bars ns = no significant difference
decrease at the 10-minute level This drop-off suggests that 10 minutes of percussion
over-treats seeds from this lot of-black locust
A selected percussion treatment intermediate in time (5 minutes) also had the
fastest germination rate for black locust (Figure A6)_ The hot water curve is
somewhat flatter indicating a slower germination rate The control curve is very flat
indicating a slow germination rate
A qualitative difference between hot water and percussion-treated seeds was
also observed Along with healthy germinants the hot water treatment produced
over-treated seeds (Figure A 7a) Examples of damaged germinants include seeds
where the radicle did not elongate (Figure A 7b) seeds where the radicle elongated
but was delayed in freeing itself from the seed coat (Figure A7c) and seeds where the
radicle broke off completely from the embryo (Figure A7d) In addition to these
examples of over-treatment there were numerous small dark seeds that failed to
imbibe water For percussion treatment nearly all seeds took up water (Figure A8a)
Germination was rapid and uniform with little damage evident (Figure A8b)
To summarize both New Mexico locust and black locust seed lots were
dormant The standard hot water scarification treatment improved germination but
only to 56 and 41 for New Mexico locust and black locust respectively Nearly
all percussion durations improved total germination as well as germination rate over
the hot water treatment Germination was greater than 90 for percussion durations
of 4 5 and 10 minutes for New Mexico locust and 3 4 and 5 minutes for black locust
58
CI)
sgt t CI) 0
igt 0
sect ttl t E
Vl 0
c3
100r==~==~~=r------------------------------------~ ___ Percussion 5 min
middotmiddotmiddot0middotmiddotmiddot Hot Water -lIf- Control
80r=========~--~~----------------------------~
60
40
20 -~-~---~---------- ---~------------I
omiddot
O
_~_----------lIf-----~bull -----_ shy---shy I
Day 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A6 Effect of selected scarification treatments on germination speed for black locust First seven days of fourteen-day experiment are shown
Day 7
a
b c d
Figure A7 Damage to New Mexico locust seeds observed during germination counts following hot water scarification A7a Hot water replicate including damaged seedlings and numerous unimbibed seeds A7b Close-up ofseed where radicle failed to elongate A7c Close-up ofseed where radicle elongated but was delayed in freeing itself from the seed coat A7d Close-up ofseed where radicle completely broke offfrom embryo
60
a
b
Figure A8 New Mexico locust seeds following percussion treatment at time of germination counts A8a Percussion replicate with uniformly healthy seedlings and near complete imbibition of seeds A8b Close-up ofpercussion-treated seedling
61
Discussion
Papilionoid seeds have a specific anatomical feature known as the strophiole
or lens-the natural site of water entry to the seed (Hamly 1932) The strophiole is
located on the cotyledonary lobe of a locust seed (Figure A9) When a papilionoid
legume seed is percussed for the appropriate amount of time repeated hits on the
integrated seed coat loosen the constrained cells of the strophiolar region without
excessively damaging the rest of the seed coat (Ballard 1976)
When a percussed seed is soaked water enters exclusively through the
strophiole in a controlled manner (Kelly and Staden 1987) This regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues This
contrasts with seeds that have been hot water or acid scarified treatments that can
randomly degrade the seed coat Unlocalized cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rehman et al 1998 Marunda 1990)
Papilionoids are the largest subfamily of legumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust and black locust these legumes often play an integral role in the
revegetation ofdisturbed lands Developing superior scarification methods to the
standard hot water and acid treatments should fadlitate the use ofthese legumes in
restoration projects
62
Figure A9 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (bc)
63
APPENDIXB
GERMINATION PERCENTS AND STANDARD ERRORS FOR SOURCE
BLOCK AND TREATMENT FOR NEW MEXICO LOCUST
Table B 1 Gennination percents and standard errors for source block and treatment for New Mexico locust
Source Block Treatment Germination Percentage Standard Error
Capitan 1 Immersion 74 437 Capitan 2 Immersion 69 462 Capitan 3 Immersion 71 454 Capitan 4 Immersion 67 470 Capitan Boiling 30 sec 79 407 Capitan 2 Boiling 30 sec 76 427 Capitan 3 Boiling 30 sec 72 449 Capitan 4 Boiling 30 sec 76 427 Capitan Boiling 60 sec 71 454 Capitan 2 Boiling 60 sec 54 498 Capitan 3 Boiling 60 sec 67 470 Capitan 4 Boiling 60 sec 76 427 Capitan I Control 5 218 Capitan 2 Control 7 255 Capitan 3 Control 4 196 Capitan 4 Control 5 218 Capitan 1 Percussion I min 47 499 Capitan 2 Percussion I min
470
Capitan Percussion I min 39 488
Capitan 4 Percussion I min 38 485 Capitan I Percussion 2 min 55 497 Capitan Capitan
2
Percussion 2 min Percussion 2 min
71 65
454 477
Capitan 4 Percussion 2 min 56 496 Capitan I Percussion 4 min 86 347 Capitan 2 Percussion 4 min 79 407 Capitan 3 Percussion 4 min 81 392 Capitan 4 Percussion 4 min 82 384 Capitan I Percussion 8 min 90 300 Capitan 2 Percussion 8 min 88 325 Capitan 3 Percussion 8 min 90 300 Capitan 4 Percussion 8 min 90 300 Gila I Immersion 71 454 Gila 2 Immersion 77 421 Gila
Immersion 75 433
Gila 4 Immersion 72 449
Gila I Boiling 30 sec 68 466
Gila 2 Boiling 30 sec 81 392
Gila 3 Boiling 30 sec 54 498
Gila 4 Boiling 30 sec 63 483
Gila I Boiling 60 sec 55 497
Gila 2 Boiling 60 sec 68 466
Gila 3 Boiling 60 sec 57 495
Gila 4 Boiling 60 sec 70 458
65
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Gila 2 Control 13 336 Gila 3 Control 13 336 Gila 4 Control 20 400 Gila 1 Percussion 1 min 66 474 Gila 2 Percussion 1 min 57 495 Gila 3 Percussion 1 min 73 444 Gila 4 Percussion 1 min 82 384 Gila 1 Percussion 2 min 81 392 Gila 2 Percussion 2 min 86 347 Gila 3 Percussion 2 min 85 357 Gila 4 Percussion 2 min 54 498 Gila 1 Percussion 4 min 79 407 Gila 2 Percussion 4 min 83 376 Gila 3 Percussion 4 min 84 367 Gila 4 Percussion 4 min 79 407 Gila 1 Percussion 8 min 51 500 Gila 2 Percussion 8 min 76 427 Gila 3 Percussion 8 min 52 500 Gila 4 Percussion 8 min 74 439 Holman 1 Immersion 83 376 Holman 2 Immersion 86 347 Holman 3 Immersion 81 392 Holman 4 Immersion 90 300 Holman 1 Boiling 30 sec 90 300 Holman 2 Boiling 30 sec 85 357 Holman 3 Boiling 30 sec 87 336 Holman 4 Boiling 30 sec 83 376 Holman 1 Boiling 60 sec 79 407 Holman 2 Boiling 60 sec 80 400 Holman 3 Boiling 60 sec 86 347 Holman 4 Boiling 60 sec 93 255 Holman Control 25 433 Holman 2 Control 19 392 Holman Control 21 407
Holman 4 Control 24 427
Holman Percussion 1 min 77 421
Holman 2 Percussion 1 min 85 357
Holman 3 Percussion 1 min 80 400
Holman 4 Percussion 1 min 89 313
Holman 1 Percussion 2 min 88 325
Holman 2 Percussion 2 min 96 196
Holman 3 Percussion 2 min 93 255
Holman 4 Percussion 2 min 96 196
Holman 1 Percussion 4 min 92 271
Holman 2 Percussion 4 min 97 171
66
I
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Holman 3 Percussion 4 min 91 286 Holman 4 Percussion 4 min 96 196 Holman 1 Percussion 8 min 88 325 Holman 2 Percussion 8 min 85 357 Holman 3 Percussion 8 min 83 376 Holman 4 Percussion 8 min 82 384 Jemez 1 Immersion 53 499 Jemez 2 Immersion 44 496 Jemez 3 Immersion 52 500 Jemez 4 Immersion 70 458 Jemez 1 Boiling 30 sec 58 494 Jemez 2 Boiling 30 sec 55 497 Jemez 3 Boiling 30 sec 48 500 Jemez 4 Boiling 30 sec 65 477 Jemez 1 Boiling 60 sec 68 466 Jemez 2 Boiling 60 sec 63 483 Jemez 3 Boiling 60 sec 73 444 Jemez 4 Boiling 60 sec 89 313 Jemez 1 Control 2 140 Jemez 2 Control 9 286 Jemez 3 Control 6 237 Jemez 4 Control 8 271 Jemez 1 Percussion 1 min 58 494 Jemez 2 Percussion 1 min 63 483 Jemez 3 Percussion 1 min 77 421 Jemez 4 Percussion 1 min 89 313 Jemez 1 Percussion 2 min 68 466 Jemez 2 Percussion 2 min 64 480 Jemez 3 Percussion 2 min 80 400 Jemez 4 Percussion 2 min 83 376 Jemez Percussion 4 min 68 466 Jemez 2 Percussion 4 min 79 407 Jemez 3 Percussion 4 min 70 458 Jemez 4 Percussion 4 min 83 376 Jemez 1 Percussion 8 min 52 500 Jemez 2 Percussion 8 min 59 492 Jemez 3 Percussion 8 min 61 488 Jemez 4 Percussion 8 min 70 458 Magdalena 1 Immersion 69 462 Magdalena 2 Immersion 69 462 Magdalena J Immersion 70 458 Magdalena 4 Immersion 85 357 Magdalena 1 Boiling 30 sec 65 477 Magdalena 2 Boiling 30 sec 71 454 Magdalena 3 Boiling 30 sec 68 466
67
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Magdalena 4 Boiling 30 sec 83 376 Magdalena 1 Boiling 60 sec 63 483 Magdalena 2 Boiling 60 sec 68 466 Magdalena 3 Boiling 60 sec 63 483 Magdalena 4 Boiling 60 sec 82 384 Magdalena 1 Control 11 313 Magdalena 2 Control 6 237 Magdalena 3 Control 5 218 Magdalena 4 Control 4 196 Magdalena Percussion 1 min 32 466 Magdalena 2 Percussion 1 min 45 497 Magdalena 3 Percussion 1 min 46 498 Magdalena 4 Percussion I min 36 480 Magdalena 1 Percussion 2 min 67 470 Magdalena 2 Percussion 2 min 68 466 Magdalena 3 Percussion 2 min 65 477 Magdalena 4 Percussion 2 min 81 392 Magdalena 1 Percussion 4 min 87 336 Magdalena 2 Percussion 4 min 97 171 Magdalena 3 Percussion 4 min 83 376 Magdalena 4 Percussion 4 min 97 171 Magdalena 1 Percussion 8 min 98 lAO Magdalena 2 Percussion 8 min 100 000 Magdalena 3 Percussion 8 min 96 196 Magdalena 4 Percussion 8 min 98 lAO Manzano 1 Immersion 67 470 Manzano 2 Immersion 57 495 Manzano 3 Immersion 72 4049 Manzano 4 Immersion 73 4044 Manzano 1 Boiling 30 sec 70 458 Manzano 2 Boiling 30 sec 64 480 Manzano 3 Boiling 30 sec 71 454 Manzano 4 Boiling 30 sec 80 400 Manzano I Boiling 60 sec 50 500 Manzano 2 Boiling 60 sec 53 499 Manzano 3 Boiling 60 sec 59 492 Manzano 4 Boiling 60 sec 74 439 Manzano I Control 4 196 Manzano 2 Control 8 271 Manzano 3 Control 1 099 Manzano 4 Control 6 237 Manzano 1 Percussion 1 min 52 500 Manzano 2 Percussion 1 min 39 488 Manzano 3 Percussion 1 min 36 480 Manzano 4 Percussion 1 min 51 500
68
Table B 1 Continued
Source Block Treatment Gerrni~ation Percentage Standard Error
Manzano Percussion 2 min 53 499 Manzano 2 Percussion 2 min 58 494 Manzano 3 Percussion 2 min 61 488 Manzano 4 Percussion 2 min 62 485 Manzano 1 Percussion 4 min 76 427 Manzano 2 Percussion 4 min 85 357 Manzano 3 Percussion 4 min 79 407 Manzano 4 Percussion 4 min 83 376 Manzano 1 Percussion 8 min 87 336 Manzano 2 Percussion 8 min 83 376 Manzano 3 Percussion 8 min 83 376 Manzano 4 Percussion 8 min 75 433 Raton 1 Immersion 54 498 Raton 2 Immersion 37 483 Raton 3 Immersion 39 488 Raton 4 Immersion 69 462 Raton 1 Boiling 30 sec 36 480 Raton 2 Boiling 30 sec 20 400 Raton 3 Boiling 30 sec 28 449 Raton 4 Boiling 30 sec 60 490 Raton 1 Boiling 60 sec 15 357 Raton 2 Boiling 60 sec 171 Raton 3 Boiling 60 sec 4 196 Raton 4 Boiling 60 sec 35 477 Raton 1 Control 4 196 Raton 2 Control 12 325 Raton 3 Control 2 140 Raton 4 Control 6 237 Raton 1 Percussion 1 min 52 500 Raton 2 Percussion I min 29 454 Raton 3 Percussion I min 35 477 Raton 4 Percussion 1 min 31 462 Raton 1 Percussion 2 min 55 497 Raton 2 Percussion 2 min 35 477 Raton 3 Percussion 2 min 33 470 Raton 4 Percussion 2 min 42 494 Raton 1 Percussion 4 min 65 477 Raton 2 Percussion 4 min 52 500 Raton Percussion 4 min 51 500 Raton 4 Percussion 4 min 53 499 Raton 1 Percussion 8 min 88 325 Raton 2 Percussion 8 min 80 400 Raton 3 Percussion 8 min 73 444 Raton 4 Percussion 8 min 74 439 Sacramento 1 Immersion 43 495
69
Table B 1 Continued
-Source Block Treatment Germination Percentage Standard Error
Sacramento 2 Immersion 30 458 Sacramento 3 Immersion 38 485 Sacramento 4 Immersion 56 496 Sacramento 1 Boiling 30 sec 47 499 Sacramento 2 Boiling 30 sec 48 500 Sacramento 3 Boiling 30 sec 48 500 Sacramento 4 Boiling 30 sec 56 496 Sacramento 1 Boiling 60 sec 52 500 Sacramento 2 Boiling 60 sec 67 470 Sacramento 3 Boiling 60 sec 43 495 Sacramento 4 Boiling 60 sec 48 500 Sacramento 1 Control 0 000 Sacramento 2 Control 3 171 Sacramento 3 Control 3 171 Sacramento 4 Control 099 Sacramento 1 Percussion 1 min 57 495 Sacramento 2 Percussion 1 min 61 488 Sacramento 3 Percussion 1 min 62 485 Sacramento 4 Percussion 1 min 55 497 Sacramento 1 Percussion 2 min 84 367 Sacramento 2 Percussion 2 min 70 458 Sacramento 3 Percussion 2 min 81 392 Sacramento 4 Percussion 2 min 69 462 Sacramento 1 Percussion 4 min 91 286 Sacramento 2 Percussion 4 min 84 367 Sacramento 3 Percussion 4 min 84 367 Sacramento 4 Percussion 4 min 94 237 Sacramento 1 Percussion 8 min 82 384 Sacramento 2 Percussion 8 min 81 392 Sacramento 3 Percussion 8 min 82 384 Sacramento 4 Percussion 8 min 81 392 Sandia 1 Immersion 76 427 Sandia 2 Immersion 70 458 Sandia 3 Immersion 70 458 Sandia 4 Immersion 81 392 Sandia 1 Boiling 30 sec 78 414 Sandia 2 Boiling 30 sec 71 454 Sandia 3 Boiling 30 sec 63 483 Sandia 4 Boiling 30 sec 78 414 Sandia 1 Boiling 60 sec 67 470 Sandia 2 Boiling 60 sec 65 477 Sandia 3 Boiling 60 sec 74 439 Sandia 4 Boiling 60 sec 66 474 Sandia 1 Control 16 367 Sandia 2 Control 7 255
70
Table B 1 Continued
Source Block Treatment Gennination Percentage Standard Error
Sandia 3 Control 8 271 Sandia 4 Control 16 367 Sandia 1 Percussion 1 min 67 470 Sandia 2 Percussion 1 min 61 488 Sandia 3 Percussion 1 min 66 474 Sandia 4 Percussion 1 min 59 492 Sandia I Percussion 2 min 82 384 Sandia 2 Percussion 2 min 83 376 Sandia 3 Percussion 2 min 81 392 Sandia 4 Percussion 2 min 84 367 Sandia 1 Percussion 4 min 89 313 Sandia 2 Percussion 4 min 86 347 Sandia 3 Percussion 4 min 89 313 Sandia 4 Percussion 4 min 85 357 Sandia 1 Percussion 8 min 80 400 Sandia 2 Percussion 8 min 77 421 Sandia 3 Percussion 8 min 69 462 Sandia 4 Percussion 8 min 66 474
LITERATURE CITED
Ashby WC Vogel WG Rogers NF 1985 Black locust in the reclamation equation General Technical Report Northeastern Forest Experiment Station USDA Forest Service NE-I05 12 p
Ballard LAT 1973 Physical barriers to gennination Seed Sci Technoll 285-303
___ 1976 Strophiolar water conduction in the seeds ofTrifolieae induced by action on the testa at nonstrophiolar sites Austr J Plant Physiol3 465-469
Barton LV 1947 Special studies on seed coat impenneability Contrib Boyce Thompson Inst 14 355-362
Baskin CC Baskin JM 1998 Seeds Ecology biogeography and evolution of donnancy and gennination Academic Press London UK 666 p
Bewley JD 1997 Seed germination and donnancy Plant Ce119 1055-1066
Bewley JD Black M 1994 Seeds physiology of development and germination 2nd ed Plenum Press New York NY 445 p
Briscoe CB 1996 Growing Nitrogen Fixing Trees in Acid Soils In MH Powell (ed) Nitrogen Fixing Trees for Acid Soils- A Field ManuaL Winrock International Morrilton AR 22 p
Copeland LO McDonald MB 1995 Principles of Seed Science and Technology 3rded Chapman amp Hall New York NY 409 p
Cox RE Klett JE 1984 Seed gennination requirements of native Colorado plants for use in the landscape Plant Propag 30(2) 6-10
Dell B 1980 Structure and function ofthe strophiolar plug in seeds ofAlbizia Lophantha Am J Bot 67 556-563
Dick-Peddie WA 1993 New Mexico Vegetation Past Present and Future University ofNew Mexico Press Albuquerque NM 244 p
Dreesen DR 2001 Propagation protocol for production ofcontainer Robinia neomexicana plants Los Lunas Plant Materials Center Los Lunas New Mexico In Native Plant Network URL httpwwwnativeplantnetworkorg (accessed 9 January 2002) Moscow (ID) University ofIdaho CoIl Natural Resources For Res Nursery
72
Dreesen DR Harrington JT 1997 Propagation of native plants for restoration projects in the Southwestern US- Preliminary Investigations In Landis TD Thompson JR tech coords National Proceedings Forest and Conservation Nursery Assocs Gen Tech Rep PNW-GTR-419 Portland OR USDA For Ser PNW Res Sta 77-88
Gottfried GJ 1980 Control ofNew Mexican locust and the effect on planted ponderosa pine in central Arizona US For Ser Res Note RM-386 Rocky Mountain For Range ExpStn Fort Collins CO
Gosling PG Samuel YK Jones SK 1995 A systematic examination of germination temperature chipping and water temperaturesoak duration pretreatments on the seeds ofLeucaena leucocephala Seed Sci amp Technol 23521-532
Hamly DH 1932 Softening of the seeds of Melilotus alba Bot Gazette 93 345shy375
Hanna PJ 1984 Anatomical features of the seed coat ofAcacia kempeana (Mueller) which relate to increased germination rate induced by heat treatment New Phytol 96 23-29
Hanks JP Dick-Peddie WA 1974 Vegetation patterns of the White Mountains New Mexico Southwest Naturalist 18(4) 371-382
Hine S Harrington JT Dreesen DR 1997 Seed scarification requirements for Robinia neomexicana In Harrington JTtech coord Proc Third SouthwestemContainer Growers Conf Feb 12-13 1997 Ft Collins CO
Hopkinson JM 1993 The strophiole of leguminous seeds Newsletter- IntI Herbage Seed Production Res Grp 18 7-8
---- 1997 Differences in hardseededness between Cunningham and Tarramba leucaena In Leucnet News The Newsletter of the International LeucaenaRes and Devlt Netwk No4
ISTA (International Seed Testing Association) 1999 International rules for seed testing rules Seed Sci amp Technol27 Supplement
Kelly KM van Staden J 1987 The lens as the site of permeability in the papilionoid seed Aspalathus linearis J Plant Physiol 128(45) 395-404
73
Keresztesi B 1988 Black locust the tree of agriculture Outlook on Agr 17(2) 77~85
Kigel 1 and Galili G 1995 Seed development and germination Marcel Dekker New York 853 p
Klemmedson 10 1994 New Mexico locust and parent material Influence on macronutrients of forest floor and soil Soil Sci Soc Am 1 58 974~980
Kuo WH Tam AW 1988 The pathway of water absorption of mung bean seeds Seed Sci amp Technol16(1) 139~144
Leadem CL 1997 Dormancy--unlocking seed secrets In Landis TD Thompson lR tech coords National Proceedings Forestand Conservation Nursery Assocs Gen Tech Rep PNW-G TR-419 Portland OR USDA For Ser PNW Res Sta 43-52
Lin Y Wagner MR Seweyestewa A Honahni MD DeGomez M McMillin JD 1996 Influence of cultural treatments on seed germination and seedling growth of Robinia neomexicana Forest Farm and Community Tree Res Reports Volume 1
Marunda CT 1990 Effects of seed pretreatment on the development ofAcacia auriculforinis and A holosericea seedlings ACIAR Proceedings Series No 28
Meginnis HG 1937 Sulphuric acid treatment to increase germination of black locust seed USDA Circ 453 34 p
Miklas PN Towensend CE Ladd SL 1987 Seedcoat anatomy and the scarification ofcicer milkvetch seed Crop Sci 27 766-772
Monsen SB 1984 Use of shrubs on mine spoils In The Challenge ofProducing Native Plants for the Intermountain Area Proceedings Intermountain Nurserymans Association 1983 conference Las Vegas NV compo PM Murphy 26-31 Ogden UT USDA For Ser Gen Tech Report INT-l68
Olson DF 1974 Robinia L Locust In Seeds of Woody Plants in the US tech coord CS Schopmeyer 728-731 USDA For Ser Agr Handbk No450
Rehman S Loescher RNl Harris P1C 1998 Dormancy breaking and germination ofAcacia salicina Lindl seeds Seed Sci amp Techno127 553-557
Rolston MP 1978 Water impermeable seed dormancy Bot Rev 44 365-396
74
SAS Institute Inc 1989 SASSTAT Users Guide Version 6 4th Edition Volume 1 Cary NC SAS Institute Inc 943 p
SerratoValenti G Conara L Ferrando M Modenesi P 1993 Structural and histochemical features ofStylosanthes scabra (Leguminosae Papilionoidae) seed coat as related to water entry Can J Bot 71 834-840
Simpson BJ 1988 A field guide to Texas trees Austin TX Texas Monthly Press 372p
Tran VN Cavanagh AK 1984 Structural aspects ofdormancy In Seed Physiology Vol 2 Murray DR (ed) p1-44 Academic Press Australia
USDA Forest Service 1948 Woody-plant seed manual USDA Misc Pub1654 416p
Vines RA 1960 Trees Shrubs and Woody Vines of the Southwest Austin Univ ofTexas Press 1104 p
Wagner MR Larson RE Craig TP 1992 Ecology and silvics ofNew Mexico locust (Robinia neomexicana) with an emphasis on the arboreal form p53-57 In JW Hanover K Miller and S Plesko (eds) Proc Intemat Confon Black Locust Biology Culture and Utilization Mich State Univ USA
Wilson JK 1944 Immersing seeds of species of Robinia in boiling water hastens germination J For 42 453-454
Zimmerman RW Carpenter SB 1980 First-year coppice production from a 5shyyear old black locust stand on surface mine spoil p309-314 Centr Hardwd For ConfProc University ofMissouri Columbia
boiling temperature for a set duration after the addition of seeds Following all
treatments test tubes with seeds cooled 24 hours in a test tube rack
Percussion treatment utilized a pneumatic paint shaker (Central Pneumatic
model 00422 Camarillo CA) Compressed air was maintained at 530 kPa plusmn30 kPa
(80 psi plusmn 5 psi) resulting in approximately 350 oscillations per minute Seeds were
placed in 118 ml (4 oz) soil tin cans (US Can Lobard IL) and the lids were secured
with duct tape Each can was used to treat one 100-seed sample To take advantage
ofgreatest lateral movement of the paint shaker a spacer (paint can) was used to
position the soil tin at the end of the shaking arm This allowed shaking to occur at
the greatest distance from the pivot point (fulcrum) Tins were placed perpendicular
to the direction of shaking motion allowing seeds to impact at greatest force by
hitting flat surfaces of the soil tin
Germination Testing
Before gennination testing all seeds were soaked 24 hours in reverse
osmosis-treated water (this was included in the process ofhot water treatment
jU~SCIlOea above) Following soaking seeds were placed in 100 cm Petri dishes lined
90 cm Whatman qualitative 1 filter paper circles (MG Scientific Pleasant
WI) One Petri dish was used for each treatment by source combination
papers were initially moistened with 5 mL reverse osmosis-treated water then
~VU1CUlJl moistened as needed to ensure adequate conditions for gennination
were monitored daily (at 300 PM) for gennination for 14 days in accordance
~UIUlnJlVUa Seed Testing Association (ISTA) standards for Robinia species
21
(1STA 1999) Tests were carried out at room temperature (22degC plusmnOSoC)
Germination was classified as protrusion of the radicle from the embryo by at least
1mm as viewed with the naked eye
X-ray Radiography
On August 132001 three lOO-seed samples from the Sacramento seed source
were treated for 14 or 8-minute percussion durations The seeds were sent
immediately by overnight delivery to the Faxitron X-Ray Corporation in Wheeling
Illinois where Dr Bob Karrfalt ofthe USDA Forest Service oversaw their imaging
Radiographs were made at SX with a Faxitron Specimen Radiography System Model
MX-20
Scanning Electron Microscope Photography
On February 192002 a Hitachi S-3200N Variable Pressure Scanning
Electron Microscope (Mountain View CA) was used to examine strophiolar cells of
untreated seeds and seeds percussed for 4 minutes Seeds were cut longitudinally
igt their entire length using a razor blade splitting the strophiolar region roughly in
Cross sections of seed samples magnifying the strophiolar region were viewed
600X under the microscope
Analysis
Categorical Analysis of Variance was used to determine treatment source and
(Le time) main effects and interactions The response variable was total
~vlI This procedure is a Chi-square test of homogeneity using the natural log
of germinated to non-germinated seed for each treatment Maximumshy
22
likelihood analysis was used to calculate Chi-square test statistics with their observed
significance levels A single contrast was conducted to cempare treatment means
against the control Observed significance levels less than or equal to 005 were
considered significant Pairwise comparisons were conducted between non-control
treatment mean percents using a conservative alp~a value of 005 divided by the
number of comparisons (Bonferroni approach) and approximate pairwise z statistics
Categorical analyses were performed using Proc Catmod (SAS Institute 1989)
RESULTS
Scarification seed source block and all interactions between factors
influenced germination (Table 3) The main effects are reported in the presence of
these interactions because the overall robustness of treatments and seed sources are of
interest Background levels ofnaturally occurring variation in seed sources are
seen in control germination percentages in Figure 2 In particular time variability
is unlikely to be under the control of the producer Appendix B details experimental
data for source by treatment gennination percents for each of the four blocks
Averaged over all treatments germination by source ranged from 40 to
79 (Figure 2) Averaged over all sources (Figure 3) and for each source (Figure 4
5) percussion scarification durations of4 or 8 minutes yielded greatest germination
Averaged over all sources germination increased with increasing durations of
percussion then leveled off for the 4- and 8-minute treatments (Figure 3) Immersion
and 30-second boiling scarification maximized germination for the hot water
treatments with a decrease in germination when seeds were boiled for 60 seconds
For all but two sources there is a similar ipcreasing trend in germination from
1to 4 minutes ofpercussion (Figure 4) The Holman source increased in germination
1 to 2 minutes then leveled off at 4 minutes The Jemez source changed little
1 to 4 minutes of treatment As treatment duration increased from 4 to 8
mutes five sources decreased in germination (Figure 4a) whereas three sources
24
Table 3 Categorical Analysis of Variance table for germination response to seed treatment seed source block and the interaction of the factors
Source of DF Chi-Square Observed Variability Significance Level
Source 8 98370 lt00001 Trt 7 233876 lt00001
contrast 1 16976 lt00001 SourceTrt 56 97077 lt00001 Block 3 5538 lt00001
SourceBlock 24 10162 lt00001 TrtBlock 21 7564 lt00001
SourceTrtBlock 168 34283 lt00001
S 4)
8 ~
0
sectg ~ 40 ~
N 0 0
20
o f0ltgt ~ ~4 jybshy ((iltgto ~~ K1ltgt 6~b-~ ~~ 0b-~~ ~~ ~b-~ ~b-~b
80
60 -I-- - ~- middotmiddotmiddotmiddotmiddot1
r -] Northern NM Sources
Central NM Sources
Southern NM Sources
sect-o ~
~b-v(i
SOURCE
Figure 2 Effect ofNew Mexico locust seed source on germination for data averaged over all scarification treatments Control germination is shown by solid line within bar
80
~ cd ~ ltl) () 60 ~ ~
N
t 0shyrti tshySltl)
40
0
20
o lmmersion Boiling 30 sec Boiling 60 sec Control 1 min 2min 4 min 8 min
Hot Water Treatments Percussion Treatments
Figure 3 Effect of scarification on germination ofNew Mexico locust for data averaged over all seed sources Means labelled with the same letter are not significantly different
100~----------------------~--~----------------~
80
60
ltU
~
40 IJ
sectgsI
l100
80
60
40
a I middotmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot middotImiddotmiddotmiddotmiddotmiddotmiddot E
_~iii-bull _ _ bull f --~--
t-- -~ ---- -shy ---- shyI ~ ---- - shy
L
fiII
~
b
- shy ---r
- Holman
Sacramento Sandia Gila Jemez
c ~
ltgt ~
Tmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot-I
j~=~=-~~-1
I middotmiddot ~ bull r
t tr
Y -------M-a-gd-a-Ie-n-a--
1 _ _
fCapitan
---- Manzano ~y -_ Raton
2 4 8
Time interval ofpercussion scarification (min)
Figure 4 Effect of seed source by treatment interaction on New Mexico locust germination for percussion scarification pattern a and pattern b
28
tv 0
~ d
~ A s o
1 c3
80
60
40
20
o
TIT
t------=middotfmiddotmiddot-middotmiddot-middotmiddot-middotmiddot 1- --- - - --=- - -shy--cE= - _--shy --shy ----shy ~ ~ ---shy ~ -~- _ --shy-------r 1
I
Raton Holman
-_ Jemez
--- Sandia Manzano Magdalena Capitan Sacramento Gila
Immersion Boiling 30 sec Boiling 60 sec
Hot Water Scarification
Figure 5 Effect of seed source by treatment interaction on New Mexico locust germination for hot water scarification
continued to increase in germination (Figure 4b) There was no difference between
the two treatment levels for the Manzano source (Figure 4b)
The effect ofhot water scarification also varied among sources with few
overall differences between hot water treatments (Figure 5) However boiling for 60
seconds yielded highest germination for the Jemez source whereas increasing
durations ofboiling decreased germination for the Raton source
In addition to consistently high germination 4- and 8-minute percussionshy
treated seeds germinated as fast or faster than all other treatments (Figure 6 Table 4)
050 measured as the number of days needed to complete 50 ofeventual
germination averaged 2 days for both ofthese treatments
X-ray radiographs were taken of seeds collected from the Sacramento source
percussed for 1 4 or 8 minutes (Figure 7) Seeds show signs of damage at 8 minutes
ofpercussion (Figure 8) Compared to an undamaged seed percussed for only 4
minutes (Figure 8a) damage at the 8-minute level includes fractured radicles pitting
of the seed coat and chips in seed coat structure (Figure 8b-d)
Scanning electron microscope (SEM) images at 600X reveal the direct effect
of 4 minutes of percussion on cells of the strophiolar region (Figure 9) Whereas
untreated seeds retain cellular organization in the strophiolar region strophiolar cells
seeds treated for 4 minutes ofpercussion separate and raise resulting in a
~-~e~U appearance
30
Umiddot-~middot--l-~---middotymiddotmiddotmiddot-----middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot--middot--middotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middotmiddotmiddotmiddotmiddotmiddotmiddot- -
050= gt1
-e-- Percussion 8 min -0- Percussion 4 min ---T- Percussion 2 min -----ltil- Percussion 1 min -- Immersion --0- Boiling 30 sec -+- Boiling 60 sec -ltgt- Control
2 4 6 8 10 12
Day Figure 6 Timecourse ofNew Mexico locust gennination following scarification
treatments G50 is the number of days to 50 of eventual germination
31
14
Table 4 Effect of seed source and treatment on G50 (days to 50 germination)
Seed Source Control Immersion B30sec B60sec Plmin P2min P4min P8min mean Raton 3 3 5 7 4 3 2 2 4 Holman 2 2 2 2 2 2 2 2 2
Jemez 4 6 6 5 3 2 2 2 4 Sandia 2 2 2 2 2 2 2 2 2
w tv Manzano 5 2 2 3 3 2 2 2 3
Magdalena 4 2 2 3 4 3 2 1 3
Capitan 5 4 2 2 3 2 2 2 3
Sacramento 3 2 2 2 4 3 2 2 3 Gila 3 2 2 2 2 2 2 2 2 mean 3 3 3 3 3 2 2 2 3 standard 11 14 16 18 09 05 0 03 08 deviation
Note Columns and rows may not agree due to rounding
Figure 7 X-ray radiographs ofSacramento seed source percussed for 1 4 or 8 minutes
33
Figure 8a-d Seed damage close-ups Figure a shows undamaged seed percussed for 4 minutes Figures b-d show seeds percussed for 8 minutes Damage includes fracturing ofradicle in b complete removal ofseed coat in c and pitting ofseed coat in d
34
Untreated Treated
Figure 9 SEM images ofuntreated vs 4-minute percussion-treated strophiolar cells ofNew Mexico locust seeds Treated cells are loosened separated and raised
DISCUSSION
Determining the Optimal Treatment
Averaging results of scarification treatmems across seed sources is a useful
tool to predict which treatment is most likely to be successful for an unknown seed
source Averaged over all sources four- and eight-minute percussion treatments
resulted in the highest germination 815 and 790 respectively and produced
germination that was as fast or faster than all other treatments Although there was no
difference between these two treatments on average an argument for 4 minutes of
percussion as a standard protocol for treating New Mexico locust seeds can be made
Descriptively 4 minutes of percussion improved germination over 8 minutes
ofpercussion for a majority of sources (five out of nine) 8 minutes of percussion
improved germination for three ofnine sources with no difference for a fmal source
For individual sources 4 minutes ofpercussion was always the best or secondmiddot best
treatment and was always equal to or better than all hot water treatments
Germination at 8 minutes ofpercussion was lower than all other percussion durations
for the Jemez and Gila sources and was lower than at least one hot water treatment for
these sources as well
The x-ray radiographs of the Sacramento seed source illustrate the excessive
internal and external seed damage that can occur with 8 minutes of percussion
germination counts 8-minute percussed seeds from Holman and Capitan
ClUll signs of damage that were visible to the naked eye (see below)
r~evertheless some of these seeds still fit the criteria for germination Neither x-ray
36
photography nor visible observations during seed counts revealed seed damage at the
4-minute percussion level In operational trials seeds percussed for 4 minutes
developed into healthy seedlings (Khadduri and Harrington unpublished data)
Strophiole and Seed Coat Behavior
Four minutes ofpercussion successfully breaks seed dormancy in a large
percentage ofNew Mexico locust seeds without excessively damaging the
surrounding seed coat or internal seed coat structures Most papilionoid legume
seeds including seeds ofNew Mexico locust have a specific region of the seed coat
known as the strophiole or lens (Hamly 1932) The strophiole is located on the
cotyledonary lobe of a locust seed (Figure 10 a-c) If seeds are not indiscriminately
damaged the strophiole is the first point of water entry to the embryo (Hopkinson
1997) When a papilionoid seed is percussed for the appropriate amount of time
repeated hits on the integrated seed coat loosen the constrained cells of the strophiolar
region without excessively damaging the rest of the seed coat (Ballard 1976) SEM
photos illuStrate that 4 minutes of percussion loosened and separated strophiolar cells
ofNew Mexico locust seeds when compared with constrained organized strophiolar
cells of untreated seeds (Figure 9) Research-has shown that water passes to the
embryo through the treated strophiole in a controlled manner and regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues
(Kelly and van Staden 1987)
37
10 Strophiole ofunimbibed New Mexico locust seed (a) and following uptake (bc)
38
In contrast hot-water treatments can excessively degrade the seed coat
(Rebman et al 1998 Marunda 1990) Random cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rebman et aI 1998 Marunda 1990) Along with a
proportionately large amount ofun-imbibed (thus under-treated) seeds (Figure l1a)
damage was observed in hot-water treated seeds from six sources during germination
counts Damage included seeds where the radicle failed to elongate (Figure 11 b)
seeds where the radicle elongated but was delayed in freeing itself from the seed coat
(Figure 11c) and seeds where the radicle completely broke off from the embryo
(Figure lId) In addition some seeds germinated in reverse (the cotyledon emerged
fIrst) If the radicle emerged completely from the seed coat they were counted as
germinated
While nearly all seeds treated for 8 minutes ofpercussion imbibed water
damage was evident in x-ray evaluations (Sacramento seed source) and in
germination counts (Holman and Capitan) at this treatment level Seed damage
during germination counts was similar to levels found in hot-water treated seeds
suggesting that over-treatment by both hot water and longer durations of percussion
may result in similar damage to seed structure
The Sacramento and Holman seed sources were two of the fIve sources that
opcrplltlpn in germination from the 4- to 8-minute durations of percussion
the benefIts ofloosening the strophiole as evidenced by imbibition of
all 8-minute treated seeds were outweighed by external and internal seed coat
39
a
b c d
11 Damage to New Mexico locust seeds observed during germination cotmts following hot water scarification 9a Hot water replicate including ~t seedlings and numerous unimbibed seeds 9b Close-up of seed where raallCle failed to elongate 9c Close-up ofseed where radicle elongated but was
-gtIn in freeing itself from the seed coat 9d Close-up ofseed where radicle broke off from embryo
40
damage (which also may be contributing to high imbibition) For the three sources
for which 8 minutes of percussion resulted in maximum germination extended
percussion of seeds necessary to loosen the strophiole may not have excessively
damaged the seed anatomy The Manzano source did not significantly change in
germination from 4 to 8 minutes of percussion suggesting that an increase in
permeable seeds at the 8-minute treatment duration was balanced by the number of
seeds that failed to germinate due to seed damage To summarize the 4-minute
percussion treatment improved germination over 8 minutes ofpercussion for a
majority of sources (five ofnine) the 8-minute percussion treatment improved
germination over 4-minutes ofpercussion for three sources and there was no
difference with the Manzano source Notably for the Raton source 8 minutes of
percussion increased germination by 235 percentage points over 4-minutes of
percussion (7875 vs 5525 respectively) the next best treatment Raton was the
only source to result in germination less than 75 for 4 minutes ofpercussion so a
grower may wish to apply longer percussion durations to lots that do not respond well
to this treatment level
Finally there was an interesting trend in the Jemez and Raton sources seed
that significantly contributed to the source by treatment interaction (Figure 12) A
~u test showed that the Jemez source had a significantly lighter seed weight than
other sources while the Raton source had an average seed weight For the Jemez
the longest duration of boiling increased germination Conversely only short
U4UVll) of percussion were needed to achieve high germination and the longest
41
80 _ Jemez a Raton
iD
~ C iD e iD
Q c 0 ~
c ~ iD 0
20
0------shy
Immersion Boiling 30 sec Boiling 60 sec
Hot Water Scarification
10degTr========~--------~------------------------------
80
60
40
20
_ Jemez Raton
0------shy
1 min 2 min 4 min 8 min
Percussion Scarification
Figure 12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources
42
b
--~----~~~~========~---- duration ofpercussion reduced germination For the Raton source longer durations
ofboiling consistently decreased germination whereas longer percussion treatments
consistently increased germination
A possible explanation is that a thick seed coat is associated with a loose
strophiole structure and that a thin seed coat is associated with a constrained
strophiole This contrast merits further study and may suggest an ecological
adaptation Further investigation ofthis phenomenon may contribute to successful
pre-screening ofunknown seed sources to determine optimal treatment leveL
43
CONCLUSION
Based on results obtained in pilot study (see Appendix) and thesis
experiments 4 minutes ofpercussion is recommended as a standard protocol for
treating an unknown source ofNew Mexico locust seed In addition to laboratory
results the 4-minute percussion treatment has resulted in germination greater than
80 and healthy seedling grow-outs for production purposes (unpublished data) In
laboratory experiments only one hundred seeds per 118 ml (40z) soil tin were
percussed at a time whereas for production we have treated up to five hundred seeds
per tin For large-scale production ofNew Mexico locust seedlings growers may
wish to experiment with larger quantities of seed and larger-size soil tins
Seed storage trials following percussion treatments were not conducted
However germination greater than 80 and healthy seedlings were obtained when
seeds were percussion-treated and subsequently stored at room-temperature (22degC)
for one week (unpublished data) Seed storage trials merit further study as the
standard hot water treatment leaves seeds in a softened condition that is not
conducive to storage
Hydroseeding is an effective method for direct seeding in reclamation
situations Percussion treatment may be conducive to hydro seeding as seeds remain
a dry relatively undamaged yet permeable condition following treatment Sulfuric
seeds also remain dry following treatment but extensive damage to seed coat
poor seedling vigor have been reported with this treatment (Kelly and van Staden
A grow-out study using a hydro seeder on a reclamation site testing percussion
44
against other treatments such as sulfuric acid and mechanical scarification also
merits further study
Papilionoids are the largest subfamily oflegumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust these legumes often play an integral role in the revegetation of
disturbed lands The pilot study (Appendix) indicates that percussion scarification
successfully treats the widely used black locust as well Developing superior
scarification methods to the standard hot water mechanical scarification and acid
treatments may facilitate the use ofpapilionoid legumes in reclamation projects
45
APPENDICES
APPENDIX A
PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR
NEW MEXICO LOCUST AND BLACK LOCUST SEEDS
Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4
Hot water and sulfuric acid soaks are traditional treatments for seeds of many
temperate woody legwnes including locusts However these scarification techniques
often produce inconsistent germination Percussion scarification where seeds are
repeatedly propelled against a hard surface was compared with hot water
scarification to evaluate treatment efficacy for New Mexico locust (Robinia
neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment
seeds were placed in a 98degC water bath which was immediately removed from the
heat source For percussion scarification seeds were placed in a soil sample tin and
agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the
control were followed by 24-hour water soaks Hot water baths resulted in 56 and
41 germination for New Mexico locust and black locust respectively For both
species nearly all durations ofpercussion increased germination over the hot water
treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3
4 and 5 minutes for black locust resulted in at least 90 germination Traditional
scarification treatments randomly degrade the entire seed coat which can lead to
tissue damage during water uptake Percussion scarification specifically weakens the
strophiole the natural source ofwater entry to the seed in papilionoid legwnes
INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom
2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031
48
Following percussion imbibition is controlled through the strophiole and underlying
tissue is protected
Keywords New Mexico locust black locust scarification percussion germination
Introduction
Locust trees (Robinia spp) are aggressive pioneer species that quickly
colonize disturbed land fix atmospheric nitrogen in the soil and stabilize slopes that
are prone to erosion (Klemmedson 1994 Ashby et al 1985) (Figure A1ab)
Recognition of these attributes has contributed to increased demand for locusts in
reclamation projects Locust seeds however exhibit physical dormancy where a
thick seed coat prevents the movement of water and gasses to the embryo (Leadem
1997)
Sulfuric acid or hot water soaks have traditionally been used to break seed
dormancy in macro-propagation of locusts Many growers are moving away from the
use of su1furic acid in treating locust seeds (Dreesen and Harrington 1997) In
addition to being dangerous acid soak durations must be specifically correlated to
seed lot (Olson 1974) Collections of native seed can vary tremendously in seed size
seed weight and hard seededness etc This variability limits the utility of sulfuric
acid soaks as even within a given lot some seeds may be damaged whereas others
are under-treated and fail to imbibe (take up water) A survey of Southwestern
container growers indicates that hot water baths are currently the most common
scarification method for locust seeds (Hine et al 1997) However hot water baths
also produce inconsistent germination (Lin et al 1996)
49
b
Figure AI New Mexico locust a primary invader following disturbance Ala Crown-sprouting at edge ofburn near Los Alamos NM--one year after Cerro Grande fire Alb Colonizing road cut Gila National Forest NM
50
Percussion scarification where seeds are repeatedly propelled against a hard
surface is an alternative dormancy-breaking method that has proven successful in
legumes related to locusts (Hamly 1932 Barton 1947) We compared this alternative
treatment to hot water scarification to evaluate treatment efficacy for New Mexico
locust and black locust seeds
Materials and Methods
Commercial New Mexico locust seeds (Western Native Seed Coaldale CO)
were collected fall 2000 in Huerfano County CO Black locust seeds were collected
September 2000 from Taos County NM by harvesting ripe pods from several trees at
a distance of up to 3 m from ground level Black locust seeds were allowed to air-dry
for 3 weeks and were threshed and separated from large chaffusing a greenhouse
fan A South Dakota Seed Blower (Chicago Illinltois) was used to remove fine chaff
from both seed lots Seeds were storedmiddotat 2-4C until the germination study was
conducted in March 2001
This study compared hot water scarification with percussion scarification
using treatment and species as experimental factors Along with a control
scarification treatments included immersion of seed in boiling water as well as
percussion durations of 1 2 3 45 or 10 minutes Four 100-seed samples were used
to test each species by scarification treatment combination
For the hot water treatment test tubes were filled with 30 mL water and
placed in a water-filled beaker (Figure A2a) The bath was raised to boiling (-98C at
51
b
c
Figure A2 Implementation ofhot water and percussion scarification A2a Hot water bath A2b Paint-shaker set-up A2c Placing soil tin containing seeds at end ofpaint shaker ann
52
Las Cruces NM elevation of 1300 meters) Each 100-seed replicate was placed in a
test tube and each test tube was immediately removed to cool at room temperature
Percussion scarification was implemented using a pneumatic paint shaker
(Central Pneumatic model 00422 Camarillo CA) (Figure A2b) This allowed
standardization of treatment intensity Compressed air pressure was maintained at
530kPa plusmn 30kPa (80psi plusmn 5psi) resulting in approximately 350 oscillations per
minute Each lOO-seed sample was placed in a 118 ml (4 oz) soil tin for shaking To
maximize lateral movement of paint shaker a paint can was used as a spacer to place
soil tins at the end of the shaking arm (Figure A2c) This allowed shaking to occur at
the greatest distance from the pivot point (fulcrum) Following all treatments
including the control seeds were soaked for twenty-four hours in distilled water prior
to plating out
Gennination was evaluated on lab benches at room temperature Each
replicate of seeds was placed in a 100 cm petri dish on moistened filter paper with
humidity maintained by enclosing the petri dishes in ziplock bags Gennination was
monitored daily for 14 days in accordance with International Seed Testing
Association standards for Robinia species (ISTA 1999) Gennination was defined as
protrusion of the radicle from the embryo by at least 1mm as viewed by the naked
eye
Categorical Analysis of Variance using Prpc Catmod (SAS Institute 1989)
was used to detennine treatment differences in germination percentages for each
source This procedure is an extension of a Chi-square test of homogeneity using the
53
natural log of the ratio of germinated to non-germinated seeds for each treatment Pshy
values less than 005 were considered significant A limited set of pairwiseshy
comparisons was conducted to compare treatment mean percents using a
conservative alpha value of 005 divided by the number of comparisons
Results
Percussion maximized germination for both New Mexico locust and black
locust There were three germination response levels for New Mexico locust (Figure
A3) First there was a low response in the control indicating a dormant seed lot
The hot water treatment and I-minute duration of percussion share a second
intermediate response Germination was highest at percussion levels from 2 to 10
minutes
A selected percussion treatment intermediate in time (5 minutes) produced the
steepest germination curve indicating the fastest germination rate (Figure A4) The
hot water treatment produced an intermediate curve or relatively slower germination
rate The control curve was very flat indicating the slowest germination rate for New
Mexico locust
Control seeds germinated poorly for black locust as well (Figure A5) Hot
water scarification improved germination over the control but all levels of percussion
improved germination over hot water The optimal durations of percussion treatment
were bracketed with a significant increase from the 1 to 2 minute percussion level a
high germination response from 2 to 5 minutes of percussion and finally a significant
54
~ d 25 CL)
p t1 0 ~ t1
g Vl ~ Vl
100~---------------------------------------------------------------
80
60
40
20
o Control Hot Water Percussion
I min Percussion
2 min Percussion
3 min
Scarification
Percussion 4 min
Figure A3 Effect of scarification on germination response for New Mexico locust
Percussion 5 min
=significant difference between adjacent treatment bars ns = no significant difference
Percussion 10 min
~ ~ () u agt
Il-t d o ~
~ ~ c3
100middotlr~~~------------------------------------e- Percussion 5min middot 0middot Hot Water -- Control
80-J=middot======
6b-middot-lmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middot--middotmiddot-middot~-middotmiddot -middotmiddot middot middotmiddot-middot 1
40
20
-----~-----------
0 0 0 middotmiddotmiddotmiddotmiddot0middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot0
0 --~-----y-----~------_---~--- T-----~
o shy -TDay 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A4 Effect of selected scarification treatments on germination speed for New Mexico locust First seven days of fourteen-day experiment are shown
Day 7
80
~ cd
5 60 ()
[) Cl d 0 1t1 d
40sect dUl
-J
20
o
ns
Scarification
Figure A5 Effect of scarification on germination response for black locust = significant difference between adjacent treatment bars ns = no significant difference
decrease at the 10-minute level This drop-off suggests that 10 minutes of percussion
over-treats seeds from this lot of-black locust
A selected percussion treatment intermediate in time (5 minutes) also had the
fastest germination rate for black locust (Figure A6)_ The hot water curve is
somewhat flatter indicating a slower germination rate The control curve is very flat
indicating a slow germination rate
A qualitative difference between hot water and percussion-treated seeds was
also observed Along with healthy germinants the hot water treatment produced
over-treated seeds (Figure A 7a) Examples of damaged germinants include seeds
where the radicle did not elongate (Figure A 7b) seeds where the radicle elongated
but was delayed in freeing itself from the seed coat (Figure A7c) and seeds where the
radicle broke off completely from the embryo (Figure A7d) In addition to these
examples of over-treatment there were numerous small dark seeds that failed to
imbibe water For percussion treatment nearly all seeds took up water (Figure A8a)
Germination was rapid and uniform with little damage evident (Figure A8b)
To summarize both New Mexico locust and black locust seed lots were
dormant The standard hot water scarification treatment improved germination but
only to 56 and 41 for New Mexico locust and black locust respectively Nearly
all percussion durations improved total germination as well as germination rate over
the hot water treatment Germination was greater than 90 for percussion durations
of 4 5 and 10 minutes for New Mexico locust and 3 4 and 5 minutes for black locust
58
CI)
sgt t CI) 0
igt 0
sect ttl t E
Vl 0
c3
100r==~==~~=r------------------------------------~ ___ Percussion 5 min
middotmiddotmiddot0middotmiddotmiddot Hot Water -lIf- Control
80r=========~--~~----------------------------~
60
40
20 -~-~---~---------- ---~------------I
omiddot
O
_~_----------lIf-----~bull -----_ shy---shy I
Day 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A6 Effect of selected scarification treatments on germination speed for black locust First seven days of fourteen-day experiment are shown
Day 7
a
b c d
Figure A7 Damage to New Mexico locust seeds observed during germination counts following hot water scarification A7a Hot water replicate including damaged seedlings and numerous unimbibed seeds A7b Close-up ofseed where radicle failed to elongate A7c Close-up ofseed where radicle elongated but was delayed in freeing itself from the seed coat A7d Close-up ofseed where radicle completely broke offfrom embryo
60
a
b
Figure A8 New Mexico locust seeds following percussion treatment at time of germination counts A8a Percussion replicate with uniformly healthy seedlings and near complete imbibition of seeds A8b Close-up ofpercussion-treated seedling
61
Discussion
Papilionoid seeds have a specific anatomical feature known as the strophiole
or lens-the natural site of water entry to the seed (Hamly 1932) The strophiole is
located on the cotyledonary lobe of a locust seed (Figure A9) When a papilionoid
legume seed is percussed for the appropriate amount of time repeated hits on the
integrated seed coat loosen the constrained cells of the strophiolar region without
excessively damaging the rest of the seed coat (Ballard 1976)
When a percussed seed is soaked water enters exclusively through the
strophiole in a controlled manner (Kelly and Staden 1987) This regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues This
contrasts with seeds that have been hot water or acid scarified treatments that can
randomly degrade the seed coat Unlocalized cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rehman et al 1998 Marunda 1990)
Papilionoids are the largest subfamily of legumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust and black locust these legumes often play an integral role in the
revegetation ofdisturbed lands Developing superior scarification methods to the
standard hot water and acid treatments should fadlitate the use ofthese legumes in
restoration projects
62
Figure A9 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (bc)
63
APPENDIXB
GERMINATION PERCENTS AND STANDARD ERRORS FOR SOURCE
BLOCK AND TREATMENT FOR NEW MEXICO LOCUST
Table B 1 Gennination percents and standard errors for source block and treatment for New Mexico locust
Source Block Treatment Germination Percentage Standard Error
Capitan 1 Immersion 74 437 Capitan 2 Immersion 69 462 Capitan 3 Immersion 71 454 Capitan 4 Immersion 67 470 Capitan Boiling 30 sec 79 407 Capitan 2 Boiling 30 sec 76 427 Capitan 3 Boiling 30 sec 72 449 Capitan 4 Boiling 30 sec 76 427 Capitan Boiling 60 sec 71 454 Capitan 2 Boiling 60 sec 54 498 Capitan 3 Boiling 60 sec 67 470 Capitan 4 Boiling 60 sec 76 427 Capitan I Control 5 218 Capitan 2 Control 7 255 Capitan 3 Control 4 196 Capitan 4 Control 5 218 Capitan 1 Percussion I min 47 499 Capitan 2 Percussion I min
470
Capitan Percussion I min 39 488
Capitan 4 Percussion I min 38 485 Capitan I Percussion 2 min 55 497 Capitan Capitan
2
Percussion 2 min Percussion 2 min
71 65
454 477
Capitan 4 Percussion 2 min 56 496 Capitan I Percussion 4 min 86 347 Capitan 2 Percussion 4 min 79 407 Capitan 3 Percussion 4 min 81 392 Capitan 4 Percussion 4 min 82 384 Capitan I Percussion 8 min 90 300 Capitan 2 Percussion 8 min 88 325 Capitan 3 Percussion 8 min 90 300 Capitan 4 Percussion 8 min 90 300 Gila I Immersion 71 454 Gila 2 Immersion 77 421 Gila
Immersion 75 433
Gila 4 Immersion 72 449
Gila I Boiling 30 sec 68 466
Gila 2 Boiling 30 sec 81 392
Gila 3 Boiling 30 sec 54 498
Gila 4 Boiling 30 sec 63 483
Gila I Boiling 60 sec 55 497
Gila 2 Boiling 60 sec 68 466
Gila 3 Boiling 60 sec 57 495
Gila 4 Boiling 60 sec 70 458
65
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Gila 2 Control 13 336 Gila 3 Control 13 336 Gila 4 Control 20 400 Gila 1 Percussion 1 min 66 474 Gila 2 Percussion 1 min 57 495 Gila 3 Percussion 1 min 73 444 Gila 4 Percussion 1 min 82 384 Gila 1 Percussion 2 min 81 392 Gila 2 Percussion 2 min 86 347 Gila 3 Percussion 2 min 85 357 Gila 4 Percussion 2 min 54 498 Gila 1 Percussion 4 min 79 407 Gila 2 Percussion 4 min 83 376 Gila 3 Percussion 4 min 84 367 Gila 4 Percussion 4 min 79 407 Gila 1 Percussion 8 min 51 500 Gila 2 Percussion 8 min 76 427 Gila 3 Percussion 8 min 52 500 Gila 4 Percussion 8 min 74 439 Holman 1 Immersion 83 376 Holman 2 Immersion 86 347 Holman 3 Immersion 81 392 Holman 4 Immersion 90 300 Holman 1 Boiling 30 sec 90 300 Holman 2 Boiling 30 sec 85 357 Holman 3 Boiling 30 sec 87 336 Holman 4 Boiling 30 sec 83 376 Holman 1 Boiling 60 sec 79 407 Holman 2 Boiling 60 sec 80 400 Holman 3 Boiling 60 sec 86 347 Holman 4 Boiling 60 sec 93 255 Holman Control 25 433 Holman 2 Control 19 392 Holman Control 21 407
Holman 4 Control 24 427
Holman Percussion 1 min 77 421
Holman 2 Percussion 1 min 85 357
Holman 3 Percussion 1 min 80 400
Holman 4 Percussion 1 min 89 313
Holman 1 Percussion 2 min 88 325
Holman 2 Percussion 2 min 96 196
Holman 3 Percussion 2 min 93 255
Holman 4 Percussion 2 min 96 196
Holman 1 Percussion 4 min 92 271
Holman 2 Percussion 4 min 97 171
66
I
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Holman 3 Percussion 4 min 91 286 Holman 4 Percussion 4 min 96 196 Holman 1 Percussion 8 min 88 325 Holman 2 Percussion 8 min 85 357 Holman 3 Percussion 8 min 83 376 Holman 4 Percussion 8 min 82 384 Jemez 1 Immersion 53 499 Jemez 2 Immersion 44 496 Jemez 3 Immersion 52 500 Jemez 4 Immersion 70 458 Jemez 1 Boiling 30 sec 58 494 Jemez 2 Boiling 30 sec 55 497 Jemez 3 Boiling 30 sec 48 500 Jemez 4 Boiling 30 sec 65 477 Jemez 1 Boiling 60 sec 68 466 Jemez 2 Boiling 60 sec 63 483 Jemez 3 Boiling 60 sec 73 444 Jemez 4 Boiling 60 sec 89 313 Jemez 1 Control 2 140 Jemez 2 Control 9 286 Jemez 3 Control 6 237 Jemez 4 Control 8 271 Jemez 1 Percussion 1 min 58 494 Jemez 2 Percussion 1 min 63 483 Jemez 3 Percussion 1 min 77 421 Jemez 4 Percussion 1 min 89 313 Jemez 1 Percussion 2 min 68 466 Jemez 2 Percussion 2 min 64 480 Jemez 3 Percussion 2 min 80 400 Jemez 4 Percussion 2 min 83 376 Jemez Percussion 4 min 68 466 Jemez 2 Percussion 4 min 79 407 Jemez 3 Percussion 4 min 70 458 Jemez 4 Percussion 4 min 83 376 Jemez 1 Percussion 8 min 52 500 Jemez 2 Percussion 8 min 59 492 Jemez 3 Percussion 8 min 61 488 Jemez 4 Percussion 8 min 70 458 Magdalena 1 Immersion 69 462 Magdalena 2 Immersion 69 462 Magdalena J Immersion 70 458 Magdalena 4 Immersion 85 357 Magdalena 1 Boiling 30 sec 65 477 Magdalena 2 Boiling 30 sec 71 454 Magdalena 3 Boiling 30 sec 68 466
67
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Magdalena 4 Boiling 30 sec 83 376 Magdalena 1 Boiling 60 sec 63 483 Magdalena 2 Boiling 60 sec 68 466 Magdalena 3 Boiling 60 sec 63 483 Magdalena 4 Boiling 60 sec 82 384 Magdalena 1 Control 11 313 Magdalena 2 Control 6 237 Magdalena 3 Control 5 218 Magdalena 4 Control 4 196 Magdalena Percussion 1 min 32 466 Magdalena 2 Percussion 1 min 45 497 Magdalena 3 Percussion 1 min 46 498 Magdalena 4 Percussion I min 36 480 Magdalena 1 Percussion 2 min 67 470 Magdalena 2 Percussion 2 min 68 466 Magdalena 3 Percussion 2 min 65 477 Magdalena 4 Percussion 2 min 81 392 Magdalena 1 Percussion 4 min 87 336 Magdalena 2 Percussion 4 min 97 171 Magdalena 3 Percussion 4 min 83 376 Magdalena 4 Percussion 4 min 97 171 Magdalena 1 Percussion 8 min 98 lAO Magdalena 2 Percussion 8 min 100 000 Magdalena 3 Percussion 8 min 96 196 Magdalena 4 Percussion 8 min 98 lAO Manzano 1 Immersion 67 470 Manzano 2 Immersion 57 495 Manzano 3 Immersion 72 4049 Manzano 4 Immersion 73 4044 Manzano 1 Boiling 30 sec 70 458 Manzano 2 Boiling 30 sec 64 480 Manzano 3 Boiling 30 sec 71 454 Manzano 4 Boiling 30 sec 80 400 Manzano I Boiling 60 sec 50 500 Manzano 2 Boiling 60 sec 53 499 Manzano 3 Boiling 60 sec 59 492 Manzano 4 Boiling 60 sec 74 439 Manzano I Control 4 196 Manzano 2 Control 8 271 Manzano 3 Control 1 099 Manzano 4 Control 6 237 Manzano 1 Percussion 1 min 52 500 Manzano 2 Percussion 1 min 39 488 Manzano 3 Percussion 1 min 36 480 Manzano 4 Percussion 1 min 51 500
68
Table B 1 Continued
Source Block Treatment Gerrni~ation Percentage Standard Error
Manzano Percussion 2 min 53 499 Manzano 2 Percussion 2 min 58 494 Manzano 3 Percussion 2 min 61 488 Manzano 4 Percussion 2 min 62 485 Manzano 1 Percussion 4 min 76 427 Manzano 2 Percussion 4 min 85 357 Manzano 3 Percussion 4 min 79 407 Manzano 4 Percussion 4 min 83 376 Manzano 1 Percussion 8 min 87 336 Manzano 2 Percussion 8 min 83 376 Manzano 3 Percussion 8 min 83 376 Manzano 4 Percussion 8 min 75 433 Raton 1 Immersion 54 498 Raton 2 Immersion 37 483 Raton 3 Immersion 39 488 Raton 4 Immersion 69 462 Raton 1 Boiling 30 sec 36 480 Raton 2 Boiling 30 sec 20 400 Raton 3 Boiling 30 sec 28 449 Raton 4 Boiling 30 sec 60 490 Raton 1 Boiling 60 sec 15 357 Raton 2 Boiling 60 sec 171 Raton 3 Boiling 60 sec 4 196 Raton 4 Boiling 60 sec 35 477 Raton 1 Control 4 196 Raton 2 Control 12 325 Raton 3 Control 2 140 Raton 4 Control 6 237 Raton 1 Percussion 1 min 52 500 Raton 2 Percussion I min 29 454 Raton 3 Percussion I min 35 477 Raton 4 Percussion 1 min 31 462 Raton 1 Percussion 2 min 55 497 Raton 2 Percussion 2 min 35 477 Raton 3 Percussion 2 min 33 470 Raton 4 Percussion 2 min 42 494 Raton 1 Percussion 4 min 65 477 Raton 2 Percussion 4 min 52 500 Raton Percussion 4 min 51 500 Raton 4 Percussion 4 min 53 499 Raton 1 Percussion 8 min 88 325 Raton 2 Percussion 8 min 80 400 Raton 3 Percussion 8 min 73 444 Raton 4 Percussion 8 min 74 439 Sacramento 1 Immersion 43 495
69
Table B 1 Continued
-Source Block Treatment Germination Percentage Standard Error
Sacramento 2 Immersion 30 458 Sacramento 3 Immersion 38 485 Sacramento 4 Immersion 56 496 Sacramento 1 Boiling 30 sec 47 499 Sacramento 2 Boiling 30 sec 48 500 Sacramento 3 Boiling 30 sec 48 500 Sacramento 4 Boiling 30 sec 56 496 Sacramento 1 Boiling 60 sec 52 500 Sacramento 2 Boiling 60 sec 67 470 Sacramento 3 Boiling 60 sec 43 495 Sacramento 4 Boiling 60 sec 48 500 Sacramento 1 Control 0 000 Sacramento 2 Control 3 171 Sacramento 3 Control 3 171 Sacramento 4 Control 099 Sacramento 1 Percussion 1 min 57 495 Sacramento 2 Percussion 1 min 61 488 Sacramento 3 Percussion 1 min 62 485 Sacramento 4 Percussion 1 min 55 497 Sacramento 1 Percussion 2 min 84 367 Sacramento 2 Percussion 2 min 70 458 Sacramento 3 Percussion 2 min 81 392 Sacramento 4 Percussion 2 min 69 462 Sacramento 1 Percussion 4 min 91 286 Sacramento 2 Percussion 4 min 84 367 Sacramento 3 Percussion 4 min 84 367 Sacramento 4 Percussion 4 min 94 237 Sacramento 1 Percussion 8 min 82 384 Sacramento 2 Percussion 8 min 81 392 Sacramento 3 Percussion 8 min 82 384 Sacramento 4 Percussion 8 min 81 392 Sandia 1 Immersion 76 427 Sandia 2 Immersion 70 458 Sandia 3 Immersion 70 458 Sandia 4 Immersion 81 392 Sandia 1 Boiling 30 sec 78 414 Sandia 2 Boiling 30 sec 71 454 Sandia 3 Boiling 30 sec 63 483 Sandia 4 Boiling 30 sec 78 414 Sandia 1 Boiling 60 sec 67 470 Sandia 2 Boiling 60 sec 65 477 Sandia 3 Boiling 60 sec 74 439 Sandia 4 Boiling 60 sec 66 474 Sandia 1 Control 16 367 Sandia 2 Control 7 255
70
Table B 1 Continued
Source Block Treatment Gennination Percentage Standard Error
Sandia 3 Control 8 271 Sandia 4 Control 16 367 Sandia 1 Percussion 1 min 67 470 Sandia 2 Percussion 1 min 61 488 Sandia 3 Percussion 1 min 66 474 Sandia 4 Percussion 1 min 59 492 Sandia I Percussion 2 min 82 384 Sandia 2 Percussion 2 min 83 376 Sandia 3 Percussion 2 min 81 392 Sandia 4 Percussion 2 min 84 367 Sandia 1 Percussion 4 min 89 313 Sandia 2 Percussion 4 min 86 347 Sandia 3 Percussion 4 min 89 313 Sandia 4 Percussion 4 min 85 357 Sandia 1 Percussion 8 min 80 400 Sandia 2 Percussion 8 min 77 421 Sandia 3 Percussion 8 min 69 462 Sandia 4 Percussion 8 min 66 474
LITERATURE CITED
Ashby WC Vogel WG Rogers NF 1985 Black locust in the reclamation equation General Technical Report Northeastern Forest Experiment Station USDA Forest Service NE-I05 12 p
Ballard LAT 1973 Physical barriers to gennination Seed Sci Technoll 285-303
___ 1976 Strophiolar water conduction in the seeds ofTrifolieae induced by action on the testa at nonstrophiolar sites Austr J Plant Physiol3 465-469
Barton LV 1947 Special studies on seed coat impenneability Contrib Boyce Thompson Inst 14 355-362
Baskin CC Baskin JM 1998 Seeds Ecology biogeography and evolution of donnancy and gennination Academic Press London UK 666 p
Bewley JD 1997 Seed germination and donnancy Plant Ce119 1055-1066
Bewley JD Black M 1994 Seeds physiology of development and germination 2nd ed Plenum Press New York NY 445 p
Briscoe CB 1996 Growing Nitrogen Fixing Trees in Acid Soils In MH Powell (ed) Nitrogen Fixing Trees for Acid Soils- A Field ManuaL Winrock International Morrilton AR 22 p
Copeland LO McDonald MB 1995 Principles of Seed Science and Technology 3rded Chapman amp Hall New York NY 409 p
Cox RE Klett JE 1984 Seed gennination requirements of native Colorado plants for use in the landscape Plant Propag 30(2) 6-10
Dell B 1980 Structure and function ofthe strophiolar plug in seeds ofAlbizia Lophantha Am J Bot 67 556-563
Dick-Peddie WA 1993 New Mexico Vegetation Past Present and Future University ofNew Mexico Press Albuquerque NM 244 p
Dreesen DR 2001 Propagation protocol for production ofcontainer Robinia neomexicana plants Los Lunas Plant Materials Center Los Lunas New Mexico In Native Plant Network URL httpwwwnativeplantnetworkorg (accessed 9 January 2002) Moscow (ID) University ofIdaho CoIl Natural Resources For Res Nursery
72
Dreesen DR Harrington JT 1997 Propagation of native plants for restoration projects in the Southwestern US- Preliminary Investigations In Landis TD Thompson JR tech coords National Proceedings Forest and Conservation Nursery Assocs Gen Tech Rep PNW-GTR-419 Portland OR USDA For Ser PNW Res Sta 77-88
Gottfried GJ 1980 Control ofNew Mexican locust and the effect on planted ponderosa pine in central Arizona US For Ser Res Note RM-386 Rocky Mountain For Range ExpStn Fort Collins CO
Gosling PG Samuel YK Jones SK 1995 A systematic examination of germination temperature chipping and water temperaturesoak duration pretreatments on the seeds ofLeucaena leucocephala Seed Sci amp Technol 23521-532
Hamly DH 1932 Softening of the seeds of Melilotus alba Bot Gazette 93 345shy375
Hanna PJ 1984 Anatomical features of the seed coat ofAcacia kempeana (Mueller) which relate to increased germination rate induced by heat treatment New Phytol 96 23-29
Hanks JP Dick-Peddie WA 1974 Vegetation patterns of the White Mountains New Mexico Southwest Naturalist 18(4) 371-382
Hine S Harrington JT Dreesen DR 1997 Seed scarification requirements for Robinia neomexicana In Harrington JTtech coord Proc Third SouthwestemContainer Growers Conf Feb 12-13 1997 Ft Collins CO
Hopkinson JM 1993 The strophiole of leguminous seeds Newsletter- IntI Herbage Seed Production Res Grp 18 7-8
---- 1997 Differences in hardseededness between Cunningham and Tarramba leucaena In Leucnet News The Newsletter of the International LeucaenaRes and Devlt Netwk No4
ISTA (International Seed Testing Association) 1999 International rules for seed testing rules Seed Sci amp Technol27 Supplement
Kelly KM van Staden J 1987 The lens as the site of permeability in the papilionoid seed Aspalathus linearis J Plant Physiol 128(45) 395-404
73
Keresztesi B 1988 Black locust the tree of agriculture Outlook on Agr 17(2) 77~85
Kigel 1 and Galili G 1995 Seed development and germination Marcel Dekker New York 853 p
Klemmedson 10 1994 New Mexico locust and parent material Influence on macronutrients of forest floor and soil Soil Sci Soc Am 1 58 974~980
Kuo WH Tam AW 1988 The pathway of water absorption of mung bean seeds Seed Sci amp Technol16(1) 139~144
Leadem CL 1997 Dormancy--unlocking seed secrets In Landis TD Thompson lR tech coords National Proceedings Forestand Conservation Nursery Assocs Gen Tech Rep PNW-G TR-419 Portland OR USDA For Ser PNW Res Sta 43-52
Lin Y Wagner MR Seweyestewa A Honahni MD DeGomez M McMillin JD 1996 Influence of cultural treatments on seed germination and seedling growth of Robinia neomexicana Forest Farm and Community Tree Res Reports Volume 1
Marunda CT 1990 Effects of seed pretreatment on the development ofAcacia auriculforinis and A holosericea seedlings ACIAR Proceedings Series No 28
Meginnis HG 1937 Sulphuric acid treatment to increase germination of black locust seed USDA Circ 453 34 p
Miklas PN Towensend CE Ladd SL 1987 Seedcoat anatomy and the scarification ofcicer milkvetch seed Crop Sci 27 766-772
Monsen SB 1984 Use of shrubs on mine spoils In The Challenge ofProducing Native Plants for the Intermountain Area Proceedings Intermountain Nurserymans Association 1983 conference Las Vegas NV compo PM Murphy 26-31 Ogden UT USDA For Ser Gen Tech Report INT-l68
Olson DF 1974 Robinia L Locust In Seeds of Woody Plants in the US tech coord CS Schopmeyer 728-731 USDA For Ser Agr Handbk No450
Rehman S Loescher RNl Harris P1C 1998 Dormancy breaking and germination ofAcacia salicina Lindl seeds Seed Sci amp Techno127 553-557
Rolston MP 1978 Water impermeable seed dormancy Bot Rev 44 365-396
74
SAS Institute Inc 1989 SASSTAT Users Guide Version 6 4th Edition Volume 1 Cary NC SAS Institute Inc 943 p
SerratoValenti G Conara L Ferrando M Modenesi P 1993 Structural and histochemical features ofStylosanthes scabra (Leguminosae Papilionoidae) seed coat as related to water entry Can J Bot 71 834-840
Simpson BJ 1988 A field guide to Texas trees Austin TX Texas Monthly Press 372p
Tran VN Cavanagh AK 1984 Structural aspects ofdormancy In Seed Physiology Vol 2 Murray DR (ed) p1-44 Academic Press Australia
USDA Forest Service 1948 Woody-plant seed manual USDA Misc Pub1654 416p
Vines RA 1960 Trees Shrubs and Woody Vines of the Southwest Austin Univ ofTexas Press 1104 p
Wagner MR Larson RE Craig TP 1992 Ecology and silvics ofNew Mexico locust (Robinia neomexicana) with an emphasis on the arboreal form p53-57 In JW Hanover K Miller and S Plesko (eds) Proc Intemat Confon Black Locust Biology Culture and Utilization Mich State Univ USA
Wilson JK 1944 Immersing seeds of species of Robinia in boiling water hastens germination J For 42 453-454
Zimmerman RW Carpenter SB 1980 First-year coppice production from a 5shyyear old black locust stand on surface mine spoil p309-314 Centr Hardwd For ConfProc University ofMissouri Columbia
(1STA 1999) Tests were carried out at room temperature (22degC plusmnOSoC)
Germination was classified as protrusion of the radicle from the embryo by at least
1mm as viewed with the naked eye
X-ray Radiography
On August 132001 three lOO-seed samples from the Sacramento seed source
were treated for 14 or 8-minute percussion durations The seeds were sent
immediately by overnight delivery to the Faxitron X-Ray Corporation in Wheeling
Illinois where Dr Bob Karrfalt ofthe USDA Forest Service oversaw their imaging
Radiographs were made at SX with a Faxitron Specimen Radiography System Model
MX-20
Scanning Electron Microscope Photography
On February 192002 a Hitachi S-3200N Variable Pressure Scanning
Electron Microscope (Mountain View CA) was used to examine strophiolar cells of
untreated seeds and seeds percussed for 4 minutes Seeds were cut longitudinally
igt their entire length using a razor blade splitting the strophiolar region roughly in
Cross sections of seed samples magnifying the strophiolar region were viewed
600X under the microscope
Analysis
Categorical Analysis of Variance was used to determine treatment source and
(Le time) main effects and interactions The response variable was total
~vlI This procedure is a Chi-square test of homogeneity using the natural log
of germinated to non-germinated seed for each treatment Maximumshy
22
likelihood analysis was used to calculate Chi-square test statistics with their observed
significance levels A single contrast was conducted to cempare treatment means
against the control Observed significance levels less than or equal to 005 were
considered significant Pairwise comparisons were conducted between non-control
treatment mean percents using a conservative alp~a value of 005 divided by the
number of comparisons (Bonferroni approach) and approximate pairwise z statistics
Categorical analyses were performed using Proc Catmod (SAS Institute 1989)
RESULTS
Scarification seed source block and all interactions between factors
influenced germination (Table 3) The main effects are reported in the presence of
these interactions because the overall robustness of treatments and seed sources are of
interest Background levels ofnaturally occurring variation in seed sources are
seen in control germination percentages in Figure 2 In particular time variability
is unlikely to be under the control of the producer Appendix B details experimental
data for source by treatment gennination percents for each of the four blocks
Averaged over all treatments germination by source ranged from 40 to
79 (Figure 2) Averaged over all sources (Figure 3) and for each source (Figure 4
5) percussion scarification durations of4 or 8 minutes yielded greatest germination
Averaged over all sources germination increased with increasing durations of
percussion then leveled off for the 4- and 8-minute treatments (Figure 3) Immersion
and 30-second boiling scarification maximized germination for the hot water
treatments with a decrease in germination when seeds were boiled for 60 seconds
For all but two sources there is a similar ipcreasing trend in germination from
1to 4 minutes ofpercussion (Figure 4) The Holman source increased in germination
1 to 2 minutes then leveled off at 4 minutes The Jemez source changed little
1 to 4 minutes of treatment As treatment duration increased from 4 to 8
mutes five sources decreased in germination (Figure 4a) whereas three sources
24
Table 3 Categorical Analysis of Variance table for germination response to seed treatment seed source block and the interaction of the factors
Source of DF Chi-Square Observed Variability Significance Level
Source 8 98370 lt00001 Trt 7 233876 lt00001
contrast 1 16976 lt00001 SourceTrt 56 97077 lt00001 Block 3 5538 lt00001
SourceBlock 24 10162 lt00001 TrtBlock 21 7564 lt00001
SourceTrtBlock 168 34283 lt00001
S 4)
8 ~
0
sectg ~ 40 ~
N 0 0
20
o f0ltgt ~ ~4 jybshy ((iltgto ~~ K1ltgt 6~b-~ ~~ 0b-~~ ~~ ~b-~ ~b-~b
80
60 -I-- - ~- middotmiddotmiddotmiddotmiddot1
r -] Northern NM Sources
Central NM Sources
Southern NM Sources
sect-o ~
~b-v(i
SOURCE
Figure 2 Effect ofNew Mexico locust seed source on germination for data averaged over all scarification treatments Control germination is shown by solid line within bar
80
~ cd ~ ltl) () 60 ~ ~
N
t 0shyrti tshySltl)
40
0
20
o lmmersion Boiling 30 sec Boiling 60 sec Control 1 min 2min 4 min 8 min
Hot Water Treatments Percussion Treatments
Figure 3 Effect of scarification on germination ofNew Mexico locust for data averaged over all seed sources Means labelled with the same letter are not significantly different
100~----------------------~--~----------------~
80
60
ltU
~
40 IJ
sectgsI
l100
80
60
40
a I middotmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot middotImiddotmiddotmiddotmiddotmiddotmiddot E
_~iii-bull _ _ bull f --~--
t-- -~ ---- -shy ---- shyI ~ ---- - shy
L
fiII
~
b
- shy ---r
- Holman
Sacramento Sandia Gila Jemez
c ~
ltgt ~
Tmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot-I
j~=~=-~~-1
I middotmiddot ~ bull r
t tr
Y -------M-a-gd-a-Ie-n-a--
1 _ _
fCapitan
---- Manzano ~y -_ Raton
2 4 8
Time interval ofpercussion scarification (min)
Figure 4 Effect of seed source by treatment interaction on New Mexico locust germination for percussion scarification pattern a and pattern b
28
tv 0
~ d
~ A s o
1 c3
80
60
40
20
o
TIT
t------=middotfmiddotmiddot-middotmiddot-middotmiddot-middotmiddot 1- --- - - --=- - -shy--cE= - _--shy --shy ----shy ~ ~ ---shy ~ -~- _ --shy-------r 1
I
Raton Holman
-_ Jemez
--- Sandia Manzano Magdalena Capitan Sacramento Gila
Immersion Boiling 30 sec Boiling 60 sec
Hot Water Scarification
Figure 5 Effect of seed source by treatment interaction on New Mexico locust germination for hot water scarification
continued to increase in germination (Figure 4b) There was no difference between
the two treatment levels for the Manzano source (Figure 4b)
The effect ofhot water scarification also varied among sources with few
overall differences between hot water treatments (Figure 5) However boiling for 60
seconds yielded highest germination for the Jemez source whereas increasing
durations ofboiling decreased germination for the Raton source
In addition to consistently high germination 4- and 8-minute percussionshy
treated seeds germinated as fast or faster than all other treatments (Figure 6 Table 4)
050 measured as the number of days needed to complete 50 ofeventual
germination averaged 2 days for both ofthese treatments
X-ray radiographs were taken of seeds collected from the Sacramento source
percussed for 1 4 or 8 minutes (Figure 7) Seeds show signs of damage at 8 minutes
ofpercussion (Figure 8) Compared to an undamaged seed percussed for only 4
minutes (Figure 8a) damage at the 8-minute level includes fractured radicles pitting
of the seed coat and chips in seed coat structure (Figure 8b-d)
Scanning electron microscope (SEM) images at 600X reveal the direct effect
of 4 minutes of percussion on cells of the strophiolar region (Figure 9) Whereas
untreated seeds retain cellular organization in the strophiolar region strophiolar cells
seeds treated for 4 minutes ofpercussion separate and raise resulting in a
~-~e~U appearance
30
Umiddot-~middot--l-~---middotymiddotmiddotmiddot-----middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot--middot--middotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middotmiddotmiddotmiddotmiddotmiddotmiddot- -
050= gt1
-e-- Percussion 8 min -0- Percussion 4 min ---T- Percussion 2 min -----ltil- Percussion 1 min -- Immersion --0- Boiling 30 sec -+- Boiling 60 sec -ltgt- Control
2 4 6 8 10 12
Day Figure 6 Timecourse ofNew Mexico locust gennination following scarification
treatments G50 is the number of days to 50 of eventual germination
31
14
Table 4 Effect of seed source and treatment on G50 (days to 50 germination)
Seed Source Control Immersion B30sec B60sec Plmin P2min P4min P8min mean Raton 3 3 5 7 4 3 2 2 4 Holman 2 2 2 2 2 2 2 2 2
Jemez 4 6 6 5 3 2 2 2 4 Sandia 2 2 2 2 2 2 2 2 2
w tv Manzano 5 2 2 3 3 2 2 2 3
Magdalena 4 2 2 3 4 3 2 1 3
Capitan 5 4 2 2 3 2 2 2 3
Sacramento 3 2 2 2 4 3 2 2 3 Gila 3 2 2 2 2 2 2 2 2 mean 3 3 3 3 3 2 2 2 3 standard 11 14 16 18 09 05 0 03 08 deviation
Note Columns and rows may not agree due to rounding
Figure 7 X-ray radiographs ofSacramento seed source percussed for 1 4 or 8 minutes
33
Figure 8a-d Seed damage close-ups Figure a shows undamaged seed percussed for 4 minutes Figures b-d show seeds percussed for 8 minutes Damage includes fracturing ofradicle in b complete removal ofseed coat in c and pitting ofseed coat in d
34
Untreated Treated
Figure 9 SEM images ofuntreated vs 4-minute percussion-treated strophiolar cells ofNew Mexico locust seeds Treated cells are loosened separated and raised
DISCUSSION
Determining the Optimal Treatment
Averaging results of scarification treatmems across seed sources is a useful
tool to predict which treatment is most likely to be successful for an unknown seed
source Averaged over all sources four- and eight-minute percussion treatments
resulted in the highest germination 815 and 790 respectively and produced
germination that was as fast or faster than all other treatments Although there was no
difference between these two treatments on average an argument for 4 minutes of
percussion as a standard protocol for treating New Mexico locust seeds can be made
Descriptively 4 minutes of percussion improved germination over 8 minutes
ofpercussion for a majority of sources (five out of nine) 8 minutes of percussion
improved germination for three ofnine sources with no difference for a fmal source
For individual sources 4 minutes ofpercussion was always the best or secondmiddot best
treatment and was always equal to or better than all hot water treatments
Germination at 8 minutes ofpercussion was lower than all other percussion durations
for the Jemez and Gila sources and was lower than at least one hot water treatment for
these sources as well
The x-ray radiographs of the Sacramento seed source illustrate the excessive
internal and external seed damage that can occur with 8 minutes of percussion
germination counts 8-minute percussed seeds from Holman and Capitan
ClUll signs of damage that were visible to the naked eye (see below)
r~evertheless some of these seeds still fit the criteria for germination Neither x-ray
36
photography nor visible observations during seed counts revealed seed damage at the
4-minute percussion level In operational trials seeds percussed for 4 minutes
developed into healthy seedlings (Khadduri and Harrington unpublished data)
Strophiole and Seed Coat Behavior
Four minutes ofpercussion successfully breaks seed dormancy in a large
percentage ofNew Mexico locust seeds without excessively damaging the
surrounding seed coat or internal seed coat structures Most papilionoid legume
seeds including seeds ofNew Mexico locust have a specific region of the seed coat
known as the strophiole or lens (Hamly 1932) The strophiole is located on the
cotyledonary lobe of a locust seed (Figure 10 a-c) If seeds are not indiscriminately
damaged the strophiole is the first point of water entry to the embryo (Hopkinson
1997) When a papilionoid seed is percussed for the appropriate amount of time
repeated hits on the integrated seed coat loosen the constrained cells of the strophiolar
region without excessively damaging the rest of the seed coat (Ballard 1976) SEM
photos illuStrate that 4 minutes of percussion loosened and separated strophiolar cells
ofNew Mexico locust seeds when compared with constrained organized strophiolar
cells of untreated seeds (Figure 9) Research-has shown that water passes to the
embryo through the treated strophiole in a controlled manner and regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues
(Kelly and van Staden 1987)
37
10 Strophiole ofunimbibed New Mexico locust seed (a) and following uptake (bc)
38
In contrast hot-water treatments can excessively degrade the seed coat
(Rebman et al 1998 Marunda 1990) Random cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rebman et aI 1998 Marunda 1990) Along with a
proportionately large amount ofun-imbibed (thus under-treated) seeds (Figure l1a)
damage was observed in hot-water treated seeds from six sources during germination
counts Damage included seeds where the radicle failed to elongate (Figure 11 b)
seeds where the radicle elongated but was delayed in freeing itself from the seed coat
(Figure 11c) and seeds where the radicle completely broke off from the embryo
(Figure lId) In addition some seeds germinated in reverse (the cotyledon emerged
fIrst) If the radicle emerged completely from the seed coat they were counted as
germinated
While nearly all seeds treated for 8 minutes ofpercussion imbibed water
damage was evident in x-ray evaluations (Sacramento seed source) and in
germination counts (Holman and Capitan) at this treatment level Seed damage
during germination counts was similar to levels found in hot-water treated seeds
suggesting that over-treatment by both hot water and longer durations of percussion
may result in similar damage to seed structure
The Sacramento and Holman seed sources were two of the fIve sources that
opcrplltlpn in germination from the 4- to 8-minute durations of percussion
the benefIts ofloosening the strophiole as evidenced by imbibition of
all 8-minute treated seeds were outweighed by external and internal seed coat
39
a
b c d
11 Damage to New Mexico locust seeds observed during germination cotmts following hot water scarification 9a Hot water replicate including ~t seedlings and numerous unimbibed seeds 9b Close-up of seed where raallCle failed to elongate 9c Close-up ofseed where radicle elongated but was
-gtIn in freeing itself from the seed coat 9d Close-up ofseed where radicle broke off from embryo
40
damage (which also may be contributing to high imbibition) For the three sources
for which 8 minutes of percussion resulted in maximum germination extended
percussion of seeds necessary to loosen the strophiole may not have excessively
damaged the seed anatomy The Manzano source did not significantly change in
germination from 4 to 8 minutes of percussion suggesting that an increase in
permeable seeds at the 8-minute treatment duration was balanced by the number of
seeds that failed to germinate due to seed damage To summarize the 4-minute
percussion treatment improved germination over 8 minutes ofpercussion for a
majority of sources (five ofnine) the 8-minute percussion treatment improved
germination over 4-minutes ofpercussion for three sources and there was no
difference with the Manzano source Notably for the Raton source 8 minutes of
percussion increased germination by 235 percentage points over 4-minutes of
percussion (7875 vs 5525 respectively) the next best treatment Raton was the
only source to result in germination less than 75 for 4 minutes ofpercussion so a
grower may wish to apply longer percussion durations to lots that do not respond well
to this treatment level
Finally there was an interesting trend in the Jemez and Raton sources seed
that significantly contributed to the source by treatment interaction (Figure 12) A
~u test showed that the Jemez source had a significantly lighter seed weight than
other sources while the Raton source had an average seed weight For the Jemez
the longest duration of boiling increased germination Conversely only short
U4UVll) of percussion were needed to achieve high germination and the longest
41
80 _ Jemez a Raton
iD
~ C iD e iD
Q c 0 ~
c ~ iD 0
20
0------shy
Immersion Boiling 30 sec Boiling 60 sec
Hot Water Scarification
10degTr========~--------~------------------------------
80
60
40
20
_ Jemez Raton
0------shy
1 min 2 min 4 min 8 min
Percussion Scarification
Figure 12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources
42
b
--~----~~~~========~---- duration ofpercussion reduced germination For the Raton source longer durations
ofboiling consistently decreased germination whereas longer percussion treatments
consistently increased germination
A possible explanation is that a thick seed coat is associated with a loose
strophiole structure and that a thin seed coat is associated with a constrained
strophiole This contrast merits further study and may suggest an ecological
adaptation Further investigation ofthis phenomenon may contribute to successful
pre-screening ofunknown seed sources to determine optimal treatment leveL
43
CONCLUSION
Based on results obtained in pilot study (see Appendix) and thesis
experiments 4 minutes ofpercussion is recommended as a standard protocol for
treating an unknown source ofNew Mexico locust seed In addition to laboratory
results the 4-minute percussion treatment has resulted in germination greater than
80 and healthy seedling grow-outs for production purposes (unpublished data) In
laboratory experiments only one hundred seeds per 118 ml (40z) soil tin were
percussed at a time whereas for production we have treated up to five hundred seeds
per tin For large-scale production ofNew Mexico locust seedlings growers may
wish to experiment with larger quantities of seed and larger-size soil tins
Seed storage trials following percussion treatments were not conducted
However germination greater than 80 and healthy seedlings were obtained when
seeds were percussion-treated and subsequently stored at room-temperature (22degC)
for one week (unpublished data) Seed storage trials merit further study as the
standard hot water treatment leaves seeds in a softened condition that is not
conducive to storage
Hydroseeding is an effective method for direct seeding in reclamation
situations Percussion treatment may be conducive to hydro seeding as seeds remain
a dry relatively undamaged yet permeable condition following treatment Sulfuric
seeds also remain dry following treatment but extensive damage to seed coat
poor seedling vigor have been reported with this treatment (Kelly and van Staden
A grow-out study using a hydro seeder on a reclamation site testing percussion
44
against other treatments such as sulfuric acid and mechanical scarification also
merits further study
Papilionoids are the largest subfamily oflegumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust these legumes often play an integral role in the revegetation of
disturbed lands The pilot study (Appendix) indicates that percussion scarification
successfully treats the widely used black locust as well Developing superior
scarification methods to the standard hot water mechanical scarification and acid
treatments may facilitate the use ofpapilionoid legumes in reclamation projects
45
APPENDICES
APPENDIX A
PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR
NEW MEXICO LOCUST AND BLACK LOCUST SEEDS
Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4
Hot water and sulfuric acid soaks are traditional treatments for seeds of many
temperate woody legwnes including locusts However these scarification techniques
often produce inconsistent germination Percussion scarification where seeds are
repeatedly propelled against a hard surface was compared with hot water
scarification to evaluate treatment efficacy for New Mexico locust (Robinia
neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment
seeds were placed in a 98degC water bath which was immediately removed from the
heat source For percussion scarification seeds were placed in a soil sample tin and
agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the
control were followed by 24-hour water soaks Hot water baths resulted in 56 and
41 germination for New Mexico locust and black locust respectively For both
species nearly all durations ofpercussion increased germination over the hot water
treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3
4 and 5 minutes for black locust resulted in at least 90 germination Traditional
scarification treatments randomly degrade the entire seed coat which can lead to
tissue damage during water uptake Percussion scarification specifically weakens the
strophiole the natural source ofwater entry to the seed in papilionoid legwnes
INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom
2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031
48
Following percussion imbibition is controlled through the strophiole and underlying
tissue is protected
Keywords New Mexico locust black locust scarification percussion germination
Introduction
Locust trees (Robinia spp) are aggressive pioneer species that quickly
colonize disturbed land fix atmospheric nitrogen in the soil and stabilize slopes that
are prone to erosion (Klemmedson 1994 Ashby et al 1985) (Figure A1ab)
Recognition of these attributes has contributed to increased demand for locusts in
reclamation projects Locust seeds however exhibit physical dormancy where a
thick seed coat prevents the movement of water and gasses to the embryo (Leadem
1997)
Sulfuric acid or hot water soaks have traditionally been used to break seed
dormancy in macro-propagation of locusts Many growers are moving away from the
use of su1furic acid in treating locust seeds (Dreesen and Harrington 1997) In
addition to being dangerous acid soak durations must be specifically correlated to
seed lot (Olson 1974) Collections of native seed can vary tremendously in seed size
seed weight and hard seededness etc This variability limits the utility of sulfuric
acid soaks as even within a given lot some seeds may be damaged whereas others
are under-treated and fail to imbibe (take up water) A survey of Southwestern
container growers indicates that hot water baths are currently the most common
scarification method for locust seeds (Hine et al 1997) However hot water baths
also produce inconsistent germination (Lin et al 1996)
49
b
Figure AI New Mexico locust a primary invader following disturbance Ala Crown-sprouting at edge ofburn near Los Alamos NM--one year after Cerro Grande fire Alb Colonizing road cut Gila National Forest NM
50
Percussion scarification where seeds are repeatedly propelled against a hard
surface is an alternative dormancy-breaking method that has proven successful in
legumes related to locusts (Hamly 1932 Barton 1947) We compared this alternative
treatment to hot water scarification to evaluate treatment efficacy for New Mexico
locust and black locust seeds
Materials and Methods
Commercial New Mexico locust seeds (Western Native Seed Coaldale CO)
were collected fall 2000 in Huerfano County CO Black locust seeds were collected
September 2000 from Taos County NM by harvesting ripe pods from several trees at
a distance of up to 3 m from ground level Black locust seeds were allowed to air-dry
for 3 weeks and were threshed and separated from large chaffusing a greenhouse
fan A South Dakota Seed Blower (Chicago Illinltois) was used to remove fine chaff
from both seed lots Seeds were storedmiddotat 2-4C until the germination study was
conducted in March 2001
This study compared hot water scarification with percussion scarification
using treatment and species as experimental factors Along with a control
scarification treatments included immersion of seed in boiling water as well as
percussion durations of 1 2 3 45 or 10 minutes Four 100-seed samples were used
to test each species by scarification treatment combination
For the hot water treatment test tubes were filled with 30 mL water and
placed in a water-filled beaker (Figure A2a) The bath was raised to boiling (-98C at
51
b
c
Figure A2 Implementation ofhot water and percussion scarification A2a Hot water bath A2b Paint-shaker set-up A2c Placing soil tin containing seeds at end ofpaint shaker ann
52
Las Cruces NM elevation of 1300 meters) Each 100-seed replicate was placed in a
test tube and each test tube was immediately removed to cool at room temperature
Percussion scarification was implemented using a pneumatic paint shaker
(Central Pneumatic model 00422 Camarillo CA) (Figure A2b) This allowed
standardization of treatment intensity Compressed air pressure was maintained at
530kPa plusmn 30kPa (80psi plusmn 5psi) resulting in approximately 350 oscillations per
minute Each lOO-seed sample was placed in a 118 ml (4 oz) soil tin for shaking To
maximize lateral movement of paint shaker a paint can was used as a spacer to place
soil tins at the end of the shaking arm (Figure A2c) This allowed shaking to occur at
the greatest distance from the pivot point (fulcrum) Following all treatments
including the control seeds were soaked for twenty-four hours in distilled water prior
to plating out
Gennination was evaluated on lab benches at room temperature Each
replicate of seeds was placed in a 100 cm petri dish on moistened filter paper with
humidity maintained by enclosing the petri dishes in ziplock bags Gennination was
monitored daily for 14 days in accordance with International Seed Testing
Association standards for Robinia species (ISTA 1999) Gennination was defined as
protrusion of the radicle from the embryo by at least 1mm as viewed by the naked
eye
Categorical Analysis of Variance using Prpc Catmod (SAS Institute 1989)
was used to detennine treatment differences in germination percentages for each
source This procedure is an extension of a Chi-square test of homogeneity using the
53
natural log of the ratio of germinated to non-germinated seeds for each treatment Pshy
values less than 005 were considered significant A limited set of pairwiseshy
comparisons was conducted to compare treatment mean percents using a
conservative alpha value of 005 divided by the number of comparisons
Results
Percussion maximized germination for both New Mexico locust and black
locust There were three germination response levels for New Mexico locust (Figure
A3) First there was a low response in the control indicating a dormant seed lot
The hot water treatment and I-minute duration of percussion share a second
intermediate response Germination was highest at percussion levels from 2 to 10
minutes
A selected percussion treatment intermediate in time (5 minutes) produced the
steepest germination curve indicating the fastest germination rate (Figure A4) The
hot water treatment produced an intermediate curve or relatively slower germination
rate The control curve was very flat indicating the slowest germination rate for New
Mexico locust
Control seeds germinated poorly for black locust as well (Figure A5) Hot
water scarification improved germination over the control but all levels of percussion
improved germination over hot water The optimal durations of percussion treatment
were bracketed with a significant increase from the 1 to 2 minute percussion level a
high germination response from 2 to 5 minutes of percussion and finally a significant
54
~ d 25 CL)
p t1 0 ~ t1
g Vl ~ Vl
100~---------------------------------------------------------------
80
60
40
20
o Control Hot Water Percussion
I min Percussion
2 min Percussion
3 min
Scarification
Percussion 4 min
Figure A3 Effect of scarification on germination response for New Mexico locust
Percussion 5 min
=significant difference between adjacent treatment bars ns = no significant difference
Percussion 10 min
~ ~ () u agt
Il-t d o ~
~ ~ c3
100middotlr~~~------------------------------------e- Percussion 5min middot 0middot Hot Water -- Control
80-J=middot======
6b-middot-lmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middot--middotmiddot-middot~-middotmiddot -middotmiddot middot middotmiddot-middot 1
40
20
-----~-----------
0 0 0 middotmiddotmiddotmiddotmiddot0middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot0
0 --~-----y-----~------_---~--- T-----~
o shy -TDay 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A4 Effect of selected scarification treatments on germination speed for New Mexico locust First seven days of fourteen-day experiment are shown
Day 7
80
~ cd
5 60 ()
[) Cl d 0 1t1 d
40sect dUl
-J
20
o
ns
Scarification
Figure A5 Effect of scarification on germination response for black locust = significant difference between adjacent treatment bars ns = no significant difference
decrease at the 10-minute level This drop-off suggests that 10 minutes of percussion
over-treats seeds from this lot of-black locust
A selected percussion treatment intermediate in time (5 minutes) also had the
fastest germination rate for black locust (Figure A6)_ The hot water curve is
somewhat flatter indicating a slower germination rate The control curve is very flat
indicating a slow germination rate
A qualitative difference between hot water and percussion-treated seeds was
also observed Along with healthy germinants the hot water treatment produced
over-treated seeds (Figure A 7a) Examples of damaged germinants include seeds
where the radicle did not elongate (Figure A 7b) seeds where the radicle elongated
but was delayed in freeing itself from the seed coat (Figure A7c) and seeds where the
radicle broke off completely from the embryo (Figure A7d) In addition to these
examples of over-treatment there were numerous small dark seeds that failed to
imbibe water For percussion treatment nearly all seeds took up water (Figure A8a)
Germination was rapid and uniform with little damage evident (Figure A8b)
To summarize both New Mexico locust and black locust seed lots were
dormant The standard hot water scarification treatment improved germination but
only to 56 and 41 for New Mexico locust and black locust respectively Nearly
all percussion durations improved total germination as well as germination rate over
the hot water treatment Germination was greater than 90 for percussion durations
of 4 5 and 10 minutes for New Mexico locust and 3 4 and 5 minutes for black locust
58
CI)
sgt t CI) 0
igt 0
sect ttl t E
Vl 0
c3
100r==~==~~=r------------------------------------~ ___ Percussion 5 min
middotmiddotmiddot0middotmiddotmiddot Hot Water -lIf- Control
80r=========~--~~----------------------------~
60
40
20 -~-~---~---------- ---~------------I
omiddot
O
_~_----------lIf-----~bull -----_ shy---shy I
Day 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A6 Effect of selected scarification treatments on germination speed for black locust First seven days of fourteen-day experiment are shown
Day 7
a
b c d
Figure A7 Damage to New Mexico locust seeds observed during germination counts following hot water scarification A7a Hot water replicate including damaged seedlings and numerous unimbibed seeds A7b Close-up ofseed where radicle failed to elongate A7c Close-up ofseed where radicle elongated but was delayed in freeing itself from the seed coat A7d Close-up ofseed where radicle completely broke offfrom embryo
60
a
b
Figure A8 New Mexico locust seeds following percussion treatment at time of germination counts A8a Percussion replicate with uniformly healthy seedlings and near complete imbibition of seeds A8b Close-up ofpercussion-treated seedling
61
Discussion
Papilionoid seeds have a specific anatomical feature known as the strophiole
or lens-the natural site of water entry to the seed (Hamly 1932) The strophiole is
located on the cotyledonary lobe of a locust seed (Figure A9) When a papilionoid
legume seed is percussed for the appropriate amount of time repeated hits on the
integrated seed coat loosen the constrained cells of the strophiolar region without
excessively damaging the rest of the seed coat (Ballard 1976)
When a percussed seed is soaked water enters exclusively through the
strophiole in a controlled manner (Kelly and Staden 1987) This regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues This
contrasts with seeds that have been hot water or acid scarified treatments that can
randomly degrade the seed coat Unlocalized cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rehman et al 1998 Marunda 1990)
Papilionoids are the largest subfamily of legumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust and black locust these legumes often play an integral role in the
revegetation ofdisturbed lands Developing superior scarification methods to the
standard hot water and acid treatments should fadlitate the use ofthese legumes in
restoration projects
62
Figure A9 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (bc)
63
APPENDIXB
GERMINATION PERCENTS AND STANDARD ERRORS FOR SOURCE
BLOCK AND TREATMENT FOR NEW MEXICO LOCUST
Table B 1 Gennination percents and standard errors for source block and treatment for New Mexico locust
Source Block Treatment Germination Percentage Standard Error
Capitan 1 Immersion 74 437 Capitan 2 Immersion 69 462 Capitan 3 Immersion 71 454 Capitan 4 Immersion 67 470 Capitan Boiling 30 sec 79 407 Capitan 2 Boiling 30 sec 76 427 Capitan 3 Boiling 30 sec 72 449 Capitan 4 Boiling 30 sec 76 427 Capitan Boiling 60 sec 71 454 Capitan 2 Boiling 60 sec 54 498 Capitan 3 Boiling 60 sec 67 470 Capitan 4 Boiling 60 sec 76 427 Capitan I Control 5 218 Capitan 2 Control 7 255 Capitan 3 Control 4 196 Capitan 4 Control 5 218 Capitan 1 Percussion I min 47 499 Capitan 2 Percussion I min
470
Capitan Percussion I min 39 488
Capitan 4 Percussion I min 38 485 Capitan I Percussion 2 min 55 497 Capitan Capitan
2
Percussion 2 min Percussion 2 min
71 65
454 477
Capitan 4 Percussion 2 min 56 496 Capitan I Percussion 4 min 86 347 Capitan 2 Percussion 4 min 79 407 Capitan 3 Percussion 4 min 81 392 Capitan 4 Percussion 4 min 82 384 Capitan I Percussion 8 min 90 300 Capitan 2 Percussion 8 min 88 325 Capitan 3 Percussion 8 min 90 300 Capitan 4 Percussion 8 min 90 300 Gila I Immersion 71 454 Gila 2 Immersion 77 421 Gila
Immersion 75 433
Gila 4 Immersion 72 449
Gila I Boiling 30 sec 68 466
Gila 2 Boiling 30 sec 81 392
Gila 3 Boiling 30 sec 54 498
Gila 4 Boiling 30 sec 63 483
Gila I Boiling 60 sec 55 497
Gila 2 Boiling 60 sec 68 466
Gila 3 Boiling 60 sec 57 495
Gila 4 Boiling 60 sec 70 458
65
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Gila 2 Control 13 336 Gila 3 Control 13 336 Gila 4 Control 20 400 Gila 1 Percussion 1 min 66 474 Gila 2 Percussion 1 min 57 495 Gila 3 Percussion 1 min 73 444 Gila 4 Percussion 1 min 82 384 Gila 1 Percussion 2 min 81 392 Gila 2 Percussion 2 min 86 347 Gila 3 Percussion 2 min 85 357 Gila 4 Percussion 2 min 54 498 Gila 1 Percussion 4 min 79 407 Gila 2 Percussion 4 min 83 376 Gila 3 Percussion 4 min 84 367 Gila 4 Percussion 4 min 79 407 Gila 1 Percussion 8 min 51 500 Gila 2 Percussion 8 min 76 427 Gila 3 Percussion 8 min 52 500 Gila 4 Percussion 8 min 74 439 Holman 1 Immersion 83 376 Holman 2 Immersion 86 347 Holman 3 Immersion 81 392 Holman 4 Immersion 90 300 Holman 1 Boiling 30 sec 90 300 Holman 2 Boiling 30 sec 85 357 Holman 3 Boiling 30 sec 87 336 Holman 4 Boiling 30 sec 83 376 Holman 1 Boiling 60 sec 79 407 Holman 2 Boiling 60 sec 80 400 Holman 3 Boiling 60 sec 86 347 Holman 4 Boiling 60 sec 93 255 Holman Control 25 433 Holman 2 Control 19 392 Holman Control 21 407
Holman 4 Control 24 427
Holman Percussion 1 min 77 421
Holman 2 Percussion 1 min 85 357
Holman 3 Percussion 1 min 80 400
Holman 4 Percussion 1 min 89 313
Holman 1 Percussion 2 min 88 325
Holman 2 Percussion 2 min 96 196
Holman 3 Percussion 2 min 93 255
Holman 4 Percussion 2 min 96 196
Holman 1 Percussion 4 min 92 271
Holman 2 Percussion 4 min 97 171
66
I
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Holman 3 Percussion 4 min 91 286 Holman 4 Percussion 4 min 96 196 Holman 1 Percussion 8 min 88 325 Holman 2 Percussion 8 min 85 357 Holman 3 Percussion 8 min 83 376 Holman 4 Percussion 8 min 82 384 Jemez 1 Immersion 53 499 Jemez 2 Immersion 44 496 Jemez 3 Immersion 52 500 Jemez 4 Immersion 70 458 Jemez 1 Boiling 30 sec 58 494 Jemez 2 Boiling 30 sec 55 497 Jemez 3 Boiling 30 sec 48 500 Jemez 4 Boiling 30 sec 65 477 Jemez 1 Boiling 60 sec 68 466 Jemez 2 Boiling 60 sec 63 483 Jemez 3 Boiling 60 sec 73 444 Jemez 4 Boiling 60 sec 89 313 Jemez 1 Control 2 140 Jemez 2 Control 9 286 Jemez 3 Control 6 237 Jemez 4 Control 8 271 Jemez 1 Percussion 1 min 58 494 Jemez 2 Percussion 1 min 63 483 Jemez 3 Percussion 1 min 77 421 Jemez 4 Percussion 1 min 89 313 Jemez 1 Percussion 2 min 68 466 Jemez 2 Percussion 2 min 64 480 Jemez 3 Percussion 2 min 80 400 Jemez 4 Percussion 2 min 83 376 Jemez Percussion 4 min 68 466 Jemez 2 Percussion 4 min 79 407 Jemez 3 Percussion 4 min 70 458 Jemez 4 Percussion 4 min 83 376 Jemez 1 Percussion 8 min 52 500 Jemez 2 Percussion 8 min 59 492 Jemez 3 Percussion 8 min 61 488 Jemez 4 Percussion 8 min 70 458 Magdalena 1 Immersion 69 462 Magdalena 2 Immersion 69 462 Magdalena J Immersion 70 458 Magdalena 4 Immersion 85 357 Magdalena 1 Boiling 30 sec 65 477 Magdalena 2 Boiling 30 sec 71 454 Magdalena 3 Boiling 30 sec 68 466
67
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Magdalena 4 Boiling 30 sec 83 376 Magdalena 1 Boiling 60 sec 63 483 Magdalena 2 Boiling 60 sec 68 466 Magdalena 3 Boiling 60 sec 63 483 Magdalena 4 Boiling 60 sec 82 384 Magdalena 1 Control 11 313 Magdalena 2 Control 6 237 Magdalena 3 Control 5 218 Magdalena 4 Control 4 196 Magdalena Percussion 1 min 32 466 Magdalena 2 Percussion 1 min 45 497 Magdalena 3 Percussion 1 min 46 498 Magdalena 4 Percussion I min 36 480 Magdalena 1 Percussion 2 min 67 470 Magdalena 2 Percussion 2 min 68 466 Magdalena 3 Percussion 2 min 65 477 Magdalena 4 Percussion 2 min 81 392 Magdalena 1 Percussion 4 min 87 336 Magdalena 2 Percussion 4 min 97 171 Magdalena 3 Percussion 4 min 83 376 Magdalena 4 Percussion 4 min 97 171 Magdalena 1 Percussion 8 min 98 lAO Magdalena 2 Percussion 8 min 100 000 Magdalena 3 Percussion 8 min 96 196 Magdalena 4 Percussion 8 min 98 lAO Manzano 1 Immersion 67 470 Manzano 2 Immersion 57 495 Manzano 3 Immersion 72 4049 Manzano 4 Immersion 73 4044 Manzano 1 Boiling 30 sec 70 458 Manzano 2 Boiling 30 sec 64 480 Manzano 3 Boiling 30 sec 71 454 Manzano 4 Boiling 30 sec 80 400 Manzano I Boiling 60 sec 50 500 Manzano 2 Boiling 60 sec 53 499 Manzano 3 Boiling 60 sec 59 492 Manzano 4 Boiling 60 sec 74 439 Manzano I Control 4 196 Manzano 2 Control 8 271 Manzano 3 Control 1 099 Manzano 4 Control 6 237 Manzano 1 Percussion 1 min 52 500 Manzano 2 Percussion 1 min 39 488 Manzano 3 Percussion 1 min 36 480 Manzano 4 Percussion 1 min 51 500
68
Table B 1 Continued
Source Block Treatment Gerrni~ation Percentage Standard Error
Manzano Percussion 2 min 53 499 Manzano 2 Percussion 2 min 58 494 Manzano 3 Percussion 2 min 61 488 Manzano 4 Percussion 2 min 62 485 Manzano 1 Percussion 4 min 76 427 Manzano 2 Percussion 4 min 85 357 Manzano 3 Percussion 4 min 79 407 Manzano 4 Percussion 4 min 83 376 Manzano 1 Percussion 8 min 87 336 Manzano 2 Percussion 8 min 83 376 Manzano 3 Percussion 8 min 83 376 Manzano 4 Percussion 8 min 75 433 Raton 1 Immersion 54 498 Raton 2 Immersion 37 483 Raton 3 Immersion 39 488 Raton 4 Immersion 69 462 Raton 1 Boiling 30 sec 36 480 Raton 2 Boiling 30 sec 20 400 Raton 3 Boiling 30 sec 28 449 Raton 4 Boiling 30 sec 60 490 Raton 1 Boiling 60 sec 15 357 Raton 2 Boiling 60 sec 171 Raton 3 Boiling 60 sec 4 196 Raton 4 Boiling 60 sec 35 477 Raton 1 Control 4 196 Raton 2 Control 12 325 Raton 3 Control 2 140 Raton 4 Control 6 237 Raton 1 Percussion 1 min 52 500 Raton 2 Percussion I min 29 454 Raton 3 Percussion I min 35 477 Raton 4 Percussion 1 min 31 462 Raton 1 Percussion 2 min 55 497 Raton 2 Percussion 2 min 35 477 Raton 3 Percussion 2 min 33 470 Raton 4 Percussion 2 min 42 494 Raton 1 Percussion 4 min 65 477 Raton 2 Percussion 4 min 52 500 Raton Percussion 4 min 51 500 Raton 4 Percussion 4 min 53 499 Raton 1 Percussion 8 min 88 325 Raton 2 Percussion 8 min 80 400 Raton 3 Percussion 8 min 73 444 Raton 4 Percussion 8 min 74 439 Sacramento 1 Immersion 43 495
69
Table B 1 Continued
-Source Block Treatment Germination Percentage Standard Error
Sacramento 2 Immersion 30 458 Sacramento 3 Immersion 38 485 Sacramento 4 Immersion 56 496 Sacramento 1 Boiling 30 sec 47 499 Sacramento 2 Boiling 30 sec 48 500 Sacramento 3 Boiling 30 sec 48 500 Sacramento 4 Boiling 30 sec 56 496 Sacramento 1 Boiling 60 sec 52 500 Sacramento 2 Boiling 60 sec 67 470 Sacramento 3 Boiling 60 sec 43 495 Sacramento 4 Boiling 60 sec 48 500 Sacramento 1 Control 0 000 Sacramento 2 Control 3 171 Sacramento 3 Control 3 171 Sacramento 4 Control 099 Sacramento 1 Percussion 1 min 57 495 Sacramento 2 Percussion 1 min 61 488 Sacramento 3 Percussion 1 min 62 485 Sacramento 4 Percussion 1 min 55 497 Sacramento 1 Percussion 2 min 84 367 Sacramento 2 Percussion 2 min 70 458 Sacramento 3 Percussion 2 min 81 392 Sacramento 4 Percussion 2 min 69 462 Sacramento 1 Percussion 4 min 91 286 Sacramento 2 Percussion 4 min 84 367 Sacramento 3 Percussion 4 min 84 367 Sacramento 4 Percussion 4 min 94 237 Sacramento 1 Percussion 8 min 82 384 Sacramento 2 Percussion 8 min 81 392 Sacramento 3 Percussion 8 min 82 384 Sacramento 4 Percussion 8 min 81 392 Sandia 1 Immersion 76 427 Sandia 2 Immersion 70 458 Sandia 3 Immersion 70 458 Sandia 4 Immersion 81 392 Sandia 1 Boiling 30 sec 78 414 Sandia 2 Boiling 30 sec 71 454 Sandia 3 Boiling 30 sec 63 483 Sandia 4 Boiling 30 sec 78 414 Sandia 1 Boiling 60 sec 67 470 Sandia 2 Boiling 60 sec 65 477 Sandia 3 Boiling 60 sec 74 439 Sandia 4 Boiling 60 sec 66 474 Sandia 1 Control 16 367 Sandia 2 Control 7 255
70
Table B 1 Continued
Source Block Treatment Gennination Percentage Standard Error
Sandia 3 Control 8 271 Sandia 4 Control 16 367 Sandia 1 Percussion 1 min 67 470 Sandia 2 Percussion 1 min 61 488 Sandia 3 Percussion 1 min 66 474 Sandia 4 Percussion 1 min 59 492 Sandia I Percussion 2 min 82 384 Sandia 2 Percussion 2 min 83 376 Sandia 3 Percussion 2 min 81 392 Sandia 4 Percussion 2 min 84 367 Sandia 1 Percussion 4 min 89 313 Sandia 2 Percussion 4 min 86 347 Sandia 3 Percussion 4 min 89 313 Sandia 4 Percussion 4 min 85 357 Sandia 1 Percussion 8 min 80 400 Sandia 2 Percussion 8 min 77 421 Sandia 3 Percussion 8 min 69 462 Sandia 4 Percussion 8 min 66 474
LITERATURE CITED
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Ballard LAT 1973 Physical barriers to gennination Seed Sci Technoll 285-303
___ 1976 Strophiolar water conduction in the seeds ofTrifolieae induced by action on the testa at nonstrophiolar sites Austr J Plant Physiol3 465-469
Barton LV 1947 Special studies on seed coat impenneability Contrib Boyce Thompson Inst 14 355-362
Baskin CC Baskin JM 1998 Seeds Ecology biogeography and evolution of donnancy and gennination Academic Press London UK 666 p
Bewley JD 1997 Seed germination and donnancy Plant Ce119 1055-1066
Bewley JD Black M 1994 Seeds physiology of development and germination 2nd ed Plenum Press New York NY 445 p
Briscoe CB 1996 Growing Nitrogen Fixing Trees in Acid Soils In MH Powell (ed) Nitrogen Fixing Trees for Acid Soils- A Field ManuaL Winrock International Morrilton AR 22 p
Copeland LO McDonald MB 1995 Principles of Seed Science and Technology 3rded Chapman amp Hall New York NY 409 p
Cox RE Klett JE 1984 Seed gennination requirements of native Colorado plants for use in the landscape Plant Propag 30(2) 6-10
Dell B 1980 Structure and function ofthe strophiolar plug in seeds ofAlbizia Lophantha Am J Bot 67 556-563
Dick-Peddie WA 1993 New Mexico Vegetation Past Present and Future University ofNew Mexico Press Albuquerque NM 244 p
Dreesen DR 2001 Propagation protocol for production ofcontainer Robinia neomexicana plants Los Lunas Plant Materials Center Los Lunas New Mexico In Native Plant Network URL httpwwwnativeplantnetworkorg (accessed 9 January 2002) Moscow (ID) University ofIdaho CoIl Natural Resources For Res Nursery
72
Dreesen DR Harrington JT 1997 Propagation of native plants for restoration projects in the Southwestern US- Preliminary Investigations In Landis TD Thompson JR tech coords National Proceedings Forest and Conservation Nursery Assocs Gen Tech Rep PNW-GTR-419 Portland OR USDA For Ser PNW Res Sta 77-88
Gottfried GJ 1980 Control ofNew Mexican locust and the effect on planted ponderosa pine in central Arizona US For Ser Res Note RM-386 Rocky Mountain For Range ExpStn Fort Collins CO
Gosling PG Samuel YK Jones SK 1995 A systematic examination of germination temperature chipping and water temperaturesoak duration pretreatments on the seeds ofLeucaena leucocephala Seed Sci amp Technol 23521-532
Hamly DH 1932 Softening of the seeds of Melilotus alba Bot Gazette 93 345shy375
Hanna PJ 1984 Anatomical features of the seed coat ofAcacia kempeana (Mueller) which relate to increased germination rate induced by heat treatment New Phytol 96 23-29
Hanks JP Dick-Peddie WA 1974 Vegetation patterns of the White Mountains New Mexico Southwest Naturalist 18(4) 371-382
Hine S Harrington JT Dreesen DR 1997 Seed scarification requirements for Robinia neomexicana In Harrington JTtech coord Proc Third SouthwestemContainer Growers Conf Feb 12-13 1997 Ft Collins CO
Hopkinson JM 1993 The strophiole of leguminous seeds Newsletter- IntI Herbage Seed Production Res Grp 18 7-8
---- 1997 Differences in hardseededness between Cunningham and Tarramba leucaena In Leucnet News The Newsletter of the International LeucaenaRes and Devlt Netwk No4
ISTA (International Seed Testing Association) 1999 International rules for seed testing rules Seed Sci amp Technol27 Supplement
Kelly KM van Staden J 1987 The lens as the site of permeability in the papilionoid seed Aspalathus linearis J Plant Physiol 128(45) 395-404
73
Keresztesi B 1988 Black locust the tree of agriculture Outlook on Agr 17(2) 77~85
Kigel 1 and Galili G 1995 Seed development and germination Marcel Dekker New York 853 p
Klemmedson 10 1994 New Mexico locust and parent material Influence on macronutrients of forest floor and soil Soil Sci Soc Am 1 58 974~980
Kuo WH Tam AW 1988 The pathway of water absorption of mung bean seeds Seed Sci amp Technol16(1) 139~144
Leadem CL 1997 Dormancy--unlocking seed secrets In Landis TD Thompson lR tech coords National Proceedings Forestand Conservation Nursery Assocs Gen Tech Rep PNW-G TR-419 Portland OR USDA For Ser PNW Res Sta 43-52
Lin Y Wagner MR Seweyestewa A Honahni MD DeGomez M McMillin JD 1996 Influence of cultural treatments on seed germination and seedling growth of Robinia neomexicana Forest Farm and Community Tree Res Reports Volume 1
Marunda CT 1990 Effects of seed pretreatment on the development ofAcacia auriculforinis and A holosericea seedlings ACIAR Proceedings Series No 28
Meginnis HG 1937 Sulphuric acid treatment to increase germination of black locust seed USDA Circ 453 34 p
Miklas PN Towensend CE Ladd SL 1987 Seedcoat anatomy and the scarification ofcicer milkvetch seed Crop Sci 27 766-772
Monsen SB 1984 Use of shrubs on mine spoils In The Challenge ofProducing Native Plants for the Intermountain Area Proceedings Intermountain Nurserymans Association 1983 conference Las Vegas NV compo PM Murphy 26-31 Ogden UT USDA For Ser Gen Tech Report INT-l68
Olson DF 1974 Robinia L Locust In Seeds of Woody Plants in the US tech coord CS Schopmeyer 728-731 USDA For Ser Agr Handbk No450
Rehman S Loescher RNl Harris P1C 1998 Dormancy breaking and germination ofAcacia salicina Lindl seeds Seed Sci amp Techno127 553-557
Rolston MP 1978 Water impermeable seed dormancy Bot Rev 44 365-396
74
SAS Institute Inc 1989 SASSTAT Users Guide Version 6 4th Edition Volume 1 Cary NC SAS Institute Inc 943 p
SerratoValenti G Conara L Ferrando M Modenesi P 1993 Structural and histochemical features ofStylosanthes scabra (Leguminosae Papilionoidae) seed coat as related to water entry Can J Bot 71 834-840
Simpson BJ 1988 A field guide to Texas trees Austin TX Texas Monthly Press 372p
Tran VN Cavanagh AK 1984 Structural aspects ofdormancy In Seed Physiology Vol 2 Murray DR (ed) p1-44 Academic Press Australia
USDA Forest Service 1948 Woody-plant seed manual USDA Misc Pub1654 416p
Vines RA 1960 Trees Shrubs and Woody Vines of the Southwest Austin Univ ofTexas Press 1104 p
Wagner MR Larson RE Craig TP 1992 Ecology and silvics ofNew Mexico locust (Robinia neomexicana) with an emphasis on the arboreal form p53-57 In JW Hanover K Miller and S Plesko (eds) Proc Intemat Confon Black Locust Biology Culture and Utilization Mich State Univ USA
Wilson JK 1944 Immersing seeds of species of Robinia in boiling water hastens germination J For 42 453-454
Zimmerman RW Carpenter SB 1980 First-year coppice production from a 5shyyear old black locust stand on surface mine spoil p309-314 Centr Hardwd For ConfProc University ofMissouri Columbia
likelihood analysis was used to calculate Chi-square test statistics with their observed
significance levels A single contrast was conducted to cempare treatment means
against the control Observed significance levels less than or equal to 005 were
considered significant Pairwise comparisons were conducted between non-control
treatment mean percents using a conservative alp~a value of 005 divided by the
number of comparisons (Bonferroni approach) and approximate pairwise z statistics
Categorical analyses were performed using Proc Catmod (SAS Institute 1989)
RESULTS
Scarification seed source block and all interactions between factors
influenced germination (Table 3) The main effects are reported in the presence of
these interactions because the overall robustness of treatments and seed sources are of
interest Background levels ofnaturally occurring variation in seed sources are
seen in control germination percentages in Figure 2 In particular time variability
is unlikely to be under the control of the producer Appendix B details experimental
data for source by treatment gennination percents for each of the four blocks
Averaged over all treatments germination by source ranged from 40 to
79 (Figure 2) Averaged over all sources (Figure 3) and for each source (Figure 4
5) percussion scarification durations of4 or 8 minutes yielded greatest germination
Averaged over all sources germination increased with increasing durations of
percussion then leveled off for the 4- and 8-minute treatments (Figure 3) Immersion
and 30-second boiling scarification maximized germination for the hot water
treatments with a decrease in germination when seeds were boiled for 60 seconds
For all but two sources there is a similar ipcreasing trend in germination from
1to 4 minutes ofpercussion (Figure 4) The Holman source increased in germination
1 to 2 minutes then leveled off at 4 minutes The Jemez source changed little
1 to 4 minutes of treatment As treatment duration increased from 4 to 8
mutes five sources decreased in germination (Figure 4a) whereas three sources
24
Table 3 Categorical Analysis of Variance table for germination response to seed treatment seed source block and the interaction of the factors
Source of DF Chi-Square Observed Variability Significance Level
Source 8 98370 lt00001 Trt 7 233876 lt00001
contrast 1 16976 lt00001 SourceTrt 56 97077 lt00001 Block 3 5538 lt00001
SourceBlock 24 10162 lt00001 TrtBlock 21 7564 lt00001
SourceTrtBlock 168 34283 lt00001
S 4)
8 ~
0
sectg ~ 40 ~
N 0 0
20
o f0ltgt ~ ~4 jybshy ((iltgto ~~ K1ltgt 6~b-~ ~~ 0b-~~ ~~ ~b-~ ~b-~b
80
60 -I-- - ~- middotmiddotmiddotmiddotmiddot1
r -] Northern NM Sources
Central NM Sources
Southern NM Sources
sect-o ~
~b-v(i
SOURCE
Figure 2 Effect ofNew Mexico locust seed source on germination for data averaged over all scarification treatments Control germination is shown by solid line within bar
80
~ cd ~ ltl) () 60 ~ ~
N
t 0shyrti tshySltl)
40
0
20
o lmmersion Boiling 30 sec Boiling 60 sec Control 1 min 2min 4 min 8 min
Hot Water Treatments Percussion Treatments
Figure 3 Effect of scarification on germination ofNew Mexico locust for data averaged over all seed sources Means labelled with the same letter are not significantly different
100~----------------------~--~----------------~
80
60
ltU
~
40 IJ
sectgsI
l100
80
60
40
a I middotmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot middotImiddotmiddotmiddotmiddotmiddotmiddot E
_~iii-bull _ _ bull f --~--
t-- -~ ---- -shy ---- shyI ~ ---- - shy
L
fiII
~
b
- shy ---r
- Holman
Sacramento Sandia Gila Jemez
c ~
ltgt ~
Tmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot-I
j~=~=-~~-1
I middotmiddot ~ bull r
t tr
Y -------M-a-gd-a-Ie-n-a--
1 _ _
fCapitan
---- Manzano ~y -_ Raton
2 4 8
Time interval ofpercussion scarification (min)
Figure 4 Effect of seed source by treatment interaction on New Mexico locust germination for percussion scarification pattern a and pattern b
28
tv 0
~ d
~ A s o
1 c3
80
60
40
20
o
TIT
t------=middotfmiddotmiddot-middotmiddot-middotmiddot-middotmiddot 1- --- - - --=- - -shy--cE= - _--shy --shy ----shy ~ ~ ---shy ~ -~- _ --shy-------r 1
I
Raton Holman
-_ Jemez
--- Sandia Manzano Magdalena Capitan Sacramento Gila
Immersion Boiling 30 sec Boiling 60 sec
Hot Water Scarification
Figure 5 Effect of seed source by treatment interaction on New Mexico locust germination for hot water scarification
continued to increase in germination (Figure 4b) There was no difference between
the two treatment levels for the Manzano source (Figure 4b)
The effect ofhot water scarification also varied among sources with few
overall differences between hot water treatments (Figure 5) However boiling for 60
seconds yielded highest germination for the Jemez source whereas increasing
durations ofboiling decreased germination for the Raton source
In addition to consistently high germination 4- and 8-minute percussionshy
treated seeds germinated as fast or faster than all other treatments (Figure 6 Table 4)
050 measured as the number of days needed to complete 50 ofeventual
germination averaged 2 days for both ofthese treatments
X-ray radiographs were taken of seeds collected from the Sacramento source
percussed for 1 4 or 8 minutes (Figure 7) Seeds show signs of damage at 8 minutes
ofpercussion (Figure 8) Compared to an undamaged seed percussed for only 4
minutes (Figure 8a) damage at the 8-minute level includes fractured radicles pitting
of the seed coat and chips in seed coat structure (Figure 8b-d)
Scanning electron microscope (SEM) images at 600X reveal the direct effect
of 4 minutes of percussion on cells of the strophiolar region (Figure 9) Whereas
untreated seeds retain cellular organization in the strophiolar region strophiolar cells
seeds treated for 4 minutes ofpercussion separate and raise resulting in a
~-~e~U appearance
30
Umiddot-~middot--l-~---middotymiddotmiddotmiddot-----middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot--middot--middotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middotmiddotmiddotmiddotmiddotmiddotmiddot- -
050= gt1
-e-- Percussion 8 min -0- Percussion 4 min ---T- Percussion 2 min -----ltil- Percussion 1 min -- Immersion --0- Boiling 30 sec -+- Boiling 60 sec -ltgt- Control
2 4 6 8 10 12
Day Figure 6 Timecourse ofNew Mexico locust gennination following scarification
treatments G50 is the number of days to 50 of eventual germination
31
14
Table 4 Effect of seed source and treatment on G50 (days to 50 germination)
Seed Source Control Immersion B30sec B60sec Plmin P2min P4min P8min mean Raton 3 3 5 7 4 3 2 2 4 Holman 2 2 2 2 2 2 2 2 2
Jemez 4 6 6 5 3 2 2 2 4 Sandia 2 2 2 2 2 2 2 2 2
w tv Manzano 5 2 2 3 3 2 2 2 3
Magdalena 4 2 2 3 4 3 2 1 3
Capitan 5 4 2 2 3 2 2 2 3
Sacramento 3 2 2 2 4 3 2 2 3 Gila 3 2 2 2 2 2 2 2 2 mean 3 3 3 3 3 2 2 2 3 standard 11 14 16 18 09 05 0 03 08 deviation
Note Columns and rows may not agree due to rounding
Figure 7 X-ray radiographs ofSacramento seed source percussed for 1 4 or 8 minutes
33
Figure 8a-d Seed damage close-ups Figure a shows undamaged seed percussed for 4 minutes Figures b-d show seeds percussed for 8 minutes Damage includes fracturing ofradicle in b complete removal ofseed coat in c and pitting ofseed coat in d
34
Untreated Treated
Figure 9 SEM images ofuntreated vs 4-minute percussion-treated strophiolar cells ofNew Mexico locust seeds Treated cells are loosened separated and raised
DISCUSSION
Determining the Optimal Treatment
Averaging results of scarification treatmems across seed sources is a useful
tool to predict which treatment is most likely to be successful for an unknown seed
source Averaged over all sources four- and eight-minute percussion treatments
resulted in the highest germination 815 and 790 respectively and produced
germination that was as fast or faster than all other treatments Although there was no
difference between these two treatments on average an argument for 4 minutes of
percussion as a standard protocol for treating New Mexico locust seeds can be made
Descriptively 4 minutes of percussion improved germination over 8 minutes
ofpercussion for a majority of sources (five out of nine) 8 minutes of percussion
improved germination for three ofnine sources with no difference for a fmal source
For individual sources 4 minutes ofpercussion was always the best or secondmiddot best
treatment and was always equal to or better than all hot water treatments
Germination at 8 minutes ofpercussion was lower than all other percussion durations
for the Jemez and Gila sources and was lower than at least one hot water treatment for
these sources as well
The x-ray radiographs of the Sacramento seed source illustrate the excessive
internal and external seed damage that can occur with 8 minutes of percussion
germination counts 8-minute percussed seeds from Holman and Capitan
ClUll signs of damage that were visible to the naked eye (see below)
r~evertheless some of these seeds still fit the criteria for germination Neither x-ray
36
photography nor visible observations during seed counts revealed seed damage at the
4-minute percussion level In operational trials seeds percussed for 4 minutes
developed into healthy seedlings (Khadduri and Harrington unpublished data)
Strophiole and Seed Coat Behavior
Four minutes ofpercussion successfully breaks seed dormancy in a large
percentage ofNew Mexico locust seeds without excessively damaging the
surrounding seed coat or internal seed coat structures Most papilionoid legume
seeds including seeds ofNew Mexico locust have a specific region of the seed coat
known as the strophiole or lens (Hamly 1932) The strophiole is located on the
cotyledonary lobe of a locust seed (Figure 10 a-c) If seeds are not indiscriminately
damaged the strophiole is the first point of water entry to the embryo (Hopkinson
1997) When a papilionoid seed is percussed for the appropriate amount of time
repeated hits on the integrated seed coat loosen the constrained cells of the strophiolar
region without excessively damaging the rest of the seed coat (Ballard 1976) SEM
photos illuStrate that 4 minutes of percussion loosened and separated strophiolar cells
ofNew Mexico locust seeds when compared with constrained organized strophiolar
cells of untreated seeds (Figure 9) Research-has shown that water passes to the
embryo through the treated strophiole in a controlled manner and regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues
(Kelly and van Staden 1987)
37
10 Strophiole ofunimbibed New Mexico locust seed (a) and following uptake (bc)
38
In contrast hot-water treatments can excessively degrade the seed coat
(Rebman et al 1998 Marunda 1990) Random cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rebman et aI 1998 Marunda 1990) Along with a
proportionately large amount ofun-imbibed (thus under-treated) seeds (Figure l1a)
damage was observed in hot-water treated seeds from six sources during germination
counts Damage included seeds where the radicle failed to elongate (Figure 11 b)
seeds where the radicle elongated but was delayed in freeing itself from the seed coat
(Figure 11c) and seeds where the radicle completely broke off from the embryo
(Figure lId) In addition some seeds germinated in reverse (the cotyledon emerged
fIrst) If the radicle emerged completely from the seed coat they were counted as
germinated
While nearly all seeds treated for 8 minutes ofpercussion imbibed water
damage was evident in x-ray evaluations (Sacramento seed source) and in
germination counts (Holman and Capitan) at this treatment level Seed damage
during germination counts was similar to levels found in hot-water treated seeds
suggesting that over-treatment by both hot water and longer durations of percussion
may result in similar damage to seed structure
The Sacramento and Holman seed sources were two of the fIve sources that
opcrplltlpn in germination from the 4- to 8-minute durations of percussion
the benefIts ofloosening the strophiole as evidenced by imbibition of
all 8-minute treated seeds were outweighed by external and internal seed coat
39
a
b c d
11 Damage to New Mexico locust seeds observed during germination cotmts following hot water scarification 9a Hot water replicate including ~t seedlings and numerous unimbibed seeds 9b Close-up of seed where raallCle failed to elongate 9c Close-up ofseed where radicle elongated but was
-gtIn in freeing itself from the seed coat 9d Close-up ofseed where radicle broke off from embryo
40
damage (which also may be contributing to high imbibition) For the three sources
for which 8 minutes of percussion resulted in maximum germination extended
percussion of seeds necessary to loosen the strophiole may not have excessively
damaged the seed anatomy The Manzano source did not significantly change in
germination from 4 to 8 minutes of percussion suggesting that an increase in
permeable seeds at the 8-minute treatment duration was balanced by the number of
seeds that failed to germinate due to seed damage To summarize the 4-minute
percussion treatment improved germination over 8 minutes ofpercussion for a
majority of sources (five ofnine) the 8-minute percussion treatment improved
germination over 4-minutes ofpercussion for three sources and there was no
difference with the Manzano source Notably for the Raton source 8 minutes of
percussion increased germination by 235 percentage points over 4-minutes of
percussion (7875 vs 5525 respectively) the next best treatment Raton was the
only source to result in germination less than 75 for 4 minutes ofpercussion so a
grower may wish to apply longer percussion durations to lots that do not respond well
to this treatment level
Finally there was an interesting trend in the Jemez and Raton sources seed
that significantly contributed to the source by treatment interaction (Figure 12) A
~u test showed that the Jemez source had a significantly lighter seed weight than
other sources while the Raton source had an average seed weight For the Jemez
the longest duration of boiling increased germination Conversely only short
U4UVll) of percussion were needed to achieve high germination and the longest
41
80 _ Jemez a Raton
iD
~ C iD e iD
Q c 0 ~
c ~ iD 0
20
0------shy
Immersion Boiling 30 sec Boiling 60 sec
Hot Water Scarification
10degTr========~--------~------------------------------
80
60
40
20
_ Jemez Raton
0------shy
1 min 2 min 4 min 8 min
Percussion Scarification
Figure 12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources
42
b
--~----~~~~========~---- duration ofpercussion reduced germination For the Raton source longer durations
ofboiling consistently decreased germination whereas longer percussion treatments
consistently increased germination
A possible explanation is that a thick seed coat is associated with a loose
strophiole structure and that a thin seed coat is associated with a constrained
strophiole This contrast merits further study and may suggest an ecological
adaptation Further investigation ofthis phenomenon may contribute to successful
pre-screening ofunknown seed sources to determine optimal treatment leveL
43
CONCLUSION
Based on results obtained in pilot study (see Appendix) and thesis
experiments 4 minutes ofpercussion is recommended as a standard protocol for
treating an unknown source ofNew Mexico locust seed In addition to laboratory
results the 4-minute percussion treatment has resulted in germination greater than
80 and healthy seedling grow-outs for production purposes (unpublished data) In
laboratory experiments only one hundred seeds per 118 ml (40z) soil tin were
percussed at a time whereas for production we have treated up to five hundred seeds
per tin For large-scale production ofNew Mexico locust seedlings growers may
wish to experiment with larger quantities of seed and larger-size soil tins
Seed storage trials following percussion treatments were not conducted
However germination greater than 80 and healthy seedlings were obtained when
seeds were percussion-treated and subsequently stored at room-temperature (22degC)
for one week (unpublished data) Seed storage trials merit further study as the
standard hot water treatment leaves seeds in a softened condition that is not
conducive to storage
Hydroseeding is an effective method for direct seeding in reclamation
situations Percussion treatment may be conducive to hydro seeding as seeds remain
a dry relatively undamaged yet permeable condition following treatment Sulfuric
seeds also remain dry following treatment but extensive damage to seed coat
poor seedling vigor have been reported with this treatment (Kelly and van Staden
A grow-out study using a hydro seeder on a reclamation site testing percussion
44
against other treatments such as sulfuric acid and mechanical scarification also
merits further study
Papilionoids are the largest subfamily oflegumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust these legumes often play an integral role in the revegetation of
disturbed lands The pilot study (Appendix) indicates that percussion scarification
successfully treats the widely used black locust as well Developing superior
scarification methods to the standard hot water mechanical scarification and acid
treatments may facilitate the use ofpapilionoid legumes in reclamation projects
45
APPENDICES
APPENDIX A
PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR
NEW MEXICO LOCUST AND BLACK LOCUST SEEDS
Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4
Hot water and sulfuric acid soaks are traditional treatments for seeds of many
temperate woody legwnes including locusts However these scarification techniques
often produce inconsistent germination Percussion scarification where seeds are
repeatedly propelled against a hard surface was compared with hot water
scarification to evaluate treatment efficacy for New Mexico locust (Robinia
neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment
seeds were placed in a 98degC water bath which was immediately removed from the
heat source For percussion scarification seeds were placed in a soil sample tin and
agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the
control were followed by 24-hour water soaks Hot water baths resulted in 56 and
41 germination for New Mexico locust and black locust respectively For both
species nearly all durations ofpercussion increased germination over the hot water
treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3
4 and 5 minutes for black locust resulted in at least 90 germination Traditional
scarification treatments randomly degrade the entire seed coat which can lead to
tissue damage during water uptake Percussion scarification specifically weakens the
strophiole the natural source ofwater entry to the seed in papilionoid legwnes
INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom
2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031
48
Following percussion imbibition is controlled through the strophiole and underlying
tissue is protected
Keywords New Mexico locust black locust scarification percussion germination
Introduction
Locust trees (Robinia spp) are aggressive pioneer species that quickly
colonize disturbed land fix atmospheric nitrogen in the soil and stabilize slopes that
are prone to erosion (Klemmedson 1994 Ashby et al 1985) (Figure A1ab)
Recognition of these attributes has contributed to increased demand for locusts in
reclamation projects Locust seeds however exhibit physical dormancy where a
thick seed coat prevents the movement of water and gasses to the embryo (Leadem
1997)
Sulfuric acid or hot water soaks have traditionally been used to break seed
dormancy in macro-propagation of locusts Many growers are moving away from the
use of su1furic acid in treating locust seeds (Dreesen and Harrington 1997) In
addition to being dangerous acid soak durations must be specifically correlated to
seed lot (Olson 1974) Collections of native seed can vary tremendously in seed size
seed weight and hard seededness etc This variability limits the utility of sulfuric
acid soaks as even within a given lot some seeds may be damaged whereas others
are under-treated and fail to imbibe (take up water) A survey of Southwestern
container growers indicates that hot water baths are currently the most common
scarification method for locust seeds (Hine et al 1997) However hot water baths
also produce inconsistent germination (Lin et al 1996)
49
b
Figure AI New Mexico locust a primary invader following disturbance Ala Crown-sprouting at edge ofburn near Los Alamos NM--one year after Cerro Grande fire Alb Colonizing road cut Gila National Forest NM
50
Percussion scarification where seeds are repeatedly propelled against a hard
surface is an alternative dormancy-breaking method that has proven successful in
legumes related to locusts (Hamly 1932 Barton 1947) We compared this alternative
treatment to hot water scarification to evaluate treatment efficacy for New Mexico
locust and black locust seeds
Materials and Methods
Commercial New Mexico locust seeds (Western Native Seed Coaldale CO)
were collected fall 2000 in Huerfano County CO Black locust seeds were collected
September 2000 from Taos County NM by harvesting ripe pods from several trees at
a distance of up to 3 m from ground level Black locust seeds were allowed to air-dry
for 3 weeks and were threshed and separated from large chaffusing a greenhouse
fan A South Dakota Seed Blower (Chicago Illinltois) was used to remove fine chaff
from both seed lots Seeds were storedmiddotat 2-4C until the germination study was
conducted in March 2001
This study compared hot water scarification with percussion scarification
using treatment and species as experimental factors Along with a control
scarification treatments included immersion of seed in boiling water as well as
percussion durations of 1 2 3 45 or 10 minutes Four 100-seed samples were used
to test each species by scarification treatment combination
For the hot water treatment test tubes were filled with 30 mL water and
placed in a water-filled beaker (Figure A2a) The bath was raised to boiling (-98C at
51
b
c
Figure A2 Implementation ofhot water and percussion scarification A2a Hot water bath A2b Paint-shaker set-up A2c Placing soil tin containing seeds at end ofpaint shaker ann
52
Las Cruces NM elevation of 1300 meters) Each 100-seed replicate was placed in a
test tube and each test tube was immediately removed to cool at room temperature
Percussion scarification was implemented using a pneumatic paint shaker
(Central Pneumatic model 00422 Camarillo CA) (Figure A2b) This allowed
standardization of treatment intensity Compressed air pressure was maintained at
530kPa plusmn 30kPa (80psi plusmn 5psi) resulting in approximately 350 oscillations per
minute Each lOO-seed sample was placed in a 118 ml (4 oz) soil tin for shaking To
maximize lateral movement of paint shaker a paint can was used as a spacer to place
soil tins at the end of the shaking arm (Figure A2c) This allowed shaking to occur at
the greatest distance from the pivot point (fulcrum) Following all treatments
including the control seeds were soaked for twenty-four hours in distilled water prior
to plating out
Gennination was evaluated on lab benches at room temperature Each
replicate of seeds was placed in a 100 cm petri dish on moistened filter paper with
humidity maintained by enclosing the petri dishes in ziplock bags Gennination was
monitored daily for 14 days in accordance with International Seed Testing
Association standards for Robinia species (ISTA 1999) Gennination was defined as
protrusion of the radicle from the embryo by at least 1mm as viewed by the naked
eye
Categorical Analysis of Variance using Prpc Catmod (SAS Institute 1989)
was used to detennine treatment differences in germination percentages for each
source This procedure is an extension of a Chi-square test of homogeneity using the
53
natural log of the ratio of germinated to non-germinated seeds for each treatment Pshy
values less than 005 were considered significant A limited set of pairwiseshy
comparisons was conducted to compare treatment mean percents using a
conservative alpha value of 005 divided by the number of comparisons
Results
Percussion maximized germination for both New Mexico locust and black
locust There were three germination response levels for New Mexico locust (Figure
A3) First there was a low response in the control indicating a dormant seed lot
The hot water treatment and I-minute duration of percussion share a second
intermediate response Germination was highest at percussion levels from 2 to 10
minutes
A selected percussion treatment intermediate in time (5 minutes) produced the
steepest germination curve indicating the fastest germination rate (Figure A4) The
hot water treatment produced an intermediate curve or relatively slower germination
rate The control curve was very flat indicating the slowest germination rate for New
Mexico locust
Control seeds germinated poorly for black locust as well (Figure A5) Hot
water scarification improved germination over the control but all levels of percussion
improved germination over hot water The optimal durations of percussion treatment
were bracketed with a significant increase from the 1 to 2 minute percussion level a
high germination response from 2 to 5 minutes of percussion and finally a significant
54
~ d 25 CL)
p t1 0 ~ t1
g Vl ~ Vl
100~---------------------------------------------------------------
80
60
40
20
o Control Hot Water Percussion
I min Percussion
2 min Percussion
3 min
Scarification
Percussion 4 min
Figure A3 Effect of scarification on germination response for New Mexico locust
Percussion 5 min
=significant difference between adjacent treatment bars ns = no significant difference
Percussion 10 min
~ ~ () u agt
Il-t d o ~
~ ~ c3
100middotlr~~~------------------------------------e- Percussion 5min middot 0middot Hot Water -- Control
80-J=middot======
6b-middot-lmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middot--middotmiddot-middot~-middotmiddot -middotmiddot middot middotmiddot-middot 1
40
20
-----~-----------
0 0 0 middotmiddotmiddotmiddotmiddot0middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot0
0 --~-----y-----~------_---~--- T-----~
o shy -TDay 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A4 Effect of selected scarification treatments on germination speed for New Mexico locust First seven days of fourteen-day experiment are shown
Day 7
80
~ cd
5 60 ()
[) Cl d 0 1t1 d
40sect dUl
-J
20
o
ns
Scarification
Figure A5 Effect of scarification on germination response for black locust = significant difference between adjacent treatment bars ns = no significant difference
decrease at the 10-minute level This drop-off suggests that 10 minutes of percussion
over-treats seeds from this lot of-black locust
A selected percussion treatment intermediate in time (5 minutes) also had the
fastest germination rate for black locust (Figure A6)_ The hot water curve is
somewhat flatter indicating a slower germination rate The control curve is very flat
indicating a slow germination rate
A qualitative difference between hot water and percussion-treated seeds was
also observed Along with healthy germinants the hot water treatment produced
over-treated seeds (Figure A 7a) Examples of damaged germinants include seeds
where the radicle did not elongate (Figure A 7b) seeds where the radicle elongated
but was delayed in freeing itself from the seed coat (Figure A7c) and seeds where the
radicle broke off completely from the embryo (Figure A7d) In addition to these
examples of over-treatment there were numerous small dark seeds that failed to
imbibe water For percussion treatment nearly all seeds took up water (Figure A8a)
Germination was rapid and uniform with little damage evident (Figure A8b)
To summarize both New Mexico locust and black locust seed lots were
dormant The standard hot water scarification treatment improved germination but
only to 56 and 41 for New Mexico locust and black locust respectively Nearly
all percussion durations improved total germination as well as germination rate over
the hot water treatment Germination was greater than 90 for percussion durations
of 4 5 and 10 minutes for New Mexico locust and 3 4 and 5 minutes for black locust
58
CI)
sgt t CI) 0
igt 0
sect ttl t E
Vl 0
c3
100r==~==~~=r------------------------------------~ ___ Percussion 5 min
middotmiddotmiddot0middotmiddotmiddot Hot Water -lIf- Control
80r=========~--~~----------------------------~
60
40
20 -~-~---~---------- ---~------------I
omiddot
O
_~_----------lIf-----~bull -----_ shy---shy I
Day 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A6 Effect of selected scarification treatments on germination speed for black locust First seven days of fourteen-day experiment are shown
Day 7
a
b c d
Figure A7 Damage to New Mexico locust seeds observed during germination counts following hot water scarification A7a Hot water replicate including damaged seedlings and numerous unimbibed seeds A7b Close-up ofseed where radicle failed to elongate A7c Close-up ofseed where radicle elongated but was delayed in freeing itself from the seed coat A7d Close-up ofseed where radicle completely broke offfrom embryo
60
a
b
Figure A8 New Mexico locust seeds following percussion treatment at time of germination counts A8a Percussion replicate with uniformly healthy seedlings and near complete imbibition of seeds A8b Close-up ofpercussion-treated seedling
61
Discussion
Papilionoid seeds have a specific anatomical feature known as the strophiole
or lens-the natural site of water entry to the seed (Hamly 1932) The strophiole is
located on the cotyledonary lobe of a locust seed (Figure A9) When a papilionoid
legume seed is percussed for the appropriate amount of time repeated hits on the
integrated seed coat loosen the constrained cells of the strophiolar region without
excessively damaging the rest of the seed coat (Ballard 1976)
When a percussed seed is soaked water enters exclusively through the
strophiole in a controlled manner (Kelly and Staden 1987) This regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues This
contrasts with seeds that have been hot water or acid scarified treatments that can
randomly degrade the seed coat Unlocalized cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rehman et al 1998 Marunda 1990)
Papilionoids are the largest subfamily of legumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust and black locust these legumes often play an integral role in the
revegetation ofdisturbed lands Developing superior scarification methods to the
standard hot water and acid treatments should fadlitate the use ofthese legumes in
restoration projects
62
Figure A9 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (bc)
63
APPENDIXB
GERMINATION PERCENTS AND STANDARD ERRORS FOR SOURCE
BLOCK AND TREATMENT FOR NEW MEXICO LOCUST
Table B 1 Gennination percents and standard errors for source block and treatment for New Mexico locust
Source Block Treatment Germination Percentage Standard Error
Capitan 1 Immersion 74 437 Capitan 2 Immersion 69 462 Capitan 3 Immersion 71 454 Capitan 4 Immersion 67 470 Capitan Boiling 30 sec 79 407 Capitan 2 Boiling 30 sec 76 427 Capitan 3 Boiling 30 sec 72 449 Capitan 4 Boiling 30 sec 76 427 Capitan Boiling 60 sec 71 454 Capitan 2 Boiling 60 sec 54 498 Capitan 3 Boiling 60 sec 67 470 Capitan 4 Boiling 60 sec 76 427 Capitan I Control 5 218 Capitan 2 Control 7 255 Capitan 3 Control 4 196 Capitan 4 Control 5 218 Capitan 1 Percussion I min 47 499 Capitan 2 Percussion I min
470
Capitan Percussion I min 39 488
Capitan 4 Percussion I min 38 485 Capitan I Percussion 2 min 55 497 Capitan Capitan
2
Percussion 2 min Percussion 2 min
71 65
454 477
Capitan 4 Percussion 2 min 56 496 Capitan I Percussion 4 min 86 347 Capitan 2 Percussion 4 min 79 407 Capitan 3 Percussion 4 min 81 392 Capitan 4 Percussion 4 min 82 384 Capitan I Percussion 8 min 90 300 Capitan 2 Percussion 8 min 88 325 Capitan 3 Percussion 8 min 90 300 Capitan 4 Percussion 8 min 90 300 Gila I Immersion 71 454 Gila 2 Immersion 77 421 Gila
Immersion 75 433
Gila 4 Immersion 72 449
Gila I Boiling 30 sec 68 466
Gila 2 Boiling 30 sec 81 392
Gila 3 Boiling 30 sec 54 498
Gila 4 Boiling 30 sec 63 483
Gila I Boiling 60 sec 55 497
Gila 2 Boiling 60 sec 68 466
Gila 3 Boiling 60 sec 57 495
Gila 4 Boiling 60 sec 70 458
65
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Gila 2 Control 13 336 Gila 3 Control 13 336 Gila 4 Control 20 400 Gila 1 Percussion 1 min 66 474 Gila 2 Percussion 1 min 57 495 Gila 3 Percussion 1 min 73 444 Gila 4 Percussion 1 min 82 384 Gila 1 Percussion 2 min 81 392 Gila 2 Percussion 2 min 86 347 Gila 3 Percussion 2 min 85 357 Gila 4 Percussion 2 min 54 498 Gila 1 Percussion 4 min 79 407 Gila 2 Percussion 4 min 83 376 Gila 3 Percussion 4 min 84 367 Gila 4 Percussion 4 min 79 407 Gila 1 Percussion 8 min 51 500 Gila 2 Percussion 8 min 76 427 Gila 3 Percussion 8 min 52 500 Gila 4 Percussion 8 min 74 439 Holman 1 Immersion 83 376 Holman 2 Immersion 86 347 Holman 3 Immersion 81 392 Holman 4 Immersion 90 300 Holman 1 Boiling 30 sec 90 300 Holman 2 Boiling 30 sec 85 357 Holman 3 Boiling 30 sec 87 336 Holman 4 Boiling 30 sec 83 376 Holman 1 Boiling 60 sec 79 407 Holman 2 Boiling 60 sec 80 400 Holman 3 Boiling 60 sec 86 347 Holman 4 Boiling 60 sec 93 255 Holman Control 25 433 Holman 2 Control 19 392 Holman Control 21 407
Holman 4 Control 24 427
Holman Percussion 1 min 77 421
Holman 2 Percussion 1 min 85 357
Holman 3 Percussion 1 min 80 400
Holman 4 Percussion 1 min 89 313
Holman 1 Percussion 2 min 88 325
Holman 2 Percussion 2 min 96 196
Holman 3 Percussion 2 min 93 255
Holman 4 Percussion 2 min 96 196
Holman 1 Percussion 4 min 92 271
Holman 2 Percussion 4 min 97 171
66
I
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Holman 3 Percussion 4 min 91 286 Holman 4 Percussion 4 min 96 196 Holman 1 Percussion 8 min 88 325 Holman 2 Percussion 8 min 85 357 Holman 3 Percussion 8 min 83 376 Holman 4 Percussion 8 min 82 384 Jemez 1 Immersion 53 499 Jemez 2 Immersion 44 496 Jemez 3 Immersion 52 500 Jemez 4 Immersion 70 458 Jemez 1 Boiling 30 sec 58 494 Jemez 2 Boiling 30 sec 55 497 Jemez 3 Boiling 30 sec 48 500 Jemez 4 Boiling 30 sec 65 477 Jemez 1 Boiling 60 sec 68 466 Jemez 2 Boiling 60 sec 63 483 Jemez 3 Boiling 60 sec 73 444 Jemez 4 Boiling 60 sec 89 313 Jemez 1 Control 2 140 Jemez 2 Control 9 286 Jemez 3 Control 6 237 Jemez 4 Control 8 271 Jemez 1 Percussion 1 min 58 494 Jemez 2 Percussion 1 min 63 483 Jemez 3 Percussion 1 min 77 421 Jemez 4 Percussion 1 min 89 313 Jemez 1 Percussion 2 min 68 466 Jemez 2 Percussion 2 min 64 480 Jemez 3 Percussion 2 min 80 400 Jemez 4 Percussion 2 min 83 376 Jemez Percussion 4 min 68 466 Jemez 2 Percussion 4 min 79 407 Jemez 3 Percussion 4 min 70 458 Jemez 4 Percussion 4 min 83 376 Jemez 1 Percussion 8 min 52 500 Jemez 2 Percussion 8 min 59 492 Jemez 3 Percussion 8 min 61 488 Jemez 4 Percussion 8 min 70 458 Magdalena 1 Immersion 69 462 Magdalena 2 Immersion 69 462 Magdalena J Immersion 70 458 Magdalena 4 Immersion 85 357 Magdalena 1 Boiling 30 sec 65 477 Magdalena 2 Boiling 30 sec 71 454 Magdalena 3 Boiling 30 sec 68 466
67
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Magdalena 4 Boiling 30 sec 83 376 Magdalena 1 Boiling 60 sec 63 483 Magdalena 2 Boiling 60 sec 68 466 Magdalena 3 Boiling 60 sec 63 483 Magdalena 4 Boiling 60 sec 82 384 Magdalena 1 Control 11 313 Magdalena 2 Control 6 237 Magdalena 3 Control 5 218 Magdalena 4 Control 4 196 Magdalena Percussion 1 min 32 466 Magdalena 2 Percussion 1 min 45 497 Magdalena 3 Percussion 1 min 46 498 Magdalena 4 Percussion I min 36 480 Magdalena 1 Percussion 2 min 67 470 Magdalena 2 Percussion 2 min 68 466 Magdalena 3 Percussion 2 min 65 477 Magdalena 4 Percussion 2 min 81 392 Magdalena 1 Percussion 4 min 87 336 Magdalena 2 Percussion 4 min 97 171 Magdalena 3 Percussion 4 min 83 376 Magdalena 4 Percussion 4 min 97 171 Magdalena 1 Percussion 8 min 98 lAO Magdalena 2 Percussion 8 min 100 000 Magdalena 3 Percussion 8 min 96 196 Magdalena 4 Percussion 8 min 98 lAO Manzano 1 Immersion 67 470 Manzano 2 Immersion 57 495 Manzano 3 Immersion 72 4049 Manzano 4 Immersion 73 4044 Manzano 1 Boiling 30 sec 70 458 Manzano 2 Boiling 30 sec 64 480 Manzano 3 Boiling 30 sec 71 454 Manzano 4 Boiling 30 sec 80 400 Manzano I Boiling 60 sec 50 500 Manzano 2 Boiling 60 sec 53 499 Manzano 3 Boiling 60 sec 59 492 Manzano 4 Boiling 60 sec 74 439 Manzano I Control 4 196 Manzano 2 Control 8 271 Manzano 3 Control 1 099 Manzano 4 Control 6 237 Manzano 1 Percussion 1 min 52 500 Manzano 2 Percussion 1 min 39 488 Manzano 3 Percussion 1 min 36 480 Manzano 4 Percussion 1 min 51 500
68
Table B 1 Continued
Source Block Treatment Gerrni~ation Percentage Standard Error
Manzano Percussion 2 min 53 499 Manzano 2 Percussion 2 min 58 494 Manzano 3 Percussion 2 min 61 488 Manzano 4 Percussion 2 min 62 485 Manzano 1 Percussion 4 min 76 427 Manzano 2 Percussion 4 min 85 357 Manzano 3 Percussion 4 min 79 407 Manzano 4 Percussion 4 min 83 376 Manzano 1 Percussion 8 min 87 336 Manzano 2 Percussion 8 min 83 376 Manzano 3 Percussion 8 min 83 376 Manzano 4 Percussion 8 min 75 433 Raton 1 Immersion 54 498 Raton 2 Immersion 37 483 Raton 3 Immersion 39 488 Raton 4 Immersion 69 462 Raton 1 Boiling 30 sec 36 480 Raton 2 Boiling 30 sec 20 400 Raton 3 Boiling 30 sec 28 449 Raton 4 Boiling 30 sec 60 490 Raton 1 Boiling 60 sec 15 357 Raton 2 Boiling 60 sec 171 Raton 3 Boiling 60 sec 4 196 Raton 4 Boiling 60 sec 35 477 Raton 1 Control 4 196 Raton 2 Control 12 325 Raton 3 Control 2 140 Raton 4 Control 6 237 Raton 1 Percussion 1 min 52 500 Raton 2 Percussion I min 29 454 Raton 3 Percussion I min 35 477 Raton 4 Percussion 1 min 31 462 Raton 1 Percussion 2 min 55 497 Raton 2 Percussion 2 min 35 477 Raton 3 Percussion 2 min 33 470 Raton 4 Percussion 2 min 42 494 Raton 1 Percussion 4 min 65 477 Raton 2 Percussion 4 min 52 500 Raton Percussion 4 min 51 500 Raton 4 Percussion 4 min 53 499 Raton 1 Percussion 8 min 88 325 Raton 2 Percussion 8 min 80 400 Raton 3 Percussion 8 min 73 444 Raton 4 Percussion 8 min 74 439 Sacramento 1 Immersion 43 495
69
Table B 1 Continued
-Source Block Treatment Germination Percentage Standard Error
Sacramento 2 Immersion 30 458 Sacramento 3 Immersion 38 485 Sacramento 4 Immersion 56 496 Sacramento 1 Boiling 30 sec 47 499 Sacramento 2 Boiling 30 sec 48 500 Sacramento 3 Boiling 30 sec 48 500 Sacramento 4 Boiling 30 sec 56 496 Sacramento 1 Boiling 60 sec 52 500 Sacramento 2 Boiling 60 sec 67 470 Sacramento 3 Boiling 60 sec 43 495 Sacramento 4 Boiling 60 sec 48 500 Sacramento 1 Control 0 000 Sacramento 2 Control 3 171 Sacramento 3 Control 3 171 Sacramento 4 Control 099 Sacramento 1 Percussion 1 min 57 495 Sacramento 2 Percussion 1 min 61 488 Sacramento 3 Percussion 1 min 62 485 Sacramento 4 Percussion 1 min 55 497 Sacramento 1 Percussion 2 min 84 367 Sacramento 2 Percussion 2 min 70 458 Sacramento 3 Percussion 2 min 81 392 Sacramento 4 Percussion 2 min 69 462 Sacramento 1 Percussion 4 min 91 286 Sacramento 2 Percussion 4 min 84 367 Sacramento 3 Percussion 4 min 84 367 Sacramento 4 Percussion 4 min 94 237 Sacramento 1 Percussion 8 min 82 384 Sacramento 2 Percussion 8 min 81 392 Sacramento 3 Percussion 8 min 82 384 Sacramento 4 Percussion 8 min 81 392 Sandia 1 Immersion 76 427 Sandia 2 Immersion 70 458 Sandia 3 Immersion 70 458 Sandia 4 Immersion 81 392 Sandia 1 Boiling 30 sec 78 414 Sandia 2 Boiling 30 sec 71 454 Sandia 3 Boiling 30 sec 63 483 Sandia 4 Boiling 30 sec 78 414 Sandia 1 Boiling 60 sec 67 470 Sandia 2 Boiling 60 sec 65 477 Sandia 3 Boiling 60 sec 74 439 Sandia 4 Boiling 60 sec 66 474 Sandia 1 Control 16 367 Sandia 2 Control 7 255
70
Table B 1 Continued
Source Block Treatment Gennination Percentage Standard Error
Sandia 3 Control 8 271 Sandia 4 Control 16 367 Sandia 1 Percussion 1 min 67 470 Sandia 2 Percussion 1 min 61 488 Sandia 3 Percussion 1 min 66 474 Sandia 4 Percussion 1 min 59 492 Sandia I Percussion 2 min 82 384 Sandia 2 Percussion 2 min 83 376 Sandia 3 Percussion 2 min 81 392 Sandia 4 Percussion 2 min 84 367 Sandia 1 Percussion 4 min 89 313 Sandia 2 Percussion 4 min 86 347 Sandia 3 Percussion 4 min 89 313 Sandia 4 Percussion 4 min 85 357 Sandia 1 Percussion 8 min 80 400 Sandia 2 Percussion 8 min 77 421 Sandia 3 Percussion 8 min 69 462 Sandia 4 Percussion 8 min 66 474
LITERATURE CITED
Ashby WC Vogel WG Rogers NF 1985 Black locust in the reclamation equation General Technical Report Northeastern Forest Experiment Station USDA Forest Service NE-I05 12 p
Ballard LAT 1973 Physical barriers to gennination Seed Sci Technoll 285-303
___ 1976 Strophiolar water conduction in the seeds ofTrifolieae induced by action on the testa at nonstrophiolar sites Austr J Plant Physiol3 465-469
Barton LV 1947 Special studies on seed coat impenneability Contrib Boyce Thompson Inst 14 355-362
Baskin CC Baskin JM 1998 Seeds Ecology biogeography and evolution of donnancy and gennination Academic Press London UK 666 p
Bewley JD 1997 Seed germination and donnancy Plant Ce119 1055-1066
Bewley JD Black M 1994 Seeds physiology of development and germination 2nd ed Plenum Press New York NY 445 p
Briscoe CB 1996 Growing Nitrogen Fixing Trees in Acid Soils In MH Powell (ed) Nitrogen Fixing Trees for Acid Soils- A Field ManuaL Winrock International Morrilton AR 22 p
Copeland LO McDonald MB 1995 Principles of Seed Science and Technology 3rded Chapman amp Hall New York NY 409 p
Cox RE Klett JE 1984 Seed gennination requirements of native Colorado plants for use in the landscape Plant Propag 30(2) 6-10
Dell B 1980 Structure and function ofthe strophiolar plug in seeds ofAlbizia Lophantha Am J Bot 67 556-563
Dick-Peddie WA 1993 New Mexico Vegetation Past Present and Future University ofNew Mexico Press Albuquerque NM 244 p
Dreesen DR 2001 Propagation protocol for production ofcontainer Robinia neomexicana plants Los Lunas Plant Materials Center Los Lunas New Mexico In Native Plant Network URL httpwwwnativeplantnetworkorg (accessed 9 January 2002) Moscow (ID) University ofIdaho CoIl Natural Resources For Res Nursery
72
Dreesen DR Harrington JT 1997 Propagation of native plants for restoration projects in the Southwestern US- Preliminary Investigations In Landis TD Thompson JR tech coords National Proceedings Forest and Conservation Nursery Assocs Gen Tech Rep PNW-GTR-419 Portland OR USDA For Ser PNW Res Sta 77-88
Gottfried GJ 1980 Control ofNew Mexican locust and the effect on planted ponderosa pine in central Arizona US For Ser Res Note RM-386 Rocky Mountain For Range ExpStn Fort Collins CO
Gosling PG Samuel YK Jones SK 1995 A systematic examination of germination temperature chipping and water temperaturesoak duration pretreatments on the seeds ofLeucaena leucocephala Seed Sci amp Technol 23521-532
Hamly DH 1932 Softening of the seeds of Melilotus alba Bot Gazette 93 345shy375
Hanna PJ 1984 Anatomical features of the seed coat ofAcacia kempeana (Mueller) which relate to increased germination rate induced by heat treatment New Phytol 96 23-29
Hanks JP Dick-Peddie WA 1974 Vegetation patterns of the White Mountains New Mexico Southwest Naturalist 18(4) 371-382
Hine S Harrington JT Dreesen DR 1997 Seed scarification requirements for Robinia neomexicana In Harrington JTtech coord Proc Third SouthwestemContainer Growers Conf Feb 12-13 1997 Ft Collins CO
Hopkinson JM 1993 The strophiole of leguminous seeds Newsletter- IntI Herbage Seed Production Res Grp 18 7-8
---- 1997 Differences in hardseededness between Cunningham and Tarramba leucaena In Leucnet News The Newsletter of the International LeucaenaRes and Devlt Netwk No4
ISTA (International Seed Testing Association) 1999 International rules for seed testing rules Seed Sci amp Technol27 Supplement
Kelly KM van Staden J 1987 The lens as the site of permeability in the papilionoid seed Aspalathus linearis J Plant Physiol 128(45) 395-404
73
Keresztesi B 1988 Black locust the tree of agriculture Outlook on Agr 17(2) 77~85
Kigel 1 and Galili G 1995 Seed development and germination Marcel Dekker New York 853 p
Klemmedson 10 1994 New Mexico locust and parent material Influence on macronutrients of forest floor and soil Soil Sci Soc Am 1 58 974~980
Kuo WH Tam AW 1988 The pathway of water absorption of mung bean seeds Seed Sci amp Technol16(1) 139~144
Leadem CL 1997 Dormancy--unlocking seed secrets In Landis TD Thompson lR tech coords National Proceedings Forestand Conservation Nursery Assocs Gen Tech Rep PNW-G TR-419 Portland OR USDA For Ser PNW Res Sta 43-52
Lin Y Wagner MR Seweyestewa A Honahni MD DeGomez M McMillin JD 1996 Influence of cultural treatments on seed germination and seedling growth of Robinia neomexicana Forest Farm and Community Tree Res Reports Volume 1
Marunda CT 1990 Effects of seed pretreatment on the development ofAcacia auriculforinis and A holosericea seedlings ACIAR Proceedings Series No 28
Meginnis HG 1937 Sulphuric acid treatment to increase germination of black locust seed USDA Circ 453 34 p
Miklas PN Towensend CE Ladd SL 1987 Seedcoat anatomy and the scarification ofcicer milkvetch seed Crop Sci 27 766-772
Monsen SB 1984 Use of shrubs on mine spoils In The Challenge ofProducing Native Plants for the Intermountain Area Proceedings Intermountain Nurserymans Association 1983 conference Las Vegas NV compo PM Murphy 26-31 Ogden UT USDA For Ser Gen Tech Report INT-l68
Olson DF 1974 Robinia L Locust In Seeds of Woody Plants in the US tech coord CS Schopmeyer 728-731 USDA For Ser Agr Handbk No450
Rehman S Loescher RNl Harris P1C 1998 Dormancy breaking and germination ofAcacia salicina Lindl seeds Seed Sci amp Techno127 553-557
Rolston MP 1978 Water impermeable seed dormancy Bot Rev 44 365-396
74
SAS Institute Inc 1989 SASSTAT Users Guide Version 6 4th Edition Volume 1 Cary NC SAS Institute Inc 943 p
SerratoValenti G Conara L Ferrando M Modenesi P 1993 Structural and histochemical features ofStylosanthes scabra (Leguminosae Papilionoidae) seed coat as related to water entry Can J Bot 71 834-840
Simpson BJ 1988 A field guide to Texas trees Austin TX Texas Monthly Press 372p
Tran VN Cavanagh AK 1984 Structural aspects ofdormancy In Seed Physiology Vol 2 Murray DR (ed) p1-44 Academic Press Australia
USDA Forest Service 1948 Woody-plant seed manual USDA Misc Pub1654 416p
Vines RA 1960 Trees Shrubs and Woody Vines of the Southwest Austin Univ ofTexas Press 1104 p
Wagner MR Larson RE Craig TP 1992 Ecology and silvics ofNew Mexico locust (Robinia neomexicana) with an emphasis on the arboreal form p53-57 In JW Hanover K Miller and S Plesko (eds) Proc Intemat Confon Black Locust Biology Culture and Utilization Mich State Univ USA
Wilson JK 1944 Immersing seeds of species of Robinia in boiling water hastens germination J For 42 453-454
Zimmerman RW Carpenter SB 1980 First-year coppice production from a 5shyyear old black locust stand on surface mine spoil p309-314 Centr Hardwd For ConfProc University ofMissouri Columbia
RESULTS
Scarification seed source block and all interactions between factors
influenced germination (Table 3) The main effects are reported in the presence of
these interactions because the overall robustness of treatments and seed sources are of
interest Background levels ofnaturally occurring variation in seed sources are
seen in control germination percentages in Figure 2 In particular time variability
is unlikely to be under the control of the producer Appendix B details experimental
data for source by treatment gennination percents for each of the four blocks
Averaged over all treatments germination by source ranged from 40 to
79 (Figure 2) Averaged over all sources (Figure 3) and for each source (Figure 4
5) percussion scarification durations of4 or 8 minutes yielded greatest germination
Averaged over all sources germination increased with increasing durations of
percussion then leveled off for the 4- and 8-minute treatments (Figure 3) Immersion
and 30-second boiling scarification maximized germination for the hot water
treatments with a decrease in germination when seeds were boiled for 60 seconds
For all but two sources there is a similar ipcreasing trend in germination from
1to 4 minutes ofpercussion (Figure 4) The Holman source increased in germination
1 to 2 minutes then leveled off at 4 minutes The Jemez source changed little
1 to 4 minutes of treatment As treatment duration increased from 4 to 8
mutes five sources decreased in germination (Figure 4a) whereas three sources
24
Table 3 Categorical Analysis of Variance table for germination response to seed treatment seed source block and the interaction of the factors
Source of DF Chi-Square Observed Variability Significance Level
Source 8 98370 lt00001 Trt 7 233876 lt00001
contrast 1 16976 lt00001 SourceTrt 56 97077 lt00001 Block 3 5538 lt00001
SourceBlock 24 10162 lt00001 TrtBlock 21 7564 lt00001
SourceTrtBlock 168 34283 lt00001
S 4)
8 ~
0
sectg ~ 40 ~
N 0 0
20
o f0ltgt ~ ~4 jybshy ((iltgto ~~ K1ltgt 6~b-~ ~~ 0b-~~ ~~ ~b-~ ~b-~b
80
60 -I-- - ~- middotmiddotmiddotmiddotmiddot1
r -] Northern NM Sources
Central NM Sources
Southern NM Sources
sect-o ~
~b-v(i
SOURCE
Figure 2 Effect ofNew Mexico locust seed source on germination for data averaged over all scarification treatments Control germination is shown by solid line within bar
80
~ cd ~ ltl) () 60 ~ ~
N
t 0shyrti tshySltl)
40
0
20
o lmmersion Boiling 30 sec Boiling 60 sec Control 1 min 2min 4 min 8 min
Hot Water Treatments Percussion Treatments
Figure 3 Effect of scarification on germination ofNew Mexico locust for data averaged over all seed sources Means labelled with the same letter are not significantly different
100~----------------------~--~----------------~
80
60
ltU
~
40 IJ
sectgsI
l100
80
60
40
a I middotmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot middotImiddotmiddotmiddotmiddotmiddotmiddot E
_~iii-bull _ _ bull f --~--
t-- -~ ---- -shy ---- shyI ~ ---- - shy
L
fiII
~
b
- shy ---r
- Holman
Sacramento Sandia Gila Jemez
c ~
ltgt ~
Tmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot-I
j~=~=-~~-1
I middotmiddot ~ bull r
t tr
Y -------M-a-gd-a-Ie-n-a--
1 _ _
fCapitan
---- Manzano ~y -_ Raton
2 4 8
Time interval ofpercussion scarification (min)
Figure 4 Effect of seed source by treatment interaction on New Mexico locust germination for percussion scarification pattern a and pattern b
28
tv 0
~ d
~ A s o
1 c3
80
60
40
20
o
TIT
t------=middotfmiddotmiddot-middotmiddot-middotmiddot-middotmiddot 1- --- - - --=- - -shy--cE= - _--shy --shy ----shy ~ ~ ---shy ~ -~- _ --shy-------r 1
I
Raton Holman
-_ Jemez
--- Sandia Manzano Magdalena Capitan Sacramento Gila
Immersion Boiling 30 sec Boiling 60 sec
Hot Water Scarification
Figure 5 Effect of seed source by treatment interaction on New Mexico locust germination for hot water scarification
continued to increase in germination (Figure 4b) There was no difference between
the two treatment levels for the Manzano source (Figure 4b)
The effect ofhot water scarification also varied among sources with few
overall differences between hot water treatments (Figure 5) However boiling for 60
seconds yielded highest germination for the Jemez source whereas increasing
durations ofboiling decreased germination for the Raton source
In addition to consistently high germination 4- and 8-minute percussionshy
treated seeds germinated as fast or faster than all other treatments (Figure 6 Table 4)
050 measured as the number of days needed to complete 50 ofeventual
germination averaged 2 days for both ofthese treatments
X-ray radiographs were taken of seeds collected from the Sacramento source
percussed for 1 4 or 8 minutes (Figure 7) Seeds show signs of damage at 8 minutes
ofpercussion (Figure 8) Compared to an undamaged seed percussed for only 4
minutes (Figure 8a) damage at the 8-minute level includes fractured radicles pitting
of the seed coat and chips in seed coat structure (Figure 8b-d)
Scanning electron microscope (SEM) images at 600X reveal the direct effect
of 4 minutes of percussion on cells of the strophiolar region (Figure 9) Whereas
untreated seeds retain cellular organization in the strophiolar region strophiolar cells
seeds treated for 4 minutes ofpercussion separate and raise resulting in a
~-~e~U appearance
30
Umiddot-~middot--l-~---middotymiddotmiddotmiddot-----middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot--middot--middotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middotmiddotmiddotmiddotmiddotmiddotmiddot- -
050= gt1
-e-- Percussion 8 min -0- Percussion 4 min ---T- Percussion 2 min -----ltil- Percussion 1 min -- Immersion --0- Boiling 30 sec -+- Boiling 60 sec -ltgt- Control
2 4 6 8 10 12
Day Figure 6 Timecourse ofNew Mexico locust gennination following scarification
treatments G50 is the number of days to 50 of eventual germination
31
14
Table 4 Effect of seed source and treatment on G50 (days to 50 germination)
Seed Source Control Immersion B30sec B60sec Plmin P2min P4min P8min mean Raton 3 3 5 7 4 3 2 2 4 Holman 2 2 2 2 2 2 2 2 2
Jemez 4 6 6 5 3 2 2 2 4 Sandia 2 2 2 2 2 2 2 2 2
w tv Manzano 5 2 2 3 3 2 2 2 3
Magdalena 4 2 2 3 4 3 2 1 3
Capitan 5 4 2 2 3 2 2 2 3
Sacramento 3 2 2 2 4 3 2 2 3 Gila 3 2 2 2 2 2 2 2 2 mean 3 3 3 3 3 2 2 2 3 standard 11 14 16 18 09 05 0 03 08 deviation
Note Columns and rows may not agree due to rounding
Figure 7 X-ray radiographs ofSacramento seed source percussed for 1 4 or 8 minutes
33
Figure 8a-d Seed damage close-ups Figure a shows undamaged seed percussed for 4 minutes Figures b-d show seeds percussed for 8 minutes Damage includes fracturing ofradicle in b complete removal ofseed coat in c and pitting ofseed coat in d
34
Untreated Treated
Figure 9 SEM images ofuntreated vs 4-minute percussion-treated strophiolar cells ofNew Mexico locust seeds Treated cells are loosened separated and raised
DISCUSSION
Determining the Optimal Treatment
Averaging results of scarification treatmems across seed sources is a useful
tool to predict which treatment is most likely to be successful for an unknown seed
source Averaged over all sources four- and eight-minute percussion treatments
resulted in the highest germination 815 and 790 respectively and produced
germination that was as fast or faster than all other treatments Although there was no
difference between these two treatments on average an argument for 4 minutes of
percussion as a standard protocol for treating New Mexico locust seeds can be made
Descriptively 4 minutes of percussion improved germination over 8 minutes
ofpercussion for a majority of sources (five out of nine) 8 minutes of percussion
improved germination for three ofnine sources with no difference for a fmal source
For individual sources 4 minutes ofpercussion was always the best or secondmiddot best
treatment and was always equal to or better than all hot water treatments
Germination at 8 minutes ofpercussion was lower than all other percussion durations
for the Jemez and Gila sources and was lower than at least one hot water treatment for
these sources as well
The x-ray radiographs of the Sacramento seed source illustrate the excessive
internal and external seed damage that can occur with 8 minutes of percussion
germination counts 8-minute percussed seeds from Holman and Capitan
ClUll signs of damage that were visible to the naked eye (see below)
r~evertheless some of these seeds still fit the criteria for germination Neither x-ray
36
photography nor visible observations during seed counts revealed seed damage at the
4-minute percussion level In operational trials seeds percussed for 4 minutes
developed into healthy seedlings (Khadduri and Harrington unpublished data)
Strophiole and Seed Coat Behavior
Four minutes ofpercussion successfully breaks seed dormancy in a large
percentage ofNew Mexico locust seeds without excessively damaging the
surrounding seed coat or internal seed coat structures Most papilionoid legume
seeds including seeds ofNew Mexico locust have a specific region of the seed coat
known as the strophiole or lens (Hamly 1932) The strophiole is located on the
cotyledonary lobe of a locust seed (Figure 10 a-c) If seeds are not indiscriminately
damaged the strophiole is the first point of water entry to the embryo (Hopkinson
1997) When a papilionoid seed is percussed for the appropriate amount of time
repeated hits on the integrated seed coat loosen the constrained cells of the strophiolar
region without excessively damaging the rest of the seed coat (Ballard 1976) SEM
photos illuStrate that 4 minutes of percussion loosened and separated strophiolar cells
ofNew Mexico locust seeds when compared with constrained organized strophiolar
cells of untreated seeds (Figure 9) Research-has shown that water passes to the
embryo through the treated strophiole in a controlled manner and regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues
(Kelly and van Staden 1987)
37
10 Strophiole ofunimbibed New Mexico locust seed (a) and following uptake (bc)
38
In contrast hot-water treatments can excessively degrade the seed coat
(Rebman et al 1998 Marunda 1990) Random cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rebman et aI 1998 Marunda 1990) Along with a
proportionately large amount ofun-imbibed (thus under-treated) seeds (Figure l1a)
damage was observed in hot-water treated seeds from six sources during germination
counts Damage included seeds where the radicle failed to elongate (Figure 11 b)
seeds where the radicle elongated but was delayed in freeing itself from the seed coat
(Figure 11c) and seeds where the radicle completely broke off from the embryo
(Figure lId) In addition some seeds germinated in reverse (the cotyledon emerged
fIrst) If the radicle emerged completely from the seed coat they were counted as
germinated
While nearly all seeds treated for 8 minutes ofpercussion imbibed water
damage was evident in x-ray evaluations (Sacramento seed source) and in
germination counts (Holman and Capitan) at this treatment level Seed damage
during germination counts was similar to levels found in hot-water treated seeds
suggesting that over-treatment by both hot water and longer durations of percussion
may result in similar damage to seed structure
The Sacramento and Holman seed sources were two of the fIve sources that
opcrplltlpn in germination from the 4- to 8-minute durations of percussion
the benefIts ofloosening the strophiole as evidenced by imbibition of
all 8-minute treated seeds were outweighed by external and internal seed coat
39
a
b c d
11 Damage to New Mexico locust seeds observed during germination cotmts following hot water scarification 9a Hot water replicate including ~t seedlings and numerous unimbibed seeds 9b Close-up of seed where raallCle failed to elongate 9c Close-up ofseed where radicle elongated but was
-gtIn in freeing itself from the seed coat 9d Close-up ofseed where radicle broke off from embryo
40
damage (which also may be contributing to high imbibition) For the three sources
for which 8 minutes of percussion resulted in maximum germination extended
percussion of seeds necessary to loosen the strophiole may not have excessively
damaged the seed anatomy The Manzano source did not significantly change in
germination from 4 to 8 minutes of percussion suggesting that an increase in
permeable seeds at the 8-minute treatment duration was balanced by the number of
seeds that failed to germinate due to seed damage To summarize the 4-minute
percussion treatment improved germination over 8 minutes ofpercussion for a
majority of sources (five ofnine) the 8-minute percussion treatment improved
germination over 4-minutes ofpercussion for three sources and there was no
difference with the Manzano source Notably for the Raton source 8 minutes of
percussion increased germination by 235 percentage points over 4-minutes of
percussion (7875 vs 5525 respectively) the next best treatment Raton was the
only source to result in germination less than 75 for 4 minutes ofpercussion so a
grower may wish to apply longer percussion durations to lots that do not respond well
to this treatment level
Finally there was an interesting trend in the Jemez and Raton sources seed
that significantly contributed to the source by treatment interaction (Figure 12) A
~u test showed that the Jemez source had a significantly lighter seed weight than
other sources while the Raton source had an average seed weight For the Jemez
the longest duration of boiling increased germination Conversely only short
U4UVll) of percussion were needed to achieve high germination and the longest
41
80 _ Jemez a Raton
iD
~ C iD e iD
Q c 0 ~
c ~ iD 0
20
0------shy
Immersion Boiling 30 sec Boiling 60 sec
Hot Water Scarification
10degTr========~--------~------------------------------
80
60
40
20
_ Jemez Raton
0------shy
1 min 2 min 4 min 8 min
Percussion Scarification
Figure 12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources
42
b
--~----~~~~========~---- duration ofpercussion reduced germination For the Raton source longer durations
ofboiling consistently decreased germination whereas longer percussion treatments
consistently increased germination
A possible explanation is that a thick seed coat is associated with a loose
strophiole structure and that a thin seed coat is associated with a constrained
strophiole This contrast merits further study and may suggest an ecological
adaptation Further investigation ofthis phenomenon may contribute to successful
pre-screening ofunknown seed sources to determine optimal treatment leveL
43
CONCLUSION
Based on results obtained in pilot study (see Appendix) and thesis
experiments 4 minutes ofpercussion is recommended as a standard protocol for
treating an unknown source ofNew Mexico locust seed In addition to laboratory
results the 4-minute percussion treatment has resulted in germination greater than
80 and healthy seedling grow-outs for production purposes (unpublished data) In
laboratory experiments only one hundred seeds per 118 ml (40z) soil tin were
percussed at a time whereas for production we have treated up to five hundred seeds
per tin For large-scale production ofNew Mexico locust seedlings growers may
wish to experiment with larger quantities of seed and larger-size soil tins
Seed storage trials following percussion treatments were not conducted
However germination greater than 80 and healthy seedlings were obtained when
seeds were percussion-treated and subsequently stored at room-temperature (22degC)
for one week (unpublished data) Seed storage trials merit further study as the
standard hot water treatment leaves seeds in a softened condition that is not
conducive to storage
Hydroseeding is an effective method for direct seeding in reclamation
situations Percussion treatment may be conducive to hydro seeding as seeds remain
a dry relatively undamaged yet permeable condition following treatment Sulfuric
seeds also remain dry following treatment but extensive damage to seed coat
poor seedling vigor have been reported with this treatment (Kelly and van Staden
A grow-out study using a hydro seeder on a reclamation site testing percussion
44
against other treatments such as sulfuric acid and mechanical scarification also
merits further study
Papilionoids are the largest subfamily oflegumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust these legumes often play an integral role in the revegetation of
disturbed lands The pilot study (Appendix) indicates that percussion scarification
successfully treats the widely used black locust as well Developing superior
scarification methods to the standard hot water mechanical scarification and acid
treatments may facilitate the use ofpapilionoid legumes in reclamation projects
45
APPENDICES
APPENDIX A
PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR
NEW MEXICO LOCUST AND BLACK LOCUST SEEDS
Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4
Hot water and sulfuric acid soaks are traditional treatments for seeds of many
temperate woody legwnes including locusts However these scarification techniques
often produce inconsistent germination Percussion scarification where seeds are
repeatedly propelled against a hard surface was compared with hot water
scarification to evaluate treatment efficacy for New Mexico locust (Robinia
neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment
seeds were placed in a 98degC water bath which was immediately removed from the
heat source For percussion scarification seeds were placed in a soil sample tin and
agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the
control were followed by 24-hour water soaks Hot water baths resulted in 56 and
41 germination for New Mexico locust and black locust respectively For both
species nearly all durations ofpercussion increased germination over the hot water
treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3
4 and 5 minutes for black locust resulted in at least 90 germination Traditional
scarification treatments randomly degrade the entire seed coat which can lead to
tissue damage during water uptake Percussion scarification specifically weakens the
strophiole the natural source ofwater entry to the seed in papilionoid legwnes
INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom
2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031
48
Following percussion imbibition is controlled through the strophiole and underlying
tissue is protected
Keywords New Mexico locust black locust scarification percussion germination
Introduction
Locust trees (Robinia spp) are aggressive pioneer species that quickly
colonize disturbed land fix atmospheric nitrogen in the soil and stabilize slopes that
are prone to erosion (Klemmedson 1994 Ashby et al 1985) (Figure A1ab)
Recognition of these attributes has contributed to increased demand for locusts in
reclamation projects Locust seeds however exhibit physical dormancy where a
thick seed coat prevents the movement of water and gasses to the embryo (Leadem
1997)
Sulfuric acid or hot water soaks have traditionally been used to break seed
dormancy in macro-propagation of locusts Many growers are moving away from the
use of su1furic acid in treating locust seeds (Dreesen and Harrington 1997) In
addition to being dangerous acid soak durations must be specifically correlated to
seed lot (Olson 1974) Collections of native seed can vary tremendously in seed size
seed weight and hard seededness etc This variability limits the utility of sulfuric
acid soaks as even within a given lot some seeds may be damaged whereas others
are under-treated and fail to imbibe (take up water) A survey of Southwestern
container growers indicates that hot water baths are currently the most common
scarification method for locust seeds (Hine et al 1997) However hot water baths
also produce inconsistent germination (Lin et al 1996)
49
b
Figure AI New Mexico locust a primary invader following disturbance Ala Crown-sprouting at edge ofburn near Los Alamos NM--one year after Cerro Grande fire Alb Colonizing road cut Gila National Forest NM
50
Percussion scarification where seeds are repeatedly propelled against a hard
surface is an alternative dormancy-breaking method that has proven successful in
legumes related to locusts (Hamly 1932 Barton 1947) We compared this alternative
treatment to hot water scarification to evaluate treatment efficacy for New Mexico
locust and black locust seeds
Materials and Methods
Commercial New Mexico locust seeds (Western Native Seed Coaldale CO)
were collected fall 2000 in Huerfano County CO Black locust seeds were collected
September 2000 from Taos County NM by harvesting ripe pods from several trees at
a distance of up to 3 m from ground level Black locust seeds were allowed to air-dry
for 3 weeks and were threshed and separated from large chaffusing a greenhouse
fan A South Dakota Seed Blower (Chicago Illinltois) was used to remove fine chaff
from both seed lots Seeds were storedmiddotat 2-4C until the germination study was
conducted in March 2001
This study compared hot water scarification with percussion scarification
using treatment and species as experimental factors Along with a control
scarification treatments included immersion of seed in boiling water as well as
percussion durations of 1 2 3 45 or 10 minutes Four 100-seed samples were used
to test each species by scarification treatment combination
For the hot water treatment test tubes were filled with 30 mL water and
placed in a water-filled beaker (Figure A2a) The bath was raised to boiling (-98C at
51
b
c
Figure A2 Implementation ofhot water and percussion scarification A2a Hot water bath A2b Paint-shaker set-up A2c Placing soil tin containing seeds at end ofpaint shaker ann
52
Las Cruces NM elevation of 1300 meters) Each 100-seed replicate was placed in a
test tube and each test tube was immediately removed to cool at room temperature
Percussion scarification was implemented using a pneumatic paint shaker
(Central Pneumatic model 00422 Camarillo CA) (Figure A2b) This allowed
standardization of treatment intensity Compressed air pressure was maintained at
530kPa plusmn 30kPa (80psi plusmn 5psi) resulting in approximately 350 oscillations per
minute Each lOO-seed sample was placed in a 118 ml (4 oz) soil tin for shaking To
maximize lateral movement of paint shaker a paint can was used as a spacer to place
soil tins at the end of the shaking arm (Figure A2c) This allowed shaking to occur at
the greatest distance from the pivot point (fulcrum) Following all treatments
including the control seeds were soaked for twenty-four hours in distilled water prior
to plating out
Gennination was evaluated on lab benches at room temperature Each
replicate of seeds was placed in a 100 cm petri dish on moistened filter paper with
humidity maintained by enclosing the petri dishes in ziplock bags Gennination was
monitored daily for 14 days in accordance with International Seed Testing
Association standards for Robinia species (ISTA 1999) Gennination was defined as
protrusion of the radicle from the embryo by at least 1mm as viewed by the naked
eye
Categorical Analysis of Variance using Prpc Catmod (SAS Institute 1989)
was used to detennine treatment differences in germination percentages for each
source This procedure is an extension of a Chi-square test of homogeneity using the
53
natural log of the ratio of germinated to non-germinated seeds for each treatment Pshy
values less than 005 were considered significant A limited set of pairwiseshy
comparisons was conducted to compare treatment mean percents using a
conservative alpha value of 005 divided by the number of comparisons
Results
Percussion maximized germination for both New Mexico locust and black
locust There were three germination response levels for New Mexico locust (Figure
A3) First there was a low response in the control indicating a dormant seed lot
The hot water treatment and I-minute duration of percussion share a second
intermediate response Germination was highest at percussion levels from 2 to 10
minutes
A selected percussion treatment intermediate in time (5 minutes) produced the
steepest germination curve indicating the fastest germination rate (Figure A4) The
hot water treatment produced an intermediate curve or relatively slower germination
rate The control curve was very flat indicating the slowest germination rate for New
Mexico locust
Control seeds germinated poorly for black locust as well (Figure A5) Hot
water scarification improved germination over the control but all levels of percussion
improved germination over hot water The optimal durations of percussion treatment
were bracketed with a significant increase from the 1 to 2 minute percussion level a
high germination response from 2 to 5 minutes of percussion and finally a significant
54
~ d 25 CL)
p t1 0 ~ t1
g Vl ~ Vl
100~---------------------------------------------------------------
80
60
40
20
o Control Hot Water Percussion
I min Percussion
2 min Percussion
3 min
Scarification
Percussion 4 min
Figure A3 Effect of scarification on germination response for New Mexico locust
Percussion 5 min
=significant difference between adjacent treatment bars ns = no significant difference
Percussion 10 min
~ ~ () u agt
Il-t d o ~
~ ~ c3
100middotlr~~~------------------------------------e- Percussion 5min middot 0middot Hot Water -- Control
80-J=middot======
6b-middot-lmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middot--middotmiddot-middot~-middotmiddot -middotmiddot middot middotmiddot-middot 1
40
20
-----~-----------
0 0 0 middotmiddotmiddotmiddotmiddot0middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot0
0 --~-----y-----~------_---~--- T-----~
o shy -TDay 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A4 Effect of selected scarification treatments on germination speed for New Mexico locust First seven days of fourteen-day experiment are shown
Day 7
80
~ cd
5 60 ()
[) Cl d 0 1t1 d
40sect dUl
-J
20
o
ns
Scarification
Figure A5 Effect of scarification on germination response for black locust = significant difference between adjacent treatment bars ns = no significant difference
decrease at the 10-minute level This drop-off suggests that 10 minutes of percussion
over-treats seeds from this lot of-black locust
A selected percussion treatment intermediate in time (5 minutes) also had the
fastest germination rate for black locust (Figure A6)_ The hot water curve is
somewhat flatter indicating a slower germination rate The control curve is very flat
indicating a slow germination rate
A qualitative difference between hot water and percussion-treated seeds was
also observed Along with healthy germinants the hot water treatment produced
over-treated seeds (Figure A 7a) Examples of damaged germinants include seeds
where the radicle did not elongate (Figure A 7b) seeds where the radicle elongated
but was delayed in freeing itself from the seed coat (Figure A7c) and seeds where the
radicle broke off completely from the embryo (Figure A7d) In addition to these
examples of over-treatment there were numerous small dark seeds that failed to
imbibe water For percussion treatment nearly all seeds took up water (Figure A8a)
Germination was rapid and uniform with little damage evident (Figure A8b)
To summarize both New Mexico locust and black locust seed lots were
dormant The standard hot water scarification treatment improved germination but
only to 56 and 41 for New Mexico locust and black locust respectively Nearly
all percussion durations improved total germination as well as germination rate over
the hot water treatment Germination was greater than 90 for percussion durations
of 4 5 and 10 minutes for New Mexico locust and 3 4 and 5 minutes for black locust
58
CI)
sgt t CI) 0
igt 0
sect ttl t E
Vl 0
c3
100r==~==~~=r------------------------------------~ ___ Percussion 5 min
middotmiddotmiddot0middotmiddotmiddot Hot Water -lIf- Control
80r=========~--~~----------------------------~
60
40
20 -~-~---~---------- ---~------------I
omiddot
O
_~_----------lIf-----~bull -----_ shy---shy I
Day 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A6 Effect of selected scarification treatments on germination speed for black locust First seven days of fourteen-day experiment are shown
Day 7
a
b c d
Figure A7 Damage to New Mexico locust seeds observed during germination counts following hot water scarification A7a Hot water replicate including damaged seedlings and numerous unimbibed seeds A7b Close-up ofseed where radicle failed to elongate A7c Close-up ofseed where radicle elongated but was delayed in freeing itself from the seed coat A7d Close-up ofseed where radicle completely broke offfrom embryo
60
a
b
Figure A8 New Mexico locust seeds following percussion treatment at time of germination counts A8a Percussion replicate with uniformly healthy seedlings and near complete imbibition of seeds A8b Close-up ofpercussion-treated seedling
61
Discussion
Papilionoid seeds have a specific anatomical feature known as the strophiole
or lens-the natural site of water entry to the seed (Hamly 1932) The strophiole is
located on the cotyledonary lobe of a locust seed (Figure A9) When a papilionoid
legume seed is percussed for the appropriate amount of time repeated hits on the
integrated seed coat loosen the constrained cells of the strophiolar region without
excessively damaging the rest of the seed coat (Ballard 1976)
When a percussed seed is soaked water enters exclusively through the
strophiole in a controlled manner (Kelly and Staden 1987) This regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues This
contrasts with seeds that have been hot water or acid scarified treatments that can
randomly degrade the seed coat Unlocalized cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rehman et al 1998 Marunda 1990)
Papilionoids are the largest subfamily of legumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust and black locust these legumes often play an integral role in the
revegetation ofdisturbed lands Developing superior scarification methods to the
standard hot water and acid treatments should fadlitate the use ofthese legumes in
restoration projects
62
Figure A9 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (bc)
63
APPENDIXB
GERMINATION PERCENTS AND STANDARD ERRORS FOR SOURCE
BLOCK AND TREATMENT FOR NEW MEXICO LOCUST
Table B 1 Gennination percents and standard errors for source block and treatment for New Mexico locust
Source Block Treatment Germination Percentage Standard Error
Capitan 1 Immersion 74 437 Capitan 2 Immersion 69 462 Capitan 3 Immersion 71 454 Capitan 4 Immersion 67 470 Capitan Boiling 30 sec 79 407 Capitan 2 Boiling 30 sec 76 427 Capitan 3 Boiling 30 sec 72 449 Capitan 4 Boiling 30 sec 76 427 Capitan Boiling 60 sec 71 454 Capitan 2 Boiling 60 sec 54 498 Capitan 3 Boiling 60 sec 67 470 Capitan 4 Boiling 60 sec 76 427 Capitan I Control 5 218 Capitan 2 Control 7 255 Capitan 3 Control 4 196 Capitan 4 Control 5 218 Capitan 1 Percussion I min 47 499 Capitan 2 Percussion I min
470
Capitan Percussion I min 39 488
Capitan 4 Percussion I min 38 485 Capitan I Percussion 2 min 55 497 Capitan Capitan
2
Percussion 2 min Percussion 2 min
71 65
454 477
Capitan 4 Percussion 2 min 56 496 Capitan I Percussion 4 min 86 347 Capitan 2 Percussion 4 min 79 407 Capitan 3 Percussion 4 min 81 392 Capitan 4 Percussion 4 min 82 384 Capitan I Percussion 8 min 90 300 Capitan 2 Percussion 8 min 88 325 Capitan 3 Percussion 8 min 90 300 Capitan 4 Percussion 8 min 90 300 Gila I Immersion 71 454 Gila 2 Immersion 77 421 Gila
Immersion 75 433
Gila 4 Immersion 72 449
Gila I Boiling 30 sec 68 466
Gila 2 Boiling 30 sec 81 392
Gila 3 Boiling 30 sec 54 498
Gila 4 Boiling 30 sec 63 483
Gila I Boiling 60 sec 55 497
Gila 2 Boiling 60 sec 68 466
Gila 3 Boiling 60 sec 57 495
Gila 4 Boiling 60 sec 70 458
65
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Gila 2 Control 13 336 Gila 3 Control 13 336 Gila 4 Control 20 400 Gila 1 Percussion 1 min 66 474 Gila 2 Percussion 1 min 57 495 Gila 3 Percussion 1 min 73 444 Gila 4 Percussion 1 min 82 384 Gila 1 Percussion 2 min 81 392 Gila 2 Percussion 2 min 86 347 Gila 3 Percussion 2 min 85 357 Gila 4 Percussion 2 min 54 498 Gila 1 Percussion 4 min 79 407 Gila 2 Percussion 4 min 83 376 Gila 3 Percussion 4 min 84 367 Gila 4 Percussion 4 min 79 407 Gila 1 Percussion 8 min 51 500 Gila 2 Percussion 8 min 76 427 Gila 3 Percussion 8 min 52 500 Gila 4 Percussion 8 min 74 439 Holman 1 Immersion 83 376 Holman 2 Immersion 86 347 Holman 3 Immersion 81 392 Holman 4 Immersion 90 300 Holman 1 Boiling 30 sec 90 300 Holman 2 Boiling 30 sec 85 357 Holman 3 Boiling 30 sec 87 336 Holman 4 Boiling 30 sec 83 376 Holman 1 Boiling 60 sec 79 407 Holman 2 Boiling 60 sec 80 400 Holman 3 Boiling 60 sec 86 347 Holman 4 Boiling 60 sec 93 255 Holman Control 25 433 Holman 2 Control 19 392 Holman Control 21 407
Holman 4 Control 24 427
Holman Percussion 1 min 77 421
Holman 2 Percussion 1 min 85 357
Holman 3 Percussion 1 min 80 400
Holman 4 Percussion 1 min 89 313
Holman 1 Percussion 2 min 88 325
Holman 2 Percussion 2 min 96 196
Holman 3 Percussion 2 min 93 255
Holman 4 Percussion 2 min 96 196
Holman 1 Percussion 4 min 92 271
Holman 2 Percussion 4 min 97 171
66
I
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Holman 3 Percussion 4 min 91 286 Holman 4 Percussion 4 min 96 196 Holman 1 Percussion 8 min 88 325 Holman 2 Percussion 8 min 85 357 Holman 3 Percussion 8 min 83 376 Holman 4 Percussion 8 min 82 384 Jemez 1 Immersion 53 499 Jemez 2 Immersion 44 496 Jemez 3 Immersion 52 500 Jemez 4 Immersion 70 458 Jemez 1 Boiling 30 sec 58 494 Jemez 2 Boiling 30 sec 55 497 Jemez 3 Boiling 30 sec 48 500 Jemez 4 Boiling 30 sec 65 477 Jemez 1 Boiling 60 sec 68 466 Jemez 2 Boiling 60 sec 63 483 Jemez 3 Boiling 60 sec 73 444 Jemez 4 Boiling 60 sec 89 313 Jemez 1 Control 2 140 Jemez 2 Control 9 286 Jemez 3 Control 6 237 Jemez 4 Control 8 271 Jemez 1 Percussion 1 min 58 494 Jemez 2 Percussion 1 min 63 483 Jemez 3 Percussion 1 min 77 421 Jemez 4 Percussion 1 min 89 313 Jemez 1 Percussion 2 min 68 466 Jemez 2 Percussion 2 min 64 480 Jemez 3 Percussion 2 min 80 400 Jemez 4 Percussion 2 min 83 376 Jemez Percussion 4 min 68 466 Jemez 2 Percussion 4 min 79 407 Jemez 3 Percussion 4 min 70 458 Jemez 4 Percussion 4 min 83 376 Jemez 1 Percussion 8 min 52 500 Jemez 2 Percussion 8 min 59 492 Jemez 3 Percussion 8 min 61 488 Jemez 4 Percussion 8 min 70 458 Magdalena 1 Immersion 69 462 Magdalena 2 Immersion 69 462 Magdalena J Immersion 70 458 Magdalena 4 Immersion 85 357 Magdalena 1 Boiling 30 sec 65 477 Magdalena 2 Boiling 30 sec 71 454 Magdalena 3 Boiling 30 sec 68 466
67
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Magdalena 4 Boiling 30 sec 83 376 Magdalena 1 Boiling 60 sec 63 483 Magdalena 2 Boiling 60 sec 68 466 Magdalena 3 Boiling 60 sec 63 483 Magdalena 4 Boiling 60 sec 82 384 Magdalena 1 Control 11 313 Magdalena 2 Control 6 237 Magdalena 3 Control 5 218 Magdalena 4 Control 4 196 Magdalena Percussion 1 min 32 466 Magdalena 2 Percussion 1 min 45 497 Magdalena 3 Percussion 1 min 46 498 Magdalena 4 Percussion I min 36 480 Magdalena 1 Percussion 2 min 67 470 Magdalena 2 Percussion 2 min 68 466 Magdalena 3 Percussion 2 min 65 477 Magdalena 4 Percussion 2 min 81 392 Magdalena 1 Percussion 4 min 87 336 Magdalena 2 Percussion 4 min 97 171 Magdalena 3 Percussion 4 min 83 376 Magdalena 4 Percussion 4 min 97 171 Magdalena 1 Percussion 8 min 98 lAO Magdalena 2 Percussion 8 min 100 000 Magdalena 3 Percussion 8 min 96 196 Magdalena 4 Percussion 8 min 98 lAO Manzano 1 Immersion 67 470 Manzano 2 Immersion 57 495 Manzano 3 Immersion 72 4049 Manzano 4 Immersion 73 4044 Manzano 1 Boiling 30 sec 70 458 Manzano 2 Boiling 30 sec 64 480 Manzano 3 Boiling 30 sec 71 454 Manzano 4 Boiling 30 sec 80 400 Manzano I Boiling 60 sec 50 500 Manzano 2 Boiling 60 sec 53 499 Manzano 3 Boiling 60 sec 59 492 Manzano 4 Boiling 60 sec 74 439 Manzano I Control 4 196 Manzano 2 Control 8 271 Manzano 3 Control 1 099 Manzano 4 Control 6 237 Manzano 1 Percussion 1 min 52 500 Manzano 2 Percussion 1 min 39 488 Manzano 3 Percussion 1 min 36 480 Manzano 4 Percussion 1 min 51 500
68
Table B 1 Continued
Source Block Treatment Gerrni~ation Percentage Standard Error
Manzano Percussion 2 min 53 499 Manzano 2 Percussion 2 min 58 494 Manzano 3 Percussion 2 min 61 488 Manzano 4 Percussion 2 min 62 485 Manzano 1 Percussion 4 min 76 427 Manzano 2 Percussion 4 min 85 357 Manzano 3 Percussion 4 min 79 407 Manzano 4 Percussion 4 min 83 376 Manzano 1 Percussion 8 min 87 336 Manzano 2 Percussion 8 min 83 376 Manzano 3 Percussion 8 min 83 376 Manzano 4 Percussion 8 min 75 433 Raton 1 Immersion 54 498 Raton 2 Immersion 37 483 Raton 3 Immersion 39 488 Raton 4 Immersion 69 462 Raton 1 Boiling 30 sec 36 480 Raton 2 Boiling 30 sec 20 400 Raton 3 Boiling 30 sec 28 449 Raton 4 Boiling 30 sec 60 490 Raton 1 Boiling 60 sec 15 357 Raton 2 Boiling 60 sec 171 Raton 3 Boiling 60 sec 4 196 Raton 4 Boiling 60 sec 35 477 Raton 1 Control 4 196 Raton 2 Control 12 325 Raton 3 Control 2 140 Raton 4 Control 6 237 Raton 1 Percussion 1 min 52 500 Raton 2 Percussion I min 29 454 Raton 3 Percussion I min 35 477 Raton 4 Percussion 1 min 31 462 Raton 1 Percussion 2 min 55 497 Raton 2 Percussion 2 min 35 477 Raton 3 Percussion 2 min 33 470 Raton 4 Percussion 2 min 42 494 Raton 1 Percussion 4 min 65 477 Raton 2 Percussion 4 min 52 500 Raton Percussion 4 min 51 500 Raton 4 Percussion 4 min 53 499 Raton 1 Percussion 8 min 88 325 Raton 2 Percussion 8 min 80 400 Raton 3 Percussion 8 min 73 444 Raton 4 Percussion 8 min 74 439 Sacramento 1 Immersion 43 495
69
Table B 1 Continued
-Source Block Treatment Germination Percentage Standard Error
Sacramento 2 Immersion 30 458 Sacramento 3 Immersion 38 485 Sacramento 4 Immersion 56 496 Sacramento 1 Boiling 30 sec 47 499 Sacramento 2 Boiling 30 sec 48 500 Sacramento 3 Boiling 30 sec 48 500 Sacramento 4 Boiling 30 sec 56 496 Sacramento 1 Boiling 60 sec 52 500 Sacramento 2 Boiling 60 sec 67 470 Sacramento 3 Boiling 60 sec 43 495 Sacramento 4 Boiling 60 sec 48 500 Sacramento 1 Control 0 000 Sacramento 2 Control 3 171 Sacramento 3 Control 3 171 Sacramento 4 Control 099 Sacramento 1 Percussion 1 min 57 495 Sacramento 2 Percussion 1 min 61 488 Sacramento 3 Percussion 1 min 62 485 Sacramento 4 Percussion 1 min 55 497 Sacramento 1 Percussion 2 min 84 367 Sacramento 2 Percussion 2 min 70 458 Sacramento 3 Percussion 2 min 81 392 Sacramento 4 Percussion 2 min 69 462 Sacramento 1 Percussion 4 min 91 286 Sacramento 2 Percussion 4 min 84 367 Sacramento 3 Percussion 4 min 84 367 Sacramento 4 Percussion 4 min 94 237 Sacramento 1 Percussion 8 min 82 384 Sacramento 2 Percussion 8 min 81 392 Sacramento 3 Percussion 8 min 82 384 Sacramento 4 Percussion 8 min 81 392 Sandia 1 Immersion 76 427 Sandia 2 Immersion 70 458 Sandia 3 Immersion 70 458 Sandia 4 Immersion 81 392 Sandia 1 Boiling 30 sec 78 414 Sandia 2 Boiling 30 sec 71 454 Sandia 3 Boiling 30 sec 63 483 Sandia 4 Boiling 30 sec 78 414 Sandia 1 Boiling 60 sec 67 470 Sandia 2 Boiling 60 sec 65 477 Sandia 3 Boiling 60 sec 74 439 Sandia 4 Boiling 60 sec 66 474 Sandia 1 Control 16 367 Sandia 2 Control 7 255
70
Table B 1 Continued
Source Block Treatment Gennination Percentage Standard Error
Sandia 3 Control 8 271 Sandia 4 Control 16 367 Sandia 1 Percussion 1 min 67 470 Sandia 2 Percussion 1 min 61 488 Sandia 3 Percussion 1 min 66 474 Sandia 4 Percussion 1 min 59 492 Sandia I Percussion 2 min 82 384 Sandia 2 Percussion 2 min 83 376 Sandia 3 Percussion 2 min 81 392 Sandia 4 Percussion 2 min 84 367 Sandia 1 Percussion 4 min 89 313 Sandia 2 Percussion 4 min 86 347 Sandia 3 Percussion 4 min 89 313 Sandia 4 Percussion 4 min 85 357 Sandia 1 Percussion 8 min 80 400 Sandia 2 Percussion 8 min 77 421 Sandia 3 Percussion 8 min 69 462 Sandia 4 Percussion 8 min 66 474
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Vines RA 1960 Trees Shrubs and Woody Vines of the Southwest Austin Univ ofTexas Press 1104 p
Wagner MR Larson RE Craig TP 1992 Ecology and silvics ofNew Mexico locust (Robinia neomexicana) with an emphasis on the arboreal form p53-57 In JW Hanover K Miller and S Plesko (eds) Proc Intemat Confon Black Locust Biology Culture and Utilization Mich State Univ USA
Wilson JK 1944 Immersing seeds of species of Robinia in boiling water hastens germination J For 42 453-454
Zimmerman RW Carpenter SB 1980 First-year coppice production from a 5shyyear old black locust stand on surface mine spoil p309-314 Centr Hardwd For ConfProc University ofMissouri Columbia
Table 3 Categorical Analysis of Variance table for germination response to seed treatment seed source block and the interaction of the factors
Source of DF Chi-Square Observed Variability Significance Level
Source 8 98370 lt00001 Trt 7 233876 lt00001
contrast 1 16976 lt00001 SourceTrt 56 97077 lt00001 Block 3 5538 lt00001
SourceBlock 24 10162 lt00001 TrtBlock 21 7564 lt00001
SourceTrtBlock 168 34283 lt00001
S 4)
8 ~
0
sectg ~ 40 ~
N 0 0
20
o f0ltgt ~ ~4 jybshy ((iltgto ~~ K1ltgt 6~b-~ ~~ 0b-~~ ~~ ~b-~ ~b-~b
80
60 -I-- - ~- middotmiddotmiddotmiddotmiddot1
r -] Northern NM Sources
Central NM Sources
Southern NM Sources
sect-o ~
~b-v(i
SOURCE
Figure 2 Effect ofNew Mexico locust seed source on germination for data averaged over all scarification treatments Control germination is shown by solid line within bar
80
~ cd ~ ltl) () 60 ~ ~
N
t 0shyrti tshySltl)
40
0
20
o lmmersion Boiling 30 sec Boiling 60 sec Control 1 min 2min 4 min 8 min
Hot Water Treatments Percussion Treatments
Figure 3 Effect of scarification on germination ofNew Mexico locust for data averaged over all seed sources Means labelled with the same letter are not significantly different
100~----------------------~--~----------------~
80
60
ltU
~
40 IJ
sectgsI
l100
80
60
40
a I middotmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot middotImiddotmiddotmiddotmiddotmiddotmiddot E
_~iii-bull _ _ bull f --~--
t-- -~ ---- -shy ---- shyI ~ ---- - shy
L
fiII
~
b
- shy ---r
- Holman
Sacramento Sandia Gila Jemez
c ~
ltgt ~
Tmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot-I
j~=~=-~~-1
I middotmiddot ~ bull r
t tr
Y -------M-a-gd-a-Ie-n-a--
1 _ _
fCapitan
---- Manzano ~y -_ Raton
2 4 8
Time interval ofpercussion scarification (min)
Figure 4 Effect of seed source by treatment interaction on New Mexico locust germination for percussion scarification pattern a and pattern b
28
tv 0
~ d
~ A s o
1 c3
80
60
40
20
o
TIT
t------=middotfmiddotmiddot-middotmiddot-middotmiddot-middotmiddot 1- --- - - --=- - -shy--cE= - _--shy --shy ----shy ~ ~ ---shy ~ -~- _ --shy-------r 1
I
Raton Holman
-_ Jemez
--- Sandia Manzano Magdalena Capitan Sacramento Gila
Immersion Boiling 30 sec Boiling 60 sec
Hot Water Scarification
Figure 5 Effect of seed source by treatment interaction on New Mexico locust germination for hot water scarification
continued to increase in germination (Figure 4b) There was no difference between
the two treatment levels for the Manzano source (Figure 4b)
The effect ofhot water scarification also varied among sources with few
overall differences between hot water treatments (Figure 5) However boiling for 60
seconds yielded highest germination for the Jemez source whereas increasing
durations ofboiling decreased germination for the Raton source
In addition to consistently high germination 4- and 8-minute percussionshy
treated seeds germinated as fast or faster than all other treatments (Figure 6 Table 4)
050 measured as the number of days needed to complete 50 ofeventual
germination averaged 2 days for both ofthese treatments
X-ray radiographs were taken of seeds collected from the Sacramento source
percussed for 1 4 or 8 minutes (Figure 7) Seeds show signs of damage at 8 minutes
ofpercussion (Figure 8) Compared to an undamaged seed percussed for only 4
minutes (Figure 8a) damage at the 8-minute level includes fractured radicles pitting
of the seed coat and chips in seed coat structure (Figure 8b-d)
Scanning electron microscope (SEM) images at 600X reveal the direct effect
of 4 minutes of percussion on cells of the strophiolar region (Figure 9) Whereas
untreated seeds retain cellular organization in the strophiolar region strophiolar cells
seeds treated for 4 minutes ofpercussion separate and raise resulting in a
~-~e~U appearance
30
Umiddot-~middot--l-~---middotymiddotmiddotmiddot-----middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot--middot--middotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middotmiddotmiddotmiddotmiddotmiddotmiddot- -
050= gt1
-e-- Percussion 8 min -0- Percussion 4 min ---T- Percussion 2 min -----ltil- Percussion 1 min -- Immersion --0- Boiling 30 sec -+- Boiling 60 sec -ltgt- Control
2 4 6 8 10 12
Day Figure 6 Timecourse ofNew Mexico locust gennination following scarification
treatments G50 is the number of days to 50 of eventual germination
31
14
Table 4 Effect of seed source and treatment on G50 (days to 50 germination)
Seed Source Control Immersion B30sec B60sec Plmin P2min P4min P8min mean Raton 3 3 5 7 4 3 2 2 4 Holman 2 2 2 2 2 2 2 2 2
Jemez 4 6 6 5 3 2 2 2 4 Sandia 2 2 2 2 2 2 2 2 2
w tv Manzano 5 2 2 3 3 2 2 2 3
Magdalena 4 2 2 3 4 3 2 1 3
Capitan 5 4 2 2 3 2 2 2 3
Sacramento 3 2 2 2 4 3 2 2 3 Gila 3 2 2 2 2 2 2 2 2 mean 3 3 3 3 3 2 2 2 3 standard 11 14 16 18 09 05 0 03 08 deviation
Note Columns and rows may not agree due to rounding
Figure 7 X-ray radiographs ofSacramento seed source percussed for 1 4 or 8 minutes
33
Figure 8a-d Seed damage close-ups Figure a shows undamaged seed percussed for 4 minutes Figures b-d show seeds percussed for 8 minutes Damage includes fracturing ofradicle in b complete removal ofseed coat in c and pitting ofseed coat in d
34
Untreated Treated
Figure 9 SEM images ofuntreated vs 4-minute percussion-treated strophiolar cells ofNew Mexico locust seeds Treated cells are loosened separated and raised
DISCUSSION
Determining the Optimal Treatment
Averaging results of scarification treatmems across seed sources is a useful
tool to predict which treatment is most likely to be successful for an unknown seed
source Averaged over all sources four- and eight-minute percussion treatments
resulted in the highest germination 815 and 790 respectively and produced
germination that was as fast or faster than all other treatments Although there was no
difference between these two treatments on average an argument for 4 minutes of
percussion as a standard protocol for treating New Mexico locust seeds can be made
Descriptively 4 minutes of percussion improved germination over 8 minutes
ofpercussion for a majority of sources (five out of nine) 8 minutes of percussion
improved germination for three ofnine sources with no difference for a fmal source
For individual sources 4 minutes ofpercussion was always the best or secondmiddot best
treatment and was always equal to or better than all hot water treatments
Germination at 8 minutes ofpercussion was lower than all other percussion durations
for the Jemez and Gila sources and was lower than at least one hot water treatment for
these sources as well
The x-ray radiographs of the Sacramento seed source illustrate the excessive
internal and external seed damage that can occur with 8 minutes of percussion
germination counts 8-minute percussed seeds from Holman and Capitan
ClUll signs of damage that were visible to the naked eye (see below)
r~evertheless some of these seeds still fit the criteria for germination Neither x-ray
36
photography nor visible observations during seed counts revealed seed damage at the
4-minute percussion level In operational trials seeds percussed for 4 minutes
developed into healthy seedlings (Khadduri and Harrington unpublished data)
Strophiole and Seed Coat Behavior
Four minutes ofpercussion successfully breaks seed dormancy in a large
percentage ofNew Mexico locust seeds without excessively damaging the
surrounding seed coat or internal seed coat structures Most papilionoid legume
seeds including seeds ofNew Mexico locust have a specific region of the seed coat
known as the strophiole or lens (Hamly 1932) The strophiole is located on the
cotyledonary lobe of a locust seed (Figure 10 a-c) If seeds are not indiscriminately
damaged the strophiole is the first point of water entry to the embryo (Hopkinson
1997) When a papilionoid seed is percussed for the appropriate amount of time
repeated hits on the integrated seed coat loosen the constrained cells of the strophiolar
region without excessively damaging the rest of the seed coat (Ballard 1976) SEM
photos illuStrate that 4 minutes of percussion loosened and separated strophiolar cells
ofNew Mexico locust seeds when compared with constrained organized strophiolar
cells of untreated seeds (Figure 9) Research-has shown that water passes to the
embryo through the treated strophiole in a controlled manner and regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues
(Kelly and van Staden 1987)
37
10 Strophiole ofunimbibed New Mexico locust seed (a) and following uptake (bc)
38
In contrast hot-water treatments can excessively degrade the seed coat
(Rebman et al 1998 Marunda 1990) Random cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rebman et aI 1998 Marunda 1990) Along with a
proportionately large amount ofun-imbibed (thus under-treated) seeds (Figure l1a)
damage was observed in hot-water treated seeds from six sources during germination
counts Damage included seeds where the radicle failed to elongate (Figure 11 b)
seeds where the radicle elongated but was delayed in freeing itself from the seed coat
(Figure 11c) and seeds where the radicle completely broke off from the embryo
(Figure lId) In addition some seeds germinated in reverse (the cotyledon emerged
fIrst) If the radicle emerged completely from the seed coat they were counted as
germinated
While nearly all seeds treated for 8 minutes ofpercussion imbibed water
damage was evident in x-ray evaluations (Sacramento seed source) and in
germination counts (Holman and Capitan) at this treatment level Seed damage
during germination counts was similar to levels found in hot-water treated seeds
suggesting that over-treatment by both hot water and longer durations of percussion
may result in similar damage to seed structure
The Sacramento and Holman seed sources were two of the fIve sources that
opcrplltlpn in germination from the 4- to 8-minute durations of percussion
the benefIts ofloosening the strophiole as evidenced by imbibition of
all 8-minute treated seeds were outweighed by external and internal seed coat
39
a
b c d
11 Damage to New Mexico locust seeds observed during germination cotmts following hot water scarification 9a Hot water replicate including ~t seedlings and numerous unimbibed seeds 9b Close-up of seed where raallCle failed to elongate 9c Close-up ofseed where radicle elongated but was
-gtIn in freeing itself from the seed coat 9d Close-up ofseed where radicle broke off from embryo
40
damage (which also may be contributing to high imbibition) For the three sources
for which 8 minutes of percussion resulted in maximum germination extended
percussion of seeds necessary to loosen the strophiole may not have excessively
damaged the seed anatomy The Manzano source did not significantly change in
germination from 4 to 8 minutes of percussion suggesting that an increase in
permeable seeds at the 8-minute treatment duration was balanced by the number of
seeds that failed to germinate due to seed damage To summarize the 4-minute
percussion treatment improved germination over 8 minutes ofpercussion for a
majority of sources (five ofnine) the 8-minute percussion treatment improved
germination over 4-minutes ofpercussion for three sources and there was no
difference with the Manzano source Notably for the Raton source 8 minutes of
percussion increased germination by 235 percentage points over 4-minutes of
percussion (7875 vs 5525 respectively) the next best treatment Raton was the
only source to result in germination less than 75 for 4 minutes ofpercussion so a
grower may wish to apply longer percussion durations to lots that do not respond well
to this treatment level
Finally there was an interesting trend in the Jemez and Raton sources seed
that significantly contributed to the source by treatment interaction (Figure 12) A
~u test showed that the Jemez source had a significantly lighter seed weight than
other sources while the Raton source had an average seed weight For the Jemez
the longest duration of boiling increased germination Conversely only short
U4UVll) of percussion were needed to achieve high germination and the longest
41
80 _ Jemez a Raton
iD
~ C iD e iD
Q c 0 ~
c ~ iD 0
20
0------shy
Immersion Boiling 30 sec Boiling 60 sec
Hot Water Scarification
10degTr========~--------~------------------------------
80
60
40
20
_ Jemez Raton
0------shy
1 min 2 min 4 min 8 min
Percussion Scarification
Figure 12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources
42
b
--~----~~~~========~---- duration ofpercussion reduced germination For the Raton source longer durations
ofboiling consistently decreased germination whereas longer percussion treatments
consistently increased germination
A possible explanation is that a thick seed coat is associated with a loose
strophiole structure and that a thin seed coat is associated with a constrained
strophiole This contrast merits further study and may suggest an ecological
adaptation Further investigation ofthis phenomenon may contribute to successful
pre-screening ofunknown seed sources to determine optimal treatment leveL
43
CONCLUSION
Based on results obtained in pilot study (see Appendix) and thesis
experiments 4 minutes ofpercussion is recommended as a standard protocol for
treating an unknown source ofNew Mexico locust seed In addition to laboratory
results the 4-minute percussion treatment has resulted in germination greater than
80 and healthy seedling grow-outs for production purposes (unpublished data) In
laboratory experiments only one hundred seeds per 118 ml (40z) soil tin were
percussed at a time whereas for production we have treated up to five hundred seeds
per tin For large-scale production ofNew Mexico locust seedlings growers may
wish to experiment with larger quantities of seed and larger-size soil tins
Seed storage trials following percussion treatments were not conducted
However germination greater than 80 and healthy seedlings were obtained when
seeds were percussion-treated and subsequently stored at room-temperature (22degC)
for one week (unpublished data) Seed storage trials merit further study as the
standard hot water treatment leaves seeds in a softened condition that is not
conducive to storage
Hydroseeding is an effective method for direct seeding in reclamation
situations Percussion treatment may be conducive to hydro seeding as seeds remain
a dry relatively undamaged yet permeable condition following treatment Sulfuric
seeds also remain dry following treatment but extensive damage to seed coat
poor seedling vigor have been reported with this treatment (Kelly and van Staden
A grow-out study using a hydro seeder on a reclamation site testing percussion
44
against other treatments such as sulfuric acid and mechanical scarification also
merits further study
Papilionoids are the largest subfamily oflegumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust these legumes often play an integral role in the revegetation of
disturbed lands The pilot study (Appendix) indicates that percussion scarification
successfully treats the widely used black locust as well Developing superior
scarification methods to the standard hot water mechanical scarification and acid
treatments may facilitate the use ofpapilionoid legumes in reclamation projects
45
APPENDICES
APPENDIX A
PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR
NEW MEXICO LOCUST AND BLACK LOCUST SEEDS
Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4
Hot water and sulfuric acid soaks are traditional treatments for seeds of many
temperate woody legwnes including locusts However these scarification techniques
often produce inconsistent germination Percussion scarification where seeds are
repeatedly propelled against a hard surface was compared with hot water
scarification to evaluate treatment efficacy for New Mexico locust (Robinia
neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment
seeds were placed in a 98degC water bath which was immediately removed from the
heat source For percussion scarification seeds were placed in a soil sample tin and
agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the
control were followed by 24-hour water soaks Hot water baths resulted in 56 and
41 germination for New Mexico locust and black locust respectively For both
species nearly all durations ofpercussion increased germination over the hot water
treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3
4 and 5 minutes for black locust resulted in at least 90 germination Traditional
scarification treatments randomly degrade the entire seed coat which can lead to
tissue damage during water uptake Percussion scarification specifically weakens the
strophiole the natural source ofwater entry to the seed in papilionoid legwnes
INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom
2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031
48
Following percussion imbibition is controlled through the strophiole and underlying
tissue is protected
Keywords New Mexico locust black locust scarification percussion germination
Introduction
Locust trees (Robinia spp) are aggressive pioneer species that quickly
colonize disturbed land fix atmospheric nitrogen in the soil and stabilize slopes that
are prone to erosion (Klemmedson 1994 Ashby et al 1985) (Figure A1ab)
Recognition of these attributes has contributed to increased demand for locusts in
reclamation projects Locust seeds however exhibit physical dormancy where a
thick seed coat prevents the movement of water and gasses to the embryo (Leadem
1997)
Sulfuric acid or hot water soaks have traditionally been used to break seed
dormancy in macro-propagation of locusts Many growers are moving away from the
use of su1furic acid in treating locust seeds (Dreesen and Harrington 1997) In
addition to being dangerous acid soak durations must be specifically correlated to
seed lot (Olson 1974) Collections of native seed can vary tremendously in seed size
seed weight and hard seededness etc This variability limits the utility of sulfuric
acid soaks as even within a given lot some seeds may be damaged whereas others
are under-treated and fail to imbibe (take up water) A survey of Southwestern
container growers indicates that hot water baths are currently the most common
scarification method for locust seeds (Hine et al 1997) However hot water baths
also produce inconsistent germination (Lin et al 1996)
49
b
Figure AI New Mexico locust a primary invader following disturbance Ala Crown-sprouting at edge ofburn near Los Alamos NM--one year after Cerro Grande fire Alb Colonizing road cut Gila National Forest NM
50
Percussion scarification where seeds are repeatedly propelled against a hard
surface is an alternative dormancy-breaking method that has proven successful in
legumes related to locusts (Hamly 1932 Barton 1947) We compared this alternative
treatment to hot water scarification to evaluate treatment efficacy for New Mexico
locust and black locust seeds
Materials and Methods
Commercial New Mexico locust seeds (Western Native Seed Coaldale CO)
were collected fall 2000 in Huerfano County CO Black locust seeds were collected
September 2000 from Taos County NM by harvesting ripe pods from several trees at
a distance of up to 3 m from ground level Black locust seeds were allowed to air-dry
for 3 weeks and were threshed and separated from large chaffusing a greenhouse
fan A South Dakota Seed Blower (Chicago Illinltois) was used to remove fine chaff
from both seed lots Seeds were storedmiddotat 2-4C until the germination study was
conducted in March 2001
This study compared hot water scarification with percussion scarification
using treatment and species as experimental factors Along with a control
scarification treatments included immersion of seed in boiling water as well as
percussion durations of 1 2 3 45 or 10 minutes Four 100-seed samples were used
to test each species by scarification treatment combination
For the hot water treatment test tubes were filled with 30 mL water and
placed in a water-filled beaker (Figure A2a) The bath was raised to boiling (-98C at
51
b
c
Figure A2 Implementation ofhot water and percussion scarification A2a Hot water bath A2b Paint-shaker set-up A2c Placing soil tin containing seeds at end ofpaint shaker ann
52
Las Cruces NM elevation of 1300 meters) Each 100-seed replicate was placed in a
test tube and each test tube was immediately removed to cool at room temperature
Percussion scarification was implemented using a pneumatic paint shaker
(Central Pneumatic model 00422 Camarillo CA) (Figure A2b) This allowed
standardization of treatment intensity Compressed air pressure was maintained at
530kPa plusmn 30kPa (80psi plusmn 5psi) resulting in approximately 350 oscillations per
minute Each lOO-seed sample was placed in a 118 ml (4 oz) soil tin for shaking To
maximize lateral movement of paint shaker a paint can was used as a spacer to place
soil tins at the end of the shaking arm (Figure A2c) This allowed shaking to occur at
the greatest distance from the pivot point (fulcrum) Following all treatments
including the control seeds were soaked for twenty-four hours in distilled water prior
to plating out
Gennination was evaluated on lab benches at room temperature Each
replicate of seeds was placed in a 100 cm petri dish on moistened filter paper with
humidity maintained by enclosing the petri dishes in ziplock bags Gennination was
monitored daily for 14 days in accordance with International Seed Testing
Association standards for Robinia species (ISTA 1999) Gennination was defined as
protrusion of the radicle from the embryo by at least 1mm as viewed by the naked
eye
Categorical Analysis of Variance using Prpc Catmod (SAS Institute 1989)
was used to detennine treatment differences in germination percentages for each
source This procedure is an extension of a Chi-square test of homogeneity using the
53
natural log of the ratio of germinated to non-germinated seeds for each treatment Pshy
values less than 005 were considered significant A limited set of pairwiseshy
comparisons was conducted to compare treatment mean percents using a
conservative alpha value of 005 divided by the number of comparisons
Results
Percussion maximized germination for both New Mexico locust and black
locust There were three germination response levels for New Mexico locust (Figure
A3) First there was a low response in the control indicating a dormant seed lot
The hot water treatment and I-minute duration of percussion share a second
intermediate response Germination was highest at percussion levels from 2 to 10
minutes
A selected percussion treatment intermediate in time (5 minutes) produced the
steepest germination curve indicating the fastest germination rate (Figure A4) The
hot water treatment produced an intermediate curve or relatively slower germination
rate The control curve was very flat indicating the slowest germination rate for New
Mexico locust
Control seeds germinated poorly for black locust as well (Figure A5) Hot
water scarification improved germination over the control but all levels of percussion
improved germination over hot water The optimal durations of percussion treatment
were bracketed with a significant increase from the 1 to 2 minute percussion level a
high germination response from 2 to 5 minutes of percussion and finally a significant
54
~ d 25 CL)
p t1 0 ~ t1
g Vl ~ Vl
100~---------------------------------------------------------------
80
60
40
20
o Control Hot Water Percussion
I min Percussion
2 min Percussion
3 min
Scarification
Percussion 4 min
Figure A3 Effect of scarification on germination response for New Mexico locust
Percussion 5 min
=significant difference between adjacent treatment bars ns = no significant difference
Percussion 10 min
~ ~ () u agt
Il-t d o ~
~ ~ c3
100middotlr~~~------------------------------------e- Percussion 5min middot 0middot Hot Water -- Control
80-J=middot======
6b-middot-lmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middot--middotmiddot-middot~-middotmiddot -middotmiddot middot middotmiddot-middot 1
40
20
-----~-----------
0 0 0 middotmiddotmiddotmiddotmiddot0middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot0
0 --~-----y-----~------_---~--- T-----~
o shy -TDay 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A4 Effect of selected scarification treatments on germination speed for New Mexico locust First seven days of fourteen-day experiment are shown
Day 7
80
~ cd
5 60 ()
[) Cl d 0 1t1 d
40sect dUl
-J
20
o
ns
Scarification
Figure A5 Effect of scarification on germination response for black locust = significant difference between adjacent treatment bars ns = no significant difference
decrease at the 10-minute level This drop-off suggests that 10 minutes of percussion
over-treats seeds from this lot of-black locust
A selected percussion treatment intermediate in time (5 minutes) also had the
fastest germination rate for black locust (Figure A6)_ The hot water curve is
somewhat flatter indicating a slower germination rate The control curve is very flat
indicating a slow germination rate
A qualitative difference between hot water and percussion-treated seeds was
also observed Along with healthy germinants the hot water treatment produced
over-treated seeds (Figure A 7a) Examples of damaged germinants include seeds
where the radicle did not elongate (Figure A 7b) seeds where the radicle elongated
but was delayed in freeing itself from the seed coat (Figure A7c) and seeds where the
radicle broke off completely from the embryo (Figure A7d) In addition to these
examples of over-treatment there were numerous small dark seeds that failed to
imbibe water For percussion treatment nearly all seeds took up water (Figure A8a)
Germination was rapid and uniform with little damage evident (Figure A8b)
To summarize both New Mexico locust and black locust seed lots were
dormant The standard hot water scarification treatment improved germination but
only to 56 and 41 for New Mexico locust and black locust respectively Nearly
all percussion durations improved total germination as well as germination rate over
the hot water treatment Germination was greater than 90 for percussion durations
of 4 5 and 10 minutes for New Mexico locust and 3 4 and 5 minutes for black locust
58
CI)
sgt t CI) 0
igt 0
sect ttl t E
Vl 0
c3
100r==~==~~=r------------------------------------~ ___ Percussion 5 min
middotmiddotmiddot0middotmiddotmiddot Hot Water -lIf- Control
80r=========~--~~----------------------------~
60
40
20 -~-~---~---------- ---~------------I
omiddot
O
_~_----------lIf-----~bull -----_ shy---shy I
Day 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A6 Effect of selected scarification treatments on germination speed for black locust First seven days of fourteen-day experiment are shown
Day 7
a
b c d
Figure A7 Damage to New Mexico locust seeds observed during germination counts following hot water scarification A7a Hot water replicate including damaged seedlings and numerous unimbibed seeds A7b Close-up ofseed where radicle failed to elongate A7c Close-up ofseed where radicle elongated but was delayed in freeing itself from the seed coat A7d Close-up ofseed where radicle completely broke offfrom embryo
60
a
b
Figure A8 New Mexico locust seeds following percussion treatment at time of germination counts A8a Percussion replicate with uniformly healthy seedlings and near complete imbibition of seeds A8b Close-up ofpercussion-treated seedling
61
Discussion
Papilionoid seeds have a specific anatomical feature known as the strophiole
or lens-the natural site of water entry to the seed (Hamly 1932) The strophiole is
located on the cotyledonary lobe of a locust seed (Figure A9) When a papilionoid
legume seed is percussed for the appropriate amount of time repeated hits on the
integrated seed coat loosen the constrained cells of the strophiolar region without
excessively damaging the rest of the seed coat (Ballard 1976)
When a percussed seed is soaked water enters exclusively through the
strophiole in a controlled manner (Kelly and Staden 1987) This regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues This
contrasts with seeds that have been hot water or acid scarified treatments that can
randomly degrade the seed coat Unlocalized cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rehman et al 1998 Marunda 1990)
Papilionoids are the largest subfamily of legumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust and black locust these legumes often play an integral role in the
revegetation ofdisturbed lands Developing superior scarification methods to the
standard hot water and acid treatments should fadlitate the use ofthese legumes in
restoration projects
62
Figure A9 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (bc)
63
APPENDIXB
GERMINATION PERCENTS AND STANDARD ERRORS FOR SOURCE
BLOCK AND TREATMENT FOR NEW MEXICO LOCUST
Table B 1 Gennination percents and standard errors for source block and treatment for New Mexico locust
Source Block Treatment Germination Percentage Standard Error
Capitan 1 Immersion 74 437 Capitan 2 Immersion 69 462 Capitan 3 Immersion 71 454 Capitan 4 Immersion 67 470 Capitan Boiling 30 sec 79 407 Capitan 2 Boiling 30 sec 76 427 Capitan 3 Boiling 30 sec 72 449 Capitan 4 Boiling 30 sec 76 427 Capitan Boiling 60 sec 71 454 Capitan 2 Boiling 60 sec 54 498 Capitan 3 Boiling 60 sec 67 470 Capitan 4 Boiling 60 sec 76 427 Capitan I Control 5 218 Capitan 2 Control 7 255 Capitan 3 Control 4 196 Capitan 4 Control 5 218 Capitan 1 Percussion I min 47 499 Capitan 2 Percussion I min
470
Capitan Percussion I min 39 488
Capitan 4 Percussion I min 38 485 Capitan I Percussion 2 min 55 497 Capitan Capitan
2
Percussion 2 min Percussion 2 min
71 65
454 477
Capitan 4 Percussion 2 min 56 496 Capitan I Percussion 4 min 86 347 Capitan 2 Percussion 4 min 79 407 Capitan 3 Percussion 4 min 81 392 Capitan 4 Percussion 4 min 82 384 Capitan I Percussion 8 min 90 300 Capitan 2 Percussion 8 min 88 325 Capitan 3 Percussion 8 min 90 300 Capitan 4 Percussion 8 min 90 300 Gila I Immersion 71 454 Gila 2 Immersion 77 421 Gila
Immersion 75 433
Gila 4 Immersion 72 449
Gila I Boiling 30 sec 68 466
Gila 2 Boiling 30 sec 81 392
Gila 3 Boiling 30 sec 54 498
Gila 4 Boiling 30 sec 63 483
Gila I Boiling 60 sec 55 497
Gila 2 Boiling 60 sec 68 466
Gila 3 Boiling 60 sec 57 495
Gila 4 Boiling 60 sec 70 458
65
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Gila 2 Control 13 336 Gila 3 Control 13 336 Gila 4 Control 20 400 Gila 1 Percussion 1 min 66 474 Gila 2 Percussion 1 min 57 495 Gila 3 Percussion 1 min 73 444 Gila 4 Percussion 1 min 82 384 Gila 1 Percussion 2 min 81 392 Gila 2 Percussion 2 min 86 347 Gila 3 Percussion 2 min 85 357 Gila 4 Percussion 2 min 54 498 Gila 1 Percussion 4 min 79 407 Gila 2 Percussion 4 min 83 376 Gila 3 Percussion 4 min 84 367 Gila 4 Percussion 4 min 79 407 Gila 1 Percussion 8 min 51 500 Gila 2 Percussion 8 min 76 427 Gila 3 Percussion 8 min 52 500 Gila 4 Percussion 8 min 74 439 Holman 1 Immersion 83 376 Holman 2 Immersion 86 347 Holman 3 Immersion 81 392 Holman 4 Immersion 90 300 Holman 1 Boiling 30 sec 90 300 Holman 2 Boiling 30 sec 85 357 Holman 3 Boiling 30 sec 87 336 Holman 4 Boiling 30 sec 83 376 Holman 1 Boiling 60 sec 79 407 Holman 2 Boiling 60 sec 80 400 Holman 3 Boiling 60 sec 86 347 Holman 4 Boiling 60 sec 93 255 Holman Control 25 433 Holman 2 Control 19 392 Holman Control 21 407
Holman 4 Control 24 427
Holman Percussion 1 min 77 421
Holman 2 Percussion 1 min 85 357
Holman 3 Percussion 1 min 80 400
Holman 4 Percussion 1 min 89 313
Holman 1 Percussion 2 min 88 325
Holman 2 Percussion 2 min 96 196
Holman 3 Percussion 2 min 93 255
Holman 4 Percussion 2 min 96 196
Holman 1 Percussion 4 min 92 271
Holman 2 Percussion 4 min 97 171
66
I
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Holman 3 Percussion 4 min 91 286 Holman 4 Percussion 4 min 96 196 Holman 1 Percussion 8 min 88 325 Holman 2 Percussion 8 min 85 357 Holman 3 Percussion 8 min 83 376 Holman 4 Percussion 8 min 82 384 Jemez 1 Immersion 53 499 Jemez 2 Immersion 44 496 Jemez 3 Immersion 52 500 Jemez 4 Immersion 70 458 Jemez 1 Boiling 30 sec 58 494 Jemez 2 Boiling 30 sec 55 497 Jemez 3 Boiling 30 sec 48 500 Jemez 4 Boiling 30 sec 65 477 Jemez 1 Boiling 60 sec 68 466 Jemez 2 Boiling 60 sec 63 483 Jemez 3 Boiling 60 sec 73 444 Jemez 4 Boiling 60 sec 89 313 Jemez 1 Control 2 140 Jemez 2 Control 9 286 Jemez 3 Control 6 237 Jemez 4 Control 8 271 Jemez 1 Percussion 1 min 58 494 Jemez 2 Percussion 1 min 63 483 Jemez 3 Percussion 1 min 77 421 Jemez 4 Percussion 1 min 89 313 Jemez 1 Percussion 2 min 68 466 Jemez 2 Percussion 2 min 64 480 Jemez 3 Percussion 2 min 80 400 Jemez 4 Percussion 2 min 83 376 Jemez Percussion 4 min 68 466 Jemez 2 Percussion 4 min 79 407 Jemez 3 Percussion 4 min 70 458 Jemez 4 Percussion 4 min 83 376 Jemez 1 Percussion 8 min 52 500 Jemez 2 Percussion 8 min 59 492 Jemez 3 Percussion 8 min 61 488 Jemez 4 Percussion 8 min 70 458 Magdalena 1 Immersion 69 462 Magdalena 2 Immersion 69 462 Magdalena J Immersion 70 458 Magdalena 4 Immersion 85 357 Magdalena 1 Boiling 30 sec 65 477 Magdalena 2 Boiling 30 sec 71 454 Magdalena 3 Boiling 30 sec 68 466
67
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Magdalena 4 Boiling 30 sec 83 376 Magdalena 1 Boiling 60 sec 63 483 Magdalena 2 Boiling 60 sec 68 466 Magdalena 3 Boiling 60 sec 63 483 Magdalena 4 Boiling 60 sec 82 384 Magdalena 1 Control 11 313 Magdalena 2 Control 6 237 Magdalena 3 Control 5 218 Magdalena 4 Control 4 196 Magdalena Percussion 1 min 32 466 Magdalena 2 Percussion 1 min 45 497 Magdalena 3 Percussion 1 min 46 498 Magdalena 4 Percussion I min 36 480 Magdalena 1 Percussion 2 min 67 470 Magdalena 2 Percussion 2 min 68 466 Magdalena 3 Percussion 2 min 65 477 Magdalena 4 Percussion 2 min 81 392 Magdalena 1 Percussion 4 min 87 336 Magdalena 2 Percussion 4 min 97 171 Magdalena 3 Percussion 4 min 83 376 Magdalena 4 Percussion 4 min 97 171 Magdalena 1 Percussion 8 min 98 lAO Magdalena 2 Percussion 8 min 100 000 Magdalena 3 Percussion 8 min 96 196 Magdalena 4 Percussion 8 min 98 lAO Manzano 1 Immersion 67 470 Manzano 2 Immersion 57 495 Manzano 3 Immersion 72 4049 Manzano 4 Immersion 73 4044 Manzano 1 Boiling 30 sec 70 458 Manzano 2 Boiling 30 sec 64 480 Manzano 3 Boiling 30 sec 71 454 Manzano 4 Boiling 30 sec 80 400 Manzano I Boiling 60 sec 50 500 Manzano 2 Boiling 60 sec 53 499 Manzano 3 Boiling 60 sec 59 492 Manzano 4 Boiling 60 sec 74 439 Manzano I Control 4 196 Manzano 2 Control 8 271 Manzano 3 Control 1 099 Manzano 4 Control 6 237 Manzano 1 Percussion 1 min 52 500 Manzano 2 Percussion 1 min 39 488 Manzano 3 Percussion 1 min 36 480 Manzano 4 Percussion 1 min 51 500
68
Table B 1 Continued
Source Block Treatment Gerrni~ation Percentage Standard Error
Manzano Percussion 2 min 53 499 Manzano 2 Percussion 2 min 58 494 Manzano 3 Percussion 2 min 61 488 Manzano 4 Percussion 2 min 62 485 Manzano 1 Percussion 4 min 76 427 Manzano 2 Percussion 4 min 85 357 Manzano 3 Percussion 4 min 79 407 Manzano 4 Percussion 4 min 83 376 Manzano 1 Percussion 8 min 87 336 Manzano 2 Percussion 8 min 83 376 Manzano 3 Percussion 8 min 83 376 Manzano 4 Percussion 8 min 75 433 Raton 1 Immersion 54 498 Raton 2 Immersion 37 483 Raton 3 Immersion 39 488 Raton 4 Immersion 69 462 Raton 1 Boiling 30 sec 36 480 Raton 2 Boiling 30 sec 20 400 Raton 3 Boiling 30 sec 28 449 Raton 4 Boiling 30 sec 60 490 Raton 1 Boiling 60 sec 15 357 Raton 2 Boiling 60 sec 171 Raton 3 Boiling 60 sec 4 196 Raton 4 Boiling 60 sec 35 477 Raton 1 Control 4 196 Raton 2 Control 12 325 Raton 3 Control 2 140 Raton 4 Control 6 237 Raton 1 Percussion 1 min 52 500 Raton 2 Percussion I min 29 454 Raton 3 Percussion I min 35 477 Raton 4 Percussion 1 min 31 462 Raton 1 Percussion 2 min 55 497 Raton 2 Percussion 2 min 35 477 Raton 3 Percussion 2 min 33 470 Raton 4 Percussion 2 min 42 494 Raton 1 Percussion 4 min 65 477 Raton 2 Percussion 4 min 52 500 Raton Percussion 4 min 51 500 Raton 4 Percussion 4 min 53 499 Raton 1 Percussion 8 min 88 325 Raton 2 Percussion 8 min 80 400 Raton 3 Percussion 8 min 73 444 Raton 4 Percussion 8 min 74 439 Sacramento 1 Immersion 43 495
69
Table B 1 Continued
-Source Block Treatment Germination Percentage Standard Error
Sacramento 2 Immersion 30 458 Sacramento 3 Immersion 38 485 Sacramento 4 Immersion 56 496 Sacramento 1 Boiling 30 sec 47 499 Sacramento 2 Boiling 30 sec 48 500 Sacramento 3 Boiling 30 sec 48 500 Sacramento 4 Boiling 30 sec 56 496 Sacramento 1 Boiling 60 sec 52 500 Sacramento 2 Boiling 60 sec 67 470 Sacramento 3 Boiling 60 sec 43 495 Sacramento 4 Boiling 60 sec 48 500 Sacramento 1 Control 0 000 Sacramento 2 Control 3 171 Sacramento 3 Control 3 171 Sacramento 4 Control 099 Sacramento 1 Percussion 1 min 57 495 Sacramento 2 Percussion 1 min 61 488 Sacramento 3 Percussion 1 min 62 485 Sacramento 4 Percussion 1 min 55 497 Sacramento 1 Percussion 2 min 84 367 Sacramento 2 Percussion 2 min 70 458 Sacramento 3 Percussion 2 min 81 392 Sacramento 4 Percussion 2 min 69 462 Sacramento 1 Percussion 4 min 91 286 Sacramento 2 Percussion 4 min 84 367 Sacramento 3 Percussion 4 min 84 367 Sacramento 4 Percussion 4 min 94 237 Sacramento 1 Percussion 8 min 82 384 Sacramento 2 Percussion 8 min 81 392 Sacramento 3 Percussion 8 min 82 384 Sacramento 4 Percussion 8 min 81 392 Sandia 1 Immersion 76 427 Sandia 2 Immersion 70 458 Sandia 3 Immersion 70 458 Sandia 4 Immersion 81 392 Sandia 1 Boiling 30 sec 78 414 Sandia 2 Boiling 30 sec 71 454 Sandia 3 Boiling 30 sec 63 483 Sandia 4 Boiling 30 sec 78 414 Sandia 1 Boiling 60 sec 67 470 Sandia 2 Boiling 60 sec 65 477 Sandia 3 Boiling 60 sec 74 439 Sandia 4 Boiling 60 sec 66 474 Sandia 1 Control 16 367 Sandia 2 Control 7 255
70
Table B 1 Continued
Source Block Treatment Gennination Percentage Standard Error
Sandia 3 Control 8 271 Sandia 4 Control 16 367 Sandia 1 Percussion 1 min 67 470 Sandia 2 Percussion 1 min 61 488 Sandia 3 Percussion 1 min 66 474 Sandia 4 Percussion 1 min 59 492 Sandia I Percussion 2 min 82 384 Sandia 2 Percussion 2 min 83 376 Sandia 3 Percussion 2 min 81 392 Sandia 4 Percussion 2 min 84 367 Sandia 1 Percussion 4 min 89 313 Sandia 2 Percussion 4 min 86 347 Sandia 3 Percussion 4 min 89 313 Sandia 4 Percussion 4 min 85 357 Sandia 1 Percussion 8 min 80 400 Sandia 2 Percussion 8 min 77 421 Sandia 3 Percussion 8 min 69 462 Sandia 4 Percussion 8 min 66 474
LITERATURE CITED
Ashby WC Vogel WG Rogers NF 1985 Black locust in the reclamation equation General Technical Report Northeastern Forest Experiment Station USDA Forest Service NE-I05 12 p
Ballard LAT 1973 Physical barriers to gennination Seed Sci Technoll 285-303
___ 1976 Strophiolar water conduction in the seeds ofTrifolieae induced by action on the testa at nonstrophiolar sites Austr J Plant Physiol3 465-469
Barton LV 1947 Special studies on seed coat impenneability Contrib Boyce Thompson Inst 14 355-362
Baskin CC Baskin JM 1998 Seeds Ecology biogeography and evolution of donnancy and gennination Academic Press London UK 666 p
Bewley JD 1997 Seed germination and donnancy Plant Ce119 1055-1066
Bewley JD Black M 1994 Seeds physiology of development and germination 2nd ed Plenum Press New York NY 445 p
Briscoe CB 1996 Growing Nitrogen Fixing Trees in Acid Soils In MH Powell (ed) Nitrogen Fixing Trees for Acid Soils- A Field ManuaL Winrock International Morrilton AR 22 p
Copeland LO McDonald MB 1995 Principles of Seed Science and Technology 3rded Chapman amp Hall New York NY 409 p
Cox RE Klett JE 1984 Seed gennination requirements of native Colorado plants for use in the landscape Plant Propag 30(2) 6-10
Dell B 1980 Structure and function ofthe strophiolar plug in seeds ofAlbizia Lophantha Am J Bot 67 556-563
Dick-Peddie WA 1993 New Mexico Vegetation Past Present and Future University ofNew Mexico Press Albuquerque NM 244 p
Dreesen DR 2001 Propagation protocol for production ofcontainer Robinia neomexicana plants Los Lunas Plant Materials Center Los Lunas New Mexico In Native Plant Network URL httpwwwnativeplantnetworkorg (accessed 9 January 2002) Moscow (ID) University ofIdaho CoIl Natural Resources For Res Nursery
72
Dreesen DR Harrington JT 1997 Propagation of native plants for restoration projects in the Southwestern US- Preliminary Investigations In Landis TD Thompson JR tech coords National Proceedings Forest and Conservation Nursery Assocs Gen Tech Rep PNW-GTR-419 Portland OR USDA For Ser PNW Res Sta 77-88
Gottfried GJ 1980 Control ofNew Mexican locust and the effect on planted ponderosa pine in central Arizona US For Ser Res Note RM-386 Rocky Mountain For Range ExpStn Fort Collins CO
Gosling PG Samuel YK Jones SK 1995 A systematic examination of germination temperature chipping and water temperaturesoak duration pretreatments on the seeds ofLeucaena leucocephala Seed Sci amp Technol 23521-532
Hamly DH 1932 Softening of the seeds of Melilotus alba Bot Gazette 93 345shy375
Hanna PJ 1984 Anatomical features of the seed coat ofAcacia kempeana (Mueller) which relate to increased germination rate induced by heat treatment New Phytol 96 23-29
Hanks JP Dick-Peddie WA 1974 Vegetation patterns of the White Mountains New Mexico Southwest Naturalist 18(4) 371-382
Hine S Harrington JT Dreesen DR 1997 Seed scarification requirements for Robinia neomexicana In Harrington JTtech coord Proc Third SouthwestemContainer Growers Conf Feb 12-13 1997 Ft Collins CO
Hopkinson JM 1993 The strophiole of leguminous seeds Newsletter- IntI Herbage Seed Production Res Grp 18 7-8
---- 1997 Differences in hardseededness between Cunningham and Tarramba leucaena In Leucnet News The Newsletter of the International LeucaenaRes and Devlt Netwk No4
ISTA (International Seed Testing Association) 1999 International rules for seed testing rules Seed Sci amp Technol27 Supplement
Kelly KM van Staden J 1987 The lens as the site of permeability in the papilionoid seed Aspalathus linearis J Plant Physiol 128(45) 395-404
73
Keresztesi B 1988 Black locust the tree of agriculture Outlook on Agr 17(2) 77~85
Kigel 1 and Galili G 1995 Seed development and germination Marcel Dekker New York 853 p
Klemmedson 10 1994 New Mexico locust and parent material Influence on macronutrients of forest floor and soil Soil Sci Soc Am 1 58 974~980
Kuo WH Tam AW 1988 The pathway of water absorption of mung bean seeds Seed Sci amp Technol16(1) 139~144
Leadem CL 1997 Dormancy--unlocking seed secrets In Landis TD Thompson lR tech coords National Proceedings Forestand Conservation Nursery Assocs Gen Tech Rep PNW-G TR-419 Portland OR USDA For Ser PNW Res Sta 43-52
Lin Y Wagner MR Seweyestewa A Honahni MD DeGomez M McMillin JD 1996 Influence of cultural treatments on seed germination and seedling growth of Robinia neomexicana Forest Farm and Community Tree Res Reports Volume 1
Marunda CT 1990 Effects of seed pretreatment on the development ofAcacia auriculforinis and A holosericea seedlings ACIAR Proceedings Series No 28
Meginnis HG 1937 Sulphuric acid treatment to increase germination of black locust seed USDA Circ 453 34 p
Miklas PN Towensend CE Ladd SL 1987 Seedcoat anatomy and the scarification ofcicer milkvetch seed Crop Sci 27 766-772
Monsen SB 1984 Use of shrubs on mine spoils In The Challenge ofProducing Native Plants for the Intermountain Area Proceedings Intermountain Nurserymans Association 1983 conference Las Vegas NV compo PM Murphy 26-31 Ogden UT USDA For Ser Gen Tech Report INT-l68
Olson DF 1974 Robinia L Locust In Seeds of Woody Plants in the US tech coord CS Schopmeyer 728-731 USDA For Ser Agr Handbk No450
Rehman S Loescher RNl Harris P1C 1998 Dormancy breaking and germination ofAcacia salicina Lindl seeds Seed Sci amp Techno127 553-557
Rolston MP 1978 Water impermeable seed dormancy Bot Rev 44 365-396
74
SAS Institute Inc 1989 SASSTAT Users Guide Version 6 4th Edition Volume 1 Cary NC SAS Institute Inc 943 p
SerratoValenti G Conara L Ferrando M Modenesi P 1993 Structural and histochemical features ofStylosanthes scabra (Leguminosae Papilionoidae) seed coat as related to water entry Can J Bot 71 834-840
Simpson BJ 1988 A field guide to Texas trees Austin TX Texas Monthly Press 372p
Tran VN Cavanagh AK 1984 Structural aspects ofdormancy In Seed Physiology Vol 2 Murray DR (ed) p1-44 Academic Press Australia
USDA Forest Service 1948 Woody-plant seed manual USDA Misc Pub1654 416p
Vines RA 1960 Trees Shrubs and Woody Vines of the Southwest Austin Univ ofTexas Press 1104 p
Wagner MR Larson RE Craig TP 1992 Ecology and silvics ofNew Mexico locust (Robinia neomexicana) with an emphasis on the arboreal form p53-57 In JW Hanover K Miller and S Plesko (eds) Proc Intemat Confon Black Locust Biology Culture and Utilization Mich State Univ USA
Wilson JK 1944 Immersing seeds of species of Robinia in boiling water hastens germination J For 42 453-454
Zimmerman RW Carpenter SB 1980 First-year coppice production from a 5shyyear old black locust stand on surface mine spoil p309-314 Centr Hardwd For ConfProc University ofMissouri Columbia
S 4)
8 ~
0
sectg ~ 40 ~
N 0 0
20
o f0ltgt ~ ~4 jybshy ((iltgto ~~ K1ltgt 6~b-~ ~~ 0b-~~ ~~ ~b-~ ~b-~b
80
60 -I-- - ~- middotmiddotmiddotmiddotmiddot1
r -] Northern NM Sources
Central NM Sources
Southern NM Sources
sect-o ~
~b-v(i
SOURCE
Figure 2 Effect ofNew Mexico locust seed source on germination for data averaged over all scarification treatments Control germination is shown by solid line within bar
80
~ cd ~ ltl) () 60 ~ ~
N
t 0shyrti tshySltl)
40
0
20
o lmmersion Boiling 30 sec Boiling 60 sec Control 1 min 2min 4 min 8 min
Hot Water Treatments Percussion Treatments
Figure 3 Effect of scarification on germination ofNew Mexico locust for data averaged over all seed sources Means labelled with the same letter are not significantly different
100~----------------------~--~----------------~
80
60
ltU
~
40 IJ
sectgsI
l100
80
60
40
a I middotmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot middotImiddotmiddotmiddotmiddotmiddotmiddot E
_~iii-bull _ _ bull f --~--
t-- -~ ---- -shy ---- shyI ~ ---- - shy
L
fiII
~
b
- shy ---r
- Holman
Sacramento Sandia Gila Jemez
c ~
ltgt ~
Tmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot-I
j~=~=-~~-1
I middotmiddot ~ bull r
t tr
Y -------M-a-gd-a-Ie-n-a--
1 _ _
fCapitan
---- Manzano ~y -_ Raton
2 4 8
Time interval ofpercussion scarification (min)
Figure 4 Effect of seed source by treatment interaction on New Mexico locust germination for percussion scarification pattern a and pattern b
28
tv 0
~ d
~ A s o
1 c3
80
60
40
20
o
TIT
t------=middotfmiddotmiddot-middotmiddot-middotmiddot-middotmiddot 1- --- - - --=- - -shy--cE= - _--shy --shy ----shy ~ ~ ---shy ~ -~- _ --shy-------r 1
I
Raton Holman
-_ Jemez
--- Sandia Manzano Magdalena Capitan Sacramento Gila
Immersion Boiling 30 sec Boiling 60 sec
Hot Water Scarification
Figure 5 Effect of seed source by treatment interaction on New Mexico locust germination for hot water scarification
continued to increase in germination (Figure 4b) There was no difference between
the two treatment levels for the Manzano source (Figure 4b)
The effect ofhot water scarification also varied among sources with few
overall differences between hot water treatments (Figure 5) However boiling for 60
seconds yielded highest germination for the Jemez source whereas increasing
durations ofboiling decreased germination for the Raton source
In addition to consistently high germination 4- and 8-minute percussionshy
treated seeds germinated as fast or faster than all other treatments (Figure 6 Table 4)
050 measured as the number of days needed to complete 50 ofeventual
germination averaged 2 days for both ofthese treatments
X-ray radiographs were taken of seeds collected from the Sacramento source
percussed for 1 4 or 8 minutes (Figure 7) Seeds show signs of damage at 8 minutes
ofpercussion (Figure 8) Compared to an undamaged seed percussed for only 4
minutes (Figure 8a) damage at the 8-minute level includes fractured radicles pitting
of the seed coat and chips in seed coat structure (Figure 8b-d)
Scanning electron microscope (SEM) images at 600X reveal the direct effect
of 4 minutes of percussion on cells of the strophiolar region (Figure 9) Whereas
untreated seeds retain cellular organization in the strophiolar region strophiolar cells
seeds treated for 4 minutes ofpercussion separate and raise resulting in a
~-~e~U appearance
30
Umiddot-~middot--l-~---middotymiddotmiddotmiddot-----middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot--middot--middotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middotmiddotmiddotmiddotmiddotmiddotmiddot- -
050= gt1
-e-- Percussion 8 min -0- Percussion 4 min ---T- Percussion 2 min -----ltil- Percussion 1 min -- Immersion --0- Boiling 30 sec -+- Boiling 60 sec -ltgt- Control
2 4 6 8 10 12
Day Figure 6 Timecourse ofNew Mexico locust gennination following scarification
treatments G50 is the number of days to 50 of eventual germination
31
14
Table 4 Effect of seed source and treatment on G50 (days to 50 germination)
Seed Source Control Immersion B30sec B60sec Plmin P2min P4min P8min mean Raton 3 3 5 7 4 3 2 2 4 Holman 2 2 2 2 2 2 2 2 2
Jemez 4 6 6 5 3 2 2 2 4 Sandia 2 2 2 2 2 2 2 2 2
w tv Manzano 5 2 2 3 3 2 2 2 3
Magdalena 4 2 2 3 4 3 2 1 3
Capitan 5 4 2 2 3 2 2 2 3
Sacramento 3 2 2 2 4 3 2 2 3 Gila 3 2 2 2 2 2 2 2 2 mean 3 3 3 3 3 2 2 2 3 standard 11 14 16 18 09 05 0 03 08 deviation
Note Columns and rows may not agree due to rounding
Figure 7 X-ray radiographs ofSacramento seed source percussed for 1 4 or 8 minutes
33
Figure 8a-d Seed damage close-ups Figure a shows undamaged seed percussed for 4 minutes Figures b-d show seeds percussed for 8 minutes Damage includes fracturing ofradicle in b complete removal ofseed coat in c and pitting ofseed coat in d
34
Untreated Treated
Figure 9 SEM images ofuntreated vs 4-minute percussion-treated strophiolar cells ofNew Mexico locust seeds Treated cells are loosened separated and raised
DISCUSSION
Determining the Optimal Treatment
Averaging results of scarification treatmems across seed sources is a useful
tool to predict which treatment is most likely to be successful for an unknown seed
source Averaged over all sources four- and eight-minute percussion treatments
resulted in the highest germination 815 and 790 respectively and produced
germination that was as fast or faster than all other treatments Although there was no
difference between these two treatments on average an argument for 4 minutes of
percussion as a standard protocol for treating New Mexico locust seeds can be made
Descriptively 4 minutes of percussion improved germination over 8 minutes
ofpercussion for a majority of sources (five out of nine) 8 minutes of percussion
improved germination for three ofnine sources with no difference for a fmal source
For individual sources 4 minutes ofpercussion was always the best or secondmiddot best
treatment and was always equal to or better than all hot water treatments
Germination at 8 minutes ofpercussion was lower than all other percussion durations
for the Jemez and Gila sources and was lower than at least one hot water treatment for
these sources as well
The x-ray radiographs of the Sacramento seed source illustrate the excessive
internal and external seed damage that can occur with 8 minutes of percussion
germination counts 8-minute percussed seeds from Holman and Capitan
ClUll signs of damage that were visible to the naked eye (see below)
r~evertheless some of these seeds still fit the criteria for germination Neither x-ray
36
photography nor visible observations during seed counts revealed seed damage at the
4-minute percussion level In operational trials seeds percussed for 4 minutes
developed into healthy seedlings (Khadduri and Harrington unpublished data)
Strophiole and Seed Coat Behavior
Four minutes ofpercussion successfully breaks seed dormancy in a large
percentage ofNew Mexico locust seeds without excessively damaging the
surrounding seed coat or internal seed coat structures Most papilionoid legume
seeds including seeds ofNew Mexico locust have a specific region of the seed coat
known as the strophiole or lens (Hamly 1932) The strophiole is located on the
cotyledonary lobe of a locust seed (Figure 10 a-c) If seeds are not indiscriminately
damaged the strophiole is the first point of water entry to the embryo (Hopkinson
1997) When a papilionoid seed is percussed for the appropriate amount of time
repeated hits on the integrated seed coat loosen the constrained cells of the strophiolar
region without excessively damaging the rest of the seed coat (Ballard 1976) SEM
photos illuStrate that 4 minutes of percussion loosened and separated strophiolar cells
ofNew Mexico locust seeds when compared with constrained organized strophiolar
cells of untreated seeds (Figure 9) Research-has shown that water passes to the
embryo through the treated strophiole in a controlled manner and regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues
(Kelly and van Staden 1987)
37
10 Strophiole ofunimbibed New Mexico locust seed (a) and following uptake (bc)
38
In contrast hot-water treatments can excessively degrade the seed coat
(Rebman et al 1998 Marunda 1990) Random cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rebman et aI 1998 Marunda 1990) Along with a
proportionately large amount ofun-imbibed (thus under-treated) seeds (Figure l1a)
damage was observed in hot-water treated seeds from six sources during germination
counts Damage included seeds where the radicle failed to elongate (Figure 11 b)
seeds where the radicle elongated but was delayed in freeing itself from the seed coat
(Figure 11c) and seeds where the radicle completely broke off from the embryo
(Figure lId) In addition some seeds germinated in reverse (the cotyledon emerged
fIrst) If the radicle emerged completely from the seed coat they were counted as
germinated
While nearly all seeds treated for 8 minutes ofpercussion imbibed water
damage was evident in x-ray evaluations (Sacramento seed source) and in
germination counts (Holman and Capitan) at this treatment level Seed damage
during germination counts was similar to levels found in hot-water treated seeds
suggesting that over-treatment by both hot water and longer durations of percussion
may result in similar damage to seed structure
The Sacramento and Holman seed sources were two of the fIve sources that
opcrplltlpn in germination from the 4- to 8-minute durations of percussion
the benefIts ofloosening the strophiole as evidenced by imbibition of
all 8-minute treated seeds were outweighed by external and internal seed coat
39
a
b c d
11 Damage to New Mexico locust seeds observed during germination cotmts following hot water scarification 9a Hot water replicate including ~t seedlings and numerous unimbibed seeds 9b Close-up of seed where raallCle failed to elongate 9c Close-up ofseed where radicle elongated but was
-gtIn in freeing itself from the seed coat 9d Close-up ofseed where radicle broke off from embryo
40
damage (which also may be contributing to high imbibition) For the three sources
for which 8 minutes of percussion resulted in maximum germination extended
percussion of seeds necessary to loosen the strophiole may not have excessively
damaged the seed anatomy The Manzano source did not significantly change in
germination from 4 to 8 minutes of percussion suggesting that an increase in
permeable seeds at the 8-minute treatment duration was balanced by the number of
seeds that failed to germinate due to seed damage To summarize the 4-minute
percussion treatment improved germination over 8 minutes ofpercussion for a
majority of sources (five ofnine) the 8-minute percussion treatment improved
germination over 4-minutes ofpercussion for three sources and there was no
difference with the Manzano source Notably for the Raton source 8 minutes of
percussion increased germination by 235 percentage points over 4-minutes of
percussion (7875 vs 5525 respectively) the next best treatment Raton was the
only source to result in germination less than 75 for 4 minutes ofpercussion so a
grower may wish to apply longer percussion durations to lots that do not respond well
to this treatment level
Finally there was an interesting trend in the Jemez and Raton sources seed
that significantly contributed to the source by treatment interaction (Figure 12) A
~u test showed that the Jemez source had a significantly lighter seed weight than
other sources while the Raton source had an average seed weight For the Jemez
the longest duration of boiling increased germination Conversely only short
U4UVll) of percussion were needed to achieve high germination and the longest
41
80 _ Jemez a Raton
iD
~ C iD e iD
Q c 0 ~
c ~ iD 0
20
0------shy
Immersion Boiling 30 sec Boiling 60 sec
Hot Water Scarification
10degTr========~--------~------------------------------
80
60
40
20
_ Jemez Raton
0------shy
1 min 2 min 4 min 8 min
Percussion Scarification
Figure 12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources
42
b
--~----~~~~========~---- duration ofpercussion reduced germination For the Raton source longer durations
ofboiling consistently decreased germination whereas longer percussion treatments
consistently increased germination
A possible explanation is that a thick seed coat is associated with a loose
strophiole structure and that a thin seed coat is associated with a constrained
strophiole This contrast merits further study and may suggest an ecological
adaptation Further investigation ofthis phenomenon may contribute to successful
pre-screening ofunknown seed sources to determine optimal treatment leveL
43
CONCLUSION
Based on results obtained in pilot study (see Appendix) and thesis
experiments 4 minutes ofpercussion is recommended as a standard protocol for
treating an unknown source ofNew Mexico locust seed In addition to laboratory
results the 4-minute percussion treatment has resulted in germination greater than
80 and healthy seedling grow-outs for production purposes (unpublished data) In
laboratory experiments only one hundred seeds per 118 ml (40z) soil tin were
percussed at a time whereas for production we have treated up to five hundred seeds
per tin For large-scale production ofNew Mexico locust seedlings growers may
wish to experiment with larger quantities of seed and larger-size soil tins
Seed storage trials following percussion treatments were not conducted
However germination greater than 80 and healthy seedlings were obtained when
seeds were percussion-treated and subsequently stored at room-temperature (22degC)
for one week (unpublished data) Seed storage trials merit further study as the
standard hot water treatment leaves seeds in a softened condition that is not
conducive to storage
Hydroseeding is an effective method for direct seeding in reclamation
situations Percussion treatment may be conducive to hydro seeding as seeds remain
a dry relatively undamaged yet permeable condition following treatment Sulfuric
seeds also remain dry following treatment but extensive damage to seed coat
poor seedling vigor have been reported with this treatment (Kelly and van Staden
A grow-out study using a hydro seeder on a reclamation site testing percussion
44
against other treatments such as sulfuric acid and mechanical scarification also
merits further study
Papilionoids are the largest subfamily oflegumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust these legumes often play an integral role in the revegetation of
disturbed lands The pilot study (Appendix) indicates that percussion scarification
successfully treats the widely used black locust as well Developing superior
scarification methods to the standard hot water mechanical scarification and acid
treatments may facilitate the use ofpapilionoid legumes in reclamation projects
45
APPENDICES
APPENDIX A
PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR
NEW MEXICO LOCUST AND BLACK LOCUST SEEDS
Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4
Hot water and sulfuric acid soaks are traditional treatments for seeds of many
temperate woody legwnes including locusts However these scarification techniques
often produce inconsistent germination Percussion scarification where seeds are
repeatedly propelled against a hard surface was compared with hot water
scarification to evaluate treatment efficacy for New Mexico locust (Robinia
neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment
seeds were placed in a 98degC water bath which was immediately removed from the
heat source For percussion scarification seeds were placed in a soil sample tin and
agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the
control were followed by 24-hour water soaks Hot water baths resulted in 56 and
41 germination for New Mexico locust and black locust respectively For both
species nearly all durations ofpercussion increased germination over the hot water
treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3
4 and 5 minutes for black locust resulted in at least 90 germination Traditional
scarification treatments randomly degrade the entire seed coat which can lead to
tissue damage during water uptake Percussion scarification specifically weakens the
strophiole the natural source ofwater entry to the seed in papilionoid legwnes
INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom
2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031
48
Following percussion imbibition is controlled through the strophiole and underlying
tissue is protected
Keywords New Mexico locust black locust scarification percussion germination
Introduction
Locust trees (Robinia spp) are aggressive pioneer species that quickly
colonize disturbed land fix atmospheric nitrogen in the soil and stabilize slopes that
are prone to erosion (Klemmedson 1994 Ashby et al 1985) (Figure A1ab)
Recognition of these attributes has contributed to increased demand for locusts in
reclamation projects Locust seeds however exhibit physical dormancy where a
thick seed coat prevents the movement of water and gasses to the embryo (Leadem
1997)
Sulfuric acid or hot water soaks have traditionally been used to break seed
dormancy in macro-propagation of locusts Many growers are moving away from the
use of su1furic acid in treating locust seeds (Dreesen and Harrington 1997) In
addition to being dangerous acid soak durations must be specifically correlated to
seed lot (Olson 1974) Collections of native seed can vary tremendously in seed size
seed weight and hard seededness etc This variability limits the utility of sulfuric
acid soaks as even within a given lot some seeds may be damaged whereas others
are under-treated and fail to imbibe (take up water) A survey of Southwestern
container growers indicates that hot water baths are currently the most common
scarification method for locust seeds (Hine et al 1997) However hot water baths
also produce inconsistent germination (Lin et al 1996)
49
b
Figure AI New Mexico locust a primary invader following disturbance Ala Crown-sprouting at edge ofburn near Los Alamos NM--one year after Cerro Grande fire Alb Colonizing road cut Gila National Forest NM
50
Percussion scarification where seeds are repeatedly propelled against a hard
surface is an alternative dormancy-breaking method that has proven successful in
legumes related to locusts (Hamly 1932 Barton 1947) We compared this alternative
treatment to hot water scarification to evaluate treatment efficacy for New Mexico
locust and black locust seeds
Materials and Methods
Commercial New Mexico locust seeds (Western Native Seed Coaldale CO)
were collected fall 2000 in Huerfano County CO Black locust seeds were collected
September 2000 from Taos County NM by harvesting ripe pods from several trees at
a distance of up to 3 m from ground level Black locust seeds were allowed to air-dry
for 3 weeks and were threshed and separated from large chaffusing a greenhouse
fan A South Dakota Seed Blower (Chicago Illinltois) was used to remove fine chaff
from both seed lots Seeds were storedmiddotat 2-4C until the germination study was
conducted in March 2001
This study compared hot water scarification with percussion scarification
using treatment and species as experimental factors Along with a control
scarification treatments included immersion of seed in boiling water as well as
percussion durations of 1 2 3 45 or 10 minutes Four 100-seed samples were used
to test each species by scarification treatment combination
For the hot water treatment test tubes were filled with 30 mL water and
placed in a water-filled beaker (Figure A2a) The bath was raised to boiling (-98C at
51
b
c
Figure A2 Implementation ofhot water and percussion scarification A2a Hot water bath A2b Paint-shaker set-up A2c Placing soil tin containing seeds at end ofpaint shaker ann
52
Las Cruces NM elevation of 1300 meters) Each 100-seed replicate was placed in a
test tube and each test tube was immediately removed to cool at room temperature
Percussion scarification was implemented using a pneumatic paint shaker
(Central Pneumatic model 00422 Camarillo CA) (Figure A2b) This allowed
standardization of treatment intensity Compressed air pressure was maintained at
530kPa plusmn 30kPa (80psi plusmn 5psi) resulting in approximately 350 oscillations per
minute Each lOO-seed sample was placed in a 118 ml (4 oz) soil tin for shaking To
maximize lateral movement of paint shaker a paint can was used as a spacer to place
soil tins at the end of the shaking arm (Figure A2c) This allowed shaking to occur at
the greatest distance from the pivot point (fulcrum) Following all treatments
including the control seeds were soaked for twenty-four hours in distilled water prior
to plating out
Gennination was evaluated on lab benches at room temperature Each
replicate of seeds was placed in a 100 cm petri dish on moistened filter paper with
humidity maintained by enclosing the petri dishes in ziplock bags Gennination was
monitored daily for 14 days in accordance with International Seed Testing
Association standards for Robinia species (ISTA 1999) Gennination was defined as
protrusion of the radicle from the embryo by at least 1mm as viewed by the naked
eye
Categorical Analysis of Variance using Prpc Catmod (SAS Institute 1989)
was used to detennine treatment differences in germination percentages for each
source This procedure is an extension of a Chi-square test of homogeneity using the
53
natural log of the ratio of germinated to non-germinated seeds for each treatment Pshy
values less than 005 were considered significant A limited set of pairwiseshy
comparisons was conducted to compare treatment mean percents using a
conservative alpha value of 005 divided by the number of comparisons
Results
Percussion maximized germination for both New Mexico locust and black
locust There were three germination response levels for New Mexico locust (Figure
A3) First there was a low response in the control indicating a dormant seed lot
The hot water treatment and I-minute duration of percussion share a second
intermediate response Germination was highest at percussion levels from 2 to 10
minutes
A selected percussion treatment intermediate in time (5 minutes) produced the
steepest germination curve indicating the fastest germination rate (Figure A4) The
hot water treatment produced an intermediate curve or relatively slower germination
rate The control curve was very flat indicating the slowest germination rate for New
Mexico locust
Control seeds germinated poorly for black locust as well (Figure A5) Hot
water scarification improved germination over the control but all levels of percussion
improved germination over hot water The optimal durations of percussion treatment
were bracketed with a significant increase from the 1 to 2 minute percussion level a
high germination response from 2 to 5 minutes of percussion and finally a significant
54
~ d 25 CL)
p t1 0 ~ t1
g Vl ~ Vl
100~---------------------------------------------------------------
80
60
40
20
o Control Hot Water Percussion
I min Percussion
2 min Percussion
3 min
Scarification
Percussion 4 min
Figure A3 Effect of scarification on germination response for New Mexico locust
Percussion 5 min
=significant difference between adjacent treatment bars ns = no significant difference
Percussion 10 min
~ ~ () u agt
Il-t d o ~
~ ~ c3
100middotlr~~~------------------------------------e- Percussion 5min middot 0middot Hot Water -- Control
80-J=middot======
6b-middot-lmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middot--middotmiddot-middot~-middotmiddot -middotmiddot middot middotmiddot-middot 1
40
20
-----~-----------
0 0 0 middotmiddotmiddotmiddotmiddot0middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot0
0 --~-----y-----~------_---~--- T-----~
o shy -TDay 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A4 Effect of selected scarification treatments on germination speed for New Mexico locust First seven days of fourteen-day experiment are shown
Day 7
80
~ cd
5 60 ()
[) Cl d 0 1t1 d
40sect dUl
-J
20
o
ns
Scarification
Figure A5 Effect of scarification on germination response for black locust = significant difference between adjacent treatment bars ns = no significant difference
decrease at the 10-minute level This drop-off suggests that 10 minutes of percussion
over-treats seeds from this lot of-black locust
A selected percussion treatment intermediate in time (5 minutes) also had the
fastest germination rate for black locust (Figure A6)_ The hot water curve is
somewhat flatter indicating a slower germination rate The control curve is very flat
indicating a slow germination rate
A qualitative difference between hot water and percussion-treated seeds was
also observed Along with healthy germinants the hot water treatment produced
over-treated seeds (Figure A 7a) Examples of damaged germinants include seeds
where the radicle did not elongate (Figure A 7b) seeds where the radicle elongated
but was delayed in freeing itself from the seed coat (Figure A7c) and seeds where the
radicle broke off completely from the embryo (Figure A7d) In addition to these
examples of over-treatment there were numerous small dark seeds that failed to
imbibe water For percussion treatment nearly all seeds took up water (Figure A8a)
Germination was rapid and uniform with little damage evident (Figure A8b)
To summarize both New Mexico locust and black locust seed lots were
dormant The standard hot water scarification treatment improved germination but
only to 56 and 41 for New Mexico locust and black locust respectively Nearly
all percussion durations improved total germination as well as germination rate over
the hot water treatment Germination was greater than 90 for percussion durations
of 4 5 and 10 minutes for New Mexico locust and 3 4 and 5 minutes for black locust
58
CI)
sgt t CI) 0
igt 0
sect ttl t E
Vl 0
c3
100r==~==~~=r------------------------------------~ ___ Percussion 5 min
middotmiddotmiddot0middotmiddotmiddot Hot Water -lIf- Control
80r=========~--~~----------------------------~
60
40
20 -~-~---~---------- ---~------------I
omiddot
O
_~_----------lIf-----~bull -----_ shy---shy I
Day 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A6 Effect of selected scarification treatments on germination speed for black locust First seven days of fourteen-day experiment are shown
Day 7
a
b c d
Figure A7 Damage to New Mexico locust seeds observed during germination counts following hot water scarification A7a Hot water replicate including damaged seedlings and numerous unimbibed seeds A7b Close-up ofseed where radicle failed to elongate A7c Close-up ofseed where radicle elongated but was delayed in freeing itself from the seed coat A7d Close-up ofseed where radicle completely broke offfrom embryo
60
a
b
Figure A8 New Mexico locust seeds following percussion treatment at time of germination counts A8a Percussion replicate with uniformly healthy seedlings and near complete imbibition of seeds A8b Close-up ofpercussion-treated seedling
61
Discussion
Papilionoid seeds have a specific anatomical feature known as the strophiole
or lens-the natural site of water entry to the seed (Hamly 1932) The strophiole is
located on the cotyledonary lobe of a locust seed (Figure A9) When a papilionoid
legume seed is percussed for the appropriate amount of time repeated hits on the
integrated seed coat loosen the constrained cells of the strophiolar region without
excessively damaging the rest of the seed coat (Ballard 1976)
When a percussed seed is soaked water enters exclusively through the
strophiole in a controlled manner (Kelly and Staden 1987) This regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues This
contrasts with seeds that have been hot water or acid scarified treatments that can
randomly degrade the seed coat Unlocalized cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rehman et al 1998 Marunda 1990)
Papilionoids are the largest subfamily of legumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust and black locust these legumes often play an integral role in the
revegetation ofdisturbed lands Developing superior scarification methods to the
standard hot water and acid treatments should fadlitate the use ofthese legumes in
restoration projects
62
Figure A9 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (bc)
63
APPENDIXB
GERMINATION PERCENTS AND STANDARD ERRORS FOR SOURCE
BLOCK AND TREATMENT FOR NEW MEXICO LOCUST
Table B 1 Gennination percents and standard errors for source block and treatment for New Mexico locust
Source Block Treatment Germination Percentage Standard Error
Capitan 1 Immersion 74 437 Capitan 2 Immersion 69 462 Capitan 3 Immersion 71 454 Capitan 4 Immersion 67 470 Capitan Boiling 30 sec 79 407 Capitan 2 Boiling 30 sec 76 427 Capitan 3 Boiling 30 sec 72 449 Capitan 4 Boiling 30 sec 76 427 Capitan Boiling 60 sec 71 454 Capitan 2 Boiling 60 sec 54 498 Capitan 3 Boiling 60 sec 67 470 Capitan 4 Boiling 60 sec 76 427 Capitan I Control 5 218 Capitan 2 Control 7 255 Capitan 3 Control 4 196 Capitan 4 Control 5 218 Capitan 1 Percussion I min 47 499 Capitan 2 Percussion I min
470
Capitan Percussion I min 39 488
Capitan 4 Percussion I min 38 485 Capitan I Percussion 2 min 55 497 Capitan Capitan
2
Percussion 2 min Percussion 2 min
71 65
454 477
Capitan 4 Percussion 2 min 56 496 Capitan I Percussion 4 min 86 347 Capitan 2 Percussion 4 min 79 407 Capitan 3 Percussion 4 min 81 392 Capitan 4 Percussion 4 min 82 384 Capitan I Percussion 8 min 90 300 Capitan 2 Percussion 8 min 88 325 Capitan 3 Percussion 8 min 90 300 Capitan 4 Percussion 8 min 90 300 Gila I Immersion 71 454 Gila 2 Immersion 77 421 Gila
Immersion 75 433
Gila 4 Immersion 72 449
Gila I Boiling 30 sec 68 466
Gila 2 Boiling 30 sec 81 392
Gila 3 Boiling 30 sec 54 498
Gila 4 Boiling 30 sec 63 483
Gila I Boiling 60 sec 55 497
Gila 2 Boiling 60 sec 68 466
Gila 3 Boiling 60 sec 57 495
Gila 4 Boiling 60 sec 70 458
65
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Gila 2 Control 13 336 Gila 3 Control 13 336 Gila 4 Control 20 400 Gila 1 Percussion 1 min 66 474 Gila 2 Percussion 1 min 57 495 Gila 3 Percussion 1 min 73 444 Gila 4 Percussion 1 min 82 384 Gila 1 Percussion 2 min 81 392 Gila 2 Percussion 2 min 86 347 Gila 3 Percussion 2 min 85 357 Gila 4 Percussion 2 min 54 498 Gila 1 Percussion 4 min 79 407 Gila 2 Percussion 4 min 83 376 Gila 3 Percussion 4 min 84 367 Gila 4 Percussion 4 min 79 407 Gila 1 Percussion 8 min 51 500 Gila 2 Percussion 8 min 76 427 Gila 3 Percussion 8 min 52 500 Gila 4 Percussion 8 min 74 439 Holman 1 Immersion 83 376 Holman 2 Immersion 86 347 Holman 3 Immersion 81 392 Holman 4 Immersion 90 300 Holman 1 Boiling 30 sec 90 300 Holman 2 Boiling 30 sec 85 357 Holman 3 Boiling 30 sec 87 336 Holman 4 Boiling 30 sec 83 376 Holman 1 Boiling 60 sec 79 407 Holman 2 Boiling 60 sec 80 400 Holman 3 Boiling 60 sec 86 347 Holman 4 Boiling 60 sec 93 255 Holman Control 25 433 Holman 2 Control 19 392 Holman Control 21 407
Holman 4 Control 24 427
Holman Percussion 1 min 77 421
Holman 2 Percussion 1 min 85 357
Holman 3 Percussion 1 min 80 400
Holman 4 Percussion 1 min 89 313
Holman 1 Percussion 2 min 88 325
Holman 2 Percussion 2 min 96 196
Holman 3 Percussion 2 min 93 255
Holman 4 Percussion 2 min 96 196
Holman 1 Percussion 4 min 92 271
Holman 2 Percussion 4 min 97 171
66
I
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Holman 3 Percussion 4 min 91 286 Holman 4 Percussion 4 min 96 196 Holman 1 Percussion 8 min 88 325 Holman 2 Percussion 8 min 85 357 Holman 3 Percussion 8 min 83 376 Holman 4 Percussion 8 min 82 384 Jemez 1 Immersion 53 499 Jemez 2 Immersion 44 496 Jemez 3 Immersion 52 500 Jemez 4 Immersion 70 458 Jemez 1 Boiling 30 sec 58 494 Jemez 2 Boiling 30 sec 55 497 Jemez 3 Boiling 30 sec 48 500 Jemez 4 Boiling 30 sec 65 477 Jemez 1 Boiling 60 sec 68 466 Jemez 2 Boiling 60 sec 63 483 Jemez 3 Boiling 60 sec 73 444 Jemez 4 Boiling 60 sec 89 313 Jemez 1 Control 2 140 Jemez 2 Control 9 286 Jemez 3 Control 6 237 Jemez 4 Control 8 271 Jemez 1 Percussion 1 min 58 494 Jemez 2 Percussion 1 min 63 483 Jemez 3 Percussion 1 min 77 421 Jemez 4 Percussion 1 min 89 313 Jemez 1 Percussion 2 min 68 466 Jemez 2 Percussion 2 min 64 480 Jemez 3 Percussion 2 min 80 400 Jemez 4 Percussion 2 min 83 376 Jemez Percussion 4 min 68 466 Jemez 2 Percussion 4 min 79 407 Jemez 3 Percussion 4 min 70 458 Jemez 4 Percussion 4 min 83 376 Jemez 1 Percussion 8 min 52 500 Jemez 2 Percussion 8 min 59 492 Jemez 3 Percussion 8 min 61 488 Jemez 4 Percussion 8 min 70 458 Magdalena 1 Immersion 69 462 Magdalena 2 Immersion 69 462 Magdalena J Immersion 70 458 Magdalena 4 Immersion 85 357 Magdalena 1 Boiling 30 sec 65 477 Magdalena 2 Boiling 30 sec 71 454 Magdalena 3 Boiling 30 sec 68 466
67
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Magdalena 4 Boiling 30 sec 83 376 Magdalena 1 Boiling 60 sec 63 483 Magdalena 2 Boiling 60 sec 68 466 Magdalena 3 Boiling 60 sec 63 483 Magdalena 4 Boiling 60 sec 82 384 Magdalena 1 Control 11 313 Magdalena 2 Control 6 237 Magdalena 3 Control 5 218 Magdalena 4 Control 4 196 Magdalena Percussion 1 min 32 466 Magdalena 2 Percussion 1 min 45 497 Magdalena 3 Percussion 1 min 46 498 Magdalena 4 Percussion I min 36 480 Magdalena 1 Percussion 2 min 67 470 Magdalena 2 Percussion 2 min 68 466 Magdalena 3 Percussion 2 min 65 477 Magdalena 4 Percussion 2 min 81 392 Magdalena 1 Percussion 4 min 87 336 Magdalena 2 Percussion 4 min 97 171 Magdalena 3 Percussion 4 min 83 376 Magdalena 4 Percussion 4 min 97 171 Magdalena 1 Percussion 8 min 98 lAO Magdalena 2 Percussion 8 min 100 000 Magdalena 3 Percussion 8 min 96 196 Magdalena 4 Percussion 8 min 98 lAO Manzano 1 Immersion 67 470 Manzano 2 Immersion 57 495 Manzano 3 Immersion 72 4049 Manzano 4 Immersion 73 4044 Manzano 1 Boiling 30 sec 70 458 Manzano 2 Boiling 30 sec 64 480 Manzano 3 Boiling 30 sec 71 454 Manzano 4 Boiling 30 sec 80 400 Manzano I Boiling 60 sec 50 500 Manzano 2 Boiling 60 sec 53 499 Manzano 3 Boiling 60 sec 59 492 Manzano 4 Boiling 60 sec 74 439 Manzano I Control 4 196 Manzano 2 Control 8 271 Manzano 3 Control 1 099 Manzano 4 Control 6 237 Manzano 1 Percussion 1 min 52 500 Manzano 2 Percussion 1 min 39 488 Manzano 3 Percussion 1 min 36 480 Manzano 4 Percussion 1 min 51 500
68
Table B 1 Continued
Source Block Treatment Gerrni~ation Percentage Standard Error
Manzano Percussion 2 min 53 499 Manzano 2 Percussion 2 min 58 494 Manzano 3 Percussion 2 min 61 488 Manzano 4 Percussion 2 min 62 485 Manzano 1 Percussion 4 min 76 427 Manzano 2 Percussion 4 min 85 357 Manzano 3 Percussion 4 min 79 407 Manzano 4 Percussion 4 min 83 376 Manzano 1 Percussion 8 min 87 336 Manzano 2 Percussion 8 min 83 376 Manzano 3 Percussion 8 min 83 376 Manzano 4 Percussion 8 min 75 433 Raton 1 Immersion 54 498 Raton 2 Immersion 37 483 Raton 3 Immersion 39 488 Raton 4 Immersion 69 462 Raton 1 Boiling 30 sec 36 480 Raton 2 Boiling 30 sec 20 400 Raton 3 Boiling 30 sec 28 449 Raton 4 Boiling 30 sec 60 490 Raton 1 Boiling 60 sec 15 357 Raton 2 Boiling 60 sec 171 Raton 3 Boiling 60 sec 4 196 Raton 4 Boiling 60 sec 35 477 Raton 1 Control 4 196 Raton 2 Control 12 325 Raton 3 Control 2 140 Raton 4 Control 6 237 Raton 1 Percussion 1 min 52 500 Raton 2 Percussion I min 29 454 Raton 3 Percussion I min 35 477 Raton 4 Percussion 1 min 31 462 Raton 1 Percussion 2 min 55 497 Raton 2 Percussion 2 min 35 477 Raton 3 Percussion 2 min 33 470 Raton 4 Percussion 2 min 42 494 Raton 1 Percussion 4 min 65 477 Raton 2 Percussion 4 min 52 500 Raton Percussion 4 min 51 500 Raton 4 Percussion 4 min 53 499 Raton 1 Percussion 8 min 88 325 Raton 2 Percussion 8 min 80 400 Raton 3 Percussion 8 min 73 444 Raton 4 Percussion 8 min 74 439 Sacramento 1 Immersion 43 495
69
Table B 1 Continued
-Source Block Treatment Germination Percentage Standard Error
Sacramento 2 Immersion 30 458 Sacramento 3 Immersion 38 485 Sacramento 4 Immersion 56 496 Sacramento 1 Boiling 30 sec 47 499 Sacramento 2 Boiling 30 sec 48 500 Sacramento 3 Boiling 30 sec 48 500 Sacramento 4 Boiling 30 sec 56 496 Sacramento 1 Boiling 60 sec 52 500 Sacramento 2 Boiling 60 sec 67 470 Sacramento 3 Boiling 60 sec 43 495 Sacramento 4 Boiling 60 sec 48 500 Sacramento 1 Control 0 000 Sacramento 2 Control 3 171 Sacramento 3 Control 3 171 Sacramento 4 Control 099 Sacramento 1 Percussion 1 min 57 495 Sacramento 2 Percussion 1 min 61 488 Sacramento 3 Percussion 1 min 62 485 Sacramento 4 Percussion 1 min 55 497 Sacramento 1 Percussion 2 min 84 367 Sacramento 2 Percussion 2 min 70 458 Sacramento 3 Percussion 2 min 81 392 Sacramento 4 Percussion 2 min 69 462 Sacramento 1 Percussion 4 min 91 286 Sacramento 2 Percussion 4 min 84 367 Sacramento 3 Percussion 4 min 84 367 Sacramento 4 Percussion 4 min 94 237 Sacramento 1 Percussion 8 min 82 384 Sacramento 2 Percussion 8 min 81 392 Sacramento 3 Percussion 8 min 82 384 Sacramento 4 Percussion 8 min 81 392 Sandia 1 Immersion 76 427 Sandia 2 Immersion 70 458 Sandia 3 Immersion 70 458 Sandia 4 Immersion 81 392 Sandia 1 Boiling 30 sec 78 414 Sandia 2 Boiling 30 sec 71 454 Sandia 3 Boiling 30 sec 63 483 Sandia 4 Boiling 30 sec 78 414 Sandia 1 Boiling 60 sec 67 470 Sandia 2 Boiling 60 sec 65 477 Sandia 3 Boiling 60 sec 74 439 Sandia 4 Boiling 60 sec 66 474 Sandia 1 Control 16 367 Sandia 2 Control 7 255
70
Table B 1 Continued
Source Block Treatment Gennination Percentage Standard Error
Sandia 3 Control 8 271 Sandia 4 Control 16 367 Sandia 1 Percussion 1 min 67 470 Sandia 2 Percussion 1 min 61 488 Sandia 3 Percussion 1 min 66 474 Sandia 4 Percussion 1 min 59 492 Sandia I Percussion 2 min 82 384 Sandia 2 Percussion 2 min 83 376 Sandia 3 Percussion 2 min 81 392 Sandia 4 Percussion 2 min 84 367 Sandia 1 Percussion 4 min 89 313 Sandia 2 Percussion 4 min 86 347 Sandia 3 Percussion 4 min 89 313 Sandia 4 Percussion 4 min 85 357 Sandia 1 Percussion 8 min 80 400 Sandia 2 Percussion 8 min 77 421 Sandia 3 Percussion 8 min 69 462 Sandia 4 Percussion 8 min 66 474
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Marunda CT 1990 Effects of seed pretreatment on the development ofAcacia auriculforinis and A holosericea seedlings ACIAR Proceedings Series No 28
Meginnis HG 1937 Sulphuric acid treatment to increase germination of black locust seed USDA Circ 453 34 p
Miklas PN Towensend CE Ladd SL 1987 Seedcoat anatomy and the scarification ofcicer milkvetch seed Crop Sci 27 766-772
Monsen SB 1984 Use of shrubs on mine spoils In The Challenge ofProducing Native Plants for the Intermountain Area Proceedings Intermountain Nurserymans Association 1983 conference Las Vegas NV compo PM Murphy 26-31 Ogden UT USDA For Ser Gen Tech Report INT-l68
Olson DF 1974 Robinia L Locust In Seeds of Woody Plants in the US tech coord CS Schopmeyer 728-731 USDA For Ser Agr Handbk No450
Rehman S Loescher RNl Harris P1C 1998 Dormancy breaking and germination ofAcacia salicina Lindl seeds Seed Sci amp Techno127 553-557
Rolston MP 1978 Water impermeable seed dormancy Bot Rev 44 365-396
74
SAS Institute Inc 1989 SASSTAT Users Guide Version 6 4th Edition Volume 1 Cary NC SAS Institute Inc 943 p
SerratoValenti G Conara L Ferrando M Modenesi P 1993 Structural and histochemical features ofStylosanthes scabra (Leguminosae Papilionoidae) seed coat as related to water entry Can J Bot 71 834-840
Simpson BJ 1988 A field guide to Texas trees Austin TX Texas Monthly Press 372p
Tran VN Cavanagh AK 1984 Structural aspects ofdormancy In Seed Physiology Vol 2 Murray DR (ed) p1-44 Academic Press Australia
USDA Forest Service 1948 Woody-plant seed manual USDA Misc Pub1654 416p
Vines RA 1960 Trees Shrubs and Woody Vines of the Southwest Austin Univ ofTexas Press 1104 p
Wagner MR Larson RE Craig TP 1992 Ecology and silvics ofNew Mexico locust (Robinia neomexicana) with an emphasis on the arboreal form p53-57 In JW Hanover K Miller and S Plesko (eds) Proc Intemat Confon Black Locust Biology Culture and Utilization Mich State Univ USA
Wilson JK 1944 Immersing seeds of species of Robinia in boiling water hastens germination J For 42 453-454
Zimmerman RW Carpenter SB 1980 First-year coppice production from a 5shyyear old black locust stand on surface mine spoil p309-314 Centr Hardwd For ConfProc University ofMissouri Columbia
80
~ cd ~ ltl) () 60 ~ ~
N
t 0shyrti tshySltl)
40
0
20
o lmmersion Boiling 30 sec Boiling 60 sec Control 1 min 2min 4 min 8 min
Hot Water Treatments Percussion Treatments
Figure 3 Effect of scarification on germination ofNew Mexico locust for data averaged over all seed sources Means labelled with the same letter are not significantly different
100~----------------------~--~----------------~
80
60
ltU
~
40 IJ
sectgsI
l100
80
60
40
a I middotmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot middotImiddotmiddotmiddotmiddotmiddotmiddot E
_~iii-bull _ _ bull f --~--
t-- -~ ---- -shy ---- shyI ~ ---- - shy
L
fiII
~
b
- shy ---r
- Holman
Sacramento Sandia Gila Jemez
c ~
ltgt ~
Tmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot-I
j~=~=-~~-1
I middotmiddot ~ bull r
t tr
Y -------M-a-gd-a-Ie-n-a--
1 _ _
fCapitan
---- Manzano ~y -_ Raton
2 4 8
Time interval ofpercussion scarification (min)
Figure 4 Effect of seed source by treatment interaction on New Mexico locust germination for percussion scarification pattern a and pattern b
28
tv 0
~ d
~ A s o
1 c3
80
60
40
20
o
TIT
t------=middotfmiddotmiddot-middotmiddot-middotmiddot-middotmiddot 1- --- - - --=- - -shy--cE= - _--shy --shy ----shy ~ ~ ---shy ~ -~- _ --shy-------r 1
I
Raton Holman
-_ Jemez
--- Sandia Manzano Magdalena Capitan Sacramento Gila
Immersion Boiling 30 sec Boiling 60 sec
Hot Water Scarification
Figure 5 Effect of seed source by treatment interaction on New Mexico locust germination for hot water scarification
continued to increase in germination (Figure 4b) There was no difference between
the two treatment levels for the Manzano source (Figure 4b)
The effect ofhot water scarification also varied among sources with few
overall differences between hot water treatments (Figure 5) However boiling for 60
seconds yielded highest germination for the Jemez source whereas increasing
durations ofboiling decreased germination for the Raton source
In addition to consistently high germination 4- and 8-minute percussionshy
treated seeds germinated as fast or faster than all other treatments (Figure 6 Table 4)
050 measured as the number of days needed to complete 50 ofeventual
germination averaged 2 days for both ofthese treatments
X-ray radiographs were taken of seeds collected from the Sacramento source
percussed for 1 4 or 8 minutes (Figure 7) Seeds show signs of damage at 8 minutes
ofpercussion (Figure 8) Compared to an undamaged seed percussed for only 4
minutes (Figure 8a) damage at the 8-minute level includes fractured radicles pitting
of the seed coat and chips in seed coat structure (Figure 8b-d)
Scanning electron microscope (SEM) images at 600X reveal the direct effect
of 4 minutes of percussion on cells of the strophiolar region (Figure 9) Whereas
untreated seeds retain cellular organization in the strophiolar region strophiolar cells
seeds treated for 4 minutes ofpercussion separate and raise resulting in a
~-~e~U appearance
30
Umiddot-~middot--l-~---middotymiddotmiddotmiddot-----middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot--middot--middotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middotmiddotmiddotmiddotmiddotmiddotmiddot- -
050= gt1
-e-- Percussion 8 min -0- Percussion 4 min ---T- Percussion 2 min -----ltil- Percussion 1 min -- Immersion --0- Boiling 30 sec -+- Boiling 60 sec -ltgt- Control
2 4 6 8 10 12
Day Figure 6 Timecourse ofNew Mexico locust gennination following scarification
treatments G50 is the number of days to 50 of eventual germination
31
14
Table 4 Effect of seed source and treatment on G50 (days to 50 germination)
Seed Source Control Immersion B30sec B60sec Plmin P2min P4min P8min mean Raton 3 3 5 7 4 3 2 2 4 Holman 2 2 2 2 2 2 2 2 2
Jemez 4 6 6 5 3 2 2 2 4 Sandia 2 2 2 2 2 2 2 2 2
w tv Manzano 5 2 2 3 3 2 2 2 3
Magdalena 4 2 2 3 4 3 2 1 3
Capitan 5 4 2 2 3 2 2 2 3
Sacramento 3 2 2 2 4 3 2 2 3 Gila 3 2 2 2 2 2 2 2 2 mean 3 3 3 3 3 2 2 2 3 standard 11 14 16 18 09 05 0 03 08 deviation
Note Columns and rows may not agree due to rounding
Figure 7 X-ray radiographs ofSacramento seed source percussed for 1 4 or 8 minutes
33
Figure 8a-d Seed damage close-ups Figure a shows undamaged seed percussed for 4 minutes Figures b-d show seeds percussed for 8 minutes Damage includes fracturing ofradicle in b complete removal ofseed coat in c and pitting ofseed coat in d
34
Untreated Treated
Figure 9 SEM images ofuntreated vs 4-minute percussion-treated strophiolar cells ofNew Mexico locust seeds Treated cells are loosened separated and raised
DISCUSSION
Determining the Optimal Treatment
Averaging results of scarification treatmems across seed sources is a useful
tool to predict which treatment is most likely to be successful for an unknown seed
source Averaged over all sources four- and eight-minute percussion treatments
resulted in the highest germination 815 and 790 respectively and produced
germination that was as fast or faster than all other treatments Although there was no
difference between these two treatments on average an argument for 4 minutes of
percussion as a standard protocol for treating New Mexico locust seeds can be made
Descriptively 4 minutes of percussion improved germination over 8 minutes
ofpercussion for a majority of sources (five out of nine) 8 minutes of percussion
improved germination for three ofnine sources with no difference for a fmal source
For individual sources 4 minutes ofpercussion was always the best or secondmiddot best
treatment and was always equal to or better than all hot water treatments
Germination at 8 minutes ofpercussion was lower than all other percussion durations
for the Jemez and Gila sources and was lower than at least one hot water treatment for
these sources as well
The x-ray radiographs of the Sacramento seed source illustrate the excessive
internal and external seed damage that can occur with 8 minutes of percussion
germination counts 8-minute percussed seeds from Holman and Capitan
ClUll signs of damage that were visible to the naked eye (see below)
r~evertheless some of these seeds still fit the criteria for germination Neither x-ray
36
photography nor visible observations during seed counts revealed seed damage at the
4-minute percussion level In operational trials seeds percussed for 4 minutes
developed into healthy seedlings (Khadduri and Harrington unpublished data)
Strophiole and Seed Coat Behavior
Four minutes ofpercussion successfully breaks seed dormancy in a large
percentage ofNew Mexico locust seeds without excessively damaging the
surrounding seed coat or internal seed coat structures Most papilionoid legume
seeds including seeds ofNew Mexico locust have a specific region of the seed coat
known as the strophiole or lens (Hamly 1932) The strophiole is located on the
cotyledonary lobe of a locust seed (Figure 10 a-c) If seeds are not indiscriminately
damaged the strophiole is the first point of water entry to the embryo (Hopkinson
1997) When a papilionoid seed is percussed for the appropriate amount of time
repeated hits on the integrated seed coat loosen the constrained cells of the strophiolar
region without excessively damaging the rest of the seed coat (Ballard 1976) SEM
photos illuStrate that 4 minutes of percussion loosened and separated strophiolar cells
ofNew Mexico locust seeds when compared with constrained organized strophiolar
cells of untreated seeds (Figure 9) Research-has shown that water passes to the
embryo through the treated strophiole in a controlled manner and regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues
(Kelly and van Staden 1987)
37
10 Strophiole ofunimbibed New Mexico locust seed (a) and following uptake (bc)
38
In contrast hot-water treatments can excessively degrade the seed coat
(Rebman et al 1998 Marunda 1990) Random cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rebman et aI 1998 Marunda 1990) Along with a
proportionately large amount ofun-imbibed (thus under-treated) seeds (Figure l1a)
damage was observed in hot-water treated seeds from six sources during germination
counts Damage included seeds where the radicle failed to elongate (Figure 11 b)
seeds where the radicle elongated but was delayed in freeing itself from the seed coat
(Figure 11c) and seeds where the radicle completely broke off from the embryo
(Figure lId) In addition some seeds germinated in reverse (the cotyledon emerged
fIrst) If the radicle emerged completely from the seed coat they were counted as
germinated
While nearly all seeds treated for 8 minutes ofpercussion imbibed water
damage was evident in x-ray evaluations (Sacramento seed source) and in
germination counts (Holman and Capitan) at this treatment level Seed damage
during germination counts was similar to levels found in hot-water treated seeds
suggesting that over-treatment by both hot water and longer durations of percussion
may result in similar damage to seed structure
The Sacramento and Holman seed sources were two of the fIve sources that
opcrplltlpn in germination from the 4- to 8-minute durations of percussion
the benefIts ofloosening the strophiole as evidenced by imbibition of
all 8-minute treated seeds were outweighed by external and internal seed coat
39
a
b c d
11 Damage to New Mexico locust seeds observed during germination cotmts following hot water scarification 9a Hot water replicate including ~t seedlings and numerous unimbibed seeds 9b Close-up of seed where raallCle failed to elongate 9c Close-up ofseed where radicle elongated but was
-gtIn in freeing itself from the seed coat 9d Close-up ofseed where radicle broke off from embryo
40
damage (which also may be contributing to high imbibition) For the three sources
for which 8 minutes of percussion resulted in maximum germination extended
percussion of seeds necessary to loosen the strophiole may not have excessively
damaged the seed anatomy The Manzano source did not significantly change in
germination from 4 to 8 minutes of percussion suggesting that an increase in
permeable seeds at the 8-minute treatment duration was balanced by the number of
seeds that failed to germinate due to seed damage To summarize the 4-minute
percussion treatment improved germination over 8 minutes ofpercussion for a
majority of sources (five ofnine) the 8-minute percussion treatment improved
germination over 4-minutes ofpercussion for three sources and there was no
difference with the Manzano source Notably for the Raton source 8 minutes of
percussion increased germination by 235 percentage points over 4-minutes of
percussion (7875 vs 5525 respectively) the next best treatment Raton was the
only source to result in germination less than 75 for 4 minutes ofpercussion so a
grower may wish to apply longer percussion durations to lots that do not respond well
to this treatment level
Finally there was an interesting trend in the Jemez and Raton sources seed
that significantly contributed to the source by treatment interaction (Figure 12) A
~u test showed that the Jemez source had a significantly lighter seed weight than
other sources while the Raton source had an average seed weight For the Jemez
the longest duration of boiling increased germination Conversely only short
U4UVll) of percussion were needed to achieve high germination and the longest
41
80 _ Jemez a Raton
iD
~ C iD e iD
Q c 0 ~
c ~ iD 0
20
0------shy
Immersion Boiling 30 sec Boiling 60 sec
Hot Water Scarification
10degTr========~--------~------------------------------
80
60
40
20
_ Jemez Raton
0------shy
1 min 2 min 4 min 8 min
Percussion Scarification
Figure 12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources
42
b
--~----~~~~========~---- duration ofpercussion reduced germination For the Raton source longer durations
ofboiling consistently decreased germination whereas longer percussion treatments
consistently increased germination
A possible explanation is that a thick seed coat is associated with a loose
strophiole structure and that a thin seed coat is associated with a constrained
strophiole This contrast merits further study and may suggest an ecological
adaptation Further investigation ofthis phenomenon may contribute to successful
pre-screening ofunknown seed sources to determine optimal treatment leveL
43
CONCLUSION
Based on results obtained in pilot study (see Appendix) and thesis
experiments 4 minutes ofpercussion is recommended as a standard protocol for
treating an unknown source ofNew Mexico locust seed In addition to laboratory
results the 4-minute percussion treatment has resulted in germination greater than
80 and healthy seedling grow-outs for production purposes (unpublished data) In
laboratory experiments only one hundred seeds per 118 ml (40z) soil tin were
percussed at a time whereas for production we have treated up to five hundred seeds
per tin For large-scale production ofNew Mexico locust seedlings growers may
wish to experiment with larger quantities of seed and larger-size soil tins
Seed storage trials following percussion treatments were not conducted
However germination greater than 80 and healthy seedlings were obtained when
seeds were percussion-treated and subsequently stored at room-temperature (22degC)
for one week (unpublished data) Seed storage trials merit further study as the
standard hot water treatment leaves seeds in a softened condition that is not
conducive to storage
Hydroseeding is an effective method for direct seeding in reclamation
situations Percussion treatment may be conducive to hydro seeding as seeds remain
a dry relatively undamaged yet permeable condition following treatment Sulfuric
seeds also remain dry following treatment but extensive damage to seed coat
poor seedling vigor have been reported with this treatment (Kelly and van Staden
A grow-out study using a hydro seeder on a reclamation site testing percussion
44
against other treatments such as sulfuric acid and mechanical scarification also
merits further study
Papilionoids are the largest subfamily oflegumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust these legumes often play an integral role in the revegetation of
disturbed lands The pilot study (Appendix) indicates that percussion scarification
successfully treats the widely used black locust as well Developing superior
scarification methods to the standard hot water mechanical scarification and acid
treatments may facilitate the use ofpapilionoid legumes in reclamation projects
45
APPENDICES
APPENDIX A
PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR
NEW MEXICO LOCUST AND BLACK LOCUST SEEDS
Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4
Hot water and sulfuric acid soaks are traditional treatments for seeds of many
temperate woody legwnes including locusts However these scarification techniques
often produce inconsistent germination Percussion scarification where seeds are
repeatedly propelled against a hard surface was compared with hot water
scarification to evaluate treatment efficacy for New Mexico locust (Robinia
neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment
seeds were placed in a 98degC water bath which was immediately removed from the
heat source For percussion scarification seeds were placed in a soil sample tin and
agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the
control were followed by 24-hour water soaks Hot water baths resulted in 56 and
41 germination for New Mexico locust and black locust respectively For both
species nearly all durations ofpercussion increased germination over the hot water
treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3
4 and 5 minutes for black locust resulted in at least 90 germination Traditional
scarification treatments randomly degrade the entire seed coat which can lead to
tissue damage during water uptake Percussion scarification specifically weakens the
strophiole the natural source ofwater entry to the seed in papilionoid legwnes
INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom
2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031
48
Following percussion imbibition is controlled through the strophiole and underlying
tissue is protected
Keywords New Mexico locust black locust scarification percussion germination
Introduction
Locust trees (Robinia spp) are aggressive pioneer species that quickly
colonize disturbed land fix atmospheric nitrogen in the soil and stabilize slopes that
are prone to erosion (Klemmedson 1994 Ashby et al 1985) (Figure A1ab)
Recognition of these attributes has contributed to increased demand for locusts in
reclamation projects Locust seeds however exhibit physical dormancy where a
thick seed coat prevents the movement of water and gasses to the embryo (Leadem
1997)
Sulfuric acid or hot water soaks have traditionally been used to break seed
dormancy in macro-propagation of locusts Many growers are moving away from the
use of su1furic acid in treating locust seeds (Dreesen and Harrington 1997) In
addition to being dangerous acid soak durations must be specifically correlated to
seed lot (Olson 1974) Collections of native seed can vary tremendously in seed size
seed weight and hard seededness etc This variability limits the utility of sulfuric
acid soaks as even within a given lot some seeds may be damaged whereas others
are under-treated and fail to imbibe (take up water) A survey of Southwestern
container growers indicates that hot water baths are currently the most common
scarification method for locust seeds (Hine et al 1997) However hot water baths
also produce inconsistent germination (Lin et al 1996)
49
b
Figure AI New Mexico locust a primary invader following disturbance Ala Crown-sprouting at edge ofburn near Los Alamos NM--one year after Cerro Grande fire Alb Colonizing road cut Gila National Forest NM
50
Percussion scarification where seeds are repeatedly propelled against a hard
surface is an alternative dormancy-breaking method that has proven successful in
legumes related to locusts (Hamly 1932 Barton 1947) We compared this alternative
treatment to hot water scarification to evaluate treatment efficacy for New Mexico
locust and black locust seeds
Materials and Methods
Commercial New Mexico locust seeds (Western Native Seed Coaldale CO)
were collected fall 2000 in Huerfano County CO Black locust seeds were collected
September 2000 from Taos County NM by harvesting ripe pods from several trees at
a distance of up to 3 m from ground level Black locust seeds were allowed to air-dry
for 3 weeks and were threshed and separated from large chaffusing a greenhouse
fan A South Dakota Seed Blower (Chicago Illinltois) was used to remove fine chaff
from both seed lots Seeds were storedmiddotat 2-4C until the germination study was
conducted in March 2001
This study compared hot water scarification with percussion scarification
using treatment and species as experimental factors Along with a control
scarification treatments included immersion of seed in boiling water as well as
percussion durations of 1 2 3 45 or 10 minutes Four 100-seed samples were used
to test each species by scarification treatment combination
For the hot water treatment test tubes were filled with 30 mL water and
placed in a water-filled beaker (Figure A2a) The bath was raised to boiling (-98C at
51
b
c
Figure A2 Implementation ofhot water and percussion scarification A2a Hot water bath A2b Paint-shaker set-up A2c Placing soil tin containing seeds at end ofpaint shaker ann
52
Las Cruces NM elevation of 1300 meters) Each 100-seed replicate was placed in a
test tube and each test tube was immediately removed to cool at room temperature
Percussion scarification was implemented using a pneumatic paint shaker
(Central Pneumatic model 00422 Camarillo CA) (Figure A2b) This allowed
standardization of treatment intensity Compressed air pressure was maintained at
530kPa plusmn 30kPa (80psi plusmn 5psi) resulting in approximately 350 oscillations per
minute Each lOO-seed sample was placed in a 118 ml (4 oz) soil tin for shaking To
maximize lateral movement of paint shaker a paint can was used as a spacer to place
soil tins at the end of the shaking arm (Figure A2c) This allowed shaking to occur at
the greatest distance from the pivot point (fulcrum) Following all treatments
including the control seeds were soaked for twenty-four hours in distilled water prior
to plating out
Gennination was evaluated on lab benches at room temperature Each
replicate of seeds was placed in a 100 cm petri dish on moistened filter paper with
humidity maintained by enclosing the petri dishes in ziplock bags Gennination was
monitored daily for 14 days in accordance with International Seed Testing
Association standards for Robinia species (ISTA 1999) Gennination was defined as
protrusion of the radicle from the embryo by at least 1mm as viewed by the naked
eye
Categorical Analysis of Variance using Prpc Catmod (SAS Institute 1989)
was used to detennine treatment differences in germination percentages for each
source This procedure is an extension of a Chi-square test of homogeneity using the
53
natural log of the ratio of germinated to non-germinated seeds for each treatment Pshy
values less than 005 were considered significant A limited set of pairwiseshy
comparisons was conducted to compare treatment mean percents using a
conservative alpha value of 005 divided by the number of comparisons
Results
Percussion maximized germination for both New Mexico locust and black
locust There were three germination response levels for New Mexico locust (Figure
A3) First there was a low response in the control indicating a dormant seed lot
The hot water treatment and I-minute duration of percussion share a second
intermediate response Germination was highest at percussion levels from 2 to 10
minutes
A selected percussion treatment intermediate in time (5 minutes) produced the
steepest germination curve indicating the fastest germination rate (Figure A4) The
hot water treatment produced an intermediate curve or relatively slower germination
rate The control curve was very flat indicating the slowest germination rate for New
Mexico locust
Control seeds germinated poorly for black locust as well (Figure A5) Hot
water scarification improved germination over the control but all levels of percussion
improved germination over hot water The optimal durations of percussion treatment
were bracketed with a significant increase from the 1 to 2 minute percussion level a
high germination response from 2 to 5 minutes of percussion and finally a significant
54
~ d 25 CL)
p t1 0 ~ t1
g Vl ~ Vl
100~---------------------------------------------------------------
80
60
40
20
o Control Hot Water Percussion
I min Percussion
2 min Percussion
3 min
Scarification
Percussion 4 min
Figure A3 Effect of scarification on germination response for New Mexico locust
Percussion 5 min
=significant difference between adjacent treatment bars ns = no significant difference
Percussion 10 min
~ ~ () u agt
Il-t d o ~
~ ~ c3
100middotlr~~~------------------------------------e- Percussion 5min middot 0middot Hot Water -- Control
80-J=middot======
6b-middot-lmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middot--middotmiddot-middot~-middotmiddot -middotmiddot middot middotmiddot-middot 1
40
20
-----~-----------
0 0 0 middotmiddotmiddotmiddotmiddot0middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot0
0 --~-----y-----~------_---~--- T-----~
o shy -TDay 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A4 Effect of selected scarification treatments on germination speed for New Mexico locust First seven days of fourteen-day experiment are shown
Day 7
80
~ cd
5 60 ()
[) Cl d 0 1t1 d
40sect dUl
-J
20
o
ns
Scarification
Figure A5 Effect of scarification on germination response for black locust = significant difference between adjacent treatment bars ns = no significant difference
decrease at the 10-minute level This drop-off suggests that 10 minutes of percussion
over-treats seeds from this lot of-black locust
A selected percussion treatment intermediate in time (5 minutes) also had the
fastest germination rate for black locust (Figure A6)_ The hot water curve is
somewhat flatter indicating a slower germination rate The control curve is very flat
indicating a slow germination rate
A qualitative difference between hot water and percussion-treated seeds was
also observed Along with healthy germinants the hot water treatment produced
over-treated seeds (Figure A 7a) Examples of damaged germinants include seeds
where the radicle did not elongate (Figure A 7b) seeds where the radicle elongated
but was delayed in freeing itself from the seed coat (Figure A7c) and seeds where the
radicle broke off completely from the embryo (Figure A7d) In addition to these
examples of over-treatment there were numerous small dark seeds that failed to
imbibe water For percussion treatment nearly all seeds took up water (Figure A8a)
Germination was rapid and uniform with little damage evident (Figure A8b)
To summarize both New Mexico locust and black locust seed lots were
dormant The standard hot water scarification treatment improved germination but
only to 56 and 41 for New Mexico locust and black locust respectively Nearly
all percussion durations improved total germination as well as germination rate over
the hot water treatment Germination was greater than 90 for percussion durations
of 4 5 and 10 minutes for New Mexico locust and 3 4 and 5 minutes for black locust
58
CI)
sgt t CI) 0
igt 0
sect ttl t E
Vl 0
c3
100r==~==~~=r------------------------------------~ ___ Percussion 5 min
middotmiddotmiddot0middotmiddotmiddot Hot Water -lIf- Control
80r=========~--~~----------------------------~
60
40
20 -~-~---~---------- ---~------------I
omiddot
O
_~_----------lIf-----~bull -----_ shy---shy I
Day 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A6 Effect of selected scarification treatments on germination speed for black locust First seven days of fourteen-day experiment are shown
Day 7
a
b c d
Figure A7 Damage to New Mexico locust seeds observed during germination counts following hot water scarification A7a Hot water replicate including damaged seedlings and numerous unimbibed seeds A7b Close-up ofseed where radicle failed to elongate A7c Close-up ofseed where radicle elongated but was delayed in freeing itself from the seed coat A7d Close-up ofseed where radicle completely broke offfrom embryo
60
a
b
Figure A8 New Mexico locust seeds following percussion treatment at time of germination counts A8a Percussion replicate with uniformly healthy seedlings and near complete imbibition of seeds A8b Close-up ofpercussion-treated seedling
61
Discussion
Papilionoid seeds have a specific anatomical feature known as the strophiole
or lens-the natural site of water entry to the seed (Hamly 1932) The strophiole is
located on the cotyledonary lobe of a locust seed (Figure A9) When a papilionoid
legume seed is percussed for the appropriate amount of time repeated hits on the
integrated seed coat loosen the constrained cells of the strophiolar region without
excessively damaging the rest of the seed coat (Ballard 1976)
When a percussed seed is soaked water enters exclusively through the
strophiole in a controlled manner (Kelly and Staden 1987) This regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues This
contrasts with seeds that have been hot water or acid scarified treatments that can
randomly degrade the seed coat Unlocalized cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rehman et al 1998 Marunda 1990)
Papilionoids are the largest subfamily of legumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust and black locust these legumes often play an integral role in the
revegetation ofdisturbed lands Developing superior scarification methods to the
standard hot water and acid treatments should fadlitate the use ofthese legumes in
restoration projects
62
Figure A9 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (bc)
63
APPENDIXB
GERMINATION PERCENTS AND STANDARD ERRORS FOR SOURCE
BLOCK AND TREATMENT FOR NEW MEXICO LOCUST
Table B 1 Gennination percents and standard errors for source block and treatment for New Mexico locust
Source Block Treatment Germination Percentage Standard Error
Capitan 1 Immersion 74 437 Capitan 2 Immersion 69 462 Capitan 3 Immersion 71 454 Capitan 4 Immersion 67 470 Capitan Boiling 30 sec 79 407 Capitan 2 Boiling 30 sec 76 427 Capitan 3 Boiling 30 sec 72 449 Capitan 4 Boiling 30 sec 76 427 Capitan Boiling 60 sec 71 454 Capitan 2 Boiling 60 sec 54 498 Capitan 3 Boiling 60 sec 67 470 Capitan 4 Boiling 60 sec 76 427 Capitan I Control 5 218 Capitan 2 Control 7 255 Capitan 3 Control 4 196 Capitan 4 Control 5 218 Capitan 1 Percussion I min 47 499 Capitan 2 Percussion I min
470
Capitan Percussion I min 39 488
Capitan 4 Percussion I min 38 485 Capitan I Percussion 2 min 55 497 Capitan Capitan
2
Percussion 2 min Percussion 2 min
71 65
454 477
Capitan 4 Percussion 2 min 56 496 Capitan I Percussion 4 min 86 347 Capitan 2 Percussion 4 min 79 407 Capitan 3 Percussion 4 min 81 392 Capitan 4 Percussion 4 min 82 384 Capitan I Percussion 8 min 90 300 Capitan 2 Percussion 8 min 88 325 Capitan 3 Percussion 8 min 90 300 Capitan 4 Percussion 8 min 90 300 Gila I Immersion 71 454 Gila 2 Immersion 77 421 Gila
Immersion 75 433
Gila 4 Immersion 72 449
Gila I Boiling 30 sec 68 466
Gila 2 Boiling 30 sec 81 392
Gila 3 Boiling 30 sec 54 498
Gila 4 Boiling 30 sec 63 483
Gila I Boiling 60 sec 55 497
Gila 2 Boiling 60 sec 68 466
Gila 3 Boiling 60 sec 57 495
Gila 4 Boiling 60 sec 70 458
65
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Gila 2 Control 13 336 Gila 3 Control 13 336 Gila 4 Control 20 400 Gila 1 Percussion 1 min 66 474 Gila 2 Percussion 1 min 57 495 Gila 3 Percussion 1 min 73 444 Gila 4 Percussion 1 min 82 384 Gila 1 Percussion 2 min 81 392 Gila 2 Percussion 2 min 86 347 Gila 3 Percussion 2 min 85 357 Gila 4 Percussion 2 min 54 498 Gila 1 Percussion 4 min 79 407 Gila 2 Percussion 4 min 83 376 Gila 3 Percussion 4 min 84 367 Gila 4 Percussion 4 min 79 407 Gila 1 Percussion 8 min 51 500 Gila 2 Percussion 8 min 76 427 Gila 3 Percussion 8 min 52 500 Gila 4 Percussion 8 min 74 439 Holman 1 Immersion 83 376 Holman 2 Immersion 86 347 Holman 3 Immersion 81 392 Holman 4 Immersion 90 300 Holman 1 Boiling 30 sec 90 300 Holman 2 Boiling 30 sec 85 357 Holman 3 Boiling 30 sec 87 336 Holman 4 Boiling 30 sec 83 376 Holman 1 Boiling 60 sec 79 407 Holman 2 Boiling 60 sec 80 400 Holman 3 Boiling 60 sec 86 347 Holman 4 Boiling 60 sec 93 255 Holman Control 25 433 Holman 2 Control 19 392 Holman Control 21 407
Holman 4 Control 24 427
Holman Percussion 1 min 77 421
Holman 2 Percussion 1 min 85 357
Holman 3 Percussion 1 min 80 400
Holman 4 Percussion 1 min 89 313
Holman 1 Percussion 2 min 88 325
Holman 2 Percussion 2 min 96 196
Holman 3 Percussion 2 min 93 255
Holman 4 Percussion 2 min 96 196
Holman 1 Percussion 4 min 92 271
Holman 2 Percussion 4 min 97 171
66
I
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Holman 3 Percussion 4 min 91 286 Holman 4 Percussion 4 min 96 196 Holman 1 Percussion 8 min 88 325 Holman 2 Percussion 8 min 85 357 Holman 3 Percussion 8 min 83 376 Holman 4 Percussion 8 min 82 384 Jemez 1 Immersion 53 499 Jemez 2 Immersion 44 496 Jemez 3 Immersion 52 500 Jemez 4 Immersion 70 458 Jemez 1 Boiling 30 sec 58 494 Jemez 2 Boiling 30 sec 55 497 Jemez 3 Boiling 30 sec 48 500 Jemez 4 Boiling 30 sec 65 477 Jemez 1 Boiling 60 sec 68 466 Jemez 2 Boiling 60 sec 63 483 Jemez 3 Boiling 60 sec 73 444 Jemez 4 Boiling 60 sec 89 313 Jemez 1 Control 2 140 Jemez 2 Control 9 286 Jemez 3 Control 6 237 Jemez 4 Control 8 271 Jemez 1 Percussion 1 min 58 494 Jemez 2 Percussion 1 min 63 483 Jemez 3 Percussion 1 min 77 421 Jemez 4 Percussion 1 min 89 313 Jemez 1 Percussion 2 min 68 466 Jemez 2 Percussion 2 min 64 480 Jemez 3 Percussion 2 min 80 400 Jemez 4 Percussion 2 min 83 376 Jemez Percussion 4 min 68 466 Jemez 2 Percussion 4 min 79 407 Jemez 3 Percussion 4 min 70 458 Jemez 4 Percussion 4 min 83 376 Jemez 1 Percussion 8 min 52 500 Jemez 2 Percussion 8 min 59 492 Jemez 3 Percussion 8 min 61 488 Jemez 4 Percussion 8 min 70 458 Magdalena 1 Immersion 69 462 Magdalena 2 Immersion 69 462 Magdalena J Immersion 70 458 Magdalena 4 Immersion 85 357 Magdalena 1 Boiling 30 sec 65 477 Magdalena 2 Boiling 30 sec 71 454 Magdalena 3 Boiling 30 sec 68 466
67
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Magdalena 4 Boiling 30 sec 83 376 Magdalena 1 Boiling 60 sec 63 483 Magdalena 2 Boiling 60 sec 68 466 Magdalena 3 Boiling 60 sec 63 483 Magdalena 4 Boiling 60 sec 82 384 Magdalena 1 Control 11 313 Magdalena 2 Control 6 237 Magdalena 3 Control 5 218 Magdalena 4 Control 4 196 Magdalena Percussion 1 min 32 466 Magdalena 2 Percussion 1 min 45 497 Magdalena 3 Percussion 1 min 46 498 Magdalena 4 Percussion I min 36 480 Magdalena 1 Percussion 2 min 67 470 Magdalena 2 Percussion 2 min 68 466 Magdalena 3 Percussion 2 min 65 477 Magdalena 4 Percussion 2 min 81 392 Magdalena 1 Percussion 4 min 87 336 Magdalena 2 Percussion 4 min 97 171 Magdalena 3 Percussion 4 min 83 376 Magdalena 4 Percussion 4 min 97 171 Magdalena 1 Percussion 8 min 98 lAO Magdalena 2 Percussion 8 min 100 000 Magdalena 3 Percussion 8 min 96 196 Magdalena 4 Percussion 8 min 98 lAO Manzano 1 Immersion 67 470 Manzano 2 Immersion 57 495 Manzano 3 Immersion 72 4049 Manzano 4 Immersion 73 4044 Manzano 1 Boiling 30 sec 70 458 Manzano 2 Boiling 30 sec 64 480 Manzano 3 Boiling 30 sec 71 454 Manzano 4 Boiling 30 sec 80 400 Manzano I Boiling 60 sec 50 500 Manzano 2 Boiling 60 sec 53 499 Manzano 3 Boiling 60 sec 59 492 Manzano 4 Boiling 60 sec 74 439 Manzano I Control 4 196 Manzano 2 Control 8 271 Manzano 3 Control 1 099 Manzano 4 Control 6 237 Manzano 1 Percussion 1 min 52 500 Manzano 2 Percussion 1 min 39 488 Manzano 3 Percussion 1 min 36 480 Manzano 4 Percussion 1 min 51 500
68
Table B 1 Continued
Source Block Treatment Gerrni~ation Percentage Standard Error
Manzano Percussion 2 min 53 499 Manzano 2 Percussion 2 min 58 494 Manzano 3 Percussion 2 min 61 488 Manzano 4 Percussion 2 min 62 485 Manzano 1 Percussion 4 min 76 427 Manzano 2 Percussion 4 min 85 357 Manzano 3 Percussion 4 min 79 407 Manzano 4 Percussion 4 min 83 376 Manzano 1 Percussion 8 min 87 336 Manzano 2 Percussion 8 min 83 376 Manzano 3 Percussion 8 min 83 376 Manzano 4 Percussion 8 min 75 433 Raton 1 Immersion 54 498 Raton 2 Immersion 37 483 Raton 3 Immersion 39 488 Raton 4 Immersion 69 462 Raton 1 Boiling 30 sec 36 480 Raton 2 Boiling 30 sec 20 400 Raton 3 Boiling 30 sec 28 449 Raton 4 Boiling 30 sec 60 490 Raton 1 Boiling 60 sec 15 357 Raton 2 Boiling 60 sec 171 Raton 3 Boiling 60 sec 4 196 Raton 4 Boiling 60 sec 35 477 Raton 1 Control 4 196 Raton 2 Control 12 325 Raton 3 Control 2 140 Raton 4 Control 6 237 Raton 1 Percussion 1 min 52 500 Raton 2 Percussion I min 29 454 Raton 3 Percussion I min 35 477 Raton 4 Percussion 1 min 31 462 Raton 1 Percussion 2 min 55 497 Raton 2 Percussion 2 min 35 477 Raton 3 Percussion 2 min 33 470 Raton 4 Percussion 2 min 42 494 Raton 1 Percussion 4 min 65 477 Raton 2 Percussion 4 min 52 500 Raton Percussion 4 min 51 500 Raton 4 Percussion 4 min 53 499 Raton 1 Percussion 8 min 88 325 Raton 2 Percussion 8 min 80 400 Raton 3 Percussion 8 min 73 444 Raton 4 Percussion 8 min 74 439 Sacramento 1 Immersion 43 495
69
Table B 1 Continued
-Source Block Treatment Germination Percentage Standard Error
Sacramento 2 Immersion 30 458 Sacramento 3 Immersion 38 485 Sacramento 4 Immersion 56 496 Sacramento 1 Boiling 30 sec 47 499 Sacramento 2 Boiling 30 sec 48 500 Sacramento 3 Boiling 30 sec 48 500 Sacramento 4 Boiling 30 sec 56 496 Sacramento 1 Boiling 60 sec 52 500 Sacramento 2 Boiling 60 sec 67 470 Sacramento 3 Boiling 60 sec 43 495 Sacramento 4 Boiling 60 sec 48 500 Sacramento 1 Control 0 000 Sacramento 2 Control 3 171 Sacramento 3 Control 3 171 Sacramento 4 Control 099 Sacramento 1 Percussion 1 min 57 495 Sacramento 2 Percussion 1 min 61 488 Sacramento 3 Percussion 1 min 62 485 Sacramento 4 Percussion 1 min 55 497 Sacramento 1 Percussion 2 min 84 367 Sacramento 2 Percussion 2 min 70 458 Sacramento 3 Percussion 2 min 81 392 Sacramento 4 Percussion 2 min 69 462 Sacramento 1 Percussion 4 min 91 286 Sacramento 2 Percussion 4 min 84 367 Sacramento 3 Percussion 4 min 84 367 Sacramento 4 Percussion 4 min 94 237 Sacramento 1 Percussion 8 min 82 384 Sacramento 2 Percussion 8 min 81 392 Sacramento 3 Percussion 8 min 82 384 Sacramento 4 Percussion 8 min 81 392 Sandia 1 Immersion 76 427 Sandia 2 Immersion 70 458 Sandia 3 Immersion 70 458 Sandia 4 Immersion 81 392 Sandia 1 Boiling 30 sec 78 414 Sandia 2 Boiling 30 sec 71 454 Sandia 3 Boiling 30 sec 63 483 Sandia 4 Boiling 30 sec 78 414 Sandia 1 Boiling 60 sec 67 470 Sandia 2 Boiling 60 sec 65 477 Sandia 3 Boiling 60 sec 74 439 Sandia 4 Boiling 60 sec 66 474 Sandia 1 Control 16 367 Sandia 2 Control 7 255
70
Table B 1 Continued
Source Block Treatment Gennination Percentage Standard Error
Sandia 3 Control 8 271 Sandia 4 Control 16 367 Sandia 1 Percussion 1 min 67 470 Sandia 2 Percussion 1 min 61 488 Sandia 3 Percussion 1 min 66 474 Sandia 4 Percussion 1 min 59 492 Sandia I Percussion 2 min 82 384 Sandia 2 Percussion 2 min 83 376 Sandia 3 Percussion 2 min 81 392 Sandia 4 Percussion 2 min 84 367 Sandia 1 Percussion 4 min 89 313 Sandia 2 Percussion 4 min 86 347 Sandia 3 Percussion 4 min 89 313 Sandia 4 Percussion 4 min 85 357 Sandia 1 Percussion 8 min 80 400 Sandia 2 Percussion 8 min 77 421 Sandia 3 Percussion 8 min 69 462 Sandia 4 Percussion 8 min 66 474
LITERATURE CITED
Ashby WC Vogel WG Rogers NF 1985 Black locust in the reclamation equation General Technical Report Northeastern Forest Experiment Station USDA Forest Service NE-I05 12 p
Ballard LAT 1973 Physical barriers to gennination Seed Sci Technoll 285-303
___ 1976 Strophiolar water conduction in the seeds ofTrifolieae induced by action on the testa at nonstrophiolar sites Austr J Plant Physiol3 465-469
Barton LV 1947 Special studies on seed coat impenneability Contrib Boyce Thompson Inst 14 355-362
Baskin CC Baskin JM 1998 Seeds Ecology biogeography and evolution of donnancy and gennination Academic Press London UK 666 p
Bewley JD 1997 Seed germination and donnancy Plant Ce119 1055-1066
Bewley JD Black M 1994 Seeds physiology of development and germination 2nd ed Plenum Press New York NY 445 p
Briscoe CB 1996 Growing Nitrogen Fixing Trees in Acid Soils In MH Powell (ed) Nitrogen Fixing Trees for Acid Soils- A Field ManuaL Winrock International Morrilton AR 22 p
Copeland LO McDonald MB 1995 Principles of Seed Science and Technology 3rded Chapman amp Hall New York NY 409 p
Cox RE Klett JE 1984 Seed gennination requirements of native Colorado plants for use in the landscape Plant Propag 30(2) 6-10
Dell B 1980 Structure and function ofthe strophiolar plug in seeds ofAlbizia Lophantha Am J Bot 67 556-563
Dick-Peddie WA 1993 New Mexico Vegetation Past Present and Future University ofNew Mexico Press Albuquerque NM 244 p
Dreesen DR 2001 Propagation protocol for production ofcontainer Robinia neomexicana plants Los Lunas Plant Materials Center Los Lunas New Mexico In Native Plant Network URL httpwwwnativeplantnetworkorg (accessed 9 January 2002) Moscow (ID) University ofIdaho CoIl Natural Resources For Res Nursery
72
Dreesen DR Harrington JT 1997 Propagation of native plants for restoration projects in the Southwestern US- Preliminary Investigations In Landis TD Thompson JR tech coords National Proceedings Forest and Conservation Nursery Assocs Gen Tech Rep PNW-GTR-419 Portland OR USDA For Ser PNW Res Sta 77-88
Gottfried GJ 1980 Control ofNew Mexican locust and the effect on planted ponderosa pine in central Arizona US For Ser Res Note RM-386 Rocky Mountain For Range ExpStn Fort Collins CO
Gosling PG Samuel YK Jones SK 1995 A systematic examination of germination temperature chipping and water temperaturesoak duration pretreatments on the seeds ofLeucaena leucocephala Seed Sci amp Technol 23521-532
Hamly DH 1932 Softening of the seeds of Melilotus alba Bot Gazette 93 345shy375
Hanna PJ 1984 Anatomical features of the seed coat ofAcacia kempeana (Mueller) which relate to increased germination rate induced by heat treatment New Phytol 96 23-29
Hanks JP Dick-Peddie WA 1974 Vegetation patterns of the White Mountains New Mexico Southwest Naturalist 18(4) 371-382
Hine S Harrington JT Dreesen DR 1997 Seed scarification requirements for Robinia neomexicana In Harrington JTtech coord Proc Third SouthwestemContainer Growers Conf Feb 12-13 1997 Ft Collins CO
Hopkinson JM 1993 The strophiole of leguminous seeds Newsletter- IntI Herbage Seed Production Res Grp 18 7-8
---- 1997 Differences in hardseededness between Cunningham and Tarramba leucaena In Leucnet News The Newsletter of the International LeucaenaRes and Devlt Netwk No4
ISTA (International Seed Testing Association) 1999 International rules for seed testing rules Seed Sci amp Technol27 Supplement
Kelly KM van Staden J 1987 The lens as the site of permeability in the papilionoid seed Aspalathus linearis J Plant Physiol 128(45) 395-404
73
Keresztesi B 1988 Black locust the tree of agriculture Outlook on Agr 17(2) 77~85
Kigel 1 and Galili G 1995 Seed development and germination Marcel Dekker New York 853 p
Klemmedson 10 1994 New Mexico locust and parent material Influence on macronutrients of forest floor and soil Soil Sci Soc Am 1 58 974~980
Kuo WH Tam AW 1988 The pathway of water absorption of mung bean seeds Seed Sci amp Technol16(1) 139~144
Leadem CL 1997 Dormancy--unlocking seed secrets In Landis TD Thompson lR tech coords National Proceedings Forestand Conservation Nursery Assocs Gen Tech Rep PNW-G TR-419 Portland OR USDA For Ser PNW Res Sta 43-52
Lin Y Wagner MR Seweyestewa A Honahni MD DeGomez M McMillin JD 1996 Influence of cultural treatments on seed germination and seedling growth of Robinia neomexicana Forest Farm and Community Tree Res Reports Volume 1
Marunda CT 1990 Effects of seed pretreatment on the development ofAcacia auriculforinis and A holosericea seedlings ACIAR Proceedings Series No 28
Meginnis HG 1937 Sulphuric acid treatment to increase germination of black locust seed USDA Circ 453 34 p
Miklas PN Towensend CE Ladd SL 1987 Seedcoat anatomy and the scarification ofcicer milkvetch seed Crop Sci 27 766-772
Monsen SB 1984 Use of shrubs on mine spoils In The Challenge ofProducing Native Plants for the Intermountain Area Proceedings Intermountain Nurserymans Association 1983 conference Las Vegas NV compo PM Murphy 26-31 Ogden UT USDA For Ser Gen Tech Report INT-l68
Olson DF 1974 Robinia L Locust In Seeds of Woody Plants in the US tech coord CS Schopmeyer 728-731 USDA For Ser Agr Handbk No450
Rehman S Loescher RNl Harris P1C 1998 Dormancy breaking and germination ofAcacia salicina Lindl seeds Seed Sci amp Techno127 553-557
Rolston MP 1978 Water impermeable seed dormancy Bot Rev 44 365-396
74
SAS Institute Inc 1989 SASSTAT Users Guide Version 6 4th Edition Volume 1 Cary NC SAS Institute Inc 943 p
SerratoValenti G Conara L Ferrando M Modenesi P 1993 Structural and histochemical features ofStylosanthes scabra (Leguminosae Papilionoidae) seed coat as related to water entry Can J Bot 71 834-840
Simpson BJ 1988 A field guide to Texas trees Austin TX Texas Monthly Press 372p
Tran VN Cavanagh AK 1984 Structural aspects ofdormancy In Seed Physiology Vol 2 Murray DR (ed) p1-44 Academic Press Australia
USDA Forest Service 1948 Woody-plant seed manual USDA Misc Pub1654 416p
Vines RA 1960 Trees Shrubs and Woody Vines of the Southwest Austin Univ ofTexas Press 1104 p
Wagner MR Larson RE Craig TP 1992 Ecology and silvics ofNew Mexico locust (Robinia neomexicana) with an emphasis on the arboreal form p53-57 In JW Hanover K Miller and S Plesko (eds) Proc Intemat Confon Black Locust Biology Culture and Utilization Mich State Univ USA
Wilson JK 1944 Immersing seeds of species of Robinia in boiling water hastens germination J For 42 453-454
Zimmerman RW Carpenter SB 1980 First-year coppice production from a 5shyyear old black locust stand on surface mine spoil p309-314 Centr Hardwd For ConfProc University ofMissouri Columbia
100~----------------------~--~----------------~
80
60
ltU
~
40 IJ
sectgsI
l100
80
60
40
a I middotmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot middotImiddotmiddotmiddotmiddotmiddotmiddot E
_~iii-bull _ _ bull f --~--
t-- -~ ---- -shy ---- shyI ~ ---- - shy
L
fiII
~
b
- shy ---r
- Holman
Sacramento Sandia Gila Jemez
c ~
ltgt ~
Tmiddotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot-I
j~=~=-~~-1
I middotmiddot ~ bull r
t tr
Y -------M-a-gd-a-Ie-n-a--
1 _ _
fCapitan
---- Manzano ~y -_ Raton
2 4 8
Time interval ofpercussion scarification (min)
Figure 4 Effect of seed source by treatment interaction on New Mexico locust germination for percussion scarification pattern a and pattern b
28
tv 0
~ d
~ A s o
1 c3
80
60
40
20
o
TIT
t------=middotfmiddotmiddot-middotmiddot-middotmiddot-middotmiddot 1- --- - - --=- - -shy--cE= - _--shy --shy ----shy ~ ~ ---shy ~ -~- _ --shy-------r 1
I
Raton Holman
-_ Jemez
--- Sandia Manzano Magdalena Capitan Sacramento Gila
Immersion Boiling 30 sec Boiling 60 sec
Hot Water Scarification
Figure 5 Effect of seed source by treatment interaction on New Mexico locust germination for hot water scarification
continued to increase in germination (Figure 4b) There was no difference between
the two treatment levels for the Manzano source (Figure 4b)
The effect ofhot water scarification also varied among sources with few
overall differences between hot water treatments (Figure 5) However boiling for 60
seconds yielded highest germination for the Jemez source whereas increasing
durations ofboiling decreased germination for the Raton source
In addition to consistently high germination 4- and 8-minute percussionshy
treated seeds germinated as fast or faster than all other treatments (Figure 6 Table 4)
050 measured as the number of days needed to complete 50 ofeventual
germination averaged 2 days for both ofthese treatments
X-ray radiographs were taken of seeds collected from the Sacramento source
percussed for 1 4 or 8 minutes (Figure 7) Seeds show signs of damage at 8 minutes
ofpercussion (Figure 8) Compared to an undamaged seed percussed for only 4
minutes (Figure 8a) damage at the 8-minute level includes fractured radicles pitting
of the seed coat and chips in seed coat structure (Figure 8b-d)
Scanning electron microscope (SEM) images at 600X reveal the direct effect
of 4 minutes of percussion on cells of the strophiolar region (Figure 9) Whereas
untreated seeds retain cellular organization in the strophiolar region strophiolar cells
seeds treated for 4 minutes ofpercussion separate and raise resulting in a
~-~e~U appearance
30
Umiddot-~middot--l-~---middotymiddotmiddotmiddot-----middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot--middot--middotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middotmiddotmiddotmiddotmiddotmiddotmiddot- -
050= gt1
-e-- Percussion 8 min -0- Percussion 4 min ---T- Percussion 2 min -----ltil- Percussion 1 min -- Immersion --0- Boiling 30 sec -+- Boiling 60 sec -ltgt- Control
2 4 6 8 10 12
Day Figure 6 Timecourse ofNew Mexico locust gennination following scarification
treatments G50 is the number of days to 50 of eventual germination
31
14
Table 4 Effect of seed source and treatment on G50 (days to 50 germination)
Seed Source Control Immersion B30sec B60sec Plmin P2min P4min P8min mean Raton 3 3 5 7 4 3 2 2 4 Holman 2 2 2 2 2 2 2 2 2
Jemez 4 6 6 5 3 2 2 2 4 Sandia 2 2 2 2 2 2 2 2 2
w tv Manzano 5 2 2 3 3 2 2 2 3
Magdalena 4 2 2 3 4 3 2 1 3
Capitan 5 4 2 2 3 2 2 2 3
Sacramento 3 2 2 2 4 3 2 2 3 Gila 3 2 2 2 2 2 2 2 2 mean 3 3 3 3 3 2 2 2 3 standard 11 14 16 18 09 05 0 03 08 deviation
Note Columns and rows may not agree due to rounding
Figure 7 X-ray radiographs ofSacramento seed source percussed for 1 4 or 8 minutes
33
Figure 8a-d Seed damage close-ups Figure a shows undamaged seed percussed for 4 minutes Figures b-d show seeds percussed for 8 minutes Damage includes fracturing ofradicle in b complete removal ofseed coat in c and pitting ofseed coat in d
34
Untreated Treated
Figure 9 SEM images ofuntreated vs 4-minute percussion-treated strophiolar cells ofNew Mexico locust seeds Treated cells are loosened separated and raised
DISCUSSION
Determining the Optimal Treatment
Averaging results of scarification treatmems across seed sources is a useful
tool to predict which treatment is most likely to be successful for an unknown seed
source Averaged over all sources four- and eight-minute percussion treatments
resulted in the highest germination 815 and 790 respectively and produced
germination that was as fast or faster than all other treatments Although there was no
difference between these two treatments on average an argument for 4 minutes of
percussion as a standard protocol for treating New Mexico locust seeds can be made
Descriptively 4 minutes of percussion improved germination over 8 minutes
ofpercussion for a majority of sources (five out of nine) 8 minutes of percussion
improved germination for three ofnine sources with no difference for a fmal source
For individual sources 4 minutes ofpercussion was always the best or secondmiddot best
treatment and was always equal to or better than all hot water treatments
Germination at 8 minutes ofpercussion was lower than all other percussion durations
for the Jemez and Gila sources and was lower than at least one hot water treatment for
these sources as well
The x-ray radiographs of the Sacramento seed source illustrate the excessive
internal and external seed damage that can occur with 8 minutes of percussion
germination counts 8-minute percussed seeds from Holman and Capitan
ClUll signs of damage that were visible to the naked eye (see below)
r~evertheless some of these seeds still fit the criteria for germination Neither x-ray
36
photography nor visible observations during seed counts revealed seed damage at the
4-minute percussion level In operational trials seeds percussed for 4 minutes
developed into healthy seedlings (Khadduri and Harrington unpublished data)
Strophiole and Seed Coat Behavior
Four minutes ofpercussion successfully breaks seed dormancy in a large
percentage ofNew Mexico locust seeds without excessively damaging the
surrounding seed coat or internal seed coat structures Most papilionoid legume
seeds including seeds ofNew Mexico locust have a specific region of the seed coat
known as the strophiole or lens (Hamly 1932) The strophiole is located on the
cotyledonary lobe of a locust seed (Figure 10 a-c) If seeds are not indiscriminately
damaged the strophiole is the first point of water entry to the embryo (Hopkinson
1997) When a papilionoid seed is percussed for the appropriate amount of time
repeated hits on the integrated seed coat loosen the constrained cells of the strophiolar
region without excessively damaging the rest of the seed coat (Ballard 1976) SEM
photos illuStrate that 4 minutes of percussion loosened and separated strophiolar cells
ofNew Mexico locust seeds when compared with constrained organized strophiolar
cells of untreated seeds (Figure 9) Research-has shown that water passes to the
embryo through the treated strophiole in a controlled manner and regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues
(Kelly and van Staden 1987)
37
10 Strophiole ofunimbibed New Mexico locust seed (a) and following uptake (bc)
38
In contrast hot-water treatments can excessively degrade the seed coat
(Rebman et al 1998 Marunda 1990) Random cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rebman et aI 1998 Marunda 1990) Along with a
proportionately large amount ofun-imbibed (thus under-treated) seeds (Figure l1a)
damage was observed in hot-water treated seeds from six sources during germination
counts Damage included seeds where the radicle failed to elongate (Figure 11 b)
seeds where the radicle elongated but was delayed in freeing itself from the seed coat
(Figure 11c) and seeds where the radicle completely broke off from the embryo
(Figure lId) In addition some seeds germinated in reverse (the cotyledon emerged
fIrst) If the radicle emerged completely from the seed coat they were counted as
germinated
While nearly all seeds treated for 8 minutes ofpercussion imbibed water
damage was evident in x-ray evaluations (Sacramento seed source) and in
germination counts (Holman and Capitan) at this treatment level Seed damage
during germination counts was similar to levels found in hot-water treated seeds
suggesting that over-treatment by both hot water and longer durations of percussion
may result in similar damage to seed structure
The Sacramento and Holman seed sources were two of the fIve sources that
opcrplltlpn in germination from the 4- to 8-minute durations of percussion
the benefIts ofloosening the strophiole as evidenced by imbibition of
all 8-minute treated seeds were outweighed by external and internal seed coat
39
a
b c d
11 Damage to New Mexico locust seeds observed during germination cotmts following hot water scarification 9a Hot water replicate including ~t seedlings and numerous unimbibed seeds 9b Close-up of seed where raallCle failed to elongate 9c Close-up ofseed where radicle elongated but was
-gtIn in freeing itself from the seed coat 9d Close-up ofseed where radicle broke off from embryo
40
damage (which also may be contributing to high imbibition) For the three sources
for which 8 minutes of percussion resulted in maximum germination extended
percussion of seeds necessary to loosen the strophiole may not have excessively
damaged the seed anatomy The Manzano source did not significantly change in
germination from 4 to 8 minutes of percussion suggesting that an increase in
permeable seeds at the 8-minute treatment duration was balanced by the number of
seeds that failed to germinate due to seed damage To summarize the 4-minute
percussion treatment improved germination over 8 minutes ofpercussion for a
majority of sources (five ofnine) the 8-minute percussion treatment improved
germination over 4-minutes ofpercussion for three sources and there was no
difference with the Manzano source Notably for the Raton source 8 minutes of
percussion increased germination by 235 percentage points over 4-minutes of
percussion (7875 vs 5525 respectively) the next best treatment Raton was the
only source to result in germination less than 75 for 4 minutes ofpercussion so a
grower may wish to apply longer percussion durations to lots that do not respond well
to this treatment level
Finally there was an interesting trend in the Jemez and Raton sources seed
that significantly contributed to the source by treatment interaction (Figure 12) A
~u test showed that the Jemez source had a significantly lighter seed weight than
other sources while the Raton source had an average seed weight For the Jemez
the longest duration of boiling increased germination Conversely only short
U4UVll) of percussion were needed to achieve high germination and the longest
41
80 _ Jemez a Raton
iD
~ C iD e iD
Q c 0 ~
c ~ iD 0
20
0------shy
Immersion Boiling 30 sec Boiling 60 sec
Hot Water Scarification
10degTr========~--------~------------------------------
80
60
40
20
_ Jemez Raton
0------shy
1 min 2 min 4 min 8 min
Percussion Scarification
Figure 12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources
42
b
--~----~~~~========~---- duration ofpercussion reduced germination For the Raton source longer durations
ofboiling consistently decreased germination whereas longer percussion treatments
consistently increased germination
A possible explanation is that a thick seed coat is associated with a loose
strophiole structure and that a thin seed coat is associated with a constrained
strophiole This contrast merits further study and may suggest an ecological
adaptation Further investigation ofthis phenomenon may contribute to successful
pre-screening ofunknown seed sources to determine optimal treatment leveL
43
CONCLUSION
Based on results obtained in pilot study (see Appendix) and thesis
experiments 4 minutes ofpercussion is recommended as a standard protocol for
treating an unknown source ofNew Mexico locust seed In addition to laboratory
results the 4-minute percussion treatment has resulted in germination greater than
80 and healthy seedling grow-outs for production purposes (unpublished data) In
laboratory experiments only one hundred seeds per 118 ml (40z) soil tin were
percussed at a time whereas for production we have treated up to five hundred seeds
per tin For large-scale production ofNew Mexico locust seedlings growers may
wish to experiment with larger quantities of seed and larger-size soil tins
Seed storage trials following percussion treatments were not conducted
However germination greater than 80 and healthy seedlings were obtained when
seeds were percussion-treated and subsequently stored at room-temperature (22degC)
for one week (unpublished data) Seed storage trials merit further study as the
standard hot water treatment leaves seeds in a softened condition that is not
conducive to storage
Hydroseeding is an effective method for direct seeding in reclamation
situations Percussion treatment may be conducive to hydro seeding as seeds remain
a dry relatively undamaged yet permeable condition following treatment Sulfuric
seeds also remain dry following treatment but extensive damage to seed coat
poor seedling vigor have been reported with this treatment (Kelly and van Staden
A grow-out study using a hydro seeder on a reclamation site testing percussion
44
against other treatments such as sulfuric acid and mechanical scarification also
merits further study
Papilionoids are the largest subfamily oflegumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust these legumes often play an integral role in the revegetation of
disturbed lands The pilot study (Appendix) indicates that percussion scarification
successfully treats the widely used black locust as well Developing superior
scarification methods to the standard hot water mechanical scarification and acid
treatments may facilitate the use ofpapilionoid legumes in reclamation projects
45
APPENDICES
APPENDIX A
PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR
NEW MEXICO LOCUST AND BLACK LOCUST SEEDS
Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4
Hot water and sulfuric acid soaks are traditional treatments for seeds of many
temperate woody legwnes including locusts However these scarification techniques
often produce inconsistent germination Percussion scarification where seeds are
repeatedly propelled against a hard surface was compared with hot water
scarification to evaluate treatment efficacy for New Mexico locust (Robinia
neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment
seeds were placed in a 98degC water bath which was immediately removed from the
heat source For percussion scarification seeds were placed in a soil sample tin and
agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the
control were followed by 24-hour water soaks Hot water baths resulted in 56 and
41 germination for New Mexico locust and black locust respectively For both
species nearly all durations ofpercussion increased germination over the hot water
treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3
4 and 5 minutes for black locust resulted in at least 90 germination Traditional
scarification treatments randomly degrade the entire seed coat which can lead to
tissue damage during water uptake Percussion scarification specifically weakens the
strophiole the natural source ofwater entry to the seed in papilionoid legwnes
INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom
2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031
48
Following percussion imbibition is controlled through the strophiole and underlying
tissue is protected
Keywords New Mexico locust black locust scarification percussion germination
Introduction
Locust trees (Robinia spp) are aggressive pioneer species that quickly
colonize disturbed land fix atmospheric nitrogen in the soil and stabilize slopes that
are prone to erosion (Klemmedson 1994 Ashby et al 1985) (Figure A1ab)
Recognition of these attributes has contributed to increased demand for locusts in
reclamation projects Locust seeds however exhibit physical dormancy where a
thick seed coat prevents the movement of water and gasses to the embryo (Leadem
1997)
Sulfuric acid or hot water soaks have traditionally been used to break seed
dormancy in macro-propagation of locusts Many growers are moving away from the
use of su1furic acid in treating locust seeds (Dreesen and Harrington 1997) In
addition to being dangerous acid soak durations must be specifically correlated to
seed lot (Olson 1974) Collections of native seed can vary tremendously in seed size
seed weight and hard seededness etc This variability limits the utility of sulfuric
acid soaks as even within a given lot some seeds may be damaged whereas others
are under-treated and fail to imbibe (take up water) A survey of Southwestern
container growers indicates that hot water baths are currently the most common
scarification method for locust seeds (Hine et al 1997) However hot water baths
also produce inconsistent germination (Lin et al 1996)
49
b
Figure AI New Mexico locust a primary invader following disturbance Ala Crown-sprouting at edge ofburn near Los Alamos NM--one year after Cerro Grande fire Alb Colonizing road cut Gila National Forest NM
50
Percussion scarification where seeds are repeatedly propelled against a hard
surface is an alternative dormancy-breaking method that has proven successful in
legumes related to locusts (Hamly 1932 Barton 1947) We compared this alternative
treatment to hot water scarification to evaluate treatment efficacy for New Mexico
locust and black locust seeds
Materials and Methods
Commercial New Mexico locust seeds (Western Native Seed Coaldale CO)
were collected fall 2000 in Huerfano County CO Black locust seeds were collected
September 2000 from Taos County NM by harvesting ripe pods from several trees at
a distance of up to 3 m from ground level Black locust seeds were allowed to air-dry
for 3 weeks and were threshed and separated from large chaffusing a greenhouse
fan A South Dakota Seed Blower (Chicago Illinltois) was used to remove fine chaff
from both seed lots Seeds were storedmiddotat 2-4C until the germination study was
conducted in March 2001
This study compared hot water scarification with percussion scarification
using treatment and species as experimental factors Along with a control
scarification treatments included immersion of seed in boiling water as well as
percussion durations of 1 2 3 45 or 10 minutes Four 100-seed samples were used
to test each species by scarification treatment combination
For the hot water treatment test tubes were filled with 30 mL water and
placed in a water-filled beaker (Figure A2a) The bath was raised to boiling (-98C at
51
b
c
Figure A2 Implementation ofhot water and percussion scarification A2a Hot water bath A2b Paint-shaker set-up A2c Placing soil tin containing seeds at end ofpaint shaker ann
52
Las Cruces NM elevation of 1300 meters) Each 100-seed replicate was placed in a
test tube and each test tube was immediately removed to cool at room temperature
Percussion scarification was implemented using a pneumatic paint shaker
(Central Pneumatic model 00422 Camarillo CA) (Figure A2b) This allowed
standardization of treatment intensity Compressed air pressure was maintained at
530kPa plusmn 30kPa (80psi plusmn 5psi) resulting in approximately 350 oscillations per
minute Each lOO-seed sample was placed in a 118 ml (4 oz) soil tin for shaking To
maximize lateral movement of paint shaker a paint can was used as a spacer to place
soil tins at the end of the shaking arm (Figure A2c) This allowed shaking to occur at
the greatest distance from the pivot point (fulcrum) Following all treatments
including the control seeds were soaked for twenty-four hours in distilled water prior
to plating out
Gennination was evaluated on lab benches at room temperature Each
replicate of seeds was placed in a 100 cm petri dish on moistened filter paper with
humidity maintained by enclosing the petri dishes in ziplock bags Gennination was
monitored daily for 14 days in accordance with International Seed Testing
Association standards for Robinia species (ISTA 1999) Gennination was defined as
protrusion of the radicle from the embryo by at least 1mm as viewed by the naked
eye
Categorical Analysis of Variance using Prpc Catmod (SAS Institute 1989)
was used to detennine treatment differences in germination percentages for each
source This procedure is an extension of a Chi-square test of homogeneity using the
53
natural log of the ratio of germinated to non-germinated seeds for each treatment Pshy
values less than 005 were considered significant A limited set of pairwiseshy
comparisons was conducted to compare treatment mean percents using a
conservative alpha value of 005 divided by the number of comparisons
Results
Percussion maximized germination for both New Mexico locust and black
locust There were three germination response levels for New Mexico locust (Figure
A3) First there was a low response in the control indicating a dormant seed lot
The hot water treatment and I-minute duration of percussion share a second
intermediate response Germination was highest at percussion levels from 2 to 10
minutes
A selected percussion treatment intermediate in time (5 minutes) produced the
steepest germination curve indicating the fastest germination rate (Figure A4) The
hot water treatment produced an intermediate curve or relatively slower germination
rate The control curve was very flat indicating the slowest germination rate for New
Mexico locust
Control seeds germinated poorly for black locust as well (Figure A5) Hot
water scarification improved germination over the control but all levels of percussion
improved germination over hot water The optimal durations of percussion treatment
were bracketed with a significant increase from the 1 to 2 minute percussion level a
high germination response from 2 to 5 minutes of percussion and finally a significant
54
~ d 25 CL)
p t1 0 ~ t1
g Vl ~ Vl
100~---------------------------------------------------------------
80
60
40
20
o Control Hot Water Percussion
I min Percussion
2 min Percussion
3 min
Scarification
Percussion 4 min
Figure A3 Effect of scarification on germination response for New Mexico locust
Percussion 5 min
=significant difference between adjacent treatment bars ns = no significant difference
Percussion 10 min
~ ~ () u agt
Il-t d o ~
~ ~ c3
100middotlr~~~------------------------------------e- Percussion 5min middot 0middot Hot Water -- Control
80-J=middot======
6b-middot-lmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middot--middotmiddot-middot~-middotmiddot -middotmiddot middot middotmiddot-middot 1
40
20
-----~-----------
0 0 0 middotmiddotmiddotmiddotmiddot0middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot0
0 --~-----y-----~------_---~--- T-----~
o shy -TDay 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A4 Effect of selected scarification treatments on germination speed for New Mexico locust First seven days of fourteen-day experiment are shown
Day 7
80
~ cd
5 60 ()
[) Cl d 0 1t1 d
40sect dUl
-J
20
o
ns
Scarification
Figure A5 Effect of scarification on germination response for black locust = significant difference between adjacent treatment bars ns = no significant difference
decrease at the 10-minute level This drop-off suggests that 10 minutes of percussion
over-treats seeds from this lot of-black locust
A selected percussion treatment intermediate in time (5 minutes) also had the
fastest germination rate for black locust (Figure A6)_ The hot water curve is
somewhat flatter indicating a slower germination rate The control curve is very flat
indicating a slow germination rate
A qualitative difference between hot water and percussion-treated seeds was
also observed Along with healthy germinants the hot water treatment produced
over-treated seeds (Figure A 7a) Examples of damaged germinants include seeds
where the radicle did not elongate (Figure A 7b) seeds where the radicle elongated
but was delayed in freeing itself from the seed coat (Figure A7c) and seeds where the
radicle broke off completely from the embryo (Figure A7d) In addition to these
examples of over-treatment there were numerous small dark seeds that failed to
imbibe water For percussion treatment nearly all seeds took up water (Figure A8a)
Germination was rapid and uniform with little damage evident (Figure A8b)
To summarize both New Mexico locust and black locust seed lots were
dormant The standard hot water scarification treatment improved germination but
only to 56 and 41 for New Mexico locust and black locust respectively Nearly
all percussion durations improved total germination as well as germination rate over
the hot water treatment Germination was greater than 90 for percussion durations
of 4 5 and 10 minutes for New Mexico locust and 3 4 and 5 minutes for black locust
58
CI)
sgt t CI) 0
igt 0
sect ttl t E
Vl 0
c3
100r==~==~~=r------------------------------------~ ___ Percussion 5 min
middotmiddotmiddot0middotmiddotmiddot Hot Water -lIf- Control
80r=========~--~~----------------------------~
60
40
20 -~-~---~---------- ---~------------I
omiddot
O
_~_----------lIf-----~bull -----_ shy---shy I
Day 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A6 Effect of selected scarification treatments on germination speed for black locust First seven days of fourteen-day experiment are shown
Day 7
a
b c d
Figure A7 Damage to New Mexico locust seeds observed during germination counts following hot water scarification A7a Hot water replicate including damaged seedlings and numerous unimbibed seeds A7b Close-up ofseed where radicle failed to elongate A7c Close-up ofseed where radicle elongated but was delayed in freeing itself from the seed coat A7d Close-up ofseed where radicle completely broke offfrom embryo
60
a
b
Figure A8 New Mexico locust seeds following percussion treatment at time of germination counts A8a Percussion replicate with uniformly healthy seedlings and near complete imbibition of seeds A8b Close-up ofpercussion-treated seedling
61
Discussion
Papilionoid seeds have a specific anatomical feature known as the strophiole
or lens-the natural site of water entry to the seed (Hamly 1932) The strophiole is
located on the cotyledonary lobe of a locust seed (Figure A9) When a papilionoid
legume seed is percussed for the appropriate amount of time repeated hits on the
integrated seed coat loosen the constrained cells of the strophiolar region without
excessively damaging the rest of the seed coat (Ballard 1976)
When a percussed seed is soaked water enters exclusively through the
strophiole in a controlled manner (Kelly and Staden 1987) This regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues This
contrasts with seeds that have been hot water or acid scarified treatments that can
randomly degrade the seed coat Unlocalized cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rehman et al 1998 Marunda 1990)
Papilionoids are the largest subfamily of legumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust and black locust these legumes often play an integral role in the
revegetation ofdisturbed lands Developing superior scarification methods to the
standard hot water and acid treatments should fadlitate the use ofthese legumes in
restoration projects
62
Figure A9 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (bc)
63
APPENDIXB
GERMINATION PERCENTS AND STANDARD ERRORS FOR SOURCE
BLOCK AND TREATMENT FOR NEW MEXICO LOCUST
Table B 1 Gennination percents and standard errors for source block and treatment for New Mexico locust
Source Block Treatment Germination Percentage Standard Error
Capitan 1 Immersion 74 437 Capitan 2 Immersion 69 462 Capitan 3 Immersion 71 454 Capitan 4 Immersion 67 470 Capitan Boiling 30 sec 79 407 Capitan 2 Boiling 30 sec 76 427 Capitan 3 Boiling 30 sec 72 449 Capitan 4 Boiling 30 sec 76 427 Capitan Boiling 60 sec 71 454 Capitan 2 Boiling 60 sec 54 498 Capitan 3 Boiling 60 sec 67 470 Capitan 4 Boiling 60 sec 76 427 Capitan I Control 5 218 Capitan 2 Control 7 255 Capitan 3 Control 4 196 Capitan 4 Control 5 218 Capitan 1 Percussion I min 47 499 Capitan 2 Percussion I min
470
Capitan Percussion I min 39 488
Capitan 4 Percussion I min 38 485 Capitan I Percussion 2 min 55 497 Capitan Capitan
2
Percussion 2 min Percussion 2 min
71 65
454 477
Capitan 4 Percussion 2 min 56 496 Capitan I Percussion 4 min 86 347 Capitan 2 Percussion 4 min 79 407 Capitan 3 Percussion 4 min 81 392 Capitan 4 Percussion 4 min 82 384 Capitan I Percussion 8 min 90 300 Capitan 2 Percussion 8 min 88 325 Capitan 3 Percussion 8 min 90 300 Capitan 4 Percussion 8 min 90 300 Gila I Immersion 71 454 Gila 2 Immersion 77 421 Gila
Immersion 75 433
Gila 4 Immersion 72 449
Gila I Boiling 30 sec 68 466
Gila 2 Boiling 30 sec 81 392
Gila 3 Boiling 30 sec 54 498
Gila 4 Boiling 30 sec 63 483
Gila I Boiling 60 sec 55 497
Gila 2 Boiling 60 sec 68 466
Gila 3 Boiling 60 sec 57 495
Gila 4 Boiling 60 sec 70 458
65
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Gila 2 Control 13 336 Gila 3 Control 13 336 Gila 4 Control 20 400 Gila 1 Percussion 1 min 66 474 Gila 2 Percussion 1 min 57 495 Gila 3 Percussion 1 min 73 444 Gila 4 Percussion 1 min 82 384 Gila 1 Percussion 2 min 81 392 Gila 2 Percussion 2 min 86 347 Gila 3 Percussion 2 min 85 357 Gila 4 Percussion 2 min 54 498 Gila 1 Percussion 4 min 79 407 Gila 2 Percussion 4 min 83 376 Gila 3 Percussion 4 min 84 367 Gila 4 Percussion 4 min 79 407 Gila 1 Percussion 8 min 51 500 Gila 2 Percussion 8 min 76 427 Gila 3 Percussion 8 min 52 500 Gila 4 Percussion 8 min 74 439 Holman 1 Immersion 83 376 Holman 2 Immersion 86 347 Holman 3 Immersion 81 392 Holman 4 Immersion 90 300 Holman 1 Boiling 30 sec 90 300 Holman 2 Boiling 30 sec 85 357 Holman 3 Boiling 30 sec 87 336 Holman 4 Boiling 30 sec 83 376 Holman 1 Boiling 60 sec 79 407 Holman 2 Boiling 60 sec 80 400 Holman 3 Boiling 60 sec 86 347 Holman 4 Boiling 60 sec 93 255 Holman Control 25 433 Holman 2 Control 19 392 Holman Control 21 407
Holman 4 Control 24 427
Holman Percussion 1 min 77 421
Holman 2 Percussion 1 min 85 357
Holman 3 Percussion 1 min 80 400
Holman 4 Percussion 1 min 89 313
Holman 1 Percussion 2 min 88 325
Holman 2 Percussion 2 min 96 196
Holman 3 Percussion 2 min 93 255
Holman 4 Percussion 2 min 96 196
Holman 1 Percussion 4 min 92 271
Holman 2 Percussion 4 min 97 171
66
I
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Holman 3 Percussion 4 min 91 286 Holman 4 Percussion 4 min 96 196 Holman 1 Percussion 8 min 88 325 Holman 2 Percussion 8 min 85 357 Holman 3 Percussion 8 min 83 376 Holman 4 Percussion 8 min 82 384 Jemez 1 Immersion 53 499 Jemez 2 Immersion 44 496 Jemez 3 Immersion 52 500 Jemez 4 Immersion 70 458 Jemez 1 Boiling 30 sec 58 494 Jemez 2 Boiling 30 sec 55 497 Jemez 3 Boiling 30 sec 48 500 Jemez 4 Boiling 30 sec 65 477 Jemez 1 Boiling 60 sec 68 466 Jemez 2 Boiling 60 sec 63 483 Jemez 3 Boiling 60 sec 73 444 Jemez 4 Boiling 60 sec 89 313 Jemez 1 Control 2 140 Jemez 2 Control 9 286 Jemez 3 Control 6 237 Jemez 4 Control 8 271 Jemez 1 Percussion 1 min 58 494 Jemez 2 Percussion 1 min 63 483 Jemez 3 Percussion 1 min 77 421 Jemez 4 Percussion 1 min 89 313 Jemez 1 Percussion 2 min 68 466 Jemez 2 Percussion 2 min 64 480 Jemez 3 Percussion 2 min 80 400 Jemez 4 Percussion 2 min 83 376 Jemez Percussion 4 min 68 466 Jemez 2 Percussion 4 min 79 407 Jemez 3 Percussion 4 min 70 458 Jemez 4 Percussion 4 min 83 376 Jemez 1 Percussion 8 min 52 500 Jemez 2 Percussion 8 min 59 492 Jemez 3 Percussion 8 min 61 488 Jemez 4 Percussion 8 min 70 458 Magdalena 1 Immersion 69 462 Magdalena 2 Immersion 69 462 Magdalena J Immersion 70 458 Magdalena 4 Immersion 85 357 Magdalena 1 Boiling 30 sec 65 477 Magdalena 2 Boiling 30 sec 71 454 Magdalena 3 Boiling 30 sec 68 466
67
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Magdalena 4 Boiling 30 sec 83 376 Magdalena 1 Boiling 60 sec 63 483 Magdalena 2 Boiling 60 sec 68 466 Magdalena 3 Boiling 60 sec 63 483 Magdalena 4 Boiling 60 sec 82 384 Magdalena 1 Control 11 313 Magdalena 2 Control 6 237 Magdalena 3 Control 5 218 Magdalena 4 Control 4 196 Magdalena Percussion 1 min 32 466 Magdalena 2 Percussion 1 min 45 497 Magdalena 3 Percussion 1 min 46 498 Magdalena 4 Percussion I min 36 480 Magdalena 1 Percussion 2 min 67 470 Magdalena 2 Percussion 2 min 68 466 Magdalena 3 Percussion 2 min 65 477 Magdalena 4 Percussion 2 min 81 392 Magdalena 1 Percussion 4 min 87 336 Magdalena 2 Percussion 4 min 97 171 Magdalena 3 Percussion 4 min 83 376 Magdalena 4 Percussion 4 min 97 171 Magdalena 1 Percussion 8 min 98 lAO Magdalena 2 Percussion 8 min 100 000 Magdalena 3 Percussion 8 min 96 196 Magdalena 4 Percussion 8 min 98 lAO Manzano 1 Immersion 67 470 Manzano 2 Immersion 57 495 Manzano 3 Immersion 72 4049 Manzano 4 Immersion 73 4044 Manzano 1 Boiling 30 sec 70 458 Manzano 2 Boiling 30 sec 64 480 Manzano 3 Boiling 30 sec 71 454 Manzano 4 Boiling 30 sec 80 400 Manzano I Boiling 60 sec 50 500 Manzano 2 Boiling 60 sec 53 499 Manzano 3 Boiling 60 sec 59 492 Manzano 4 Boiling 60 sec 74 439 Manzano I Control 4 196 Manzano 2 Control 8 271 Manzano 3 Control 1 099 Manzano 4 Control 6 237 Manzano 1 Percussion 1 min 52 500 Manzano 2 Percussion 1 min 39 488 Manzano 3 Percussion 1 min 36 480 Manzano 4 Percussion 1 min 51 500
68
Table B 1 Continued
Source Block Treatment Gerrni~ation Percentage Standard Error
Manzano Percussion 2 min 53 499 Manzano 2 Percussion 2 min 58 494 Manzano 3 Percussion 2 min 61 488 Manzano 4 Percussion 2 min 62 485 Manzano 1 Percussion 4 min 76 427 Manzano 2 Percussion 4 min 85 357 Manzano 3 Percussion 4 min 79 407 Manzano 4 Percussion 4 min 83 376 Manzano 1 Percussion 8 min 87 336 Manzano 2 Percussion 8 min 83 376 Manzano 3 Percussion 8 min 83 376 Manzano 4 Percussion 8 min 75 433 Raton 1 Immersion 54 498 Raton 2 Immersion 37 483 Raton 3 Immersion 39 488 Raton 4 Immersion 69 462 Raton 1 Boiling 30 sec 36 480 Raton 2 Boiling 30 sec 20 400 Raton 3 Boiling 30 sec 28 449 Raton 4 Boiling 30 sec 60 490 Raton 1 Boiling 60 sec 15 357 Raton 2 Boiling 60 sec 171 Raton 3 Boiling 60 sec 4 196 Raton 4 Boiling 60 sec 35 477 Raton 1 Control 4 196 Raton 2 Control 12 325 Raton 3 Control 2 140 Raton 4 Control 6 237 Raton 1 Percussion 1 min 52 500 Raton 2 Percussion I min 29 454 Raton 3 Percussion I min 35 477 Raton 4 Percussion 1 min 31 462 Raton 1 Percussion 2 min 55 497 Raton 2 Percussion 2 min 35 477 Raton 3 Percussion 2 min 33 470 Raton 4 Percussion 2 min 42 494 Raton 1 Percussion 4 min 65 477 Raton 2 Percussion 4 min 52 500 Raton Percussion 4 min 51 500 Raton 4 Percussion 4 min 53 499 Raton 1 Percussion 8 min 88 325 Raton 2 Percussion 8 min 80 400 Raton 3 Percussion 8 min 73 444 Raton 4 Percussion 8 min 74 439 Sacramento 1 Immersion 43 495
69
Table B 1 Continued
-Source Block Treatment Germination Percentage Standard Error
Sacramento 2 Immersion 30 458 Sacramento 3 Immersion 38 485 Sacramento 4 Immersion 56 496 Sacramento 1 Boiling 30 sec 47 499 Sacramento 2 Boiling 30 sec 48 500 Sacramento 3 Boiling 30 sec 48 500 Sacramento 4 Boiling 30 sec 56 496 Sacramento 1 Boiling 60 sec 52 500 Sacramento 2 Boiling 60 sec 67 470 Sacramento 3 Boiling 60 sec 43 495 Sacramento 4 Boiling 60 sec 48 500 Sacramento 1 Control 0 000 Sacramento 2 Control 3 171 Sacramento 3 Control 3 171 Sacramento 4 Control 099 Sacramento 1 Percussion 1 min 57 495 Sacramento 2 Percussion 1 min 61 488 Sacramento 3 Percussion 1 min 62 485 Sacramento 4 Percussion 1 min 55 497 Sacramento 1 Percussion 2 min 84 367 Sacramento 2 Percussion 2 min 70 458 Sacramento 3 Percussion 2 min 81 392 Sacramento 4 Percussion 2 min 69 462 Sacramento 1 Percussion 4 min 91 286 Sacramento 2 Percussion 4 min 84 367 Sacramento 3 Percussion 4 min 84 367 Sacramento 4 Percussion 4 min 94 237 Sacramento 1 Percussion 8 min 82 384 Sacramento 2 Percussion 8 min 81 392 Sacramento 3 Percussion 8 min 82 384 Sacramento 4 Percussion 8 min 81 392 Sandia 1 Immersion 76 427 Sandia 2 Immersion 70 458 Sandia 3 Immersion 70 458 Sandia 4 Immersion 81 392 Sandia 1 Boiling 30 sec 78 414 Sandia 2 Boiling 30 sec 71 454 Sandia 3 Boiling 30 sec 63 483 Sandia 4 Boiling 30 sec 78 414 Sandia 1 Boiling 60 sec 67 470 Sandia 2 Boiling 60 sec 65 477 Sandia 3 Boiling 60 sec 74 439 Sandia 4 Boiling 60 sec 66 474 Sandia 1 Control 16 367 Sandia 2 Control 7 255
70
Table B 1 Continued
Source Block Treatment Gennination Percentage Standard Error
Sandia 3 Control 8 271 Sandia 4 Control 16 367 Sandia 1 Percussion 1 min 67 470 Sandia 2 Percussion 1 min 61 488 Sandia 3 Percussion 1 min 66 474 Sandia 4 Percussion 1 min 59 492 Sandia I Percussion 2 min 82 384 Sandia 2 Percussion 2 min 83 376 Sandia 3 Percussion 2 min 81 392 Sandia 4 Percussion 2 min 84 367 Sandia 1 Percussion 4 min 89 313 Sandia 2 Percussion 4 min 86 347 Sandia 3 Percussion 4 min 89 313 Sandia 4 Percussion 4 min 85 357 Sandia 1 Percussion 8 min 80 400 Sandia 2 Percussion 8 min 77 421 Sandia 3 Percussion 8 min 69 462 Sandia 4 Percussion 8 min 66 474
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74
SAS Institute Inc 1989 SASSTAT Users Guide Version 6 4th Edition Volume 1 Cary NC SAS Institute Inc 943 p
SerratoValenti G Conara L Ferrando M Modenesi P 1993 Structural and histochemical features ofStylosanthes scabra (Leguminosae Papilionoidae) seed coat as related to water entry Can J Bot 71 834-840
Simpson BJ 1988 A field guide to Texas trees Austin TX Texas Monthly Press 372p
Tran VN Cavanagh AK 1984 Structural aspects ofdormancy In Seed Physiology Vol 2 Murray DR (ed) p1-44 Academic Press Australia
USDA Forest Service 1948 Woody-plant seed manual USDA Misc Pub1654 416p
Vines RA 1960 Trees Shrubs and Woody Vines of the Southwest Austin Univ ofTexas Press 1104 p
Wagner MR Larson RE Craig TP 1992 Ecology and silvics ofNew Mexico locust (Robinia neomexicana) with an emphasis on the arboreal form p53-57 In JW Hanover K Miller and S Plesko (eds) Proc Intemat Confon Black Locust Biology Culture and Utilization Mich State Univ USA
Wilson JK 1944 Immersing seeds of species of Robinia in boiling water hastens germination J For 42 453-454
Zimmerman RW Carpenter SB 1980 First-year coppice production from a 5shyyear old black locust stand on surface mine spoil p309-314 Centr Hardwd For ConfProc University ofMissouri Columbia
tv 0
~ d
~ A s o
1 c3
80
60
40
20
o
TIT
t------=middotfmiddotmiddot-middotmiddot-middotmiddot-middotmiddot 1- --- - - --=- - -shy--cE= - _--shy --shy ----shy ~ ~ ---shy ~ -~- _ --shy-------r 1
I
Raton Holman
-_ Jemez
--- Sandia Manzano Magdalena Capitan Sacramento Gila
Immersion Boiling 30 sec Boiling 60 sec
Hot Water Scarification
Figure 5 Effect of seed source by treatment interaction on New Mexico locust germination for hot water scarification
continued to increase in germination (Figure 4b) There was no difference between
the two treatment levels for the Manzano source (Figure 4b)
The effect ofhot water scarification also varied among sources with few
overall differences between hot water treatments (Figure 5) However boiling for 60
seconds yielded highest germination for the Jemez source whereas increasing
durations ofboiling decreased germination for the Raton source
In addition to consistently high germination 4- and 8-minute percussionshy
treated seeds germinated as fast or faster than all other treatments (Figure 6 Table 4)
050 measured as the number of days needed to complete 50 ofeventual
germination averaged 2 days for both ofthese treatments
X-ray radiographs were taken of seeds collected from the Sacramento source
percussed for 1 4 or 8 minutes (Figure 7) Seeds show signs of damage at 8 minutes
ofpercussion (Figure 8) Compared to an undamaged seed percussed for only 4
minutes (Figure 8a) damage at the 8-minute level includes fractured radicles pitting
of the seed coat and chips in seed coat structure (Figure 8b-d)
Scanning electron microscope (SEM) images at 600X reveal the direct effect
of 4 minutes of percussion on cells of the strophiolar region (Figure 9) Whereas
untreated seeds retain cellular organization in the strophiolar region strophiolar cells
seeds treated for 4 minutes ofpercussion separate and raise resulting in a
~-~e~U appearance
30
Umiddot-~middot--l-~---middotymiddotmiddotmiddot-----middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot--middot--middotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middotmiddotmiddotmiddotmiddotmiddotmiddot- -
050= gt1
-e-- Percussion 8 min -0- Percussion 4 min ---T- Percussion 2 min -----ltil- Percussion 1 min -- Immersion --0- Boiling 30 sec -+- Boiling 60 sec -ltgt- Control
2 4 6 8 10 12
Day Figure 6 Timecourse ofNew Mexico locust gennination following scarification
treatments G50 is the number of days to 50 of eventual germination
31
14
Table 4 Effect of seed source and treatment on G50 (days to 50 germination)
Seed Source Control Immersion B30sec B60sec Plmin P2min P4min P8min mean Raton 3 3 5 7 4 3 2 2 4 Holman 2 2 2 2 2 2 2 2 2
Jemez 4 6 6 5 3 2 2 2 4 Sandia 2 2 2 2 2 2 2 2 2
w tv Manzano 5 2 2 3 3 2 2 2 3
Magdalena 4 2 2 3 4 3 2 1 3
Capitan 5 4 2 2 3 2 2 2 3
Sacramento 3 2 2 2 4 3 2 2 3 Gila 3 2 2 2 2 2 2 2 2 mean 3 3 3 3 3 2 2 2 3 standard 11 14 16 18 09 05 0 03 08 deviation
Note Columns and rows may not agree due to rounding
Figure 7 X-ray radiographs ofSacramento seed source percussed for 1 4 or 8 minutes
33
Figure 8a-d Seed damage close-ups Figure a shows undamaged seed percussed for 4 minutes Figures b-d show seeds percussed for 8 minutes Damage includes fracturing ofradicle in b complete removal ofseed coat in c and pitting ofseed coat in d
34
Untreated Treated
Figure 9 SEM images ofuntreated vs 4-minute percussion-treated strophiolar cells ofNew Mexico locust seeds Treated cells are loosened separated and raised
DISCUSSION
Determining the Optimal Treatment
Averaging results of scarification treatmems across seed sources is a useful
tool to predict which treatment is most likely to be successful for an unknown seed
source Averaged over all sources four- and eight-minute percussion treatments
resulted in the highest germination 815 and 790 respectively and produced
germination that was as fast or faster than all other treatments Although there was no
difference between these two treatments on average an argument for 4 minutes of
percussion as a standard protocol for treating New Mexico locust seeds can be made
Descriptively 4 minutes of percussion improved germination over 8 minutes
ofpercussion for a majority of sources (five out of nine) 8 minutes of percussion
improved germination for three ofnine sources with no difference for a fmal source
For individual sources 4 minutes ofpercussion was always the best or secondmiddot best
treatment and was always equal to or better than all hot water treatments
Germination at 8 minutes ofpercussion was lower than all other percussion durations
for the Jemez and Gila sources and was lower than at least one hot water treatment for
these sources as well
The x-ray radiographs of the Sacramento seed source illustrate the excessive
internal and external seed damage that can occur with 8 minutes of percussion
germination counts 8-minute percussed seeds from Holman and Capitan
ClUll signs of damage that were visible to the naked eye (see below)
r~evertheless some of these seeds still fit the criteria for germination Neither x-ray
36
photography nor visible observations during seed counts revealed seed damage at the
4-minute percussion level In operational trials seeds percussed for 4 minutes
developed into healthy seedlings (Khadduri and Harrington unpublished data)
Strophiole and Seed Coat Behavior
Four minutes ofpercussion successfully breaks seed dormancy in a large
percentage ofNew Mexico locust seeds without excessively damaging the
surrounding seed coat or internal seed coat structures Most papilionoid legume
seeds including seeds ofNew Mexico locust have a specific region of the seed coat
known as the strophiole or lens (Hamly 1932) The strophiole is located on the
cotyledonary lobe of a locust seed (Figure 10 a-c) If seeds are not indiscriminately
damaged the strophiole is the first point of water entry to the embryo (Hopkinson
1997) When a papilionoid seed is percussed for the appropriate amount of time
repeated hits on the integrated seed coat loosen the constrained cells of the strophiolar
region without excessively damaging the rest of the seed coat (Ballard 1976) SEM
photos illuStrate that 4 minutes of percussion loosened and separated strophiolar cells
ofNew Mexico locust seeds when compared with constrained organized strophiolar
cells of untreated seeds (Figure 9) Research-has shown that water passes to the
embryo through the treated strophiole in a controlled manner and regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues
(Kelly and van Staden 1987)
37
10 Strophiole ofunimbibed New Mexico locust seed (a) and following uptake (bc)
38
In contrast hot-water treatments can excessively degrade the seed coat
(Rebman et al 1998 Marunda 1990) Random cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rebman et aI 1998 Marunda 1990) Along with a
proportionately large amount ofun-imbibed (thus under-treated) seeds (Figure l1a)
damage was observed in hot-water treated seeds from six sources during germination
counts Damage included seeds where the radicle failed to elongate (Figure 11 b)
seeds where the radicle elongated but was delayed in freeing itself from the seed coat
(Figure 11c) and seeds where the radicle completely broke off from the embryo
(Figure lId) In addition some seeds germinated in reverse (the cotyledon emerged
fIrst) If the radicle emerged completely from the seed coat they were counted as
germinated
While nearly all seeds treated for 8 minutes ofpercussion imbibed water
damage was evident in x-ray evaluations (Sacramento seed source) and in
germination counts (Holman and Capitan) at this treatment level Seed damage
during germination counts was similar to levels found in hot-water treated seeds
suggesting that over-treatment by both hot water and longer durations of percussion
may result in similar damage to seed structure
The Sacramento and Holman seed sources were two of the fIve sources that
opcrplltlpn in germination from the 4- to 8-minute durations of percussion
the benefIts ofloosening the strophiole as evidenced by imbibition of
all 8-minute treated seeds were outweighed by external and internal seed coat
39
a
b c d
11 Damage to New Mexico locust seeds observed during germination cotmts following hot water scarification 9a Hot water replicate including ~t seedlings and numerous unimbibed seeds 9b Close-up of seed where raallCle failed to elongate 9c Close-up ofseed where radicle elongated but was
-gtIn in freeing itself from the seed coat 9d Close-up ofseed where radicle broke off from embryo
40
damage (which also may be contributing to high imbibition) For the three sources
for which 8 minutes of percussion resulted in maximum germination extended
percussion of seeds necessary to loosen the strophiole may not have excessively
damaged the seed anatomy The Manzano source did not significantly change in
germination from 4 to 8 minutes of percussion suggesting that an increase in
permeable seeds at the 8-minute treatment duration was balanced by the number of
seeds that failed to germinate due to seed damage To summarize the 4-minute
percussion treatment improved germination over 8 minutes ofpercussion for a
majority of sources (five ofnine) the 8-minute percussion treatment improved
germination over 4-minutes ofpercussion for three sources and there was no
difference with the Manzano source Notably for the Raton source 8 minutes of
percussion increased germination by 235 percentage points over 4-minutes of
percussion (7875 vs 5525 respectively) the next best treatment Raton was the
only source to result in germination less than 75 for 4 minutes ofpercussion so a
grower may wish to apply longer percussion durations to lots that do not respond well
to this treatment level
Finally there was an interesting trend in the Jemez and Raton sources seed
that significantly contributed to the source by treatment interaction (Figure 12) A
~u test showed that the Jemez source had a significantly lighter seed weight than
other sources while the Raton source had an average seed weight For the Jemez
the longest duration of boiling increased germination Conversely only short
U4UVll) of percussion were needed to achieve high germination and the longest
41
80 _ Jemez a Raton
iD
~ C iD e iD
Q c 0 ~
c ~ iD 0
20
0------shy
Immersion Boiling 30 sec Boiling 60 sec
Hot Water Scarification
10degTr========~--------~------------------------------
80
60
40
20
_ Jemez Raton
0------shy
1 min 2 min 4 min 8 min
Percussion Scarification
Figure 12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources
42
b
--~----~~~~========~---- duration ofpercussion reduced germination For the Raton source longer durations
ofboiling consistently decreased germination whereas longer percussion treatments
consistently increased germination
A possible explanation is that a thick seed coat is associated with a loose
strophiole structure and that a thin seed coat is associated with a constrained
strophiole This contrast merits further study and may suggest an ecological
adaptation Further investigation ofthis phenomenon may contribute to successful
pre-screening ofunknown seed sources to determine optimal treatment leveL
43
CONCLUSION
Based on results obtained in pilot study (see Appendix) and thesis
experiments 4 minutes ofpercussion is recommended as a standard protocol for
treating an unknown source ofNew Mexico locust seed In addition to laboratory
results the 4-minute percussion treatment has resulted in germination greater than
80 and healthy seedling grow-outs for production purposes (unpublished data) In
laboratory experiments only one hundred seeds per 118 ml (40z) soil tin were
percussed at a time whereas for production we have treated up to five hundred seeds
per tin For large-scale production ofNew Mexico locust seedlings growers may
wish to experiment with larger quantities of seed and larger-size soil tins
Seed storage trials following percussion treatments were not conducted
However germination greater than 80 and healthy seedlings were obtained when
seeds were percussion-treated and subsequently stored at room-temperature (22degC)
for one week (unpublished data) Seed storage trials merit further study as the
standard hot water treatment leaves seeds in a softened condition that is not
conducive to storage
Hydroseeding is an effective method for direct seeding in reclamation
situations Percussion treatment may be conducive to hydro seeding as seeds remain
a dry relatively undamaged yet permeable condition following treatment Sulfuric
seeds also remain dry following treatment but extensive damage to seed coat
poor seedling vigor have been reported with this treatment (Kelly and van Staden
A grow-out study using a hydro seeder on a reclamation site testing percussion
44
against other treatments such as sulfuric acid and mechanical scarification also
merits further study
Papilionoids are the largest subfamily oflegumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust these legumes often play an integral role in the revegetation of
disturbed lands The pilot study (Appendix) indicates that percussion scarification
successfully treats the widely used black locust as well Developing superior
scarification methods to the standard hot water mechanical scarification and acid
treatments may facilitate the use ofpapilionoid legumes in reclamation projects
45
APPENDICES
APPENDIX A
PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR
NEW MEXICO LOCUST AND BLACK LOCUST SEEDS
Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4
Hot water and sulfuric acid soaks are traditional treatments for seeds of many
temperate woody legwnes including locusts However these scarification techniques
often produce inconsistent germination Percussion scarification where seeds are
repeatedly propelled against a hard surface was compared with hot water
scarification to evaluate treatment efficacy for New Mexico locust (Robinia
neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment
seeds were placed in a 98degC water bath which was immediately removed from the
heat source For percussion scarification seeds were placed in a soil sample tin and
agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the
control were followed by 24-hour water soaks Hot water baths resulted in 56 and
41 germination for New Mexico locust and black locust respectively For both
species nearly all durations ofpercussion increased germination over the hot water
treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3
4 and 5 minutes for black locust resulted in at least 90 germination Traditional
scarification treatments randomly degrade the entire seed coat which can lead to
tissue damage during water uptake Percussion scarification specifically weakens the
strophiole the natural source ofwater entry to the seed in papilionoid legwnes
INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom
2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031
48
Following percussion imbibition is controlled through the strophiole and underlying
tissue is protected
Keywords New Mexico locust black locust scarification percussion germination
Introduction
Locust trees (Robinia spp) are aggressive pioneer species that quickly
colonize disturbed land fix atmospheric nitrogen in the soil and stabilize slopes that
are prone to erosion (Klemmedson 1994 Ashby et al 1985) (Figure A1ab)
Recognition of these attributes has contributed to increased demand for locusts in
reclamation projects Locust seeds however exhibit physical dormancy where a
thick seed coat prevents the movement of water and gasses to the embryo (Leadem
1997)
Sulfuric acid or hot water soaks have traditionally been used to break seed
dormancy in macro-propagation of locusts Many growers are moving away from the
use of su1furic acid in treating locust seeds (Dreesen and Harrington 1997) In
addition to being dangerous acid soak durations must be specifically correlated to
seed lot (Olson 1974) Collections of native seed can vary tremendously in seed size
seed weight and hard seededness etc This variability limits the utility of sulfuric
acid soaks as even within a given lot some seeds may be damaged whereas others
are under-treated and fail to imbibe (take up water) A survey of Southwestern
container growers indicates that hot water baths are currently the most common
scarification method for locust seeds (Hine et al 1997) However hot water baths
also produce inconsistent germination (Lin et al 1996)
49
b
Figure AI New Mexico locust a primary invader following disturbance Ala Crown-sprouting at edge ofburn near Los Alamos NM--one year after Cerro Grande fire Alb Colonizing road cut Gila National Forest NM
50
Percussion scarification where seeds are repeatedly propelled against a hard
surface is an alternative dormancy-breaking method that has proven successful in
legumes related to locusts (Hamly 1932 Barton 1947) We compared this alternative
treatment to hot water scarification to evaluate treatment efficacy for New Mexico
locust and black locust seeds
Materials and Methods
Commercial New Mexico locust seeds (Western Native Seed Coaldale CO)
were collected fall 2000 in Huerfano County CO Black locust seeds were collected
September 2000 from Taos County NM by harvesting ripe pods from several trees at
a distance of up to 3 m from ground level Black locust seeds were allowed to air-dry
for 3 weeks and were threshed and separated from large chaffusing a greenhouse
fan A South Dakota Seed Blower (Chicago Illinltois) was used to remove fine chaff
from both seed lots Seeds were storedmiddotat 2-4C until the germination study was
conducted in March 2001
This study compared hot water scarification with percussion scarification
using treatment and species as experimental factors Along with a control
scarification treatments included immersion of seed in boiling water as well as
percussion durations of 1 2 3 45 or 10 minutes Four 100-seed samples were used
to test each species by scarification treatment combination
For the hot water treatment test tubes were filled with 30 mL water and
placed in a water-filled beaker (Figure A2a) The bath was raised to boiling (-98C at
51
b
c
Figure A2 Implementation ofhot water and percussion scarification A2a Hot water bath A2b Paint-shaker set-up A2c Placing soil tin containing seeds at end ofpaint shaker ann
52
Las Cruces NM elevation of 1300 meters) Each 100-seed replicate was placed in a
test tube and each test tube was immediately removed to cool at room temperature
Percussion scarification was implemented using a pneumatic paint shaker
(Central Pneumatic model 00422 Camarillo CA) (Figure A2b) This allowed
standardization of treatment intensity Compressed air pressure was maintained at
530kPa plusmn 30kPa (80psi plusmn 5psi) resulting in approximately 350 oscillations per
minute Each lOO-seed sample was placed in a 118 ml (4 oz) soil tin for shaking To
maximize lateral movement of paint shaker a paint can was used as a spacer to place
soil tins at the end of the shaking arm (Figure A2c) This allowed shaking to occur at
the greatest distance from the pivot point (fulcrum) Following all treatments
including the control seeds were soaked for twenty-four hours in distilled water prior
to plating out
Gennination was evaluated on lab benches at room temperature Each
replicate of seeds was placed in a 100 cm petri dish on moistened filter paper with
humidity maintained by enclosing the petri dishes in ziplock bags Gennination was
monitored daily for 14 days in accordance with International Seed Testing
Association standards for Robinia species (ISTA 1999) Gennination was defined as
protrusion of the radicle from the embryo by at least 1mm as viewed by the naked
eye
Categorical Analysis of Variance using Prpc Catmod (SAS Institute 1989)
was used to detennine treatment differences in germination percentages for each
source This procedure is an extension of a Chi-square test of homogeneity using the
53
natural log of the ratio of germinated to non-germinated seeds for each treatment Pshy
values less than 005 were considered significant A limited set of pairwiseshy
comparisons was conducted to compare treatment mean percents using a
conservative alpha value of 005 divided by the number of comparisons
Results
Percussion maximized germination for both New Mexico locust and black
locust There were three germination response levels for New Mexico locust (Figure
A3) First there was a low response in the control indicating a dormant seed lot
The hot water treatment and I-minute duration of percussion share a second
intermediate response Germination was highest at percussion levels from 2 to 10
minutes
A selected percussion treatment intermediate in time (5 minutes) produced the
steepest germination curve indicating the fastest germination rate (Figure A4) The
hot water treatment produced an intermediate curve or relatively slower germination
rate The control curve was very flat indicating the slowest germination rate for New
Mexico locust
Control seeds germinated poorly for black locust as well (Figure A5) Hot
water scarification improved germination over the control but all levels of percussion
improved germination over hot water The optimal durations of percussion treatment
were bracketed with a significant increase from the 1 to 2 minute percussion level a
high germination response from 2 to 5 minutes of percussion and finally a significant
54
~ d 25 CL)
p t1 0 ~ t1
g Vl ~ Vl
100~---------------------------------------------------------------
80
60
40
20
o Control Hot Water Percussion
I min Percussion
2 min Percussion
3 min
Scarification
Percussion 4 min
Figure A3 Effect of scarification on germination response for New Mexico locust
Percussion 5 min
=significant difference between adjacent treatment bars ns = no significant difference
Percussion 10 min
~ ~ () u agt
Il-t d o ~
~ ~ c3
100middotlr~~~------------------------------------e- Percussion 5min middot 0middot Hot Water -- Control
80-J=middot======
6b-middot-lmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middot--middotmiddot-middot~-middotmiddot -middotmiddot middot middotmiddot-middot 1
40
20
-----~-----------
0 0 0 middotmiddotmiddotmiddotmiddot0middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot0
0 --~-----y-----~------_---~--- T-----~
o shy -TDay 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A4 Effect of selected scarification treatments on germination speed for New Mexico locust First seven days of fourteen-day experiment are shown
Day 7
80
~ cd
5 60 ()
[) Cl d 0 1t1 d
40sect dUl
-J
20
o
ns
Scarification
Figure A5 Effect of scarification on germination response for black locust = significant difference between adjacent treatment bars ns = no significant difference
decrease at the 10-minute level This drop-off suggests that 10 minutes of percussion
over-treats seeds from this lot of-black locust
A selected percussion treatment intermediate in time (5 minutes) also had the
fastest germination rate for black locust (Figure A6)_ The hot water curve is
somewhat flatter indicating a slower germination rate The control curve is very flat
indicating a slow germination rate
A qualitative difference between hot water and percussion-treated seeds was
also observed Along with healthy germinants the hot water treatment produced
over-treated seeds (Figure A 7a) Examples of damaged germinants include seeds
where the radicle did not elongate (Figure A 7b) seeds where the radicle elongated
but was delayed in freeing itself from the seed coat (Figure A7c) and seeds where the
radicle broke off completely from the embryo (Figure A7d) In addition to these
examples of over-treatment there were numerous small dark seeds that failed to
imbibe water For percussion treatment nearly all seeds took up water (Figure A8a)
Germination was rapid and uniform with little damage evident (Figure A8b)
To summarize both New Mexico locust and black locust seed lots were
dormant The standard hot water scarification treatment improved germination but
only to 56 and 41 for New Mexico locust and black locust respectively Nearly
all percussion durations improved total germination as well as germination rate over
the hot water treatment Germination was greater than 90 for percussion durations
of 4 5 and 10 minutes for New Mexico locust and 3 4 and 5 minutes for black locust
58
CI)
sgt t CI) 0
igt 0
sect ttl t E
Vl 0
c3
100r==~==~~=r------------------------------------~ ___ Percussion 5 min
middotmiddotmiddot0middotmiddotmiddot Hot Water -lIf- Control
80r=========~--~~----------------------------~
60
40
20 -~-~---~---------- ---~------------I
omiddot
O
_~_----------lIf-----~bull -----_ shy---shy I
Day 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A6 Effect of selected scarification treatments on germination speed for black locust First seven days of fourteen-day experiment are shown
Day 7
a
b c d
Figure A7 Damage to New Mexico locust seeds observed during germination counts following hot water scarification A7a Hot water replicate including damaged seedlings and numerous unimbibed seeds A7b Close-up ofseed where radicle failed to elongate A7c Close-up ofseed where radicle elongated but was delayed in freeing itself from the seed coat A7d Close-up ofseed where radicle completely broke offfrom embryo
60
a
b
Figure A8 New Mexico locust seeds following percussion treatment at time of germination counts A8a Percussion replicate with uniformly healthy seedlings and near complete imbibition of seeds A8b Close-up ofpercussion-treated seedling
61
Discussion
Papilionoid seeds have a specific anatomical feature known as the strophiole
or lens-the natural site of water entry to the seed (Hamly 1932) The strophiole is
located on the cotyledonary lobe of a locust seed (Figure A9) When a papilionoid
legume seed is percussed for the appropriate amount of time repeated hits on the
integrated seed coat loosen the constrained cells of the strophiolar region without
excessively damaging the rest of the seed coat (Ballard 1976)
When a percussed seed is soaked water enters exclusively through the
strophiole in a controlled manner (Kelly and Staden 1987) This regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues This
contrasts with seeds that have been hot water or acid scarified treatments that can
randomly degrade the seed coat Unlocalized cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rehman et al 1998 Marunda 1990)
Papilionoids are the largest subfamily of legumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust and black locust these legumes often play an integral role in the
revegetation ofdisturbed lands Developing superior scarification methods to the
standard hot water and acid treatments should fadlitate the use ofthese legumes in
restoration projects
62
Figure A9 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (bc)
63
APPENDIXB
GERMINATION PERCENTS AND STANDARD ERRORS FOR SOURCE
BLOCK AND TREATMENT FOR NEW MEXICO LOCUST
Table B 1 Gennination percents and standard errors for source block and treatment for New Mexico locust
Source Block Treatment Germination Percentage Standard Error
Capitan 1 Immersion 74 437 Capitan 2 Immersion 69 462 Capitan 3 Immersion 71 454 Capitan 4 Immersion 67 470 Capitan Boiling 30 sec 79 407 Capitan 2 Boiling 30 sec 76 427 Capitan 3 Boiling 30 sec 72 449 Capitan 4 Boiling 30 sec 76 427 Capitan Boiling 60 sec 71 454 Capitan 2 Boiling 60 sec 54 498 Capitan 3 Boiling 60 sec 67 470 Capitan 4 Boiling 60 sec 76 427 Capitan I Control 5 218 Capitan 2 Control 7 255 Capitan 3 Control 4 196 Capitan 4 Control 5 218 Capitan 1 Percussion I min 47 499 Capitan 2 Percussion I min
470
Capitan Percussion I min 39 488
Capitan 4 Percussion I min 38 485 Capitan I Percussion 2 min 55 497 Capitan Capitan
2
Percussion 2 min Percussion 2 min
71 65
454 477
Capitan 4 Percussion 2 min 56 496 Capitan I Percussion 4 min 86 347 Capitan 2 Percussion 4 min 79 407 Capitan 3 Percussion 4 min 81 392 Capitan 4 Percussion 4 min 82 384 Capitan I Percussion 8 min 90 300 Capitan 2 Percussion 8 min 88 325 Capitan 3 Percussion 8 min 90 300 Capitan 4 Percussion 8 min 90 300 Gila I Immersion 71 454 Gila 2 Immersion 77 421 Gila
Immersion 75 433
Gila 4 Immersion 72 449
Gila I Boiling 30 sec 68 466
Gila 2 Boiling 30 sec 81 392
Gila 3 Boiling 30 sec 54 498
Gila 4 Boiling 30 sec 63 483
Gila I Boiling 60 sec 55 497
Gila 2 Boiling 60 sec 68 466
Gila 3 Boiling 60 sec 57 495
Gila 4 Boiling 60 sec 70 458
65
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Gila 2 Control 13 336 Gila 3 Control 13 336 Gila 4 Control 20 400 Gila 1 Percussion 1 min 66 474 Gila 2 Percussion 1 min 57 495 Gila 3 Percussion 1 min 73 444 Gila 4 Percussion 1 min 82 384 Gila 1 Percussion 2 min 81 392 Gila 2 Percussion 2 min 86 347 Gila 3 Percussion 2 min 85 357 Gila 4 Percussion 2 min 54 498 Gila 1 Percussion 4 min 79 407 Gila 2 Percussion 4 min 83 376 Gila 3 Percussion 4 min 84 367 Gila 4 Percussion 4 min 79 407 Gila 1 Percussion 8 min 51 500 Gila 2 Percussion 8 min 76 427 Gila 3 Percussion 8 min 52 500 Gila 4 Percussion 8 min 74 439 Holman 1 Immersion 83 376 Holman 2 Immersion 86 347 Holman 3 Immersion 81 392 Holman 4 Immersion 90 300 Holman 1 Boiling 30 sec 90 300 Holman 2 Boiling 30 sec 85 357 Holman 3 Boiling 30 sec 87 336 Holman 4 Boiling 30 sec 83 376 Holman 1 Boiling 60 sec 79 407 Holman 2 Boiling 60 sec 80 400 Holman 3 Boiling 60 sec 86 347 Holman 4 Boiling 60 sec 93 255 Holman Control 25 433 Holman 2 Control 19 392 Holman Control 21 407
Holman 4 Control 24 427
Holman Percussion 1 min 77 421
Holman 2 Percussion 1 min 85 357
Holman 3 Percussion 1 min 80 400
Holman 4 Percussion 1 min 89 313
Holman 1 Percussion 2 min 88 325
Holman 2 Percussion 2 min 96 196
Holman 3 Percussion 2 min 93 255
Holman 4 Percussion 2 min 96 196
Holman 1 Percussion 4 min 92 271
Holman 2 Percussion 4 min 97 171
66
I
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Holman 3 Percussion 4 min 91 286 Holman 4 Percussion 4 min 96 196 Holman 1 Percussion 8 min 88 325 Holman 2 Percussion 8 min 85 357 Holman 3 Percussion 8 min 83 376 Holman 4 Percussion 8 min 82 384 Jemez 1 Immersion 53 499 Jemez 2 Immersion 44 496 Jemez 3 Immersion 52 500 Jemez 4 Immersion 70 458 Jemez 1 Boiling 30 sec 58 494 Jemez 2 Boiling 30 sec 55 497 Jemez 3 Boiling 30 sec 48 500 Jemez 4 Boiling 30 sec 65 477 Jemez 1 Boiling 60 sec 68 466 Jemez 2 Boiling 60 sec 63 483 Jemez 3 Boiling 60 sec 73 444 Jemez 4 Boiling 60 sec 89 313 Jemez 1 Control 2 140 Jemez 2 Control 9 286 Jemez 3 Control 6 237 Jemez 4 Control 8 271 Jemez 1 Percussion 1 min 58 494 Jemez 2 Percussion 1 min 63 483 Jemez 3 Percussion 1 min 77 421 Jemez 4 Percussion 1 min 89 313 Jemez 1 Percussion 2 min 68 466 Jemez 2 Percussion 2 min 64 480 Jemez 3 Percussion 2 min 80 400 Jemez 4 Percussion 2 min 83 376 Jemez Percussion 4 min 68 466 Jemez 2 Percussion 4 min 79 407 Jemez 3 Percussion 4 min 70 458 Jemez 4 Percussion 4 min 83 376 Jemez 1 Percussion 8 min 52 500 Jemez 2 Percussion 8 min 59 492 Jemez 3 Percussion 8 min 61 488 Jemez 4 Percussion 8 min 70 458 Magdalena 1 Immersion 69 462 Magdalena 2 Immersion 69 462 Magdalena J Immersion 70 458 Magdalena 4 Immersion 85 357 Magdalena 1 Boiling 30 sec 65 477 Magdalena 2 Boiling 30 sec 71 454 Magdalena 3 Boiling 30 sec 68 466
67
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Magdalena 4 Boiling 30 sec 83 376 Magdalena 1 Boiling 60 sec 63 483 Magdalena 2 Boiling 60 sec 68 466 Magdalena 3 Boiling 60 sec 63 483 Magdalena 4 Boiling 60 sec 82 384 Magdalena 1 Control 11 313 Magdalena 2 Control 6 237 Magdalena 3 Control 5 218 Magdalena 4 Control 4 196 Magdalena Percussion 1 min 32 466 Magdalena 2 Percussion 1 min 45 497 Magdalena 3 Percussion 1 min 46 498 Magdalena 4 Percussion I min 36 480 Magdalena 1 Percussion 2 min 67 470 Magdalena 2 Percussion 2 min 68 466 Magdalena 3 Percussion 2 min 65 477 Magdalena 4 Percussion 2 min 81 392 Magdalena 1 Percussion 4 min 87 336 Magdalena 2 Percussion 4 min 97 171 Magdalena 3 Percussion 4 min 83 376 Magdalena 4 Percussion 4 min 97 171 Magdalena 1 Percussion 8 min 98 lAO Magdalena 2 Percussion 8 min 100 000 Magdalena 3 Percussion 8 min 96 196 Magdalena 4 Percussion 8 min 98 lAO Manzano 1 Immersion 67 470 Manzano 2 Immersion 57 495 Manzano 3 Immersion 72 4049 Manzano 4 Immersion 73 4044 Manzano 1 Boiling 30 sec 70 458 Manzano 2 Boiling 30 sec 64 480 Manzano 3 Boiling 30 sec 71 454 Manzano 4 Boiling 30 sec 80 400 Manzano I Boiling 60 sec 50 500 Manzano 2 Boiling 60 sec 53 499 Manzano 3 Boiling 60 sec 59 492 Manzano 4 Boiling 60 sec 74 439 Manzano I Control 4 196 Manzano 2 Control 8 271 Manzano 3 Control 1 099 Manzano 4 Control 6 237 Manzano 1 Percussion 1 min 52 500 Manzano 2 Percussion 1 min 39 488 Manzano 3 Percussion 1 min 36 480 Manzano 4 Percussion 1 min 51 500
68
Table B 1 Continued
Source Block Treatment Gerrni~ation Percentage Standard Error
Manzano Percussion 2 min 53 499 Manzano 2 Percussion 2 min 58 494 Manzano 3 Percussion 2 min 61 488 Manzano 4 Percussion 2 min 62 485 Manzano 1 Percussion 4 min 76 427 Manzano 2 Percussion 4 min 85 357 Manzano 3 Percussion 4 min 79 407 Manzano 4 Percussion 4 min 83 376 Manzano 1 Percussion 8 min 87 336 Manzano 2 Percussion 8 min 83 376 Manzano 3 Percussion 8 min 83 376 Manzano 4 Percussion 8 min 75 433 Raton 1 Immersion 54 498 Raton 2 Immersion 37 483 Raton 3 Immersion 39 488 Raton 4 Immersion 69 462 Raton 1 Boiling 30 sec 36 480 Raton 2 Boiling 30 sec 20 400 Raton 3 Boiling 30 sec 28 449 Raton 4 Boiling 30 sec 60 490 Raton 1 Boiling 60 sec 15 357 Raton 2 Boiling 60 sec 171 Raton 3 Boiling 60 sec 4 196 Raton 4 Boiling 60 sec 35 477 Raton 1 Control 4 196 Raton 2 Control 12 325 Raton 3 Control 2 140 Raton 4 Control 6 237 Raton 1 Percussion 1 min 52 500 Raton 2 Percussion I min 29 454 Raton 3 Percussion I min 35 477 Raton 4 Percussion 1 min 31 462 Raton 1 Percussion 2 min 55 497 Raton 2 Percussion 2 min 35 477 Raton 3 Percussion 2 min 33 470 Raton 4 Percussion 2 min 42 494 Raton 1 Percussion 4 min 65 477 Raton 2 Percussion 4 min 52 500 Raton Percussion 4 min 51 500 Raton 4 Percussion 4 min 53 499 Raton 1 Percussion 8 min 88 325 Raton 2 Percussion 8 min 80 400 Raton 3 Percussion 8 min 73 444 Raton 4 Percussion 8 min 74 439 Sacramento 1 Immersion 43 495
69
Table B 1 Continued
-Source Block Treatment Germination Percentage Standard Error
Sacramento 2 Immersion 30 458 Sacramento 3 Immersion 38 485 Sacramento 4 Immersion 56 496 Sacramento 1 Boiling 30 sec 47 499 Sacramento 2 Boiling 30 sec 48 500 Sacramento 3 Boiling 30 sec 48 500 Sacramento 4 Boiling 30 sec 56 496 Sacramento 1 Boiling 60 sec 52 500 Sacramento 2 Boiling 60 sec 67 470 Sacramento 3 Boiling 60 sec 43 495 Sacramento 4 Boiling 60 sec 48 500 Sacramento 1 Control 0 000 Sacramento 2 Control 3 171 Sacramento 3 Control 3 171 Sacramento 4 Control 099 Sacramento 1 Percussion 1 min 57 495 Sacramento 2 Percussion 1 min 61 488 Sacramento 3 Percussion 1 min 62 485 Sacramento 4 Percussion 1 min 55 497 Sacramento 1 Percussion 2 min 84 367 Sacramento 2 Percussion 2 min 70 458 Sacramento 3 Percussion 2 min 81 392 Sacramento 4 Percussion 2 min 69 462 Sacramento 1 Percussion 4 min 91 286 Sacramento 2 Percussion 4 min 84 367 Sacramento 3 Percussion 4 min 84 367 Sacramento 4 Percussion 4 min 94 237 Sacramento 1 Percussion 8 min 82 384 Sacramento 2 Percussion 8 min 81 392 Sacramento 3 Percussion 8 min 82 384 Sacramento 4 Percussion 8 min 81 392 Sandia 1 Immersion 76 427 Sandia 2 Immersion 70 458 Sandia 3 Immersion 70 458 Sandia 4 Immersion 81 392 Sandia 1 Boiling 30 sec 78 414 Sandia 2 Boiling 30 sec 71 454 Sandia 3 Boiling 30 sec 63 483 Sandia 4 Boiling 30 sec 78 414 Sandia 1 Boiling 60 sec 67 470 Sandia 2 Boiling 60 sec 65 477 Sandia 3 Boiling 60 sec 74 439 Sandia 4 Boiling 60 sec 66 474 Sandia 1 Control 16 367 Sandia 2 Control 7 255
70
Table B 1 Continued
Source Block Treatment Gennination Percentage Standard Error
Sandia 3 Control 8 271 Sandia 4 Control 16 367 Sandia 1 Percussion 1 min 67 470 Sandia 2 Percussion 1 min 61 488 Sandia 3 Percussion 1 min 66 474 Sandia 4 Percussion 1 min 59 492 Sandia I Percussion 2 min 82 384 Sandia 2 Percussion 2 min 83 376 Sandia 3 Percussion 2 min 81 392 Sandia 4 Percussion 2 min 84 367 Sandia 1 Percussion 4 min 89 313 Sandia 2 Percussion 4 min 86 347 Sandia 3 Percussion 4 min 89 313 Sandia 4 Percussion 4 min 85 357 Sandia 1 Percussion 8 min 80 400 Sandia 2 Percussion 8 min 77 421 Sandia 3 Percussion 8 min 69 462 Sandia 4 Percussion 8 min 66 474
LITERATURE CITED
Ashby WC Vogel WG Rogers NF 1985 Black locust in the reclamation equation General Technical Report Northeastern Forest Experiment Station USDA Forest Service NE-I05 12 p
Ballard LAT 1973 Physical barriers to gennination Seed Sci Technoll 285-303
___ 1976 Strophiolar water conduction in the seeds ofTrifolieae induced by action on the testa at nonstrophiolar sites Austr J Plant Physiol3 465-469
Barton LV 1947 Special studies on seed coat impenneability Contrib Boyce Thompson Inst 14 355-362
Baskin CC Baskin JM 1998 Seeds Ecology biogeography and evolution of donnancy and gennination Academic Press London UK 666 p
Bewley JD 1997 Seed germination and donnancy Plant Ce119 1055-1066
Bewley JD Black M 1994 Seeds physiology of development and germination 2nd ed Plenum Press New York NY 445 p
Briscoe CB 1996 Growing Nitrogen Fixing Trees in Acid Soils In MH Powell (ed) Nitrogen Fixing Trees for Acid Soils- A Field ManuaL Winrock International Morrilton AR 22 p
Copeland LO McDonald MB 1995 Principles of Seed Science and Technology 3rded Chapman amp Hall New York NY 409 p
Cox RE Klett JE 1984 Seed gennination requirements of native Colorado plants for use in the landscape Plant Propag 30(2) 6-10
Dell B 1980 Structure and function ofthe strophiolar plug in seeds ofAlbizia Lophantha Am J Bot 67 556-563
Dick-Peddie WA 1993 New Mexico Vegetation Past Present and Future University ofNew Mexico Press Albuquerque NM 244 p
Dreesen DR 2001 Propagation protocol for production ofcontainer Robinia neomexicana plants Los Lunas Plant Materials Center Los Lunas New Mexico In Native Plant Network URL httpwwwnativeplantnetworkorg (accessed 9 January 2002) Moscow (ID) University ofIdaho CoIl Natural Resources For Res Nursery
72
Dreesen DR Harrington JT 1997 Propagation of native plants for restoration projects in the Southwestern US- Preliminary Investigations In Landis TD Thompson JR tech coords National Proceedings Forest and Conservation Nursery Assocs Gen Tech Rep PNW-GTR-419 Portland OR USDA For Ser PNW Res Sta 77-88
Gottfried GJ 1980 Control ofNew Mexican locust and the effect on planted ponderosa pine in central Arizona US For Ser Res Note RM-386 Rocky Mountain For Range ExpStn Fort Collins CO
Gosling PG Samuel YK Jones SK 1995 A systematic examination of germination temperature chipping and water temperaturesoak duration pretreatments on the seeds ofLeucaena leucocephala Seed Sci amp Technol 23521-532
Hamly DH 1932 Softening of the seeds of Melilotus alba Bot Gazette 93 345shy375
Hanna PJ 1984 Anatomical features of the seed coat ofAcacia kempeana (Mueller) which relate to increased germination rate induced by heat treatment New Phytol 96 23-29
Hanks JP Dick-Peddie WA 1974 Vegetation patterns of the White Mountains New Mexico Southwest Naturalist 18(4) 371-382
Hine S Harrington JT Dreesen DR 1997 Seed scarification requirements for Robinia neomexicana In Harrington JTtech coord Proc Third SouthwestemContainer Growers Conf Feb 12-13 1997 Ft Collins CO
Hopkinson JM 1993 The strophiole of leguminous seeds Newsletter- IntI Herbage Seed Production Res Grp 18 7-8
---- 1997 Differences in hardseededness between Cunningham and Tarramba leucaena In Leucnet News The Newsletter of the International LeucaenaRes and Devlt Netwk No4
ISTA (International Seed Testing Association) 1999 International rules for seed testing rules Seed Sci amp Technol27 Supplement
Kelly KM van Staden J 1987 The lens as the site of permeability in the papilionoid seed Aspalathus linearis J Plant Physiol 128(45) 395-404
73
Keresztesi B 1988 Black locust the tree of agriculture Outlook on Agr 17(2) 77~85
Kigel 1 and Galili G 1995 Seed development and germination Marcel Dekker New York 853 p
Klemmedson 10 1994 New Mexico locust and parent material Influence on macronutrients of forest floor and soil Soil Sci Soc Am 1 58 974~980
Kuo WH Tam AW 1988 The pathway of water absorption of mung bean seeds Seed Sci amp Technol16(1) 139~144
Leadem CL 1997 Dormancy--unlocking seed secrets In Landis TD Thompson lR tech coords National Proceedings Forestand Conservation Nursery Assocs Gen Tech Rep PNW-G TR-419 Portland OR USDA For Ser PNW Res Sta 43-52
Lin Y Wagner MR Seweyestewa A Honahni MD DeGomez M McMillin JD 1996 Influence of cultural treatments on seed germination and seedling growth of Robinia neomexicana Forest Farm and Community Tree Res Reports Volume 1
Marunda CT 1990 Effects of seed pretreatment on the development ofAcacia auriculforinis and A holosericea seedlings ACIAR Proceedings Series No 28
Meginnis HG 1937 Sulphuric acid treatment to increase germination of black locust seed USDA Circ 453 34 p
Miklas PN Towensend CE Ladd SL 1987 Seedcoat anatomy and the scarification ofcicer milkvetch seed Crop Sci 27 766-772
Monsen SB 1984 Use of shrubs on mine spoils In The Challenge ofProducing Native Plants for the Intermountain Area Proceedings Intermountain Nurserymans Association 1983 conference Las Vegas NV compo PM Murphy 26-31 Ogden UT USDA For Ser Gen Tech Report INT-l68
Olson DF 1974 Robinia L Locust In Seeds of Woody Plants in the US tech coord CS Schopmeyer 728-731 USDA For Ser Agr Handbk No450
Rehman S Loescher RNl Harris P1C 1998 Dormancy breaking and germination ofAcacia salicina Lindl seeds Seed Sci amp Techno127 553-557
Rolston MP 1978 Water impermeable seed dormancy Bot Rev 44 365-396
74
SAS Institute Inc 1989 SASSTAT Users Guide Version 6 4th Edition Volume 1 Cary NC SAS Institute Inc 943 p
SerratoValenti G Conara L Ferrando M Modenesi P 1993 Structural and histochemical features ofStylosanthes scabra (Leguminosae Papilionoidae) seed coat as related to water entry Can J Bot 71 834-840
Simpson BJ 1988 A field guide to Texas trees Austin TX Texas Monthly Press 372p
Tran VN Cavanagh AK 1984 Structural aspects ofdormancy In Seed Physiology Vol 2 Murray DR (ed) p1-44 Academic Press Australia
USDA Forest Service 1948 Woody-plant seed manual USDA Misc Pub1654 416p
Vines RA 1960 Trees Shrubs and Woody Vines of the Southwest Austin Univ ofTexas Press 1104 p
Wagner MR Larson RE Craig TP 1992 Ecology and silvics ofNew Mexico locust (Robinia neomexicana) with an emphasis on the arboreal form p53-57 In JW Hanover K Miller and S Plesko (eds) Proc Intemat Confon Black Locust Biology Culture and Utilization Mich State Univ USA
Wilson JK 1944 Immersing seeds of species of Robinia in boiling water hastens germination J For 42 453-454
Zimmerman RW Carpenter SB 1980 First-year coppice production from a 5shyyear old black locust stand on surface mine spoil p309-314 Centr Hardwd For ConfProc University ofMissouri Columbia
continued to increase in germination (Figure 4b) There was no difference between
the two treatment levels for the Manzano source (Figure 4b)
The effect ofhot water scarification also varied among sources with few
overall differences between hot water treatments (Figure 5) However boiling for 60
seconds yielded highest germination for the Jemez source whereas increasing
durations ofboiling decreased germination for the Raton source
In addition to consistently high germination 4- and 8-minute percussionshy
treated seeds germinated as fast or faster than all other treatments (Figure 6 Table 4)
050 measured as the number of days needed to complete 50 ofeventual
germination averaged 2 days for both ofthese treatments
X-ray radiographs were taken of seeds collected from the Sacramento source
percussed for 1 4 or 8 minutes (Figure 7) Seeds show signs of damage at 8 minutes
ofpercussion (Figure 8) Compared to an undamaged seed percussed for only 4
minutes (Figure 8a) damage at the 8-minute level includes fractured radicles pitting
of the seed coat and chips in seed coat structure (Figure 8b-d)
Scanning electron microscope (SEM) images at 600X reveal the direct effect
of 4 minutes of percussion on cells of the strophiolar region (Figure 9) Whereas
untreated seeds retain cellular organization in the strophiolar region strophiolar cells
seeds treated for 4 minutes ofpercussion separate and raise resulting in a
~-~e~U appearance
30
Umiddot-~middot--l-~---middotymiddotmiddotmiddot-----middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot--middot--middotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middotmiddotmiddotmiddotmiddotmiddotmiddot- -
050= gt1
-e-- Percussion 8 min -0- Percussion 4 min ---T- Percussion 2 min -----ltil- Percussion 1 min -- Immersion --0- Boiling 30 sec -+- Boiling 60 sec -ltgt- Control
2 4 6 8 10 12
Day Figure 6 Timecourse ofNew Mexico locust gennination following scarification
treatments G50 is the number of days to 50 of eventual germination
31
14
Table 4 Effect of seed source and treatment on G50 (days to 50 germination)
Seed Source Control Immersion B30sec B60sec Plmin P2min P4min P8min mean Raton 3 3 5 7 4 3 2 2 4 Holman 2 2 2 2 2 2 2 2 2
Jemez 4 6 6 5 3 2 2 2 4 Sandia 2 2 2 2 2 2 2 2 2
w tv Manzano 5 2 2 3 3 2 2 2 3
Magdalena 4 2 2 3 4 3 2 1 3
Capitan 5 4 2 2 3 2 2 2 3
Sacramento 3 2 2 2 4 3 2 2 3 Gila 3 2 2 2 2 2 2 2 2 mean 3 3 3 3 3 2 2 2 3 standard 11 14 16 18 09 05 0 03 08 deviation
Note Columns and rows may not agree due to rounding
Figure 7 X-ray radiographs ofSacramento seed source percussed for 1 4 or 8 minutes
33
Figure 8a-d Seed damage close-ups Figure a shows undamaged seed percussed for 4 minutes Figures b-d show seeds percussed for 8 minutes Damage includes fracturing ofradicle in b complete removal ofseed coat in c and pitting ofseed coat in d
34
Untreated Treated
Figure 9 SEM images ofuntreated vs 4-minute percussion-treated strophiolar cells ofNew Mexico locust seeds Treated cells are loosened separated and raised
DISCUSSION
Determining the Optimal Treatment
Averaging results of scarification treatmems across seed sources is a useful
tool to predict which treatment is most likely to be successful for an unknown seed
source Averaged over all sources four- and eight-minute percussion treatments
resulted in the highest germination 815 and 790 respectively and produced
germination that was as fast or faster than all other treatments Although there was no
difference between these two treatments on average an argument for 4 minutes of
percussion as a standard protocol for treating New Mexico locust seeds can be made
Descriptively 4 minutes of percussion improved germination over 8 minutes
ofpercussion for a majority of sources (five out of nine) 8 minutes of percussion
improved germination for three ofnine sources with no difference for a fmal source
For individual sources 4 minutes ofpercussion was always the best or secondmiddot best
treatment and was always equal to or better than all hot water treatments
Germination at 8 minutes ofpercussion was lower than all other percussion durations
for the Jemez and Gila sources and was lower than at least one hot water treatment for
these sources as well
The x-ray radiographs of the Sacramento seed source illustrate the excessive
internal and external seed damage that can occur with 8 minutes of percussion
germination counts 8-minute percussed seeds from Holman and Capitan
ClUll signs of damage that were visible to the naked eye (see below)
r~evertheless some of these seeds still fit the criteria for germination Neither x-ray
36
photography nor visible observations during seed counts revealed seed damage at the
4-minute percussion level In operational trials seeds percussed for 4 minutes
developed into healthy seedlings (Khadduri and Harrington unpublished data)
Strophiole and Seed Coat Behavior
Four minutes ofpercussion successfully breaks seed dormancy in a large
percentage ofNew Mexico locust seeds without excessively damaging the
surrounding seed coat or internal seed coat structures Most papilionoid legume
seeds including seeds ofNew Mexico locust have a specific region of the seed coat
known as the strophiole or lens (Hamly 1932) The strophiole is located on the
cotyledonary lobe of a locust seed (Figure 10 a-c) If seeds are not indiscriminately
damaged the strophiole is the first point of water entry to the embryo (Hopkinson
1997) When a papilionoid seed is percussed for the appropriate amount of time
repeated hits on the integrated seed coat loosen the constrained cells of the strophiolar
region without excessively damaging the rest of the seed coat (Ballard 1976) SEM
photos illuStrate that 4 minutes of percussion loosened and separated strophiolar cells
ofNew Mexico locust seeds when compared with constrained organized strophiolar
cells of untreated seeds (Figure 9) Research-has shown that water passes to the
embryo through the treated strophiole in a controlled manner and regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues
(Kelly and van Staden 1987)
37
10 Strophiole ofunimbibed New Mexico locust seed (a) and following uptake (bc)
38
In contrast hot-water treatments can excessively degrade the seed coat
(Rebman et al 1998 Marunda 1990) Random cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rebman et aI 1998 Marunda 1990) Along with a
proportionately large amount ofun-imbibed (thus under-treated) seeds (Figure l1a)
damage was observed in hot-water treated seeds from six sources during germination
counts Damage included seeds where the radicle failed to elongate (Figure 11 b)
seeds where the radicle elongated but was delayed in freeing itself from the seed coat
(Figure 11c) and seeds where the radicle completely broke off from the embryo
(Figure lId) In addition some seeds germinated in reverse (the cotyledon emerged
fIrst) If the radicle emerged completely from the seed coat they were counted as
germinated
While nearly all seeds treated for 8 minutes ofpercussion imbibed water
damage was evident in x-ray evaluations (Sacramento seed source) and in
germination counts (Holman and Capitan) at this treatment level Seed damage
during germination counts was similar to levels found in hot-water treated seeds
suggesting that over-treatment by both hot water and longer durations of percussion
may result in similar damage to seed structure
The Sacramento and Holman seed sources were two of the fIve sources that
opcrplltlpn in germination from the 4- to 8-minute durations of percussion
the benefIts ofloosening the strophiole as evidenced by imbibition of
all 8-minute treated seeds were outweighed by external and internal seed coat
39
a
b c d
11 Damage to New Mexico locust seeds observed during germination cotmts following hot water scarification 9a Hot water replicate including ~t seedlings and numerous unimbibed seeds 9b Close-up of seed where raallCle failed to elongate 9c Close-up ofseed where radicle elongated but was
-gtIn in freeing itself from the seed coat 9d Close-up ofseed where radicle broke off from embryo
40
damage (which also may be contributing to high imbibition) For the three sources
for which 8 minutes of percussion resulted in maximum germination extended
percussion of seeds necessary to loosen the strophiole may not have excessively
damaged the seed anatomy The Manzano source did not significantly change in
germination from 4 to 8 minutes of percussion suggesting that an increase in
permeable seeds at the 8-minute treatment duration was balanced by the number of
seeds that failed to germinate due to seed damage To summarize the 4-minute
percussion treatment improved germination over 8 minutes ofpercussion for a
majority of sources (five ofnine) the 8-minute percussion treatment improved
germination over 4-minutes ofpercussion for three sources and there was no
difference with the Manzano source Notably for the Raton source 8 minutes of
percussion increased germination by 235 percentage points over 4-minutes of
percussion (7875 vs 5525 respectively) the next best treatment Raton was the
only source to result in germination less than 75 for 4 minutes ofpercussion so a
grower may wish to apply longer percussion durations to lots that do not respond well
to this treatment level
Finally there was an interesting trend in the Jemez and Raton sources seed
that significantly contributed to the source by treatment interaction (Figure 12) A
~u test showed that the Jemez source had a significantly lighter seed weight than
other sources while the Raton source had an average seed weight For the Jemez
the longest duration of boiling increased germination Conversely only short
U4UVll) of percussion were needed to achieve high germination and the longest
41
80 _ Jemez a Raton
iD
~ C iD e iD
Q c 0 ~
c ~ iD 0
20
0------shy
Immersion Boiling 30 sec Boiling 60 sec
Hot Water Scarification
10degTr========~--------~------------------------------
80
60
40
20
_ Jemez Raton
0------shy
1 min 2 min 4 min 8 min
Percussion Scarification
Figure 12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources
42
b
--~----~~~~========~---- duration ofpercussion reduced germination For the Raton source longer durations
ofboiling consistently decreased germination whereas longer percussion treatments
consistently increased germination
A possible explanation is that a thick seed coat is associated with a loose
strophiole structure and that a thin seed coat is associated with a constrained
strophiole This contrast merits further study and may suggest an ecological
adaptation Further investigation ofthis phenomenon may contribute to successful
pre-screening ofunknown seed sources to determine optimal treatment leveL
43
CONCLUSION
Based on results obtained in pilot study (see Appendix) and thesis
experiments 4 minutes ofpercussion is recommended as a standard protocol for
treating an unknown source ofNew Mexico locust seed In addition to laboratory
results the 4-minute percussion treatment has resulted in germination greater than
80 and healthy seedling grow-outs for production purposes (unpublished data) In
laboratory experiments only one hundred seeds per 118 ml (40z) soil tin were
percussed at a time whereas for production we have treated up to five hundred seeds
per tin For large-scale production ofNew Mexico locust seedlings growers may
wish to experiment with larger quantities of seed and larger-size soil tins
Seed storage trials following percussion treatments were not conducted
However germination greater than 80 and healthy seedlings were obtained when
seeds were percussion-treated and subsequently stored at room-temperature (22degC)
for one week (unpublished data) Seed storage trials merit further study as the
standard hot water treatment leaves seeds in a softened condition that is not
conducive to storage
Hydroseeding is an effective method for direct seeding in reclamation
situations Percussion treatment may be conducive to hydro seeding as seeds remain
a dry relatively undamaged yet permeable condition following treatment Sulfuric
seeds also remain dry following treatment but extensive damage to seed coat
poor seedling vigor have been reported with this treatment (Kelly and van Staden
A grow-out study using a hydro seeder on a reclamation site testing percussion
44
against other treatments such as sulfuric acid and mechanical scarification also
merits further study
Papilionoids are the largest subfamily oflegumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust these legumes often play an integral role in the revegetation of
disturbed lands The pilot study (Appendix) indicates that percussion scarification
successfully treats the widely used black locust as well Developing superior
scarification methods to the standard hot water mechanical scarification and acid
treatments may facilitate the use ofpapilionoid legumes in reclamation projects
45
APPENDICES
APPENDIX A
PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR
NEW MEXICO LOCUST AND BLACK LOCUST SEEDS
Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4
Hot water and sulfuric acid soaks are traditional treatments for seeds of many
temperate woody legwnes including locusts However these scarification techniques
often produce inconsistent germination Percussion scarification where seeds are
repeatedly propelled against a hard surface was compared with hot water
scarification to evaluate treatment efficacy for New Mexico locust (Robinia
neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment
seeds were placed in a 98degC water bath which was immediately removed from the
heat source For percussion scarification seeds were placed in a soil sample tin and
agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the
control were followed by 24-hour water soaks Hot water baths resulted in 56 and
41 germination for New Mexico locust and black locust respectively For both
species nearly all durations ofpercussion increased germination over the hot water
treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3
4 and 5 minutes for black locust resulted in at least 90 germination Traditional
scarification treatments randomly degrade the entire seed coat which can lead to
tissue damage during water uptake Percussion scarification specifically weakens the
strophiole the natural source ofwater entry to the seed in papilionoid legwnes
INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom
2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031
48
Following percussion imbibition is controlled through the strophiole and underlying
tissue is protected
Keywords New Mexico locust black locust scarification percussion germination
Introduction
Locust trees (Robinia spp) are aggressive pioneer species that quickly
colonize disturbed land fix atmospheric nitrogen in the soil and stabilize slopes that
are prone to erosion (Klemmedson 1994 Ashby et al 1985) (Figure A1ab)
Recognition of these attributes has contributed to increased demand for locusts in
reclamation projects Locust seeds however exhibit physical dormancy where a
thick seed coat prevents the movement of water and gasses to the embryo (Leadem
1997)
Sulfuric acid or hot water soaks have traditionally been used to break seed
dormancy in macro-propagation of locusts Many growers are moving away from the
use of su1furic acid in treating locust seeds (Dreesen and Harrington 1997) In
addition to being dangerous acid soak durations must be specifically correlated to
seed lot (Olson 1974) Collections of native seed can vary tremendously in seed size
seed weight and hard seededness etc This variability limits the utility of sulfuric
acid soaks as even within a given lot some seeds may be damaged whereas others
are under-treated and fail to imbibe (take up water) A survey of Southwestern
container growers indicates that hot water baths are currently the most common
scarification method for locust seeds (Hine et al 1997) However hot water baths
also produce inconsistent germination (Lin et al 1996)
49
b
Figure AI New Mexico locust a primary invader following disturbance Ala Crown-sprouting at edge ofburn near Los Alamos NM--one year after Cerro Grande fire Alb Colonizing road cut Gila National Forest NM
50
Percussion scarification where seeds are repeatedly propelled against a hard
surface is an alternative dormancy-breaking method that has proven successful in
legumes related to locusts (Hamly 1932 Barton 1947) We compared this alternative
treatment to hot water scarification to evaluate treatment efficacy for New Mexico
locust and black locust seeds
Materials and Methods
Commercial New Mexico locust seeds (Western Native Seed Coaldale CO)
were collected fall 2000 in Huerfano County CO Black locust seeds were collected
September 2000 from Taos County NM by harvesting ripe pods from several trees at
a distance of up to 3 m from ground level Black locust seeds were allowed to air-dry
for 3 weeks and were threshed and separated from large chaffusing a greenhouse
fan A South Dakota Seed Blower (Chicago Illinltois) was used to remove fine chaff
from both seed lots Seeds were storedmiddotat 2-4C until the germination study was
conducted in March 2001
This study compared hot water scarification with percussion scarification
using treatment and species as experimental factors Along with a control
scarification treatments included immersion of seed in boiling water as well as
percussion durations of 1 2 3 45 or 10 minutes Four 100-seed samples were used
to test each species by scarification treatment combination
For the hot water treatment test tubes were filled with 30 mL water and
placed in a water-filled beaker (Figure A2a) The bath was raised to boiling (-98C at
51
b
c
Figure A2 Implementation ofhot water and percussion scarification A2a Hot water bath A2b Paint-shaker set-up A2c Placing soil tin containing seeds at end ofpaint shaker ann
52
Las Cruces NM elevation of 1300 meters) Each 100-seed replicate was placed in a
test tube and each test tube was immediately removed to cool at room temperature
Percussion scarification was implemented using a pneumatic paint shaker
(Central Pneumatic model 00422 Camarillo CA) (Figure A2b) This allowed
standardization of treatment intensity Compressed air pressure was maintained at
530kPa plusmn 30kPa (80psi plusmn 5psi) resulting in approximately 350 oscillations per
minute Each lOO-seed sample was placed in a 118 ml (4 oz) soil tin for shaking To
maximize lateral movement of paint shaker a paint can was used as a spacer to place
soil tins at the end of the shaking arm (Figure A2c) This allowed shaking to occur at
the greatest distance from the pivot point (fulcrum) Following all treatments
including the control seeds were soaked for twenty-four hours in distilled water prior
to plating out
Gennination was evaluated on lab benches at room temperature Each
replicate of seeds was placed in a 100 cm petri dish on moistened filter paper with
humidity maintained by enclosing the petri dishes in ziplock bags Gennination was
monitored daily for 14 days in accordance with International Seed Testing
Association standards for Robinia species (ISTA 1999) Gennination was defined as
protrusion of the radicle from the embryo by at least 1mm as viewed by the naked
eye
Categorical Analysis of Variance using Prpc Catmod (SAS Institute 1989)
was used to detennine treatment differences in germination percentages for each
source This procedure is an extension of a Chi-square test of homogeneity using the
53
natural log of the ratio of germinated to non-germinated seeds for each treatment Pshy
values less than 005 were considered significant A limited set of pairwiseshy
comparisons was conducted to compare treatment mean percents using a
conservative alpha value of 005 divided by the number of comparisons
Results
Percussion maximized germination for both New Mexico locust and black
locust There were three germination response levels for New Mexico locust (Figure
A3) First there was a low response in the control indicating a dormant seed lot
The hot water treatment and I-minute duration of percussion share a second
intermediate response Germination was highest at percussion levels from 2 to 10
minutes
A selected percussion treatment intermediate in time (5 minutes) produced the
steepest germination curve indicating the fastest germination rate (Figure A4) The
hot water treatment produced an intermediate curve or relatively slower germination
rate The control curve was very flat indicating the slowest germination rate for New
Mexico locust
Control seeds germinated poorly for black locust as well (Figure A5) Hot
water scarification improved germination over the control but all levels of percussion
improved germination over hot water The optimal durations of percussion treatment
were bracketed with a significant increase from the 1 to 2 minute percussion level a
high germination response from 2 to 5 minutes of percussion and finally a significant
54
~ d 25 CL)
p t1 0 ~ t1
g Vl ~ Vl
100~---------------------------------------------------------------
80
60
40
20
o Control Hot Water Percussion
I min Percussion
2 min Percussion
3 min
Scarification
Percussion 4 min
Figure A3 Effect of scarification on germination response for New Mexico locust
Percussion 5 min
=significant difference between adjacent treatment bars ns = no significant difference
Percussion 10 min
~ ~ () u agt
Il-t d o ~
~ ~ c3
100middotlr~~~------------------------------------e- Percussion 5min middot 0middot Hot Water -- Control
80-J=middot======
6b-middot-lmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middot--middotmiddot-middot~-middotmiddot -middotmiddot middot middotmiddot-middot 1
40
20
-----~-----------
0 0 0 middotmiddotmiddotmiddotmiddot0middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot0
0 --~-----y-----~------_---~--- T-----~
o shy -TDay 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A4 Effect of selected scarification treatments on germination speed for New Mexico locust First seven days of fourteen-day experiment are shown
Day 7
80
~ cd
5 60 ()
[) Cl d 0 1t1 d
40sect dUl
-J
20
o
ns
Scarification
Figure A5 Effect of scarification on germination response for black locust = significant difference between adjacent treatment bars ns = no significant difference
decrease at the 10-minute level This drop-off suggests that 10 minutes of percussion
over-treats seeds from this lot of-black locust
A selected percussion treatment intermediate in time (5 minutes) also had the
fastest germination rate for black locust (Figure A6)_ The hot water curve is
somewhat flatter indicating a slower germination rate The control curve is very flat
indicating a slow germination rate
A qualitative difference between hot water and percussion-treated seeds was
also observed Along with healthy germinants the hot water treatment produced
over-treated seeds (Figure A 7a) Examples of damaged germinants include seeds
where the radicle did not elongate (Figure A 7b) seeds where the radicle elongated
but was delayed in freeing itself from the seed coat (Figure A7c) and seeds where the
radicle broke off completely from the embryo (Figure A7d) In addition to these
examples of over-treatment there were numerous small dark seeds that failed to
imbibe water For percussion treatment nearly all seeds took up water (Figure A8a)
Germination was rapid and uniform with little damage evident (Figure A8b)
To summarize both New Mexico locust and black locust seed lots were
dormant The standard hot water scarification treatment improved germination but
only to 56 and 41 for New Mexico locust and black locust respectively Nearly
all percussion durations improved total germination as well as germination rate over
the hot water treatment Germination was greater than 90 for percussion durations
of 4 5 and 10 minutes for New Mexico locust and 3 4 and 5 minutes for black locust
58
CI)
sgt t CI) 0
igt 0
sect ttl t E
Vl 0
c3
100r==~==~~=r------------------------------------~ ___ Percussion 5 min
middotmiddotmiddot0middotmiddotmiddot Hot Water -lIf- Control
80r=========~--~~----------------------------~
60
40
20 -~-~---~---------- ---~------------I
omiddot
O
_~_----------lIf-----~bull -----_ shy---shy I
Day 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A6 Effect of selected scarification treatments on germination speed for black locust First seven days of fourteen-day experiment are shown
Day 7
a
b c d
Figure A7 Damage to New Mexico locust seeds observed during germination counts following hot water scarification A7a Hot water replicate including damaged seedlings and numerous unimbibed seeds A7b Close-up ofseed where radicle failed to elongate A7c Close-up ofseed where radicle elongated but was delayed in freeing itself from the seed coat A7d Close-up ofseed where radicle completely broke offfrom embryo
60
a
b
Figure A8 New Mexico locust seeds following percussion treatment at time of germination counts A8a Percussion replicate with uniformly healthy seedlings and near complete imbibition of seeds A8b Close-up ofpercussion-treated seedling
61
Discussion
Papilionoid seeds have a specific anatomical feature known as the strophiole
or lens-the natural site of water entry to the seed (Hamly 1932) The strophiole is
located on the cotyledonary lobe of a locust seed (Figure A9) When a papilionoid
legume seed is percussed for the appropriate amount of time repeated hits on the
integrated seed coat loosen the constrained cells of the strophiolar region without
excessively damaging the rest of the seed coat (Ballard 1976)
When a percussed seed is soaked water enters exclusively through the
strophiole in a controlled manner (Kelly and Staden 1987) This regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues This
contrasts with seeds that have been hot water or acid scarified treatments that can
randomly degrade the seed coat Unlocalized cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rehman et al 1998 Marunda 1990)
Papilionoids are the largest subfamily of legumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust and black locust these legumes often play an integral role in the
revegetation ofdisturbed lands Developing superior scarification methods to the
standard hot water and acid treatments should fadlitate the use ofthese legumes in
restoration projects
62
Figure A9 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (bc)
63
APPENDIXB
GERMINATION PERCENTS AND STANDARD ERRORS FOR SOURCE
BLOCK AND TREATMENT FOR NEW MEXICO LOCUST
Table B 1 Gennination percents and standard errors for source block and treatment for New Mexico locust
Source Block Treatment Germination Percentage Standard Error
Capitan 1 Immersion 74 437 Capitan 2 Immersion 69 462 Capitan 3 Immersion 71 454 Capitan 4 Immersion 67 470 Capitan Boiling 30 sec 79 407 Capitan 2 Boiling 30 sec 76 427 Capitan 3 Boiling 30 sec 72 449 Capitan 4 Boiling 30 sec 76 427 Capitan Boiling 60 sec 71 454 Capitan 2 Boiling 60 sec 54 498 Capitan 3 Boiling 60 sec 67 470 Capitan 4 Boiling 60 sec 76 427 Capitan I Control 5 218 Capitan 2 Control 7 255 Capitan 3 Control 4 196 Capitan 4 Control 5 218 Capitan 1 Percussion I min 47 499 Capitan 2 Percussion I min
470
Capitan Percussion I min 39 488
Capitan 4 Percussion I min 38 485 Capitan I Percussion 2 min 55 497 Capitan Capitan
2
Percussion 2 min Percussion 2 min
71 65
454 477
Capitan 4 Percussion 2 min 56 496 Capitan I Percussion 4 min 86 347 Capitan 2 Percussion 4 min 79 407 Capitan 3 Percussion 4 min 81 392 Capitan 4 Percussion 4 min 82 384 Capitan I Percussion 8 min 90 300 Capitan 2 Percussion 8 min 88 325 Capitan 3 Percussion 8 min 90 300 Capitan 4 Percussion 8 min 90 300 Gila I Immersion 71 454 Gila 2 Immersion 77 421 Gila
Immersion 75 433
Gila 4 Immersion 72 449
Gila I Boiling 30 sec 68 466
Gila 2 Boiling 30 sec 81 392
Gila 3 Boiling 30 sec 54 498
Gila 4 Boiling 30 sec 63 483
Gila I Boiling 60 sec 55 497
Gila 2 Boiling 60 sec 68 466
Gila 3 Boiling 60 sec 57 495
Gila 4 Boiling 60 sec 70 458
65
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Gila 2 Control 13 336 Gila 3 Control 13 336 Gila 4 Control 20 400 Gila 1 Percussion 1 min 66 474 Gila 2 Percussion 1 min 57 495 Gila 3 Percussion 1 min 73 444 Gila 4 Percussion 1 min 82 384 Gila 1 Percussion 2 min 81 392 Gila 2 Percussion 2 min 86 347 Gila 3 Percussion 2 min 85 357 Gila 4 Percussion 2 min 54 498 Gila 1 Percussion 4 min 79 407 Gila 2 Percussion 4 min 83 376 Gila 3 Percussion 4 min 84 367 Gila 4 Percussion 4 min 79 407 Gila 1 Percussion 8 min 51 500 Gila 2 Percussion 8 min 76 427 Gila 3 Percussion 8 min 52 500 Gila 4 Percussion 8 min 74 439 Holman 1 Immersion 83 376 Holman 2 Immersion 86 347 Holman 3 Immersion 81 392 Holman 4 Immersion 90 300 Holman 1 Boiling 30 sec 90 300 Holman 2 Boiling 30 sec 85 357 Holman 3 Boiling 30 sec 87 336 Holman 4 Boiling 30 sec 83 376 Holman 1 Boiling 60 sec 79 407 Holman 2 Boiling 60 sec 80 400 Holman 3 Boiling 60 sec 86 347 Holman 4 Boiling 60 sec 93 255 Holman Control 25 433 Holman 2 Control 19 392 Holman Control 21 407
Holman 4 Control 24 427
Holman Percussion 1 min 77 421
Holman 2 Percussion 1 min 85 357
Holman 3 Percussion 1 min 80 400
Holman 4 Percussion 1 min 89 313
Holman 1 Percussion 2 min 88 325
Holman 2 Percussion 2 min 96 196
Holman 3 Percussion 2 min 93 255
Holman 4 Percussion 2 min 96 196
Holman 1 Percussion 4 min 92 271
Holman 2 Percussion 4 min 97 171
66
I
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Holman 3 Percussion 4 min 91 286 Holman 4 Percussion 4 min 96 196 Holman 1 Percussion 8 min 88 325 Holman 2 Percussion 8 min 85 357 Holman 3 Percussion 8 min 83 376 Holman 4 Percussion 8 min 82 384 Jemez 1 Immersion 53 499 Jemez 2 Immersion 44 496 Jemez 3 Immersion 52 500 Jemez 4 Immersion 70 458 Jemez 1 Boiling 30 sec 58 494 Jemez 2 Boiling 30 sec 55 497 Jemez 3 Boiling 30 sec 48 500 Jemez 4 Boiling 30 sec 65 477 Jemez 1 Boiling 60 sec 68 466 Jemez 2 Boiling 60 sec 63 483 Jemez 3 Boiling 60 sec 73 444 Jemez 4 Boiling 60 sec 89 313 Jemez 1 Control 2 140 Jemez 2 Control 9 286 Jemez 3 Control 6 237 Jemez 4 Control 8 271 Jemez 1 Percussion 1 min 58 494 Jemez 2 Percussion 1 min 63 483 Jemez 3 Percussion 1 min 77 421 Jemez 4 Percussion 1 min 89 313 Jemez 1 Percussion 2 min 68 466 Jemez 2 Percussion 2 min 64 480 Jemez 3 Percussion 2 min 80 400 Jemez 4 Percussion 2 min 83 376 Jemez Percussion 4 min 68 466 Jemez 2 Percussion 4 min 79 407 Jemez 3 Percussion 4 min 70 458 Jemez 4 Percussion 4 min 83 376 Jemez 1 Percussion 8 min 52 500 Jemez 2 Percussion 8 min 59 492 Jemez 3 Percussion 8 min 61 488 Jemez 4 Percussion 8 min 70 458 Magdalena 1 Immersion 69 462 Magdalena 2 Immersion 69 462 Magdalena J Immersion 70 458 Magdalena 4 Immersion 85 357 Magdalena 1 Boiling 30 sec 65 477 Magdalena 2 Boiling 30 sec 71 454 Magdalena 3 Boiling 30 sec 68 466
67
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Magdalena 4 Boiling 30 sec 83 376 Magdalena 1 Boiling 60 sec 63 483 Magdalena 2 Boiling 60 sec 68 466 Magdalena 3 Boiling 60 sec 63 483 Magdalena 4 Boiling 60 sec 82 384 Magdalena 1 Control 11 313 Magdalena 2 Control 6 237 Magdalena 3 Control 5 218 Magdalena 4 Control 4 196 Magdalena Percussion 1 min 32 466 Magdalena 2 Percussion 1 min 45 497 Magdalena 3 Percussion 1 min 46 498 Magdalena 4 Percussion I min 36 480 Magdalena 1 Percussion 2 min 67 470 Magdalena 2 Percussion 2 min 68 466 Magdalena 3 Percussion 2 min 65 477 Magdalena 4 Percussion 2 min 81 392 Magdalena 1 Percussion 4 min 87 336 Magdalena 2 Percussion 4 min 97 171 Magdalena 3 Percussion 4 min 83 376 Magdalena 4 Percussion 4 min 97 171 Magdalena 1 Percussion 8 min 98 lAO Magdalena 2 Percussion 8 min 100 000 Magdalena 3 Percussion 8 min 96 196 Magdalena 4 Percussion 8 min 98 lAO Manzano 1 Immersion 67 470 Manzano 2 Immersion 57 495 Manzano 3 Immersion 72 4049 Manzano 4 Immersion 73 4044 Manzano 1 Boiling 30 sec 70 458 Manzano 2 Boiling 30 sec 64 480 Manzano 3 Boiling 30 sec 71 454 Manzano 4 Boiling 30 sec 80 400 Manzano I Boiling 60 sec 50 500 Manzano 2 Boiling 60 sec 53 499 Manzano 3 Boiling 60 sec 59 492 Manzano 4 Boiling 60 sec 74 439 Manzano I Control 4 196 Manzano 2 Control 8 271 Manzano 3 Control 1 099 Manzano 4 Control 6 237 Manzano 1 Percussion 1 min 52 500 Manzano 2 Percussion 1 min 39 488 Manzano 3 Percussion 1 min 36 480 Manzano 4 Percussion 1 min 51 500
68
Table B 1 Continued
Source Block Treatment Gerrni~ation Percentage Standard Error
Manzano Percussion 2 min 53 499 Manzano 2 Percussion 2 min 58 494 Manzano 3 Percussion 2 min 61 488 Manzano 4 Percussion 2 min 62 485 Manzano 1 Percussion 4 min 76 427 Manzano 2 Percussion 4 min 85 357 Manzano 3 Percussion 4 min 79 407 Manzano 4 Percussion 4 min 83 376 Manzano 1 Percussion 8 min 87 336 Manzano 2 Percussion 8 min 83 376 Manzano 3 Percussion 8 min 83 376 Manzano 4 Percussion 8 min 75 433 Raton 1 Immersion 54 498 Raton 2 Immersion 37 483 Raton 3 Immersion 39 488 Raton 4 Immersion 69 462 Raton 1 Boiling 30 sec 36 480 Raton 2 Boiling 30 sec 20 400 Raton 3 Boiling 30 sec 28 449 Raton 4 Boiling 30 sec 60 490 Raton 1 Boiling 60 sec 15 357 Raton 2 Boiling 60 sec 171 Raton 3 Boiling 60 sec 4 196 Raton 4 Boiling 60 sec 35 477 Raton 1 Control 4 196 Raton 2 Control 12 325 Raton 3 Control 2 140 Raton 4 Control 6 237 Raton 1 Percussion 1 min 52 500 Raton 2 Percussion I min 29 454 Raton 3 Percussion I min 35 477 Raton 4 Percussion 1 min 31 462 Raton 1 Percussion 2 min 55 497 Raton 2 Percussion 2 min 35 477 Raton 3 Percussion 2 min 33 470 Raton 4 Percussion 2 min 42 494 Raton 1 Percussion 4 min 65 477 Raton 2 Percussion 4 min 52 500 Raton Percussion 4 min 51 500 Raton 4 Percussion 4 min 53 499 Raton 1 Percussion 8 min 88 325 Raton 2 Percussion 8 min 80 400 Raton 3 Percussion 8 min 73 444 Raton 4 Percussion 8 min 74 439 Sacramento 1 Immersion 43 495
69
Table B 1 Continued
-Source Block Treatment Germination Percentage Standard Error
Sacramento 2 Immersion 30 458 Sacramento 3 Immersion 38 485 Sacramento 4 Immersion 56 496 Sacramento 1 Boiling 30 sec 47 499 Sacramento 2 Boiling 30 sec 48 500 Sacramento 3 Boiling 30 sec 48 500 Sacramento 4 Boiling 30 sec 56 496 Sacramento 1 Boiling 60 sec 52 500 Sacramento 2 Boiling 60 sec 67 470 Sacramento 3 Boiling 60 sec 43 495 Sacramento 4 Boiling 60 sec 48 500 Sacramento 1 Control 0 000 Sacramento 2 Control 3 171 Sacramento 3 Control 3 171 Sacramento 4 Control 099 Sacramento 1 Percussion 1 min 57 495 Sacramento 2 Percussion 1 min 61 488 Sacramento 3 Percussion 1 min 62 485 Sacramento 4 Percussion 1 min 55 497 Sacramento 1 Percussion 2 min 84 367 Sacramento 2 Percussion 2 min 70 458 Sacramento 3 Percussion 2 min 81 392 Sacramento 4 Percussion 2 min 69 462 Sacramento 1 Percussion 4 min 91 286 Sacramento 2 Percussion 4 min 84 367 Sacramento 3 Percussion 4 min 84 367 Sacramento 4 Percussion 4 min 94 237 Sacramento 1 Percussion 8 min 82 384 Sacramento 2 Percussion 8 min 81 392 Sacramento 3 Percussion 8 min 82 384 Sacramento 4 Percussion 8 min 81 392 Sandia 1 Immersion 76 427 Sandia 2 Immersion 70 458 Sandia 3 Immersion 70 458 Sandia 4 Immersion 81 392 Sandia 1 Boiling 30 sec 78 414 Sandia 2 Boiling 30 sec 71 454 Sandia 3 Boiling 30 sec 63 483 Sandia 4 Boiling 30 sec 78 414 Sandia 1 Boiling 60 sec 67 470 Sandia 2 Boiling 60 sec 65 477 Sandia 3 Boiling 60 sec 74 439 Sandia 4 Boiling 60 sec 66 474 Sandia 1 Control 16 367 Sandia 2 Control 7 255
70
Table B 1 Continued
Source Block Treatment Gennination Percentage Standard Error
Sandia 3 Control 8 271 Sandia 4 Control 16 367 Sandia 1 Percussion 1 min 67 470 Sandia 2 Percussion 1 min 61 488 Sandia 3 Percussion 1 min 66 474 Sandia 4 Percussion 1 min 59 492 Sandia I Percussion 2 min 82 384 Sandia 2 Percussion 2 min 83 376 Sandia 3 Percussion 2 min 81 392 Sandia 4 Percussion 2 min 84 367 Sandia 1 Percussion 4 min 89 313 Sandia 2 Percussion 4 min 86 347 Sandia 3 Percussion 4 min 89 313 Sandia 4 Percussion 4 min 85 357 Sandia 1 Percussion 8 min 80 400 Sandia 2 Percussion 8 min 77 421 Sandia 3 Percussion 8 min 69 462 Sandia 4 Percussion 8 min 66 474
LITERATURE CITED
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Ballard LAT 1973 Physical barriers to gennination Seed Sci Technoll 285-303
___ 1976 Strophiolar water conduction in the seeds ofTrifolieae induced by action on the testa at nonstrophiolar sites Austr J Plant Physiol3 465-469
Barton LV 1947 Special studies on seed coat impenneability Contrib Boyce Thompson Inst 14 355-362
Baskin CC Baskin JM 1998 Seeds Ecology biogeography and evolution of donnancy and gennination Academic Press London UK 666 p
Bewley JD 1997 Seed germination and donnancy Plant Ce119 1055-1066
Bewley JD Black M 1994 Seeds physiology of development and germination 2nd ed Plenum Press New York NY 445 p
Briscoe CB 1996 Growing Nitrogen Fixing Trees in Acid Soils In MH Powell (ed) Nitrogen Fixing Trees for Acid Soils- A Field ManuaL Winrock International Morrilton AR 22 p
Copeland LO McDonald MB 1995 Principles of Seed Science and Technology 3rded Chapman amp Hall New York NY 409 p
Cox RE Klett JE 1984 Seed gennination requirements of native Colorado plants for use in the landscape Plant Propag 30(2) 6-10
Dell B 1980 Structure and function ofthe strophiolar plug in seeds ofAlbizia Lophantha Am J Bot 67 556-563
Dick-Peddie WA 1993 New Mexico Vegetation Past Present and Future University ofNew Mexico Press Albuquerque NM 244 p
Dreesen DR 2001 Propagation protocol for production ofcontainer Robinia neomexicana plants Los Lunas Plant Materials Center Los Lunas New Mexico In Native Plant Network URL httpwwwnativeplantnetworkorg (accessed 9 January 2002) Moscow (ID) University ofIdaho CoIl Natural Resources For Res Nursery
72
Dreesen DR Harrington JT 1997 Propagation of native plants for restoration projects in the Southwestern US- Preliminary Investigations In Landis TD Thompson JR tech coords National Proceedings Forest and Conservation Nursery Assocs Gen Tech Rep PNW-GTR-419 Portland OR USDA For Ser PNW Res Sta 77-88
Gottfried GJ 1980 Control ofNew Mexican locust and the effect on planted ponderosa pine in central Arizona US For Ser Res Note RM-386 Rocky Mountain For Range ExpStn Fort Collins CO
Gosling PG Samuel YK Jones SK 1995 A systematic examination of germination temperature chipping and water temperaturesoak duration pretreatments on the seeds ofLeucaena leucocephala Seed Sci amp Technol 23521-532
Hamly DH 1932 Softening of the seeds of Melilotus alba Bot Gazette 93 345shy375
Hanna PJ 1984 Anatomical features of the seed coat ofAcacia kempeana (Mueller) which relate to increased germination rate induced by heat treatment New Phytol 96 23-29
Hanks JP Dick-Peddie WA 1974 Vegetation patterns of the White Mountains New Mexico Southwest Naturalist 18(4) 371-382
Hine S Harrington JT Dreesen DR 1997 Seed scarification requirements for Robinia neomexicana In Harrington JTtech coord Proc Third SouthwestemContainer Growers Conf Feb 12-13 1997 Ft Collins CO
Hopkinson JM 1993 The strophiole of leguminous seeds Newsletter- IntI Herbage Seed Production Res Grp 18 7-8
---- 1997 Differences in hardseededness between Cunningham and Tarramba leucaena In Leucnet News The Newsletter of the International LeucaenaRes and Devlt Netwk No4
ISTA (International Seed Testing Association) 1999 International rules for seed testing rules Seed Sci amp Technol27 Supplement
Kelly KM van Staden J 1987 The lens as the site of permeability in the papilionoid seed Aspalathus linearis J Plant Physiol 128(45) 395-404
73
Keresztesi B 1988 Black locust the tree of agriculture Outlook on Agr 17(2) 77~85
Kigel 1 and Galili G 1995 Seed development and germination Marcel Dekker New York 853 p
Klemmedson 10 1994 New Mexico locust and parent material Influence on macronutrients of forest floor and soil Soil Sci Soc Am 1 58 974~980
Kuo WH Tam AW 1988 The pathway of water absorption of mung bean seeds Seed Sci amp Technol16(1) 139~144
Leadem CL 1997 Dormancy--unlocking seed secrets In Landis TD Thompson lR tech coords National Proceedings Forestand Conservation Nursery Assocs Gen Tech Rep PNW-G TR-419 Portland OR USDA For Ser PNW Res Sta 43-52
Lin Y Wagner MR Seweyestewa A Honahni MD DeGomez M McMillin JD 1996 Influence of cultural treatments on seed germination and seedling growth of Robinia neomexicana Forest Farm and Community Tree Res Reports Volume 1
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Meginnis HG 1937 Sulphuric acid treatment to increase germination of black locust seed USDA Circ 453 34 p
Miklas PN Towensend CE Ladd SL 1987 Seedcoat anatomy and the scarification ofcicer milkvetch seed Crop Sci 27 766-772
Monsen SB 1984 Use of shrubs on mine spoils In The Challenge ofProducing Native Plants for the Intermountain Area Proceedings Intermountain Nurserymans Association 1983 conference Las Vegas NV compo PM Murphy 26-31 Ogden UT USDA For Ser Gen Tech Report INT-l68
Olson DF 1974 Robinia L Locust In Seeds of Woody Plants in the US tech coord CS Schopmeyer 728-731 USDA For Ser Agr Handbk No450
Rehman S Loescher RNl Harris P1C 1998 Dormancy breaking and germination ofAcacia salicina Lindl seeds Seed Sci amp Techno127 553-557
Rolston MP 1978 Water impermeable seed dormancy Bot Rev 44 365-396
74
SAS Institute Inc 1989 SASSTAT Users Guide Version 6 4th Edition Volume 1 Cary NC SAS Institute Inc 943 p
SerratoValenti G Conara L Ferrando M Modenesi P 1993 Structural and histochemical features ofStylosanthes scabra (Leguminosae Papilionoidae) seed coat as related to water entry Can J Bot 71 834-840
Simpson BJ 1988 A field guide to Texas trees Austin TX Texas Monthly Press 372p
Tran VN Cavanagh AK 1984 Structural aspects ofdormancy In Seed Physiology Vol 2 Murray DR (ed) p1-44 Academic Press Australia
USDA Forest Service 1948 Woody-plant seed manual USDA Misc Pub1654 416p
Vines RA 1960 Trees Shrubs and Woody Vines of the Southwest Austin Univ ofTexas Press 1104 p
Wagner MR Larson RE Craig TP 1992 Ecology and silvics ofNew Mexico locust (Robinia neomexicana) with an emphasis on the arboreal form p53-57 In JW Hanover K Miller and S Plesko (eds) Proc Intemat Confon Black Locust Biology Culture and Utilization Mich State Univ USA
Wilson JK 1944 Immersing seeds of species of Robinia in boiling water hastens germination J For 42 453-454
Zimmerman RW Carpenter SB 1980 First-year coppice production from a 5shyyear old black locust stand on surface mine spoil p309-314 Centr Hardwd For ConfProc University ofMissouri Columbia
Umiddot-~middot--l-~---middotymiddotmiddotmiddot-----middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot--middot--middotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middotmiddotmiddotmiddotmiddotmiddotmiddot- -
050= gt1
-e-- Percussion 8 min -0- Percussion 4 min ---T- Percussion 2 min -----ltil- Percussion 1 min -- Immersion --0- Boiling 30 sec -+- Boiling 60 sec -ltgt- Control
2 4 6 8 10 12
Day Figure 6 Timecourse ofNew Mexico locust gennination following scarification
treatments G50 is the number of days to 50 of eventual germination
31
14
Table 4 Effect of seed source and treatment on G50 (days to 50 germination)
Seed Source Control Immersion B30sec B60sec Plmin P2min P4min P8min mean Raton 3 3 5 7 4 3 2 2 4 Holman 2 2 2 2 2 2 2 2 2
Jemez 4 6 6 5 3 2 2 2 4 Sandia 2 2 2 2 2 2 2 2 2
w tv Manzano 5 2 2 3 3 2 2 2 3
Magdalena 4 2 2 3 4 3 2 1 3
Capitan 5 4 2 2 3 2 2 2 3
Sacramento 3 2 2 2 4 3 2 2 3 Gila 3 2 2 2 2 2 2 2 2 mean 3 3 3 3 3 2 2 2 3 standard 11 14 16 18 09 05 0 03 08 deviation
Note Columns and rows may not agree due to rounding
Figure 7 X-ray radiographs ofSacramento seed source percussed for 1 4 or 8 minutes
33
Figure 8a-d Seed damage close-ups Figure a shows undamaged seed percussed for 4 minutes Figures b-d show seeds percussed for 8 minutes Damage includes fracturing ofradicle in b complete removal ofseed coat in c and pitting ofseed coat in d
34
Untreated Treated
Figure 9 SEM images ofuntreated vs 4-minute percussion-treated strophiolar cells ofNew Mexico locust seeds Treated cells are loosened separated and raised
DISCUSSION
Determining the Optimal Treatment
Averaging results of scarification treatmems across seed sources is a useful
tool to predict which treatment is most likely to be successful for an unknown seed
source Averaged over all sources four- and eight-minute percussion treatments
resulted in the highest germination 815 and 790 respectively and produced
germination that was as fast or faster than all other treatments Although there was no
difference between these two treatments on average an argument for 4 minutes of
percussion as a standard protocol for treating New Mexico locust seeds can be made
Descriptively 4 minutes of percussion improved germination over 8 minutes
ofpercussion for a majority of sources (five out of nine) 8 minutes of percussion
improved germination for three ofnine sources with no difference for a fmal source
For individual sources 4 minutes ofpercussion was always the best or secondmiddot best
treatment and was always equal to or better than all hot water treatments
Germination at 8 minutes ofpercussion was lower than all other percussion durations
for the Jemez and Gila sources and was lower than at least one hot water treatment for
these sources as well
The x-ray radiographs of the Sacramento seed source illustrate the excessive
internal and external seed damage that can occur with 8 minutes of percussion
germination counts 8-minute percussed seeds from Holman and Capitan
ClUll signs of damage that were visible to the naked eye (see below)
r~evertheless some of these seeds still fit the criteria for germination Neither x-ray
36
photography nor visible observations during seed counts revealed seed damage at the
4-minute percussion level In operational trials seeds percussed for 4 minutes
developed into healthy seedlings (Khadduri and Harrington unpublished data)
Strophiole and Seed Coat Behavior
Four minutes ofpercussion successfully breaks seed dormancy in a large
percentage ofNew Mexico locust seeds without excessively damaging the
surrounding seed coat or internal seed coat structures Most papilionoid legume
seeds including seeds ofNew Mexico locust have a specific region of the seed coat
known as the strophiole or lens (Hamly 1932) The strophiole is located on the
cotyledonary lobe of a locust seed (Figure 10 a-c) If seeds are not indiscriminately
damaged the strophiole is the first point of water entry to the embryo (Hopkinson
1997) When a papilionoid seed is percussed for the appropriate amount of time
repeated hits on the integrated seed coat loosen the constrained cells of the strophiolar
region without excessively damaging the rest of the seed coat (Ballard 1976) SEM
photos illuStrate that 4 minutes of percussion loosened and separated strophiolar cells
ofNew Mexico locust seeds when compared with constrained organized strophiolar
cells of untreated seeds (Figure 9) Research-has shown that water passes to the
embryo through the treated strophiole in a controlled manner and regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues
(Kelly and van Staden 1987)
37
10 Strophiole ofunimbibed New Mexico locust seed (a) and following uptake (bc)
38
In contrast hot-water treatments can excessively degrade the seed coat
(Rebman et al 1998 Marunda 1990) Random cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rebman et aI 1998 Marunda 1990) Along with a
proportionately large amount ofun-imbibed (thus under-treated) seeds (Figure l1a)
damage was observed in hot-water treated seeds from six sources during germination
counts Damage included seeds where the radicle failed to elongate (Figure 11 b)
seeds where the radicle elongated but was delayed in freeing itself from the seed coat
(Figure 11c) and seeds where the radicle completely broke off from the embryo
(Figure lId) In addition some seeds germinated in reverse (the cotyledon emerged
fIrst) If the radicle emerged completely from the seed coat they were counted as
germinated
While nearly all seeds treated for 8 minutes ofpercussion imbibed water
damage was evident in x-ray evaluations (Sacramento seed source) and in
germination counts (Holman and Capitan) at this treatment level Seed damage
during germination counts was similar to levels found in hot-water treated seeds
suggesting that over-treatment by both hot water and longer durations of percussion
may result in similar damage to seed structure
The Sacramento and Holman seed sources were two of the fIve sources that
opcrplltlpn in germination from the 4- to 8-minute durations of percussion
the benefIts ofloosening the strophiole as evidenced by imbibition of
all 8-minute treated seeds were outweighed by external and internal seed coat
39
a
b c d
11 Damage to New Mexico locust seeds observed during germination cotmts following hot water scarification 9a Hot water replicate including ~t seedlings and numerous unimbibed seeds 9b Close-up of seed where raallCle failed to elongate 9c Close-up ofseed where radicle elongated but was
-gtIn in freeing itself from the seed coat 9d Close-up ofseed where radicle broke off from embryo
40
damage (which also may be contributing to high imbibition) For the three sources
for which 8 minutes of percussion resulted in maximum germination extended
percussion of seeds necessary to loosen the strophiole may not have excessively
damaged the seed anatomy The Manzano source did not significantly change in
germination from 4 to 8 minutes of percussion suggesting that an increase in
permeable seeds at the 8-minute treatment duration was balanced by the number of
seeds that failed to germinate due to seed damage To summarize the 4-minute
percussion treatment improved germination over 8 minutes ofpercussion for a
majority of sources (five ofnine) the 8-minute percussion treatment improved
germination over 4-minutes ofpercussion for three sources and there was no
difference with the Manzano source Notably for the Raton source 8 minutes of
percussion increased germination by 235 percentage points over 4-minutes of
percussion (7875 vs 5525 respectively) the next best treatment Raton was the
only source to result in germination less than 75 for 4 minutes ofpercussion so a
grower may wish to apply longer percussion durations to lots that do not respond well
to this treatment level
Finally there was an interesting trend in the Jemez and Raton sources seed
that significantly contributed to the source by treatment interaction (Figure 12) A
~u test showed that the Jemez source had a significantly lighter seed weight than
other sources while the Raton source had an average seed weight For the Jemez
the longest duration of boiling increased germination Conversely only short
U4UVll) of percussion were needed to achieve high germination and the longest
41
80 _ Jemez a Raton
iD
~ C iD e iD
Q c 0 ~
c ~ iD 0
20
0------shy
Immersion Boiling 30 sec Boiling 60 sec
Hot Water Scarification
10degTr========~--------~------------------------------
80
60
40
20
_ Jemez Raton
0------shy
1 min 2 min 4 min 8 min
Percussion Scarification
Figure 12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources
42
b
--~----~~~~========~---- duration ofpercussion reduced germination For the Raton source longer durations
ofboiling consistently decreased germination whereas longer percussion treatments
consistently increased germination
A possible explanation is that a thick seed coat is associated with a loose
strophiole structure and that a thin seed coat is associated with a constrained
strophiole This contrast merits further study and may suggest an ecological
adaptation Further investigation ofthis phenomenon may contribute to successful
pre-screening ofunknown seed sources to determine optimal treatment leveL
43
CONCLUSION
Based on results obtained in pilot study (see Appendix) and thesis
experiments 4 minutes ofpercussion is recommended as a standard protocol for
treating an unknown source ofNew Mexico locust seed In addition to laboratory
results the 4-minute percussion treatment has resulted in germination greater than
80 and healthy seedling grow-outs for production purposes (unpublished data) In
laboratory experiments only one hundred seeds per 118 ml (40z) soil tin were
percussed at a time whereas for production we have treated up to five hundred seeds
per tin For large-scale production ofNew Mexico locust seedlings growers may
wish to experiment with larger quantities of seed and larger-size soil tins
Seed storage trials following percussion treatments were not conducted
However germination greater than 80 and healthy seedlings were obtained when
seeds were percussion-treated and subsequently stored at room-temperature (22degC)
for one week (unpublished data) Seed storage trials merit further study as the
standard hot water treatment leaves seeds in a softened condition that is not
conducive to storage
Hydroseeding is an effective method for direct seeding in reclamation
situations Percussion treatment may be conducive to hydro seeding as seeds remain
a dry relatively undamaged yet permeable condition following treatment Sulfuric
seeds also remain dry following treatment but extensive damage to seed coat
poor seedling vigor have been reported with this treatment (Kelly and van Staden
A grow-out study using a hydro seeder on a reclamation site testing percussion
44
against other treatments such as sulfuric acid and mechanical scarification also
merits further study
Papilionoids are the largest subfamily oflegumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust these legumes often play an integral role in the revegetation of
disturbed lands The pilot study (Appendix) indicates that percussion scarification
successfully treats the widely used black locust as well Developing superior
scarification methods to the standard hot water mechanical scarification and acid
treatments may facilitate the use ofpapilionoid legumes in reclamation projects
45
APPENDICES
APPENDIX A
PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR
NEW MEXICO LOCUST AND BLACK LOCUST SEEDS
Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4
Hot water and sulfuric acid soaks are traditional treatments for seeds of many
temperate woody legwnes including locusts However these scarification techniques
often produce inconsistent germination Percussion scarification where seeds are
repeatedly propelled against a hard surface was compared with hot water
scarification to evaluate treatment efficacy for New Mexico locust (Robinia
neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment
seeds were placed in a 98degC water bath which was immediately removed from the
heat source For percussion scarification seeds were placed in a soil sample tin and
agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the
control were followed by 24-hour water soaks Hot water baths resulted in 56 and
41 germination for New Mexico locust and black locust respectively For both
species nearly all durations ofpercussion increased germination over the hot water
treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3
4 and 5 minutes for black locust resulted in at least 90 germination Traditional
scarification treatments randomly degrade the entire seed coat which can lead to
tissue damage during water uptake Percussion scarification specifically weakens the
strophiole the natural source ofwater entry to the seed in papilionoid legwnes
INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom
2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031
48
Following percussion imbibition is controlled through the strophiole and underlying
tissue is protected
Keywords New Mexico locust black locust scarification percussion germination
Introduction
Locust trees (Robinia spp) are aggressive pioneer species that quickly
colonize disturbed land fix atmospheric nitrogen in the soil and stabilize slopes that
are prone to erosion (Klemmedson 1994 Ashby et al 1985) (Figure A1ab)
Recognition of these attributes has contributed to increased demand for locusts in
reclamation projects Locust seeds however exhibit physical dormancy where a
thick seed coat prevents the movement of water and gasses to the embryo (Leadem
1997)
Sulfuric acid or hot water soaks have traditionally been used to break seed
dormancy in macro-propagation of locusts Many growers are moving away from the
use of su1furic acid in treating locust seeds (Dreesen and Harrington 1997) In
addition to being dangerous acid soak durations must be specifically correlated to
seed lot (Olson 1974) Collections of native seed can vary tremendously in seed size
seed weight and hard seededness etc This variability limits the utility of sulfuric
acid soaks as even within a given lot some seeds may be damaged whereas others
are under-treated and fail to imbibe (take up water) A survey of Southwestern
container growers indicates that hot water baths are currently the most common
scarification method for locust seeds (Hine et al 1997) However hot water baths
also produce inconsistent germination (Lin et al 1996)
49
b
Figure AI New Mexico locust a primary invader following disturbance Ala Crown-sprouting at edge ofburn near Los Alamos NM--one year after Cerro Grande fire Alb Colonizing road cut Gila National Forest NM
50
Percussion scarification where seeds are repeatedly propelled against a hard
surface is an alternative dormancy-breaking method that has proven successful in
legumes related to locusts (Hamly 1932 Barton 1947) We compared this alternative
treatment to hot water scarification to evaluate treatment efficacy for New Mexico
locust and black locust seeds
Materials and Methods
Commercial New Mexico locust seeds (Western Native Seed Coaldale CO)
were collected fall 2000 in Huerfano County CO Black locust seeds were collected
September 2000 from Taos County NM by harvesting ripe pods from several trees at
a distance of up to 3 m from ground level Black locust seeds were allowed to air-dry
for 3 weeks and were threshed and separated from large chaffusing a greenhouse
fan A South Dakota Seed Blower (Chicago Illinltois) was used to remove fine chaff
from both seed lots Seeds were storedmiddotat 2-4C until the germination study was
conducted in March 2001
This study compared hot water scarification with percussion scarification
using treatment and species as experimental factors Along with a control
scarification treatments included immersion of seed in boiling water as well as
percussion durations of 1 2 3 45 or 10 minutes Four 100-seed samples were used
to test each species by scarification treatment combination
For the hot water treatment test tubes were filled with 30 mL water and
placed in a water-filled beaker (Figure A2a) The bath was raised to boiling (-98C at
51
b
c
Figure A2 Implementation ofhot water and percussion scarification A2a Hot water bath A2b Paint-shaker set-up A2c Placing soil tin containing seeds at end ofpaint shaker ann
52
Las Cruces NM elevation of 1300 meters) Each 100-seed replicate was placed in a
test tube and each test tube was immediately removed to cool at room temperature
Percussion scarification was implemented using a pneumatic paint shaker
(Central Pneumatic model 00422 Camarillo CA) (Figure A2b) This allowed
standardization of treatment intensity Compressed air pressure was maintained at
530kPa plusmn 30kPa (80psi plusmn 5psi) resulting in approximately 350 oscillations per
minute Each lOO-seed sample was placed in a 118 ml (4 oz) soil tin for shaking To
maximize lateral movement of paint shaker a paint can was used as a spacer to place
soil tins at the end of the shaking arm (Figure A2c) This allowed shaking to occur at
the greatest distance from the pivot point (fulcrum) Following all treatments
including the control seeds were soaked for twenty-four hours in distilled water prior
to plating out
Gennination was evaluated on lab benches at room temperature Each
replicate of seeds was placed in a 100 cm petri dish on moistened filter paper with
humidity maintained by enclosing the petri dishes in ziplock bags Gennination was
monitored daily for 14 days in accordance with International Seed Testing
Association standards for Robinia species (ISTA 1999) Gennination was defined as
protrusion of the radicle from the embryo by at least 1mm as viewed by the naked
eye
Categorical Analysis of Variance using Prpc Catmod (SAS Institute 1989)
was used to detennine treatment differences in germination percentages for each
source This procedure is an extension of a Chi-square test of homogeneity using the
53
natural log of the ratio of germinated to non-germinated seeds for each treatment Pshy
values less than 005 were considered significant A limited set of pairwiseshy
comparisons was conducted to compare treatment mean percents using a
conservative alpha value of 005 divided by the number of comparisons
Results
Percussion maximized germination for both New Mexico locust and black
locust There were three germination response levels for New Mexico locust (Figure
A3) First there was a low response in the control indicating a dormant seed lot
The hot water treatment and I-minute duration of percussion share a second
intermediate response Germination was highest at percussion levels from 2 to 10
minutes
A selected percussion treatment intermediate in time (5 minutes) produced the
steepest germination curve indicating the fastest germination rate (Figure A4) The
hot water treatment produced an intermediate curve or relatively slower germination
rate The control curve was very flat indicating the slowest germination rate for New
Mexico locust
Control seeds germinated poorly for black locust as well (Figure A5) Hot
water scarification improved germination over the control but all levels of percussion
improved germination over hot water The optimal durations of percussion treatment
were bracketed with a significant increase from the 1 to 2 minute percussion level a
high germination response from 2 to 5 minutes of percussion and finally a significant
54
~ d 25 CL)
p t1 0 ~ t1
g Vl ~ Vl
100~---------------------------------------------------------------
80
60
40
20
o Control Hot Water Percussion
I min Percussion
2 min Percussion
3 min
Scarification
Percussion 4 min
Figure A3 Effect of scarification on germination response for New Mexico locust
Percussion 5 min
=significant difference between adjacent treatment bars ns = no significant difference
Percussion 10 min
~ ~ () u agt
Il-t d o ~
~ ~ c3
100middotlr~~~------------------------------------e- Percussion 5min middot 0middot Hot Water -- Control
80-J=middot======
6b-middot-lmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middot--middotmiddot-middot~-middotmiddot -middotmiddot middot middotmiddot-middot 1
40
20
-----~-----------
0 0 0 middotmiddotmiddotmiddotmiddot0middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot0
0 --~-----y-----~------_---~--- T-----~
o shy -TDay 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A4 Effect of selected scarification treatments on germination speed for New Mexico locust First seven days of fourteen-day experiment are shown
Day 7
80
~ cd
5 60 ()
[) Cl d 0 1t1 d
40sect dUl
-J
20
o
ns
Scarification
Figure A5 Effect of scarification on germination response for black locust = significant difference between adjacent treatment bars ns = no significant difference
decrease at the 10-minute level This drop-off suggests that 10 minutes of percussion
over-treats seeds from this lot of-black locust
A selected percussion treatment intermediate in time (5 minutes) also had the
fastest germination rate for black locust (Figure A6)_ The hot water curve is
somewhat flatter indicating a slower germination rate The control curve is very flat
indicating a slow germination rate
A qualitative difference between hot water and percussion-treated seeds was
also observed Along with healthy germinants the hot water treatment produced
over-treated seeds (Figure A 7a) Examples of damaged germinants include seeds
where the radicle did not elongate (Figure A 7b) seeds where the radicle elongated
but was delayed in freeing itself from the seed coat (Figure A7c) and seeds where the
radicle broke off completely from the embryo (Figure A7d) In addition to these
examples of over-treatment there were numerous small dark seeds that failed to
imbibe water For percussion treatment nearly all seeds took up water (Figure A8a)
Germination was rapid and uniform with little damage evident (Figure A8b)
To summarize both New Mexico locust and black locust seed lots were
dormant The standard hot water scarification treatment improved germination but
only to 56 and 41 for New Mexico locust and black locust respectively Nearly
all percussion durations improved total germination as well as germination rate over
the hot water treatment Germination was greater than 90 for percussion durations
of 4 5 and 10 minutes for New Mexico locust and 3 4 and 5 minutes for black locust
58
CI)
sgt t CI) 0
igt 0
sect ttl t E
Vl 0
c3
100r==~==~~=r------------------------------------~ ___ Percussion 5 min
middotmiddotmiddot0middotmiddotmiddot Hot Water -lIf- Control
80r=========~--~~----------------------------~
60
40
20 -~-~---~---------- ---~------------I
omiddot
O
_~_----------lIf-----~bull -----_ shy---shy I
Day 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A6 Effect of selected scarification treatments on germination speed for black locust First seven days of fourteen-day experiment are shown
Day 7
a
b c d
Figure A7 Damage to New Mexico locust seeds observed during germination counts following hot water scarification A7a Hot water replicate including damaged seedlings and numerous unimbibed seeds A7b Close-up ofseed where radicle failed to elongate A7c Close-up ofseed where radicle elongated but was delayed in freeing itself from the seed coat A7d Close-up ofseed where radicle completely broke offfrom embryo
60
a
b
Figure A8 New Mexico locust seeds following percussion treatment at time of germination counts A8a Percussion replicate with uniformly healthy seedlings and near complete imbibition of seeds A8b Close-up ofpercussion-treated seedling
61
Discussion
Papilionoid seeds have a specific anatomical feature known as the strophiole
or lens-the natural site of water entry to the seed (Hamly 1932) The strophiole is
located on the cotyledonary lobe of a locust seed (Figure A9) When a papilionoid
legume seed is percussed for the appropriate amount of time repeated hits on the
integrated seed coat loosen the constrained cells of the strophiolar region without
excessively damaging the rest of the seed coat (Ballard 1976)
When a percussed seed is soaked water enters exclusively through the
strophiole in a controlled manner (Kelly and Staden 1987) This regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues This
contrasts with seeds that have been hot water or acid scarified treatments that can
randomly degrade the seed coat Unlocalized cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rehman et al 1998 Marunda 1990)
Papilionoids are the largest subfamily of legumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust and black locust these legumes often play an integral role in the
revegetation ofdisturbed lands Developing superior scarification methods to the
standard hot water and acid treatments should fadlitate the use ofthese legumes in
restoration projects
62
Figure A9 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (bc)
63
APPENDIXB
GERMINATION PERCENTS AND STANDARD ERRORS FOR SOURCE
BLOCK AND TREATMENT FOR NEW MEXICO LOCUST
Table B 1 Gennination percents and standard errors for source block and treatment for New Mexico locust
Source Block Treatment Germination Percentage Standard Error
Capitan 1 Immersion 74 437 Capitan 2 Immersion 69 462 Capitan 3 Immersion 71 454 Capitan 4 Immersion 67 470 Capitan Boiling 30 sec 79 407 Capitan 2 Boiling 30 sec 76 427 Capitan 3 Boiling 30 sec 72 449 Capitan 4 Boiling 30 sec 76 427 Capitan Boiling 60 sec 71 454 Capitan 2 Boiling 60 sec 54 498 Capitan 3 Boiling 60 sec 67 470 Capitan 4 Boiling 60 sec 76 427 Capitan I Control 5 218 Capitan 2 Control 7 255 Capitan 3 Control 4 196 Capitan 4 Control 5 218 Capitan 1 Percussion I min 47 499 Capitan 2 Percussion I min
470
Capitan Percussion I min 39 488
Capitan 4 Percussion I min 38 485 Capitan I Percussion 2 min 55 497 Capitan Capitan
2
Percussion 2 min Percussion 2 min
71 65
454 477
Capitan 4 Percussion 2 min 56 496 Capitan I Percussion 4 min 86 347 Capitan 2 Percussion 4 min 79 407 Capitan 3 Percussion 4 min 81 392 Capitan 4 Percussion 4 min 82 384 Capitan I Percussion 8 min 90 300 Capitan 2 Percussion 8 min 88 325 Capitan 3 Percussion 8 min 90 300 Capitan 4 Percussion 8 min 90 300 Gila I Immersion 71 454 Gila 2 Immersion 77 421 Gila
Immersion 75 433
Gila 4 Immersion 72 449
Gila I Boiling 30 sec 68 466
Gila 2 Boiling 30 sec 81 392
Gila 3 Boiling 30 sec 54 498
Gila 4 Boiling 30 sec 63 483
Gila I Boiling 60 sec 55 497
Gila 2 Boiling 60 sec 68 466
Gila 3 Boiling 60 sec 57 495
Gila 4 Boiling 60 sec 70 458
65
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Gila 2 Control 13 336 Gila 3 Control 13 336 Gila 4 Control 20 400 Gila 1 Percussion 1 min 66 474 Gila 2 Percussion 1 min 57 495 Gila 3 Percussion 1 min 73 444 Gila 4 Percussion 1 min 82 384 Gila 1 Percussion 2 min 81 392 Gila 2 Percussion 2 min 86 347 Gila 3 Percussion 2 min 85 357 Gila 4 Percussion 2 min 54 498 Gila 1 Percussion 4 min 79 407 Gila 2 Percussion 4 min 83 376 Gila 3 Percussion 4 min 84 367 Gila 4 Percussion 4 min 79 407 Gila 1 Percussion 8 min 51 500 Gila 2 Percussion 8 min 76 427 Gila 3 Percussion 8 min 52 500 Gila 4 Percussion 8 min 74 439 Holman 1 Immersion 83 376 Holman 2 Immersion 86 347 Holman 3 Immersion 81 392 Holman 4 Immersion 90 300 Holman 1 Boiling 30 sec 90 300 Holman 2 Boiling 30 sec 85 357 Holman 3 Boiling 30 sec 87 336 Holman 4 Boiling 30 sec 83 376 Holman 1 Boiling 60 sec 79 407 Holman 2 Boiling 60 sec 80 400 Holman 3 Boiling 60 sec 86 347 Holman 4 Boiling 60 sec 93 255 Holman Control 25 433 Holman 2 Control 19 392 Holman Control 21 407
Holman 4 Control 24 427
Holman Percussion 1 min 77 421
Holman 2 Percussion 1 min 85 357
Holman 3 Percussion 1 min 80 400
Holman 4 Percussion 1 min 89 313
Holman 1 Percussion 2 min 88 325
Holman 2 Percussion 2 min 96 196
Holman 3 Percussion 2 min 93 255
Holman 4 Percussion 2 min 96 196
Holman 1 Percussion 4 min 92 271
Holman 2 Percussion 4 min 97 171
66
I
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Holman 3 Percussion 4 min 91 286 Holman 4 Percussion 4 min 96 196 Holman 1 Percussion 8 min 88 325 Holman 2 Percussion 8 min 85 357 Holman 3 Percussion 8 min 83 376 Holman 4 Percussion 8 min 82 384 Jemez 1 Immersion 53 499 Jemez 2 Immersion 44 496 Jemez 3 Immersion 52 500 Jemez 4 Immersion 70 458 Jemez 1 Boiling 30 sec 58 494 Jemez 2 Boiling 30 sec 55 497 Jemez 3 Boiling 30 sec 48 500 Jemez 4 Boiling 30 sec 65 477 Jemez 1 Boiling 60 sec 68 466 Jemez 2 Boiling 60 sec 63 483 Jemez 3 Boiling 60 sec 73 444 Jemez 4 Boiling 60 sec 89 313 Jemez 1 Control 2 140 Jemez 2 Control 9 286 Jemez 3 Control 6 237 Jemez 4 Control 8 271 Jemez 1 Percussion 1 min 58 494 Jemez 2 Percussion 1 min 63 483 Jemez 3 Percussion 1 min 77 421 Jemez 4 Percussion 1 min 89 313 Jemez 1 Percussion 2 min 68 466 Jemez 2 Percussion 2 min 64 480 Jemez 3 Percussion 2 min 80 400 Jemez 4 Percussion 2 min 83 376 Jemez Percussion 4 min 68 466 Jemez 2 Percussion 4 min 79 407 Jemez 3 Percussion 4 min 70 458 Jemez 4 Percussion 4 min 83 376 Jemez 1 Percussion 8 min 52 500 Jemez 2 Percussion 8 min 59 492 Jemez 3 Percussion 8 min 61 488 Jemez 4 Percussion 8 min 70 458 Magdalena 1 Immersion 69 462 Magdalena 2 Immersion 69 462 Magdalena J Immersion 70 458 Magdalena 4 Immersion 85 357 Magdalena 1 Boiling 30 sec 65 477 Magdalena 2 Boiling 30 sec 71 454 Magdalena 3 Boiling 30 sec 68 466
67
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Magdalena 4 Boiling 30 sec 83 376 Magdalena 1 Boiling 60 sec 63 483 Magdalena 2 Boiling 60 sec 68 466 Magdalena 3 Boiling 60 sec 63 483 Magdalena 4 Boiling 60 sec 82 384 Magdalena 1 Control 11 313 Magdalena 2 Control 6 237 Magdalena 3 Control 5 218 Magdalena 4 Control 4 196 Magdalena Percussion 1 min 32 466 Magdalena 2 Percussion 1 min 45 497 Magdalena 3 Percussion 1 min 46 498 Magdalena 4 Percussion I min 36 480 Magdalena 1 Percussion 2 min 67 470 Magdalena 2 Percussion 2 min 68 466 Magdalena 3 Percussion 2 min 65 477 Magdalena 4 Percussion 2 min 81 392 Magdalena 1 Percussion 4 min 87 336 Magdalena 2 Percussion 4 min 97 171 Magdalena 3 Percussion 4 min 83 376 Magdalena 4 Percussion 4 min 97 171 Magdalena 1 Percussion 8 min 98 lAO Magdalena 2 Percussion 8 min 100 000 Magdalena 3 Percussion 8 min 96 196 Magdalena 4 Percussion 8 min 98 lAO Manzano 1 Immersion 67 470 Manzano 2 Immersion 57 495 Manzano 3 Immersion 72 4049 Manzano 4 Immersion 73 4044 Manzano 1 Boiling 30 sec 70 458 Manzano 2 Boiling 30 sec 64 480 Manzano 3 Boiling 30 sec 71 454 Manzano 4 Boiling 30 sec 80 400 Manzano I Boiling 60 sec 50 500 Manzano 2 Boiling 60 sec 53 499 Manzano 3 Boiling 60 sec 59 492 Manzano 4 Boiling 60 sec 74 439 Manzano I Control 4 196 Manzano 2 Control 8 271 Manzano 3 Control 1 099 Manzano 4 Control 6 237 Manzano 1 Percussion 1 min 52 500 Manzano 2 Percussion 1 min 39 488 Manzano 3 Percussion 1 min 36 480 Manzano 4 Percussion 1 min 51 500
68
Table B 1 Continued
Source Block Treatment Gerrni~ation Percentage Standard Error
Manzano Percussion 2 min 53 499 Manzano 2 Percussion 2 min 58 494 Manzano 3 Percussion 2 min 61 488 Manzano 4 Percussion 2 min 62 485 Manzano 1 Percussion 4 min 76 427 Manzano 2 Percussion 4 min 85 357 Manzano 3 Percussion 4 min 79 407 Manzano 4 Percussion 4 min 83 376 Manzano 1 Percussion 8 min 87 336 Manzano 2 Percussion 8 min 83 376 Manzano 3 Percussion 8 min 83 376 Manzano 4 Percussion 8 min 75 433 Raton 1 Immersion 54 498 Raton 2 Immersion 37 483 Raton 3 Immersion 39 488 Raton 4 Immersion 69 462 Raton 1 Boiling 30 sec 36 480 Raton 2 Boiling 30 sec 20 400 Raton 3 Boiling 30 sec 28 449 Raton 4 Boiling 30 sec 60 490 Raton 1 Boiling 60 sec 15 357 Raton 2 Boiling 60 sec 171 Raton 3 Boiling 60 sec 4 196 Raton 4 Boiling 60 sec 35 477 Raton 1 Control 4 196 Raton 2 Control 12 325 Raton 3 Control 2 140 Raton 4 Control 6 237 Raton 1 Percussion 1 min 52 500 Raton 2 Percussion I min 29 454 Raton 3 Percussion I min 35 477 Raton 4 Percussion 1 min 31 462 Raton 1 Percussion 2 min 55 497 Raton 2 Percussion 2 min 35 477 Raton 3 Percussion 2 min 33 470 Raton 4 Percussion 2 min 42 494 Raton 1 Percussion 4 min 65 477 Raton 2 Percussion 4 min 52 500 Raton Percussion 4 min 51 500 Raton 4 Percussion 4 min 53 499 Raton 1 Percussion 8 min 88 325 Raton 2 Percussion 8 min 80 400 Raton 3 Percussion 8 min 73 444 Raton 4 Percussion 8 min 74 439 Sacramento 1 Immersion 43 495
69
Table B 1 Continued
-Source Block Treatment Germination Percentage Standard Error
Sacramento 2 Immersion 30 458 Sacramento 3 Immersion 38 485 Sacramento 4 Immersion 56 496 Sacramento 1 Boiling 30 sec 47 499 Sacramento 2 Boiling 30 sec 48 500 Sacramento 3 Boiling 30 sec 48 500 Sacramento 4 Boiling 30 sec 56 496 Sacramento 1 Boiling 60 sec 52 500 Sacramento 2 Boiling 60 sec 67 470 Sacramento 3 Boiling 60 sec 43 495 Sacramento 4 Boiling 60 sec 48 500 Sacramento 1 Control 0 000 Sacramento 2 Control 3 171 Sacramento 3 Control 3 171 Sacramento 4 Control 099 Sacramento 1 Percussion 1 min 57 495 Sacramento 2 Percussion 1 min 61 488 Sacramento 3 Percussion 1 min 62 485 Sacramento 4 Percussion 1 min 55 497 Sacramento 1 Percussion 2 min 84 367 Sacramento 2 Percussion 2 min 70 458 Sacramento 3 Percussion 2 min 81 392 Sacramento 4 Percussion 2 min 69 462 Sacramento 1 Percussion 4 min 91 286 Sacramento 2 Percussion 4 min 84 367 Sacramento 3 Percussion 4 min 84 367 Sacramento 4 Percussion 4 min 94 237 Sacramento 1 Percussion 8 min 82 384 Sacramento 2 Percussion 8 min 81 392 Sacramento 3 Percussion 8 min 82 384 Sacramento 4 Percussion 8 min 81 392 Sandia 1 Immersion 76 427 Sandia 2 Immersion 70 458 Sandia 3 Immersion 70 458 Sandia 4 Immersion 81 392 Sandia 1 Boiling 30 sec 78 414 Sandia 2 Boiling 30 sec 71 454 Sandia 3 Boiling 30 sec 63 483 Sandia 4 Boiling 30 sec 78 414 Sandia 1 Boiling 60 sec 67 470 Sandia 2 Boiling 60 sec 65 477 Sandia 3 Boiling 60 sec 74 439 Sandia 4 Boiling 60 sec 66 474 Sandia 1 Control 16 367 Sandia 2 Control 7 255
70
Table B 1 Continued
Source Block Treatment Gennination Percentage Standard Error
Sandia 3 Control 8 271 Sandia 4 Control 16 367 Sandia 1 Percussion 1 min 67 470 Sandia 2 Percussion 1 min 61 488 Sandia 3 Percussion 1 min 66 474 Sandia 4 Percussion 1 min 59 492 Sandia I Percussion 2 min 82 384 Sandia 2 Percussion 2 min 83 376 Sandia 3 Percussion 2 min 81 392 Sandia 4 Percussion 2 min 84 367 Sandia 1 Percussion 4 min 89 313 Sandia 2 Percussion 4 min 86 347 Sandia 3 Percussion 4 min 89 313 Sandia 4 Percussion 4 min 85 357 Sandia 1 Percussion 8 min 80 400 Sandia 2 Percussion 8 min 77 421 Sandia 3 Percussion 8 min 69 462 Sandia 4 Percussion 8 min 66 474
LITERATURE CITED
Ashby WC Vogel WG Rogers NF 1985 Black locust in the reclamation equation General Technical Report Northeastern Forest Experiment Station USDA Forest Service NE-I05 12 p
Ballard LAT 1973 Physical barriers to gennination Seed Sci Technoll 285-303
___ 1976 Strophiolar water conduction in the seeds ofTrifolieae induced by action on the testa at nonstrophiolar sites Austr J Plant Physiol3 465-469
Barton LV 1947 Special studies on seed coat impenneability Contrib Boyce Thompson Inst 14 355-362
Baskin CC Baskin JM 1998 Seeds Ecology biogeography and evolution of donnancy and gennination Academic Press London UK 666 p
Bewley JD 1997 Seed germination and donnancy Plant Ce119 1055-1066
Bewley JD Black M 1994 Seeds physiology of development and germination 2nd ed Plenum Press New York NY 445 p
Briscoe CB 1996 Growing Nitrogen Fixing Trees in Acid Soils In MH Powell (ed) Nitrogen Fixing Trees for Acid Soils- A Field ManuaL Winrock International Morrilton AR 22 p
Copeland LO McDonald MB 1995 Principles of Seed Science and Technology 3rded Chapman amp Hall New York NY 409 p
Cox RE Klett JE 1984 Seed gennination requirements of native Colorado plants for use in the landscape Plant Propag 30(2) 6-10
Dell B 1980 Structure and function ofthe strophiolar plug in seeds ofAlbizia Lophantha Am J Bot 67 556-563
Dick-Peddie WA 1993 New Mexico Vegetation Past Present and Future University ofNew Mexico Press Albuquerque NM 244 p
Dreesen DR 2001 Propagation protocol for production ofcontainer Robinia neomexicana plants Los Lunas Plant Materials Center Los Lunas New Mexico In Native Plant Network URL httpwwwnativeplantnetworkorg (accessed 9 January 2002) Moscow (ID) University ofIdaho CoIl Natural Resources For Res Nursery
72
Dreesen DR Harrington JT 1997 Propagation of native plants for restoration projects in the Southwestern US- Preliminary Investigations In Landis TD Thompson JR tech coords National Proceedings Forest and Conservation Nursery Assocs Gen Tech Rep PNW-GTR-419 Portland OR USDA For Ser PNW Res Sta 77-88
Gottfried GJ 1980 Control ofNew Mexican locust and the effect on planted ponderosa pine in central Arizona US For Ser Res Note RM-386 Rocky Mountain For Range ExpStn Fort Collins CO
Gosling PG Samuel YK Jones SK 1995 A systematic examination of germination temperature chipping and water temperaturesoak duration pretreatments on the seeds ofLeucaena leucocephala Seed Sci amp Technol 23521-532
Hamly DH 1932 Softening of the seeds of Melilotus alba Bot Gazette 93 345shy375
Hanna PJ 1984 Anatomical features of the seed coat ofAcacia kempeana (Mueller) which relate to increased germination rate induced by heat treatment New Phytol 96 23-29
Hanks JP Dick-Peddie WA 1974 Vegetation patterns of the White Mountains New Mexico Southwest Naturalist 18(4) 371-382
Hine S Harrington JT Dreesen DR 1997 Seed scarification requirements for Robinia neomexicana In Harrington JTtech coord Proc Third SouthwestemContainer Growers Conf Feb 12-13 1997 Ft Collins CO
Hopkinson JM 1993 The strophiole of leguminous seeds Newsletter- IntI Herbage Seed Production Res Grp 18 7-8
---- 1997 Differences in hardseededness between Cunningham and Tarramba leucaena In Leucnet News The Newsletter of the International LeucaenaRes and Devlt Netwk No4
ISTA (International Seed Testing Association) 1999 International rules for seed testing rules Seed Sci amp Technol27 Supplement
Kelly KM van Staden J 1987 The lens as the site of permeability in the papilionoid seed Aspalathus linearis J Plant Physiol 128(45) 395-404
73
Keresztesi B 1988 Black locust the tree of agriculture Outlook on Agr 17(2) 77~85
Kigel 1 and Galili G 1995 Seed development and germination Marcel Dekker New York 853 p
Klemmedson 10 1994 New Mexico locust and parent material Influence on macronutrients of forest floor and soil Soil Sci Soc Am 1 58 974~980
Kuo WH Tam AW 1988 The pathway of water absorption of mung bean seeds Seed Sci amp Technol16(1) 139~144
Leadem CL 1997 Dormancy--unlocking seed secrets In Landis TD Thompson lR tech coords National Proceedings Forestand Conservation Nursery Assocs Gen Tech Rep PNW-G TR-419 Portland OR USDA For Ser PNW Res Sta 43-52
Lin Y Wagner MR Seweyestewa A Honahni MD DeGomez M McMillin JD 1996 Influence of cultural treatments on seed germination and seedling growth of Robinia neomexicana Forest Farm and Community Tree Res Reports Volume 1
Marunda CT 1990 Effects of seed pretreatment on the development ofAcacia auriculforinis and A holosericea seedlings ACIAR Proceedings Series No 28
Meginnis HG 1937 Sulphuric acid treatment to increase germination of black locust seed USDA Circ 453 34 p
Miklas PN Towensend CE Ladd SL 1987 Seedcoat anatomy and the scarification ofcicer milkvetch seed Crop Sci 27 766-772
Monsen SB 1984 Use of shrubs on mine spoils In The Challenge ofProducing Native Plants for the Intermountain Area Proceedings Intermountain Nurserymans Association 1983 conference Las Vegas NV compo PM Murphy 26-31 Ogden UT USDA For Ser Gen Tech Report INT-l68
Olson DF 1974 Robinia L Locust In Seeds of Woody Plants in the US tech coord CS Schopmeyer 728-731 USDA For Ser Agr Handbk No450
Rehman S Loescher RNl Harris P1C 1998 Dormancy breaking and germination ofAcacia salicina Lindl seeds Seed Sci amp Techno127 553-557
Rolston MP 1978 Water impermeable seed dormancy Bot Rev 44 365-396
74
SAS Institute Inc 1989 SASSTAT Users Guide Version 6 4th Edition Volume 1 Cary NC SAS Institute Inc 943 p
SerratoValenti G Conara L Ferrando M Modenesi P 1993 Structural and histochemical features ofStylosanthes scabra (Leguminosae Papilionoidae) seed coat as related to water entry Can J Bot 71 834-840
Simpson BJ 1988 A field guide to Texas trees Austin TX Texas Monthly Press 372p
Tran VN Cavanagh AK 1984 Structural aspects ofdormancy In Seed Physiology Vol 2 Murray DR (ed) p1-44 Academic Press Australia
USDA Forest Service 1948 Woody-plant seed manual USDA Misc Pub1654 416p
Vines RA 1960 Trees Shrubs and Woody Vines of the Southwest Austin Univ ofTexas Press 1104 p
Wagner MR Larson RE Craig TP 1992 Ecology and silvics ofNew Mexico locust (Robinia neomexicana) with an emphasis on the arboreal form p53-57 In JW Hanover K Miller and S Plesko (eds) Proc Intemat Confon Black Locust Biology Culture and Utilization Mich State Univ USA
Wilson JK 1944 Immersing seeds of species of Robinia in boiling water hastens germination J For 42 453-454
Zimmerman RW Carpenter SB 1980 First-year coppice production from a 5shyyear old black locust stand on surface mine spoil p309-314 Centr Hardwd For ConfProc University ofMissouri Columbia
Table 4 Effect of seed source and treatment on G50 (days to 50 germination)
Seed Source Control Immersion B30sec B60sec Plmin P2min P4min P8min mean Raton 3 3 5 7 4 3 2 2 4 Holman 2 2 2 2 2 2 2 2 2
Jemez 4 6 6 5 3 2 2 2 4 Sandia 2 2 2 2 2 2 2 2 2
w tv Manzano 5 2 2 3 3 2 2 2 3
Magdalena 4 2 2 3 4 3 2 1 3
Capitan 5 4 2 2 3 2 2 2 3
Sacramento 3 2 2 2 4 3 2 2 3 Gila 3 2 2 2 2 2 2 2 2 mean 3 3 3 3 3 2 2 2 3 standard 11 14 16 18 09 05 0 03 08 deviation
Note Columns and rows may not agree due to rounding
Figure 7 X-ray radiographs ofSacramento seed source percussed for 1 4 or 8 minutes
33
Figure 8a-d Seed damage close-ups Figure a shows undamaged seed percussed for 4 minutes Figures b-d show seeds percussed for 8 minutes Damage includes fracturing ofradicle in b complete removal ofseed coat in c and pitting ofseed coat in d
34
Untreated Treated
Figure 9 SEM images ofuntreated vs 4-minute percussion-treated strophiolar cells ofNew Mexico locust seeds Treated cells are loosened separated and raised
DISCUSSION
Determining the Optimal Treatment
Averaging results of scarification treatmems across seed sources is a useful
tool to predict which treatment is most likely to be successful for an unknown seed
source Averaged over all sources four- and eight-minute percussion treatments
resulted in the highest germination 815 and 790 respectively and produced
germination that was as fast or faster than all other treatments Although there was no
difference between these two treatments on average an argument for 4 minutes of
percussion as a standard protocol for treating New Mexico locust seeds can be made
Descriptively 4 minutes of percussion improved germination over 8 minutes
ofpercussion for a majority of sources (five out of nine) 8 minutes of percussion
improved germination for three ofnine sources with no difference for a fmal source
For individual sources 4 minutes ofpercussion was always the best or secondmiddot best
treatment and was always equal to or better than all hot water treatments
Germination at 8 minutes ofpercussion was lower than all other percussion durations
for the Jemez and Gila sources and was lower than at least one hot water treatment for
these sources as well
The x-ray radiographs of the Sacramento seed source illustrate the excessive
internal and external seed damage that can occur with 8 minutes of percussion
germination counts 8-minute percussed seeds from Holman and Capitan
ClUll signs of damage that were visible to the naked eye (see below)
r~evertheless some of these seeds still fit the criteria for germination Neither x-ray
36
photography nor visible observations during seed counts revealed seed damage at the
4-minute percussion level In operational trials seeds percussed for 4 minutes
developed into healthy seedlings (Khadduri and Harrington unpublished data)
Strophiole and Seed Coat Behavior
Four minutes ofpercussion successfully breaks seed dormancy in a large
percentage ofNew Mexico locust seeds without excessively damaging the
surrounding seed coat or internal seed coat structures Most papilionoid legume
seeds including seeds ofNew Mexico locust have a specific region of the seed coat
known as the strophiole or lens (Hamly 1932) The strophiole is located on the
cotyledonary lobe of a locust seed (Figure 10 a-c) If seeds are not indiscriminately
damaged the strophiole is the first point of water entry to the embryo (Hopkinson
1997) When a papilionoid seed is percussed for the appropriate amount of time
repeated hits on the integrated seed coat loosen the constrained cells of the strophiolar
region without excessively damaging the rest of the seed coat (Ballard 1976) SEM
photos illuStrate that 4 minutes of percussion loosened and separated strophiolar cells
ofNew Mexico locust seeds when compared with constrained organized strophiolar
cells of untreated seeds (Figure 9) Research-has shown that water passes to the
embryo through the treated strophiole in a controlled manner and regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues
(Kelly and van Staden 1987)
37
10 Strophiole ofunimbibed New Mexico locust seed (a) and following uptake (bc)
38
In contrast hot-water treatments can excessively degrade the seed coat
(Rebman et al 1998 Marunda 1990) Random cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rebman et aI 1998 Marunda 1990) Along with a
proportionately large amount ofun-imbibed (thus under-treated) seeds (Figure l1a)
damage was observed in hot-water treated seeds from six sources during germination
counts Damage included seeds where the radicle failed to elongate (Figure 11 b)
seeds where the radicle elongated but was delayed in freeing itself from the seed coat
(Figure 11c) and seeds where the radicle completely broke off from the embryo
(Figure lId) In addition some seeds germinated in reverse (the cotyledon emerged
fIrst) If the radicle emerged completely from the seed coat they were counted as
germinated
While nearly all seeds treated for 8 minutes ofpercussion imbibed water
damage was evident in x-ray evaluations (Sacramento seed source) and in
germination counts (Holman and Capitan) at this treatment level Seed damage
during germination counts was similar to levels found in hot-water treated seeds
suggesting that over-treatment by both hot water and longer durations of percussion
may result in similar damage to seed structure
The Sacramento and Holman seed sources were two of the fIve sources that
opcrplltlpn in germination from the 4- to 8-minute durations of percussion
the benefIts ofloosening the strophiole as evidenced by imbibition of
all 8-minute treated seeds were outweighed by external and internal seed coat
39
a
b c d
11 Damage to New Mexico locust seeds observed during germination cotmts following hot water scarification 9a Hot water replicate including ~t seedlings and numerous unimbibed seeds 9b Close-up of seed where raallCle failed to elongate 9c Close-up ofseed where radicle elongated but was
-gtIn in freeing itself from the seed coat 9d Close-up ofseed where radicle broke off from embryo
40
damage (which also may be contributing to high imbibition) For the three sources
for which 8 minutes of percussion resulted in maximum germination extended
percussion of seeds necessary to loosen the strophiole may not have excessively
damaged the seed anatomy The Manzano source did not significantly change in
germination from 4 to 8 minutes of percussion suggesting that an increase in
permeable seeds at the 8-minute treatment duration was balanced by the number of
seeds that failed to germinate due to seed damage To summarize the 4-minute
percussion treatment improved germination over 8 minutes ofpercussion for a
majority of sources (five ofnine) the 8-minute percussion treatment improved
germination over 4-minutes ofpercussion for three sources and there was no
difference with the Manzano source Notably for the Raton source 8 minutes of
percussion increased germination by 235 percentage points over 4-minutes of
percussion (7875 vs 5525 respectively) the next best treatment Raton was the
only source to result in germination less than 75 for 4 minutes ofpercussion so a
grower may wish to apply longer percussion durations to lots that do not respond well
to this treatment level
Finally there was an interesting trend in the Jemez and Raton sources seed
that significantly contributed to the source by treatment interaction (Figure 12) A
~u test showed that the Jemez source had a significantly lighter seed weight than
other sources while the Raton source had an average seed weight For the Jemez
the longest duration of boiling increased germination Conversely only short
U4UVll) of percussion were needed to achieve high germination and the longest
41
80 _ Jemez a Raton
iD
~ C iD e iD
Q c 0 ~
c ~ iD 0
20
0------shy
Immersion Boiling 30 sec Boiling 60 sec
Hot Water Scarification
10degTr========~--------~------------------------------
80
60
40
20
_ Jemez Raton
0------shy
1 min 2 min 4 min 8 min
Percussion Scarification
Figure 12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources
42
b
--~----~~~~========~---- duration ofpercussion reduced germination For the Raton source longer durations
ofboiling consistently decreased germination whereas longer percussion treatments
consistently increased germination
A possible explanation is that a thick seed coat is associated with a loose
strophiole structure and that a thin seed coat is associated with a constrained
strophiole This contrast merits further study and may suggest an ecological
adaptation Further investigation ofthis phenomenon may contribute to successful
pre-screening ofunknown seed sources to determine optimal treatment leveL
43
CONCLUSION
Based on results obtained in pilot study (see Appendix) and thesis
experiments 4 minutes ofpercussion is recommended as a standard protocol for
treating an unknown source ofNew Mexico locust seed In addition to laboratory
results the 4-minute percussion treatment has resulted in germination greater than
80 and healthy seedling grow-outs for production purposes (unpublished data) In
laboratory experiments only one hundred seeds per 118 ml (40z) soil tin were
percussed at a time whereas for production we have treated up to five hundred seeds
per tin For large-scale production ofNew Mexico locust seedlings growers may
wish to experiment with larger quantities of seed and larger-size soil tins
Seed storage trials following percussion treatments were not conducted
However germination greater than 80 and healthy seedlings were obtained when
seeds were percussion-treated and subsequently stored at room-temperature (22degC)
for one week (unpublished data) Seed storage trials merit further study as the
standard hot water treatment leaves seeds in a softened condition that is not
conducive to storage
Hydroseeding is an effective method for direct seeding in reclamation
situations Percussion treatment may be conducive to hydro seeding as seeds remain
a dry relatively undamaged yet permeable condition following treatment Sulfuric
seeds also remain dry following treatment but extensive damage to seed coat
poor seedling vigor have been reported with this treatment (Kelly and van Staden
A grow-out study using a hydro seeder on a reclamation site testing percussion
44
against other treatments such as sulfuric acid and mechanical scarification also
merits further study
Papilionoids are the largest subfamily oflegumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust these legumes often play an integral role in the revegetation of
disturbed lands The pilot study (Appendix) indicates that percussion scarification
successfully treats the widely used black locust as well Developing superior
scarification methods to the standard hot water mechanical scarification and acid
treatments may facilitate the use ofpapilionoid legumes in reclamation projects
45
APPENDICES
APPENDIX A
PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR
NEW MEXICO LOCUST AND BLACK LOCUST SEEDS
Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4
Hot water and sulfuric acid soaks are traditional treatments for seeds of many
temperate woody legwnes including locusts However these scarification techniques
often produce inconsistent germination Percussion scarification where seeds are
repeatedly propelled against a hard surface was compared with hot water
scarification to evaluate treatment efficacy for New Mexico locust (Robinia
neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment
seeds were placed in a 98degC water bath which was immediately removed from the
heat source For percussion scarification seeds were placed in a soil sample tin and
agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the
control were followed by 24-hour water soaks Hot water baths resulted in 56 and
41 germination for New Mexico locust and black locust respectively For both
species nearly all durations ofpercussion increased germination over the hot water
treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3
4 and 5 minutes for black locust resulted in at least 90 germination Traditional
scarification treatments randomly degrade the entire seed coat which can lead to
tissue damage during water uptake Percussion scarification specifically weakens the
strophiole the natural source ofwater entry to the seed in papilionoid legwnes
INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom
2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031
48
Following percussion imbibition is controlled through the strophiole and underlying
tissue is protected
Keywords New Mexico locust black locust scarification percussion germination
Introduction
Locust trees (Robinia spp) are aggressive pioneer species that quickly
colonize disturbed land fix atmospheric nitrogen in the soil and stabilize slopes that
are prone to erosion (Klemmedson 1994 Ashby et al 1985) (Figure A1ab)
Recognition of these attributes has contributed to increased demand for locusts in
reclamation projects Locust seeds however exhibit physical dormancy where a
thick seed coat prevents the movement of water and gasses to the embryo (Leadem
1997)
Sulfuric acid or hot water soaks have traditionally been used to break seed
dormancy in macro-propagation of locusts Many growers are moving away from the
use of su1furic acid in treating locust seeds (Dreesen and Harrington 1997) In
addition to being dangerous acid soak durations must be specifically correlated to
seed lot (Olson 1974) Collections of native seed can vary tremendously in seed size
seed weight and hard seededness etc This variability limits the utility of sulfuric
acid soaks as even within a given lot some seeds may be damaged whereas others
are under-treated and fail to imbibe (take up water) A survey of Southwestern
container growers indicates that hot water baths are currently the most common
scarification method for locust seeds (Hine et al 1997) However hot water baths
also produce inconsistent germination (Lin et al 1996)
49
b
Figure AI New Mexico locust a primary invader following disturbance Ala Crown-sprouting at edge ofburn near Los Alamos NM--one year after Cerro Grande fire Alb Colonizing road cut Gila National Forest NM
50
Percussion scarification where seeds are repeatedly propelled against a hard
surface is an alternative dormancy-breaking method that has proven successful in
legumes related to locusts (Hamly 1932 Barton 1947) We compared this alternative
treatment to hot water scarification to evaluate treatment efficacy for New Mexico
locust and black locust seeds
Materials and Methods
Commercial New Mexico locust seeds (Western Native Seed Coaldale CO)
were collected fall 2000 in Huerfano County CO Black locust seeds were collected
September 2000 from Taos County NM by harvesting ripe pods from several trees at
a distance of up to 3 m from ground level Black locust seeds were allowed to air-dry
for 3 weeks and were threshed and separated from large chaffusing a greenhouse
fan A South Dakota Seed Blower (Chicago Illinltois) was used to remove fine chaff
from both seed lots Seeds were storedmiddotat 2-4C until the germination study was
conducted in March 2001
This study compared hot water scarification with percussion scarification
using treatment and species as experimental factors Along with a control
scarification treatments included immersion of seed in boiling water as well as
percussion durations of 1 2 3 45 or 10 minutes Four 100-seed samples were used
to test each species by scarification treatment combination
For the hot water treatment test tubes were filled with 30 mL water and
placed in a water-filled beaker (Figure A2a) The bath was raised to boiling (-98C at
51
b
c
Figure A2 Implementation ofhot water and percussion scarification A2a Hot water bath A2b Paint-shaker set-up A2c Placing soil tin containing seeds at end ofpaint shaker ann
52
Las Cruces NM elevation of 1300 meters) Each 100-seed replicate was placed in a
test tube and each test tube was immediately removed to cool at room temperature
Percussion scarification was implemented using a pneumatic paint shaker
(Central Pneumatic model 00422 Camarillo CA) (Figure A2b) This allowed
standardization of treatment intensity Compressed air pressure was maintained at
530kPa plusmn 30kPa (80psi plusmn 5psi) resulting in approximately 350 oscillations per
minute Each lOO-seed sample was placed in a 118 ml (4 oz) soil tin for shaking To
maximize lateral movement of paint shaker a paint can was used as a spacer to place
soil tins at the end of the shaking arm (Figure A2c) This allowed shaking to occur at
the greatest distance from the pivot point (fulcrum) Following all treatments
including the control seeds were soaked for twenty-four hours in distilled water prior
to plating out
Gennination was evaluated on lab benches at room temperature Each
replicate of seeds was placed in a 100 cm petri dish on moistened filter paper with
humidity maintained by enclosing the petri dishes in ziplock bags Gennination was
monitored daily for 14 days in accordance with International Seed Testing
Association standards for Robinia species (ISTA 1999) Gennination was defined as
protrusion of the radicle from the embryo by at least 1mm as viewed by the naked
eye
Categorical Analysis of Variance using Prpc Catmod (SAS Institute 1989)
was used to detennine treatment differences in germination percentages for each
source This procedure is an extension of a Chi-square test of homogeneity using the
53
natural log of the ratio of germinated to non-germinated seeds for each treatment Pshy
values less than 005 were considered significant A limited set of pairwiseshy
comparisons was conducted to compare treatment mean percents using a
conservative alpha value of 005 divided by the number of comparisons
Results
Percussion maximized germination for both New Mexico locust and black
locust There were three germination response levels for New Mexico locust (Figure
A3) First there was a low response in the control indicating a dormant seed lot
The hot water treatment and I-minute duration of percussion share a second
intermediate response Germination was highest at percussion levels from 2 to 10
minutes
A selected percussion treatment intermediate in time (5 minutes) produced the
steepest germination curve indicating the fastest germination rate (Figure A4) The
hot water treatment produced an intermediate curve or relatively slower germination
rate The control curve was very flat indicating the slowest germination rate for New
Mexico locust
Control seeds germinated poorly for black locust as well (Figure A5) Hot
water scarification improved germination over the control but all levels of percussion
improved germination over hot water The optimal durations of percussion treatment
were bracketed with a significant increase from the 1 to 2 minute percussion level a
high germination response from 2 to 5 minutes of percussion and finally a significant
54
~ d 25 CL)
p t1 0 ~ t1
g Vl ~ Vl
100~---------------------------------------------------------------
80
60
40
20
o Control Hot Water Percussion
I min Percussion
2 min Percussion
3 min
Scarification
Percussion 4 min
Figure A3 Effect of scarification on germination response for New Mexico locust
Percussion 5 min
=significant difference between adjacent treatment bars ns = no significant difference
Percussion 10 min
~ ~ () u agt
Il-t d o ~
~ ~ c3
100middotlr~~~------------------------------------e- Percussion 5min middot 0middot Hot Water -- Control
80-J=middot======
6b-middot-lmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middot--middotmiddot-middot~-middotmiddot -middotmiddot middot middotmiddot-middot 1
40
20
-----~-----------
0 0 0 middotmiddotmiddotmiddotmiddot0middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot0
0 --~-----y-----~------_---~--- T-----~
o shy -TDay 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A4 Effect of selected scarification treatments on germination speed for New Mexico locust First seven days of fourteen-day experiment are shown
Day 7
80
~ cd
5 60 ()
[) Cl d 0 1t1 d
40sect dUl
-J
20
o
ns
Scarification
Figure A5 Effect of scarification on germination response for black locust = significant difference between adjacent treatment bars ns = no significant difference
decrease at the 10-minute level This drop-off suggests that 10 minutes of percussion
over-treats seeds from this lot of-black locust
A selected percussion treatment intermediate in time (5 minutes) also had the
fastest germination rate for black locust (Figure A6)_ The hot water curve is
somewhat flatter indicating a slower germination rate The control curve is very flat
indicating a slow germination rate
A qualitative difference between hot water and percussion-treated seeds was
also observed Along with healthy germinants the hot water treatment produced
over-treated seeds (Figure A 7a) Examples of damaged germinants include seeds
where the radicle did not elongate (Figure A 7b) seeds where the radicle elongated
but was delayed in freeing itself from the seed coat (Figure A7c) and seeds where the
radicle broke off completely from the embryo (Figure A7d) In addition to these
examples of over-treatment there were numerous small dark seeds that failed to
imbibe water For percussion treatment nearly all seeds took up water (Figure A8a)
Germination was rapid and uniform with little damage evident (Figure A8b)
To summarize both New Mexico locust and black locust seed lots were
dormant The standard hot water scarification treatment improved germination but
only to 56 and 41 for New Mexico locust and black locust respectively Nearly
all percussion durations improved total germination as well as germination rate over
the hot water treatment Germination was greater than 90 for percussion durations
of 4 5 and 10 minutes for New Mexico locust and 3 4 and 5 minutes for black locust
58
CI)
sgt t CI) 0
igt 0
sect ttl t E
Vl 0
c3
100r==~==~~=r------------------------------------~ ___ Percussion 5 min
middotmiddotmiddot0middotmiddotmiddot Hot Water -lIf- Control
80r=========~--~~----------------------------~
60
40
20 -~-~---~---------- ---~------------I
omiddot
O
_~_----------lIf-----~bull -----_ shy---shy I
Day 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A6 Effect of selected scarification treatments on germination speed for black locust First seven days of fourteen-day experiment are shown
Day 7
a
b c d
Figure A7 Damage to New Mexico locust seeds observed during germination counts following hot water scarification A7a Hot water replicate including damaged seedlings and numerous unimbibed seeds A7b Close-up ofseed where radicle failed to elongate A7c Close-up ofseed where radicle elongated but was delayed in freeing itself from the seed coat A7d Close-up ofseed where radicle completely broke offfrom embryo
60
a
b
Figure A8 New Mexico locust seeds following percussion treatment at time of germination counts A8a Percussion replicate with uniformly healthy seedlings and near complete imbibition of seeds A8b Close-up ofpercussion-treated seedling
61
Discussion
Papilionoid seeds have a specific anatomical feature known as the strophiole
or lens-the natural site of water entry to the seed (Hamly 1932) The strophiole is
located on the cotyledonary lobe of a locust seed (Figure A9) When a papilionoid
legume seed is percussed for the appropriate amount of time repeated hits on the
integrated seed coat loosen the constrained cells of the strophiolar region without
excessively damaging the rest of the seed coat (Ballard 1976)
When a percussed seed is soaked water enters exclusively through the
strophiole in a controlled manner (Kelly and Staden 1987) This regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues This
contrasts with seeds that have been hot water or acid scarified treatments that can
randomly degrade the seed coat Unlocalized cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rehman et al 1998 Marunda 1990)
Papilionoids are the largest subfamily of legumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust and black locust these legumes often play an integral role in the
revegetation ofdisturbed lands Developing superior scarification methods to the
standard hot water and acid treatments should fadlitate the use ofthese legumes in
restoration projects
62
Figure A9 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (bc)
63
APPENDIXB
GERMINATION PERCENTS AND STANDARD ERRORS FOR SOURCE
BLOCK AND TREATMENT FOR NEW MEXICO LOCUST
Table B 1 Gennination percents and standard errors for source block and treatment for New Mexico locust
Source Block Treatment Germination Percentage Standard Error
Capitan 1 Immersion 74 437 Capitan 2 Immersion 69 462 Capitan 3 Immersion 71 454 Capitan 4 Immersion 67 470 Capitan Boiling 30 sec 79 407 Capitan 2 Boiling 30 sec 76 427 Capitan 3 Boiling 30 sec 72 449 Capitan 4 Boiling 30 sec 76 427 Capitan Boiling 60 sec 71 454 Capitan 2 Boiling 60 sec 54 498 Capitan 3 Boiling 60 sec 67 470 Capitan 4 Boiling 60 sec 76 427 Capitan I Control 5 218 Capitan 2 Control 7 255 Capitan 3 Control 4 196 Capitan 4 Control 5 218 Capitan 1 Percussion I min 47 499 Capitan 2 Percussion I min
470
Capitan Percussion I min 39 488
Capitan 4 Percussion I min 38 485 Capitan I Percussion 2 min 55 497 Capitan Capitan
2
Percussion 2 min Percussion 2 min
71 65
454 477
Capitan 4 Percussion 2 min 56 496 Capitan I Percussion 4 min 86 347 Capitan 2 Percussion 4 min 79 407 Capitan 3 Percussion 4 min 81 392 Capitan 4 Percussion 4 min 82 384 Capitan I Percussion 8 min 90 300 Capitan 2 Percussion 8 min 88 325 Capitan 3 Percussion 8 min 90 300 Capitan 4 Percussion 8 min 90 300 Gila I Immersion 71 454 Gila 2 Immersion 77 421 Gila
Immersion 75 433
Gila 4 Immersion 72 449
Gila I Boiling 30 sec 68 466
Gila 2 Boiling 30 sec 81 392
Gila 3 Boiling 30 sec 54 498
Gila 4 Boiling 30 sec 63 483
Gila I Boiling 60 sec 55 497
Gila 2 Boiling 60 sec 68 466
Gila 3 Boiling 60 sec 57 495
Gila 4 Boiling 60 sec 70 458
65
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Gila 2 Control 13 336 Gila 3 Control 13 336 Gila 4 Control 20 400 Gila 1 Percussion 1 min 66 474 Gila 2 Percussion 1 min 57 495 Gila 3 Percussion 1 min 73 444 Gila 4 Percussion 1 min 82 384 Gila 1 Percussion 2 min 81 392 Gila 2 Percussion 2 min 86 347 Gila 3 Percussion 2 min 85 357 Gila 4 Percussion 2 min 54 498 Gila 1 Percussion 4 min 79 407 Gila 2 Percussion 4 min 83 376 Gila 3 Percussion 4 min 84 367 Gila 4 Percussion 4 min 79 407 Gila 1 Percussion 8 min 51 500 Gila 2 Percussion 8 min 76 427 Gila 3 Percussion 8 min 52 500 Gila 4 Percussion 8 min 74 439 Holman 1 Immersion 83 376 Holman 2 Immersion 86 347 Holman 3 Immersion 81 392 Holman 4 Immersion 90 300 Holman 1 Boiling 30 sec 90 300 Holman 2 Boiling 30 sec 85 357 Holman 3 Boiling 30 sec 87 336 Holman 4 Boiling 30 sec 83 376 Holman 1 Boiling 60 sec 79 407 Holman 2 Boiling 60 sec 80 400 Holman 3 Boiling 60 sec 86 347 Holman 4 Boiling 60 sec 93 255 Holman Control 25 433 Holman 2 Control 19 392 Holman Control 21 407
Holman 4 Control 24 427
Holman Percussion 1 min 77 421
Holman 2 Percussion 1 min 85 357
Holman 3 Percussion 1 min 80 400
Holman 4 Percussion 1 min 89 313
Holman 1 Percussion 2 min 88 325
Holman 2 Percussion 2 min 96 196
Holman 3 Percussion 2 min 93 255
Holman 4 Percussion 2 min 96 196
Holman 1 Percussion 4 min 92 271
Holman 2 Percussion 4 min 97 171
66
I
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Holman 3 Percussion 4 min 91 286 Holman 4 Percussion 4 min 96 196 Holman 1 Percussion 8 min 88 325 Holman 2 Percussion 8 min 85 357 Holman 3 Percussion 8 min 83 376 Holman 4 Percussion 8 min 82 384 Jemez 1 Immersion 53 499 Jemez 2 Immersion 44 496 Jemez 3 Immersion 52 500 Jemez 4 Immersion 70 458 Jemez 1 Boiling 30 sec 58 494 Jemez 2 Boiling 30 sec 55 497 Jemez 3 Boiling 30 sec 48 500 Jemez 4 Boiling 30 sec 65 477 Jemez 1 Boiling 60 sec 68 466 Jemez 2 Boiling 60 sec 63 483 Jemez 3 Boiling 60 sec 73 444 Jemez 4 Boiling 60 sec 89 313 Jemez 1 Control 2 140 Jemez 2 Control 9 286 Jemez 3 Control 6 237 Jemez 4 Control 8 271 Jemez 1 Percussion 1 min 58 494 Jemez 2 Percussion 1 min 63 483 Jemez 3 Percussion 1 min 77 421 Jemez 4 Percussion 1 min 89 313 Jemez 1 Percussion 2 min 68 466 Jemez 2 Percussion 2 min 64 480 Jemez 3 Percussion 2 min 80 400 Jemez 4 Percussion 2 min 83 376 Jemez Percussion 4 min 68 466 Jemez 2 Percussion 4 min 79 407 Jemez 3 Percussion 4 min 70 458 Jemez 4 Percussion 4 min 83 376 Jemez 1 Percussion 8 min 52 500 Jemez 2 Percussion 8 min 59 492 Jemez 3 Percussion 8 min 61 488 Jemez 4 Percussion 8 min 70 458 Magdalena 1 Immersion 69 462 Magdalena 2 Immersion 69 462 Magdalena J Immersion 70 458 Magdalena 4 Immersion 85 357 Magdalena 1 Boiling 30 sec 65 477 Magdalena 2 Boiling 30 sec 71 454 Magdalena 3 Boiling 30 sec 68 466
67
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Magdalena 4 Boiling 30 sec 83 376 Magdalena 1 Boiling 60 sec 63 483 Magdalena 2 Boiling 60 sec 68 466 Magdalena 3 Boiling 60 sec 63 483 Magdalena 4 Boiling 60 sec 82 384 Magdalena 1 Control 11 313 Magdalena 2 Control 6 237 Magdalena 3 Control 5 218 Magdalena 4 Control 4 196 Magdalena Percussion 1 min 32 466 Magdalena 2 Percussion 1 min 45 497 Magdalena 3 Percussion 1 min 46 498 Magdalena 4 Percussion I min 36 480 Magdalena 1 Percussion 2 min 67 470 Magdalena 2 Percussion 2 min 68 466 Magdalena 3 Percussion 2 min 65 477 Magdalena 4 Percussion 2 min 81 392 Magdalena 1 Percussion 4 min 87 336 Magdalena 2 Percussion 4 min 97 171 Magdalena 3 Percussion 4 min 83 376 Magdalena 4 Percussion 4 min 97 171 Magdalena 1 Percussion 8 min 98 lAO Magdalena 2 Percussion 8 min 100 000 Magdalena 3 Percussion 8 min 96 196 Magdalena 4 Percussion 8 min 98 lAO Manzano 1 Immersion 67 470 Manzano 2 Immersion 57 495 Manzano 3 Immersion 72 4049 Manzano 4 Immersion 73 4044 Manzano 1 Boiling 30 sec 70 458 Manzano 2 Boiling 30 sec 64 480 Manzano 3 Boiling 30 sec 71 454 Manzano 4 Boiling 30 sec 80 400 Manzano I Boiling 60 sec 50 500 Manzano 2 Boiling 60 sec 53 499 Manzano 3 Boiling 60 sec 59 492 Manzano 4 Boiling 60 sec 74 439 Manzano I Control 4 196 Manzano 2 Control 8 271 Manzano 3 Control 1 099 Manzano 4 Control 6 237 Manzano 1 Percussion 1 min 52 500 Manzano 2 Percussion 1 min 39 488 Manzano 3 Percussion 1 min 36 480 Manzano 4 Percussion 1 min 51 500
68
Table B 1 Continued
Source Block Treatment Gerrni~ation Percentage Standard Error
Manzano Percussion 2 min 53 499 Manzano 2 Percussion 2 min 58 494 Manzano 3 Percussion 2 min 61 488 Manzano 4 Percussion 2 min 62 485 Manzano 1 Percussion 4 min 76 427 Manzano 2 Percussion 4 min 85 357 Manzano 3 Percussion 4 min 79 407 Manzano 4 Percussion 4 min 83 376 Manzano 1 Percussion 8 min 87 336 Manzano 2 Percussion 8 min 83 376 Manzano 3 Percussion 8 min 83 376 Manzano 4 Percussion 8 min 75 433 Raton 1 Immersion 54 498 Raton 2 Immersion 37 483 Raton 3 Immersion 39 488 Raton 4 Immersion 69 462 Raton 1 Boiling 30 sec 36 480 Raton 2 Boiling 30 sec 20 400 Raton 3 Boiling 30 sec 28 449 Raton 4 Boiling 30 sec 60 490 Raton 1 Boiling 60 sec 15 357 Raton 2 Boiling 60 sec 171 Raton 3 Boiling 60 sec 4 196 Raton 4 Boiling 60 sec 35 477 Raton 1 Control 4 196 Raton 2 Control 12 325 Raton 3 Control 2 140 Raton 4 Control 6 237 Raton 1 Percussion 1 min 52 500 Raton 2 Percussion I min 29 454 Raton 3 Percussion I min 35 477 Raton 4 Percussion 1 min 31 462 Raton 1 Percussion 2 min 55 497 Raton 2 Percussion 2 min 35 477 Raton 3 Percussion 2 min 33 470 Raton 4 Percussion 2 min 42 494 Raton 1 Percussion 4 min 65 477 Raton 2 Percussion 4 min 52 500 Raton Percussion 4 min 51 500 Raton 4 Percussion 4 min 53 499 Raton 1 Percussion 8 min 88 325 Raton 2 Percussion 8 min 80 400 Raton 3 Percussion 8 min 73 444 Raton 4 Percussion 8 min 74 439 Sacramento 1 Immersion 43 495
69
Table B 1 Continued
-Source Block Treatment Germination Percentage Standard Error
Sacramento 2 Immersion 30 458 Sacramento 3 Immersion 38 485 Sacramento 4 Immersion 56 496 Sacramento 1 Boiling 30 sec 47 499 Sacramento 2 Boiling 30 sec 48 500 Sacramento 3 Boiling 30 sec 48 500 Sacramento 4 Boiling 30 sec 56 496 Sacramento 1 Boiling 60 sec 52 500 Sacramento 2 Boiling 60 sec 67 470 Sacramento 3 Boiling 60 sec 43 495 Sacramento 4 Boiling 60 sec 48 500 Sacramento 1 Control 0 000 Sacramento 2 Control 3 171 Sacramento 3 Control 3 171 Sacramento 4 Control 099 Sacramento 1 Percussion 1 min 57 495 Sacramento 2 Percussion 1 min 61 488 Sacramento 3 Percussion 1 min 62 485 Sacramento 4 Percussion 1 min 55 497 Sacramento 1 Percussion 2 min 84 367 Sacramento 2 Percussion 2 min 70 458 Sacramento 3 Percussion 2 min 81 392 Sacramento 4 Percussion 2 min 69 462 Sacramento 1 Percussion 4 min 91 286 Sacramento 2 Percussion 4 min 84 367 Sacramento 3 Percussion 4 min 84 367 Sacramento 4 Percussion 4 min 94 237 Sacramento 1 Percussion 8 min 82 384 Sacramento 2 Percussion 8 min 81 392 Sacramento 3 Percussion 8 min 82 384 Sacramento 4 Percussion 8 min 81 392 Sandia 1 Immersion 76 427 Sandia 2 Immersion 70 458 Sandia 3 Immersion 70 458 Sandia 4 Immersion 81 392 Sandia 1 Boiling 30 sec 78 414 Sandia 2 Boiling 30 sec 71 454 Sandia 3 Boiling 30 sec 63 483 Sandia 4 Boiling 30 sec 78 414 Sandia 1 Boiling 60 sec 67 470 Sandia 2 Boiling 60 sec 65 477 Sandia 3 Boiling 60 sec 74 439 Sandia 4 Boiling 60 sec 66 474 Sandia 1 Control 16 367 Sandia 2 Control 7 255
70
Table B 1 Continued
Source Block Treatment Gennination Percentage Standard Error
Sandia 3 Control 8 271 Sandia 4 Control 16 367 Sandia 1 Percussion 1 min 67 470 Sandia 2 Percussion 1 min 61 488 Sandia 3 Percussion 1 min 66 474 Sandia 4 Percussion 1 min 59 492 Sandia I Percussion 2 min 82 384 Sandia 2 Percussion 2 min 83 376 Sandia 3 Percussion 2 min 81 392 Sandia 4 Percussion 2 min 84 367 Sandia 1 Percussion 4 min 89 313 Sandia 2 Percussion 4 min 86 347 Sandia 3 Percussion 4 min 89 313 Sandia 4 Percussion 4 min 85 357 Sandia 1 Percussion 8 min 80 400 Sandia 2 Percussion 8 min 77 421 Sandia 3 Percussion 8 min 69 462 Sandia 4 Percussion 8 min 66 474
LITERATURE CITED
Ashby WC Vogel WG Rogers NF 1985 Black locust in the reclamation equation General Technical Report Northeastern Forest Experiment Station USDA Forest Service NE-I05 12 p
Ballard LAT 1973 Physical barriers to gennination Seed Sci Technoll 285-303
___ 1976 Strophiolar water conduction in the seeds ofTrifolieae induced by action on the testa at nonstrophiolar sites Austr J Plant Physiol3 465-469
Barton LV 1947 Special studies on seed coat impenneability Contrib Boyce Thompson Inst 14 355-362
Baskin CC Baskin JM 1998 Seeds Ecology biogeography and evolution of donnancy and gennination Academic Press London UK 666 p
Bewley JD 1997 Seed germination and donnancy Plant Ce119 1055-1066
Bewley JD Black M 1994 Seeds physiology of development and germination 2nd ed Plenum Press New York NY 445 p
Briscoe CB 1996 Growing Nitrogen Fixing Trees in Acid Soils In MH Powell (ed) Nitrogen Fixing Trees for Acid Soils- A Field ManuaL Winrock International Morrilton AR 22 p
Copeland LO McDonald MB 1995 Principles of Seed Science and Technology 3rded Chapman amp Hall New York NY 409 p
Cox RE Klett JE 1984 Seed gennination requirements of native Colorado plants for use in the landscape Plant Propag 30(2) 6-10
Dell B 1980 Structure and function ofthe strophiolar plug in seeds ofAlbizia Lophantha Am J Bot 67 556-563
Dick-Peddie WA 1993 New Mexico Vegetation Past Present and Future University ofNew Mexico Press Albuquerque NM 244 p
Dreesen DR 2001 Propagation protocol for production ofcontainer Robinia neomexicana plants Los Lunas Plant Materials Center Los Lunas New Mexico In Native Plant Network URL httpwwwnativeplantnetworkorg (accessed 9 January 2002) Moscow (ID) University ofIdaho CoIl Natural Resources For Res Nursery
72
Dreesen DR Harrington JT 1997 Propagation of native plants for restoration projects in the Southwestern US- Preliminary Investigations In Landis TD Thompson JR tech coords National Proceedings Forest and Conservation Nursery Assocs Gen Tech Rep PNW-GTR-419 Portland OR USDA For Ser PNW Res Sta 77-88
Gottfried GJ 1980 Control ofNew Mexican locust and the effect on planted ponderosa pine in central Arizona US For Ser Res Note RM-386 Rocky Mountain For Range ExpStn Fort Collins CO
Gosling PG Samuel YK Jones SK 1995 A systematic examination of germination temperature chipping and water temperaturesoak duration pretreatments on the seeds ofLeucaena leucocephala Seed Sci amp Technol 23521-532
Hamly DH 1932 Softening of the seeds of Melilotus alba Bot Gazette 93 345shy375
Hanna PJ 1984 Anatomical features of the seed coat ofAcacia kempeana (Mueller) which relate to increased germination rate induced by heat treatment New Phytol 96 23-29
Hanks JP Dick-Peddie WA 1974 Vegetation patterns of the White Mountains New Mexico Southwest Naturalist 18(4) 371-382
Hine S Harrington JT Dreesen DR 1997 Seed scarification requirements for Robinia neomexicana In Harrington JTtech coord Proc Third SouthwestemContainer Growers Conf Feb 12-13 1997 Ft Collins CO
Hopkinson JM 1993 The strophiole of leguminous seeds Newsletter- IntI Herbage Seed Production Res Grp 18 7-8
---- 1997 Differences in hardseededness between Cunningham and Tarramba leucaena In Leucnet News The Newsletter of the International LeucaenaRes and Devlt Netwk No4
ISTA (International Seed Testing Association) 1999 International rules for seed testing rules Seed Sci amp Technol27 Supplement
Kelly KM van Staden J 1987 The lens as the site of permeability in the papilionoid seed Aspalathus linearis J Plant Physiol 128(45) 395-404
73
Keresztesi B 1988 Black locust the tree of agriculture Outlook on Agr 17(2) 77~85
Kigel 1 and Galili G 1995 Seed development and germination Marcel Dekker New York 853 p
Klemmedson 10 1994 New Mexico locust and parent material Influence on macronutrients of forest floor and soil Soil Sci Soc Am 1 58 974~980
Kuo WH Tam AW 1988 The pathway of water absorption of mung bean seeds Seed Sci amp Technol16(1) 139~144
Leadem CL 1997 Dormancy--unlocking seed secrets In Landis TD Thompson lR tech coords National Proceedings Forestand Conservation Nursery Assocs Gen Tech Rep PNW-G TR-419 Portland OR USDA For Ser PNW Res Sta 43-52
Lin Y Wagner MR Seweyestewa A Honahni MD DeGomez M McMillin JD 1996 Influence of cultural treatments on seed germination and seedling growth of Robinia neomexicana Forest Farm and Community Tree Res Reports Volume 1
Marunda CT 1990 Effects of seed pretreatment on the development ofAcacia auriculforinis and A holosericea seedlings ACIAR Proceedings Series No 28
Meginnis HG 1937 Sulphuric acid treatment to increase germination of black locust seed USDA Circ 453 34 p
Miklas PN Towensend CE Ladd SL 1987 Seedcoat anatomy and the scarification ofcicer milkvetch seed Crop Sci 27 766-772
Monsen SB 1984 Use of shrubs on mine spoils In The Challenge ofProducing Native Plants for the Intermountain Area Proceedings Intermountain Nurserymans Association 1983 conference Las Vegas NV compo PM Murphy 26-31 Ogden UT USDA For Ser Gen Tech Report INT-l68
Olson DF 1974 Robinia L Locust In Seeds of Woody Plants in the US tech coord CS Schopmeyer 728-731 USDA For Ser Agr Handbk No450
Rehman S Loescher RNl Harris P1C 1998 Dormancy breaking and germination ofAcacia salicina Lindl seeds Seed Sci amp Techno127 553-557
Rolston MP 1978 Water impermeable seed dormancy Bot Rev 44 365-396
74
SAS Institute Inc 1989 SASSTAT Users Guide Version 6 4th Edition Volume 1 Cary NC SAS Institute Inc 943 p
SerratoValenti G Conara L Ferrando M Modenesi P 1993 Structural and histochemical features ofStylosanthes scabra (Leguminosae Papilionoidae) seed coat as related to water entry Can J Bot 71 834-840
Simpson BJ 1988 A field guide to Texas trees Austin TX Texas Monthly Press 372p
Tran VN Cavanagh AK 1984 Structural aspects ofdormancy In Seed Physiology Vol 2 Murray DR (ed) p1-44 Academic Press Australia
USDA Forest Service 1948 Woody-plant seed manual USDA Misc Pub1654 416p
Vines RA 1960 Trees Shrubs and Woody Vines of the Southwest Austin Univ ofTexas Press 1104 p
Wagner MR Larson RE Craig TP 1992 Ecology and silvics ofNew Mexico locust (Robinia neomexicana) with an emphasis on the arboreal form p53-57 In JW Hanover K Miller and S Plesko (eds) Proc Intemat Confon Black Locust Biology Culture and Utilization Mich State Univ USA
Wilson JK 1944 Immersing seeds of species of Robinia in boiling water hastens germination J For 42 453-454
Zimmerman RW Carpenter SB 1980 First-year coppice production from a 5shyyear old black locust stand on surface mine spoil p309-314 Centr Hardwd For ConfProc University ofMissouri Columbia
Figure 7 X-ray radiographs ofSacramento seed source percussed for 1 4 or 8 minutes
33
Figure 8a-d Seed damage close-ups Figure a shows undamaged seed percussed for 4 minutes Figures b-d show seeds percussed for 8 minutes Damage includes fracturing ofradicle in b complete removal ofseed coat in c and pitting ofseed coat in d
34
Untreated Treated
Figure 9 SEM images ofuntreated vs 4-minute percussion-treated strophiolar cells ofNew Mexico locust seeds Treated cells are loosened separated and raised
DISCUSSION
Determining the Optimal Treatment
Averaging results of scarification treatmems across seed sources is a useful
tool to predict which treatment is most likely to be successful for an unknown seed
source Averaged over all sources four- and eight-minute percussion treatments
resulted in the highest germination 815 and 790 respectively and produced
germination that was as fast or faster than all other treatments Although there was no
difference between these two treatments on average an argument for 4 minutes of
percussion as a standard protocol for treating New Mexico locust seeds can be made
Descriptively 4 minutes of percussion improved germination over 8 minutes
ofpercussion for a majority of sources (five out of nine) 8 minutes of percussion
improved germination for three ofnine sources with no difference for a fmal source
For individual sources 4 minutes ofpercussion was always the best or secondmiddot best
treatment and was always equal to or better than all hot water treatments
Germination at 8 minutes ofpercussion was lower than all other percussion durations
for the Jemez and Gila sources and was lower than at least one hot water treatment for
these sources as well
The x-ray radiographs of the Sacramento seed source illustrate the excessive
internal and external seed damage that can occur with 8 minutes of percussion
germination counts 8-minute percussed seeds from Holman and Capitan
ClUll signs of damage that were visible to the naked eye (see below)
r~evertheless some of these seeds still fit the criteria for germination Neither x-ray
36
photography nor visible observations during seed counts revealed seed damage at the
4-minute percussion level In operational trials seeds percussed for 4 minutes
developed into healthy seedlings (Khadduri and Harrington unpublished data)
Strophiole and Seed Coat Behavior
Four minutes ofpercussion successfully breaks seed dormancy in a large
percentage ofNew Mexico locust seeds without excessively damaging the
surrounding seed coat or internal seed coat structures Most papilionoid legume
seeds including seeds ofNew Mexico locust have a specific region of the seed coat
known as the strophiole or lens (Hamly 1932) The strophiole is located on the
cotyledonary lobe of a locust seed (Figure 10 a-c) If seeds are not indiscriminately
damaged the strophiole is the first point of water entry to the embryo (Hopkinson
1997) When a papilionoid seed is percussed for the appropriate amount of time
repeated hits on the integrated seed coat loosen the constrained cells of the strophiolar
region without excessively damaging the rest of the seed coat (Ballard 1976) SEM
photos illuStrate that 4 minutes of percussion loosened and separated strophiolar cells
ofNew Mexico locust seeds when compared with constrained organized strophiolar
cells of untreated seeds (Figure 9) Research-has shown that water passes to the
embryo through the treated strophiole in a controlled manner and regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues
(Kelly and van Staden 1987)
37
10 Strophiole ofunimbibed New Mexico locust seed (a) and following uptake (bc)
38
In contrast hot-water treatments can excessively degrade the seed coat
(Rebman et al 1998 Marunda 1990) Random cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rebman et aI 1998 Marunda 1990) Along with a
proportionately large amount ofun-imbibed (thus under-treated) seeds (Figure l1a)
damage was observed in hot-water treated seeds from six sources during germination
counts Damage included seeds where the radicle failed to elongate (Figure 11 b)
seeds where the radicle elongated but was delayed in freeing itself from the seed coat
(Figure 11c) and seeds where the radicle completely broke off from the embryo
(Figure lId) In addition some seeds germinated in reverse (the cotyledon emerged
fIrst) If the radicle emerged completely from the seed coat they were counted as
germinated
While nearly all seeds treated for 8 minutes ofpercussion imbibed water
damage was evident in x-ray evaluations (Sacramento seed source) and in
germination counts (Holman and Capitan) at this treatment level Seed damage
during germination counts was similar to levels found in hot-water treated seeds
suggesting that over-treatment by both hot water and longer durations of percussion
may result in similar damage to seed structure
The Sacramento and Holman seed sources were two of the fIve sources that
opcrplltlpn in germination from the 4- to 8-minute durations of percussion
the benefIts ofloosening the strophiole as evidenced by imbibition of
all 8-minute treated seeds were outweighed by external and internal seed coat
39
a
b c d
11 Damage to New Mexico locust seeds observed during germination cotmts following hot water scarification 9a Hot water replicate including ~t seedlings and numerous unimbibed seeds 9b Close-up of seed where raallCle failed to elongate 9c Close-up ofseed where radicle elongated but was
-gtIn in freeing itself from the seed coat 9d Close-up ofseed where radicle broke off from embryo
40
damage (which also may be contributing to high imbibition) For the three sources
for which 8 minutes of percussion resulted in maximum germination extended
percussion of seeds necessary to loosen the strophiole may not have excessively
damaged the seed anatomy The Manzano source did not significantly change in
germination from 4 to 8 minutes of percussion suggesting that an increase in
permeable seeds at the 8-minute treatment duration was balanced by the number of
seeds that failed to germinate due to seed damage To summarize the 4-minute
percussion treatment improved germination over 8 minutes ofpercussion for a
majority of sources (five ofnine) the 8-minute percussion treatment improved
germination over 4-minutes ofpercussion for three sources and there was no
difference with the Manzano source Notably for the Raton source 8 minutes of
percussion increased germination by 235 percentage points over 4-minutes of
percussion (7875 vs 5525 respectively) the next best treatment Raton was the
only source to result in germination less than 75 for 4 minutes ofpercussion so a
grower may wish to apply longer percussion durations to lots that do not respond well
to this treatment level
Finally there was an interesting trend in the Jemez and Raton sources seed
that significantly contributed to the source by treatment interaction (Figure 12) A
~u test showed that the Jemez source had a significantly lighter seed weight than
other sources while the Raton source had an average seed weight For the Jemez
the longest duration of boiling increased germination Conversely only short
U4UVll) of percussion were needed to achieve high germination and the longest
41
80 _ Jemez a Raton
iD
~ C iD e iD
Q c 0 ~
c ~ iD 0
20
0------shy
Immersion Boiling 30 sec Boiling 60 sec
Hot Water Scarification
10degTr========~--------~------------------------------
80
60
40
20
_ Jemez Raton
0------shy
1 min 2 min 4 min 8 min
Percussion Scarification
Figure 12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources
42
b
--~----~~~~========~---- duration ofpercussion reduced germination For the Raton source longer durations
ofboiling consistently decreased germination whereas longer percussion treatments
consistently increased germination
A possible explanation is that a thick seed coat is associated with a loose
strophiole structure and that a thin seed coat is associated with a constrained
strophiole This contrast merits further study and may suggest an ecological
adaptation Further investigation ofthis phenomenon may contribute to successful
pre-screening ofunknown seed sources to determine optimal treatment leveL
43
CONCLUSION
Based on results obtained in pilot study (see Appendix) and thesis
experiments 4 minutes ofpercussion is recommended as a standard protocol for
treating an unknown source ofNew Mexico locust seed In addition to laboratory
results the 4-minute percussion treatment has resulted in germination greater than
80 and healthy seedling grow-outs for production purposes (unpublished data) In
laboratory experiments only one hundred seeds per 118 ml (40z) soil tin were
percussed at a time whereas for production we have treated up to five hundred seeds
per tin For large-scale production ofNew Mexico locust seedlings growers may
wish to experiment with larger quantities of seed and larger-size soil tins
Seed storage trials following percussion treatments were not conducted
However germination greater than 80 and healthy seedlings were obtained when
seeds were percussion-treated and subsequently stored at room-temperature (22degC)
for one week (unpublished data) Seed storage trials merit further study as the
standard hot water treatment leaves seeds in a softened condition that is not
conducive to storage
Hydroseeding is an effective method for direct seeding in reclamation
situations Percussion treatment may be conducive to hydro seeding as seeds remain
a dry relatively undamaged yet permeable condition following treatment Sulfuric
seeds also remain dry following treatment but extensive damage to seed coat
poor seedling vigor have been reported with this treatment (Kelly and van Staden
A grow-out study using a hydro seeder on a reclamation site testing percussion
44
against other treatments such as sulfuric acid and mechanical scarification also
merits further study
Papilionoids are the largest subfamily oflegumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust these legumes often play an integral role in the revegetation of
disturbed lands The pilot study (Appendix) indicates that percussion scarification
successfully treats the widely used black locust as well Developing superior
scarification methods to the standard hot water mechanical scarification and acid
treatments may facilitate the use ofpapilionoid legumes in reclamation projects
45
APPENDICES
APPENDIX A
PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR
NEW MEXICO LOCUST AND BLACK LOCUST SEEDS
Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4
Hot water and sulfuric acid soaks are traditional treatments for seeds of many
temperate woody legwnes including locusts However these scarification techniques
often produce inconsistent germination Percussion scarification where seeds are
repeatedly propelled against a hard surface was compared with hot water
scarification to evaluate treatment efficacy for New Mexico locust (Robinia
neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment
seeds were placed in a 98degC water bath which was immediately removed from the
heat source For percussion scarification seeds were placed in a soil sample tin and
agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the
control were followed by 24-hour water soaks Hot water baths resulted in 56 and
41 germination for New Mexico locust and black locust respectively For both
species nearly all durations ofpercussion increased germination over the hot water
treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3
4 and 5 minutes for black locust resulted in at least 90 germination Traditional
scarification treatments randomly degrade the entire seed coat which can lead to
tissue damage during water uptake Percussion scarification specifically weakens the
strophiole the natural source ofwater entry to the seed in papilionoid legwnes
INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom
2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031
48
Following percussion imbibition is controlled through the strophiole and underlying
tissue is protected
Keywords New Mexico locust black locust scarification percussion germination
Introduction
Locust trees (Robinia spp) are aggressive pioneer species that quickly
colonize disturbed land fix atmospheric nitrogen in the soil and stabilize slopes that
are prone to erosion (Klemmedson 1994 Ashby et al 1985) (Figure A1ab)
Recognition of these attributes has contributed to increased demand for locusts in
reclamation projects Locust seeds however exhibit physical dormancy where a
thick seed coat prevents the movement of water and gasses to the embryo (Leadem
1997)
Sulfuric acid or hot water soaks have traditionally been used to break seed
dormancy in macro-propagation of locusts Many growers are moving away from the
use of su1furic acid in treating locust seeds (Dreesen and Harrington 1997) In
addition to being dangerous acid soak durations must be specifically correlated to
seed lot (Olson 1974) Collections of native seed can vary tremendously in seed size
seed weight and hard seededness etc This variability limits the utility of sulfuric
acid soaks as even within a given lot some seeds may be damaged whereas others
are under-treated and fail to imbibe (take up water) A survey of Southwestern
container growers indicates that hot water baths are currently the most common
scarification method for locust seeds (Hine et al 1997) However hot water baths
also produce inconsistent germination (Lin et al 1996)
49
b
Figure AI New Mexico locust a primary invader following disturbance Ala Crown-sprouting at edge ofburn near Los Alamos NM--one year after Cerro Grande fire Alb Colonizing road cut Gila National Forest NM
50
Percussion scarification where seeds are repeatedly propelled against a hard
surface is an alternative dormancy-breaking method that has proven successful in
legumes related to locusts (Hamly 1932 Barton 1947) We compared this alternative
treatment to hot water scarification to evaluate treatment efficacy for New Mexico
locust and black locust seeds
Materials and Methods
Commercial New Mexico locust seeds (Western Native Seed Coaldale CO)
were collected fall 2000 in Huerfano County CO Black locust seeds were collected
September 2000 from Taos County NM by harvesting ripe pods from several trees at
a distance of up to 3 m from ground level Black locust seeds were allowed to air-dry
for 3 weeks and were threshed and separated from large chaffusing a greenhouse
fan A South Dakota Seed Blower (Chicago Illinltois) was used to remove fine chaff
from both seed lots Seeds were storedmiddotat 2-4C until the germination study was
conducted in March 2001
This study compared hot water scarification with percussion scarification
using treatment and species as experimental factors Along with a control
scarification treatments included immersion of seed in boiling water as well as
percussion durations of 1 2 3 45 or 10 minutes Four 100-seed samples were used
to test each species by scarification treatment combination
For the hot water treatment test tubes were filled with 30 mL water and
placed in a water-filled beaker (Figure A2a) The bath was raised to boiling (-98C at
51
b
c
Figure A2 Implementation ofhot water and percussion scarification A2a Hot water bath A2b Paint-shaker set-up A2c Placing soil tin containing seeds at end ofpaint shaker ann
52
Las Cruces NM elevation of 1300 meters) Each 100-seed replicate was placed in a
test tube and each test tube was immediately removed to cool at room temperature
Percussion scarification was implemented using a pneumatic paint shaker
(Central Pneumatic model 00422 Camarillo CA) (Figure A2b) This allowed
standardization of treatment intensity Compressed air pressure was maintained at
530kPa plusmn 30kPa (80psi plusmn 5psi) resulting in approximately 350 oscillations per
minute Each lOO-seed sample was placed in a 118 ml (4 oz) soil tin for shaking To
maximize lateral movement of paint shaker a paint can was used as a spacer to place
soil tins at the end of the shaking arm (Figure A2c) This allowed shaking to occur at
the greatest distance from the pivot point (fulcrum) Following all treatments
including the control seeds were soaked for twenty-four hours in distilled water prior
to plating out
Gennination was evaluated on lab benches at room temperature Each
replicate of seeds was placed in a 100 cm petri dish on moistened filter paper with
humidity maintained by enclosing the petri dishes in ziplock bags Gennination was
monitored daily for 14 days in accordance with International Seed Testing
Association standards for Robinia species (ISTA 1999) Gennination was defined as
protrusion of the radicle from the embryo by at least 1mm as viewed by the naked
eye
Categorical Analysis of Variance using Prpc Catmod (SAS Institute 1989)
was used to detennine treatment differences in germination percentages for each
source This procedure is an extension of a Chi-square test of homogeneity using the
53
natural log of the ratio of germinated to non-germinated seeds for each treatment Pshy
values less than 005 were considered significant A limited set of pairwiseshy
comparisons was conducted to compare treatment mean percents using a
conservative alpha value of 005 divided by the number of comparisons
Results
Percussion maximized germination for both New Mexico locust and black
locust There were three germination response levels for New Mexico locust (Figure
A3) First there was a low response in the control indicating a dormant seed lot
The hot water treatment and I-minute duration of percussion share a second
intermediate response Germination was highest at percussion levels from 2 to 10
minutes
A selected percussion treatment intermediate in time (5 minutes) produced the
steepest germination curve indicating the fastest germination rate (Figure A4) The
hot water treatment produced an intermediate curve or relatively slower germination
rate The control curve was very flat indicating the slowest germination rate for New
Mexico locust
Control seeds germinated poorly for black locust as well (Figure A5) Hot
water scarification improved germination over the control but all levels of percussion
improved germination over hot water The optimal durations of percussion treatment
were bracketed with a significant increase from the 1 to 2 minute percussion level a
high germination response from 2 to 5 minutes of percussion and finally a significant
54
~ d 25 CL)
p t1 0 ~ t1
g Vl ~ Vl
100~---------------------------------------------------------------
80
60
40
20
o Control Hot Water Percussion
I min Percussion
2 min Percussion
3 min
Scarification
Percussion 4 min
Figure A3 Effect of scarification on germination response for New Mexico locust
Percussion 5 min
=significant difference between adjacent treatment bars ns = no significant difference
Percussion 10 min
~ ~ () u agt
Il-t d o ~
~ ~ c3
100middotlr~~~------------------------------------e- Percussion 5min middot 0middot Hot Water -- Control
80-J=middot======
6b-middot-lmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middot--middotmiddot-middot~-middotmiddot -middotmiddot middot middotmiddot-middot 1
40
20
-----~-----------
0 0 0 middotmiddotmiddotmiddotmiddot0middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot0
0 --~-----y-----~------_---~--- T-----~
o shy -TDay 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A4 Effect of selected scarification treatments on germination speed for New Mexico locust First seven days of fourteen-day experiment are shown
Day 7
80
~ cd
5 60 ()
[) Cl d 0 1t1 d
40sect dUl
-J
20
o
ns
Scarification
Figure A5 Effect of scarification on germination response for black locust = significant difference between adjacent treatment bars ns = no significant difference
decrease at the 10-minute level This drop-off suggests that 10 minutes of percussion
over-treats seeds from this lot of-black locust
A selected percussion treatment intermediate in time (5 minutes) also had the
fastest germination rate for black locust (Figure A6)_ The hot water curve is
somewhat flatter indicating a slower germination rate The control curve is very flat
indicating a slow germination rate
A qualitative difference between hot water and percussion-treated seeds was
also observed Along with healthy germinants the hot water treatment produced
over-treated seeds (Figure A 7a) Examples of damaged germinants include seeds
where the radicle did not elongate (Figure A 7b) seeds where the radicle elongated
but was delayed in freeing itself from the seed coat (Figure A7c) and seeds where the
radicle broke off completely from the embryo (Figure A7d) In addition to these
examples of over-treatment there were numerous small dark seeds that failed to
imbibe water For percussion treatment nearly all seeds took up water (Figure A8a)
Germination was rapid and uniform with little damage evident (Figure A8b)
To summarize both New Mexico locust and black locust seed lots were
dormant The standard hot water scarification treatment improved germination but
only to 56 and 41 for New Mexico locust and black locust respectively Nearly
all percussion durations improved total germination as well as germination rate over
the hot water treatment Germination was greater than 90 for percussion durations
of 4 5 and 10 minutes for New Mexico locust and 3 4 and 5 minutes for black locust
58
CI)
sgt t CI) 0
igt 0
sect ttl t E
Vl 0
c3
100r==~==~~=r------------------------------------~ ___ Percussion 5 min
middotmiddotmiddot0middotmiddotmiddot Hot Water -lIf- Control
80r=========~--~~----------------------------~
60
40
20 -~-~---~---------- ---~------------I
omiddot
O
_~_----------lIf-----~bull -----_ shy---shy I
Day 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A6 Effect of selected scarification treatments on germination speed for black locust First seven days of fourteen-day experiment are shown
Day 7
a
b c d
Figure A7 Damage to New Mexico locust seeds observed during germination counts following hot water scarification A7a Hot water replicate including damaged seedlings and numerous unimbibed seeds A7b Close-up ofseed where radicle failed to elongate A7c Close-up ofseed where radicle elongated but was delayed in freeing itself from the seed coat A7d Close-up ofseed where radicle completely broke offfrom embryo
60
a
b
Figure A8 New Mexico locust seeds following percussion treatment at time of germination counts A8a Percussion replicate with uniformly healthy seedlings and near complete imbibition of seeds A8b Close-up ofpercussion-treated seedling
61
Discussion
Papilionoid seeds have a specific anatomical feature known as the strophiole
or lens-the natural site of water entry to the seed (Hamly 1932) The strophiole is
located on the cotyledonary lobe of a locust seed (Figure A9) When a papilionoid
legume seed is percussed for the appropriate amount of time repeated hits on the
integrated seed coat loosen the constrained cells of the strophiolar region without
excessively damaging the rest of the seed coat (Ballard 1976)
When a percussed seed is soaked water enters exclusively through the
strophiole in a controlled manner (Kelly and Staden 1987) This regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues This
contrasts with seeds that have been hot water or acid scarified treatments that can
randomly degrade the seed coat Unlocalized cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rehman et al 1998 Marunda 1990)
Papilionoids are the largest subfamily of legumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust and black locust these legumes often play an integral role in the
revegetation ofdisturbed lands Developing superior scarification methods to the
standard hot water and acid treatments should fadlitate the use ofthese legumes in
restoration projects
62
Figure A9 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (bc)
63
APPENDIXB
GERMINATION PERCENTS AND STANDARD ERRORS FOR SOURCE
BLOCK AND TREATMENT FOR NEW MEXICO LOCUST
Table B 1 Gennination percents and standard errors for source block and treatment for New Mexico locust
Source Block Treatment Germination Percentage Standard Error
Capitan 1 Immersion 74 437 Capitan 2 Immersion 69 462 Capitan 3 Immersion 71 454 Capitan 4 Immersion 67 470 Capitan Boiling 30 sec 79 407 Capitan 2 Boiling 30 sec 76 427 Capitan 3 Boiling 30 sec 72 449 Capitan 4 Boiling 30 sec 76 427 Capitan Boiling 60 sec 71 454 Capitan 2 Boiling 60 sec 54 498 Capitan 3 Boiling 60 sec 67 470 Capitan 4 Boiling 60 sec 76 427 Capitan I Control 5 218 Capitan 2 Control 7 255 Capitan 3 Control 4 196 Capitan 4 Control 5 218 Capitan 1 Percussion I min 47 499 Capitan 2 Percussion I min
470
Capitan Percussion I min 39 488
Capitan 4 Percussion I min 38 485 Capitan I Percussion 2 min 55 497 Capitan Capitan
2
Percussion 2 min Percussion 2 min
71 65
454 477
Capitan 4 Percussion 2 min 56 496 Capitan I Percussion 4 min 86 347 Capitan 2 Percussion 4 min 79 407 Capitan 3 Percussion 4 min 81 392 Capitan 4 Percussion 4 min 82 384 Capitan I Percussion 8 min 90 300 Capitan 2 Percussion 8 min 88 325 Capitan 3 Percussion 8 min 90 300 Capitan 4 Percussion 8 min 90 300 Gila I Immersion 71 454 Gila 2 Immersion 77 421 Gila
Immersion 75 433
Gila 4 Immersion 72 449
Gila I Boiling 30 sec 68 466
Gila 2 Boiling 30 sec 81 392
Gila 3 Boiling 30 sec 54 498
Gila 4 Boiling 30 sec 63 483
Gila I Boiling 60 sec 55 497
Gila 2 Boiling 60 sec 68 466
Gila 3 Boiling 60 sec 57 495
Gila 4 Boiling 60 sec 70 458
65
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Gila 2 Control 13 336 Gila 3 Control 13 336 Gila 4 Control 20 400 Gila 1 Percussion 1 min 66 474 Gila 2 Percussion 1 min 57 495 Gila 3 Percussion 1 min 73 444 Gila 4 Percussion 1 min 82 384 Gila 1 Percussion 2 min 81 392 Gila 2 Percussion 2 min 86 347 Gila 3 Percussion 2 min 85 357 Gila 4 Percussion 2 min 54 498 Gila 1 Percussion 4 min 79 407 Gila 2 Percussion 4 min 83 376 Gila 3 Percussion 4 min 84 367 Gila 4 Percussion 4 min 79 407 Gila 1 Percussion 8 min 51 500 Gila 2 Percussion 8 min 76 427 Gila 3 Percussion 8 min 52 500 Gila 4 Percussion 8 min 74 439 Holman 1 Immersion 83 376 Holman 2 Immersion 86 347 Holman 3 Immersion 81 392 Holman 4 Immersion 90 300 Holman 1 Boiling 30 sec 90 300 Holman 2 Boiling 30 sec 85 357 Holman 3 Boiling 30 sec 87 336 Holman 4 Boiling 30 sec 83 376 Holman 1 Boiling 60 sec 79 407 Holman 2 Boiling 60 sec 80 400 Holman 3 Boiling 60 sec 86 347 Holman 4 Boiling 60 sec 93 255 Holman Control 25 433 Holman 2 Control 19 392 Holman Control 21 407
Holman 4 Control 24 427
Holman Percussion 1 min 77 421
Holman 2 Percussion 1 min 85 357
Holman 3 Percussion 1 min 80 400
Holman 4 Percussion 1 min 89 313
Holman 1 Percussion 2 min 88 325
Holman 2 Percussion 2 min 96 196
Holman 3 Percussion 2 min 93 255
Holman 4 Percussion 2 min 96 196
Holman 1 Percussion 4 min 92 271
Holman 2 Percussion 4 min 97 171
66
I
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Holman 3 Percussion 4 min 91 286 Holman 4 Percussion 4 min 96 196 Holman 1 Percussion 8 min 88 325 Holman 2 Percussion 8 min 85 357 Holman 3 Percussion 8 min 83 376 Holman 4 Percussion 8 min 82 384 Jemez 1 Immersion 53 499 Jemez 2 Immersion 44 496 Jemez 3 Immersion 52 500 Jemez 4 Immersion 70 458 Jemez 1 Boiling 30 sec 58 494 Jemez 2 Boiling 30 sec 55 497 Jemez 3 Boiling 30 sec 48 500 Jemez 4 Boiling 30 sec 65 477 Jemez 1 Boiling 60 sec 68 466 Jemez 2 Boiling 60 sec 63 483 Jemez 3 Boiling 60 sec 73 444 Jemez 4 Boiling 60 sec 89 313 Jemez 1 Control 2 140 Jemez 2 Control 9 286 Jemez 3 Control 6 237 Jemez 4 Control 8 271 Jemez 1 Percussion 1 min 58 494 Jemez 2 Percussion 1 min 63 483 Jemez 3 Percussion 1 min 77 421 Jemez 4 Percussion 1 min 89 313 Jemez 1 Percussion 2 min 68 466 Jemez 2 Percussion 2 min 64 480 Jemez 3 Percussion 2 min 80 400 Jemez 4 Percussion 2 min 83 376 Jemez Percussion 4 min 68 466 Jemez 2 Percussion 4 min 79 407 Jemez 3 Percussion 4 min 70 458 Jemez 4 Percussion 4 min 83 376 Jemez 1 Percussion 8 min 52 500 Jemez 2 Percussion 8 min 59 492 Jemez 3 Percussion 8 min 61 488 Jemez 4 Percussion 8 min 70 458 Magdalena 1 Immersion 69 462 Magdalena 2 Immersion 69 462 Magdalena J Immersion 70 458 Magdalena 4 Immersion 85 357 Magdalena 1 Boiling 30 sec 65 477 Magdalena 2 Boiling 30 sec 71 454 Magdalena 3 Boiling 30 sec 68 466
67
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Magdalena 4 Boiling 30 sec 83 376 Magdalena 1 Boiling 60 sec 63 483 Magdalena 2 Boiling 60 sec 68 466 Magdalena 3 Boiling 60 sec 63 483 Magdalena 4 Boiling 60 sec 82 384 Magdalena 1 Control 11 313 Magdalena 2 Control 6 237 Magdalena 3 Control 5 218 Magdalena 4 Control 4 196 Magdalena Percussion 1 min 32 466 Magdalena 2 Percussion 1 min 45 497 Magdalena 3 Percussion 1 min 46 498 Magdalena 4 Percussion I min 36 480 Magdalena 1 Percussion 2 min 67 470 Magdalena 2 Percussion 2 min 68 466 Magdalena 3 Percussion 2 min 65 477 Magdalena 4 Percussion 2 min 81 392 Magdalena 1 Percussion 4 min 87 336 Magdalena 2 Percussion 4 min 97 171 Magdalena 3 Percussion 4 min 83 376 Magdalena 4 Percussion 4 min 97 171 Magdalena 1 Percussion 8 min 98 lAO Magdalena 2 Percussion 8 min 100 000 Magdalena 3 Percussion 8 min 96 196 Magdalena 4 Percussion 8 min 98 lAO Manzano 1 Immersion 67 470 Manzano 2 Immersion 57 495 Manzano 3 Immersion 72 4049 Manzano 4 Immersion 73 4044 Manzano 1 Boiling 30 sec 70 458 Manzano 2 Boiling 30 sec 64 480 Manzano 3 Boiling 30 sec 71 454 Manzano 4 Boiling 30 sec 80 400 Manzano I Boiling 60 sec 50 500 Manzano 2 Boiling 60 sec 53 499 Manzano 3 Boiling 60 sec 59 492 Manzano 4 Boiling 60 sec 74 439 Manzano I Control 4 196 Manzano 2 Control 8 271 Manzano 3 Control 1 099 Manzano 4 Control 6 237 Manzano 1 Percussion 1 min 52 500 Manzano 2 Percussion 1 min 39 488 Manzano 3 Percussion 1 min 36 480 Manzano 4 Percussion 1 min 51 500
68
Table B 1 Continued
Source Block Treatment Gerrni~ation Percentage Standard Error
Manzano Percussion 2 min 53 499 Manzano 2 Percussion 2 min 58 494 Manzano 3 Percussion 2 min 61 488 Manzano 4 Percussion 2 min 62 485 Manzano 1 Percussion 4 min 76 427 Manzano 2 Percussion 4 min 85 357 Manzano 3 Percussion 4 min 79 407 Manzano 4 Percussion 4 min 83 376 Manzano 1 Percussion 8 min 87 336 Manzano 2 Percussion 8 min 83 376 Manzano 3 Percussion 8 min 83 376 Manzano 4 Percussion 8 min 75 433 Raton 1 Immersion 54 498 Raton 2 Immersion 37 483 Raton 3 Immersion 39 488 Raton 4 Immersion 69 462 Raton 1 Boiling 30 sec 36 480 Raton 2 Boiling 30 sec 20 400 Raton 3 Boiling 30 sec 28 449 Raton 4 Boiling 30 sec 60 490 Raton 1 Boiling 60 sec 15 357 Raton 2 Boiling 60 sec 171 Raton 3 Boiling 60 sec 4 196 Raton 4 Boiling 60 sec 35 477 Raton 1 Control 4 196 Raton 2 Control 12 325 Raton 3 Control 2 140 Raton 4 Control 6 237 Raton 1 Percussion 1 min 52 500 Raton 2 Percussion I min 29 454 Raton 3 Percussion I min 35 477 Raton 4 Percussion 1 min 31 462 Raton 1 Percussion 2 min 55 497 Raton 2 Percussion 2 min 35 477 Raton 3 Percussion 2 min 33 470 Raton 4 Percussion 2 min 42 494 Raton 1 Percussion 4 min 65 477 Raton 2 Percussion 4 min 52 500 Raton Percussion 4 min 51 500 Raton 4 Percussion 4 min 53 499 Raton 1 Percussion 8 min 88 325 Raton 2 Percussion 8 min 80 400 Raton 3 Percussion 8 min 73 444 Raton 4 Percussion 8 min 74 439 Sacramento 1 Immersion 43 495
69
Table B 1 Continued
-Source Block Treatment Germination Percentage Standard Error
Sacramento 2 Immersion 30 458 Sacramento 3 Immersion 38 485 Sacramento 4 Immersion 56 496 Sacramento 1 Boiling 30 sec 47 499 Sacramento 2 Boiling 30 sec 48 500 Sacramento 3 Boiling 30 sec 48 500 Sacramento 4 Boiling 30 sec 56 496 Sacramento 1 Boiling 60 sec 52 500 Sacramento 2 Boiling 60 sec 67 470 Sacramento 3 Boiling 60 sec 43 495 Sacramento 4 Boiling 60 sec 48 500 Sacramento 1 Control 0 000 Sacramento 2 Control 3 171 Sacramento 3 Control 3 171 Sacramento 4 Control 099 Sacramento 1 Percussion 1 min 57 495 Sacramento 2 Percussion 1 min 61 488 Sacramento 3 Percussion 1 min 62 485 Sacramento 4 Percussion 1 min 55 497 Sacramento 1 Percussion 2 min 84 367 Sacramento 2 Percussion 2 min 70 458 Sacramento 3 Percussion 2 min 81 392 Sacramento 4 Percussion 2 min 69 462 Sacramento 1 Percussion 4 min 91 286 Sacramento 2 Percussion 4 min 84 367 Sacramento 3 Percussion 4 min 84 367 Sacramento 4 Percussion 4 min 94 237 Sacramento 1 Percussion 8 min 82 384 Sacramento 2 Percussion 8 min 81 392 Sacramento 3 Percussion 8 min 82 384 Sacramento 4 Percussion 8 min 81 392 Sandia 1 Immersion 76 427 Sandia 2 Immersion 70 458 Sandia 3 Immersion 70 458 Sandia 4 Immersion 81 392 Sandia 1 Boiling 30 sec 78 414 Sandia 2 Boiling 30 sec 71 454 Sandia 3 Boiling 30 sec 63 483 Sandia 4 Boiling 30 sec 78 414 Sandia 1 Boiling 60 sec 67 470 Sandia 2 Boiling 60 sec 65 477 Sandia 3 Boiling 60 sec 74 439 Sandia 4 Boiling 60 sec 66 474 Sandia 1 Control 16 367 Sandia 2 Control 7 255
70
Table B 1 Continued
Source Block Treatment Gennination Percentage Standard Error
Sandia 3 Control 8 271 Sandia 4 Control 16 367 Sandia 1 Percussion 1 min 67 470 Sandia 2 Percussion 1 min 61 488 Sandia 3 Percussion 1 min 66 474 Sandia 4 Percussion 1 min 59 492 Sandia I Percussion 2 min 82 384 Sandia 2 Percussion 2 min 83 376 Sandia 3 Percussion 2 min 81 392 Sandia 4 Percussion 2 min 84 367 Sandia 1 Percussion 4 min 89 313 Sandia 2 Percussion 4 min 86 347 Sandia 3 Percussion 4 min 89 313 Sandia 4 Percussion 4 min 85 357 Sandia 1 Percussion 8 min 80 400 Sandia 2 Percussion 8 min 77 421 Sandia 3 Percussion 8 min 69 462 Sandia 4 Percussion 8 min 66 474
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Wilson JK 1944 Immersing seeds of species of Robinia in boiling water hastens germination J For 42 453-454
Zimmerman RW Carpenter SB 1980 First-year coppice production from a 5shyyear old black locust stand on surface mine spoil p309-314 Centr Hardwd For ConfProc University ofMissouri Columbia
Figure 8a-d Seed damage close-ups Figure a shows undamaged seed percussed for 4 minutes Figures b-d show seeds percussed for 8 minutes Damage includes fracturing ofradicle in b complete removal ofseed coat in c and pitting ofseed coat in d
34
Untreated Treated
Figure 9 SEM images ofuntreated vs 4-minute percussion-treated strophiolar cells ofNew Mexico locust seeds Treated cells are loosened separated and raised
DISCUSSION
Determining the Optimal Treatment
Averaging results of scarification treatmems across seed sources is a useful
tool to predict which treatment is most likely to be successful for an unknown seed
source Averaged over all sources four- and eight-minute percussion treatments
resulted in the highest germination 815 and 790 respectively and produced
germination that was as fast or faster than all other treatments Although there was no
difference between these two treatments on average an argument for 4 minutes of
percussion as a standard protocol for treating New Mexico locust seeds can be made
Descriptively 4 minutes of percussion improved germination over 8 minutes
ofpercussion for a majority of sources (five out of nine) 8 minutes of percussion
improved germination for three ofnine sources with no difference for a fmal source
For individual sources 4 minutes ofpercussion was always the best or secondmiddot best
treatment and was always equal to or better than all hot water treatments
Germination at 8 minutes ofpercussion was lower than all other percussion durations
for the Jemez and Gila sources and was lower than at least one hot water treatment for
these sources as well
The x-ray radiographs of the Sacramento seed source illustrate the excessive
internal and external seed damage that can occur with 8 minutes of percussion
germination counts 8-minute percussed seeds from Holman and Capitan
ClUll signs of damage that were visible to the naked eye (see below)
r~evertheless some of these seeds still fit the criteria for germination Neither x-ray
36
photography nor visible observations during seed counts revealed seed damage at the
4-minute percussion level In operational trials seeds percussed for 4 minutes
developed into healthy seedlings (Khadduri and Harrington unpublished data)
Strophiole and Seed Coat Behavior
Four minutes ofpercussion successfully breaks seed dormancy in a large
percentage ofNew Mexico locust seeds without excessively damaging the
surrounding seed coat or internal seed coat structures Most papilionoid legume
seeds including seeds ofNew Mexico locust have a specific region of the seed coat
known as the strophiole or lens (Hamly 1932) The strophiole is located on the
cotyledonary lobe of a locust seed (Figure 10 a-c) If seeds are not indiscriminately
damaged the strophiole is the first point of water entry to the embryo (Hopkinson
1997) When a papilionoid seed is percussed for the appropriate amount of time
repeated hits on the integrated seed coat loosen the constrained cells of the strophiolar
region without excessively damaging the rest of the seed coat (Ballard 1976) SEM
photos illuStrate that 4 minutes of percussion loosened and separated strophiolar cells
ofNew Mexico locust seeds when compared with constrained organized strophiolar
cells of untreated seeds (Figure 9) Research-has shown that water passes to the
embryo through the treated strophiole in a controlled manner and regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues
(Kelly and van Staden 1987)
37
10 Strophiole ofunimbibed New Mexico locust seed (a) and following uptake (bc)
38
In contrast hot-water treatments can excessively degrade the seed coat
(Rebman et al 1998 Marunda 1990) Random cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rebman et aI 1998 Marunda 1990) Along with a
proportionately large amount ofun-imbibed (thus under-treated) seeds (Figure l1a)
damage was observed in hot-water treated seeds from six sources during germination
counts Damage included seeds where the radicle failed to elongate (Figure 11 b)
seeds where the radicle elongated but was delayed in freeing itself from the seed coat
(Figure 11c) and seeds where the radicle completely broke off from the embryo
(Figure lId) In addition some seeds germinated in reverse (the cotyledon emerged
fIrst) If the radicle emerged completely from the seed coat they were counted as
germinated
While nearly all seeds treated for 8 minutes ofpercussion imbibed water
damage was evident in x-ray evaluations (Sacramento seed source) and in
germination counts (Holman and Capitan) at this treatment level Seed damage
during germination counts was similar to levels found in hot-water treated seeds
suggesting that over-treatment by both hot water and longer durations of percussion
may result in similar damage to seed structure
The Sacramento and Holman seed sources were two of the fIve sources that
opcrplltlpn in germination from the 4- to 8-minute durations of percussion
the benefIts ofloosening the strophiole as evidenced by imbibition of
all 8-minute treated seeds were outweighed by external and internal seed coat
39
a
b c d
11 Damage to New Mexico locust seeds observed during germination cotmts following hot water scarification 9a Hot water replicate including ~t seedlings and numerous unimbibed seeds 9b Close-up of seed where raallCle failed to elongate 9c Close-up ofseed where radicle elongated but was
-gtIn in freeing itself from the seed coat 9d Close-up ofseed where radicle broke off from embryo
40
damage (which also may be contributing to high imbibition) For the three sources
for which 8 minutes of percussion resulted in maximum germination extended
percussion of seeds necessary to loosen the strophiole may not have excessively
damaged the seed anatomy The Manzano source did not significantly change in
germination from 4 to 8 minutes of percussion suggesting that an increase in
permeable seeds at the 8-minute treatment duration was balanced by the number of
seeds that failed to germinate due to seed damage To summarize the 4-minute
percussion treatment improved germination over 8 minutes ofpercussion for a
majority of sources (five ofnine) the 8-minute percussion treatment improved
germination over 4-minutes ofpercussion for three sources and there was no
difference with the Manzano source Notably for the Raton source 8 minutes of
percussion increased germination by 235 percentage points over 4-minutes of
percussion (7875 vs 5525 respectively) the next best treatment Raton was the
only source to result in germination less than 75 for 4 minutes ofpercussion so a
grower may wish to apply longer percussion durations to lots that do not respond well
to this treatment level
Finally there was an interesting trend in the Jemez and Raton sources seed
that significantly contributed to the source by treatment interaction (Figure 12) A
~u test showed that the Jemez source had a significantly lighter seed weight than
other sources while the Raton source had an average seed weight For the Jemez
the longest duration of boiling increased germination Conversely only short
U4UVll) of percussion were needed to achieve high germination and the longest
41
80 _ Jemez a Raton
iD
~ C iD e iD
Q c 0 ~
c ~ iD 0
20
0------shy
Immersion Boiling 30 sec Boiling 60 sec
Hot Water Scarification
10degTr========~--------~------------------------------
80
60
40
20
_ Jemez Raton
0------shy
1 min 2 min 4 min 8 min
Percussion Scarification
Figure 12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources
42
b
--~----~~~~========~---- duration ofpercussion reduced germination For the Raton source longer durations
ofboiling consistently decreased germination whereas longer percussion treatments
consistently increased germination
A possible explanation is that a thick seed coat is associated with a loose
strophiole structure and that a thin seed coat is associated with a constrained
strophiole This contrast merits further study and may suggest an ecological
adaptation Further investigation ofthis phenomenon may contribute to successful
pre-screening ofunknown seed sources to determine optimal treatment leveL
43
CONCLUSION
Based on results obtained in pilot study (see Appendix) and thesis
experiments 4 minutes ofpercussion is recommended as a standard protocol for
treating an unknown source ofNew Mexico locust seed In addition to laboratory
results the 4-minute percussion treatment has resulted in germination greater than
80 and healthy seedling grow-outs for production purposes (unpublished data) In
laboratory experiments only one hundred seeds per 118 ml (40z) soil tin were
percussed at a time whereas for production we have treated up to five hundred seeds
per tin For large-scale production ofNew Mexico locust seedlings growers may
wish to experiment with larger quantities of seed and larger-size soil tins
Seed storage trials following percussion treatments were not conducted
However germination greater than 80 and healthy seedlings were obtained when
seeds were percussion-treated and subsequently stored at room-temperature (22degC)
for one week (unpublished data) Seed storage trials merit further study as the
standard hot water treatment leaves seeds in a softened condition that is not
conducive to storage
Hydroseeding is an effective method for direct seeding in reclamation
situations Percussion treatment may be conducive to hydro seeding as seeds remain
a dry relatively undamaged yet permeable condition following treatment Sulfuric
seeds also remain dry following treatment but extensive damage to seed coat
poor seedling vigor have been reported with this treatment (Kelly and van Staden
A grow-out study using a hydro seeder on a reclamation site testing percussion
44
against other treatments such as sulfuric acid and mechanical scarification also
merits further study
Papilionoids are the largest subfamily oflegumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust these legumes often play an integral role in the revegetation of
disturbed lands The pilot study (Appendix) indicates that percussion scarification
successfully treats the widely used black locust as well Developing superior
scarification methods to the standard hot water mechanical scarification and acid
treatments may facilitate the use ofpapilionoid legumes in reclamation projects
45
APPENDICES
APPENDIX A
PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR
NEW MEXICO LOCUST AND BLACK LOCUST SEEDS
Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4
Hot water and sulfuric acid soaks are traditional treatments for seeds of many
temperate woody legwnes including locusts However these scarification techniques
often produce inconsistent germination Percussion scarification where seeds are
repeatedly propelled against a hard surface was compared with hot water
scarification to evaluate treatment efficacy for New Mexico locust (Robinia
neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment
seeds were placed in a 98degC water bath which was immediately removed from the
heat source For percussion scarification seeds were placed in a soil sample tin and
agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the
control were followed by 24-hour water soaks Hot water baths resulted in 56 and
41 germination for New Mexico locust and black locust respectively For both
species nearly all durations ofpercussion increased germination over the hot water
treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3
4 and 5 minutes for black locust resulted in at least 90 germination Traditional
scarification treatments randomly degrade the entire seed coat which can lead to
tissue damage during water uptake Percussion scarification specifically weakens the
strophiole the natural source ofwater entry to the seed in papilionoid legwnes
INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom
2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031
48
Following percussion imbibition is controlled through the strophiole and underlying
tissue is protected
Keywords New Mexico locust black locust scarification percussion germination
Introduction
Locust trees (Robinia spp) are aggressive pioneer species that quickly
colonize disturbed land fix atmospheric nitrogen in the soil and stabilize slopes that
are prone to erosion (Klemmedson 1994 Ashby et al 1985) (Figure A1ab)
Recognition of these attributes has contributed to increased demand for locusts in
reclamation projects Locust seeds however exhibit physical dormancy where a
thick seed coat prevents the movement of water and gasses to the embryo (Leadem
1997)
Sulfuric acid or hot water soaks have traditionally been used to break seed
dormancy in macro-propagation of locusts Many growers are moving away from the
use of su1furic acid in treating locust seeds (Dreesen and Harrington 1997) In
addition to being dangerous acid soak durations must be specifically correlated to
seed lot (Olson 1974) Collections of native seed can vary tremendously in seed size
seed weight and hard seededness etc This variability limits the utility of sulfuric
acid soaks as even within a given lot some seeds may be damaged whereas others
are under-treated and fail to imbibe (take up water) A survey of Southwestern
container growers indicates that hot water baths are currently the most common
scarification method for locust seeds (Hine et al 1997) However hot water baths
also produce inconsistent germination (Lin et al 1996)
49
b
Figure AI New Mexico locust a primary invader following disturbance Ala Crown-sprouting at edge ofburn near Los Alamos NM--one year after Cerro Grande fire Alb Colonizing road cut Gila National Forest NM
50
Percussion scarification where seeds are repeatedly propelled against a hard
surface is an alternative dormancy-breaking method that has proven successful in
legumes related to locusts (Hamly 1932 Barton 1947) We compared this alternative
treatment to hot water scarification to evaluate treatment efficacy for New Mexico
locust and black locust seeds
Materials and Methods
Commercial New Mexico locust seeds (Western Native Seed Coaldale CO)
were collected fall 2000 in Huerfano County CO Black locust seeds were collected
September 2000 from Taos County NM by harvesting ripe pods from several trees at
a distance of up to 3 m from ground level Black locust seeds were allowed to air-dry
for 3 weeks and were threshed and separated from large chaffusing a greenhouse
fan A South Dakota Seed Blower (Chicago Illinltois) was used to remove fine chaff
from both seed lots Seeds were storedmiddotat 2-4C until the germination study was
conducted in March 2001
This study compared hot water scarification with percussion scarification
using treatment and species as experimental factors Along with a control
scarification treatments included immersion of seed in boiling water as well as
percussion durations of 1 2 3 45 or 10 minutes Four 100-seed samples were used
to test each species by scarification treatment combination
For the hot water treatment test tubes were filled with 30 mL water and
placed in a water-filled beaker (Figure A2a) The bath was raised to boiling (-98C at
51
b
c
Figure A2 Implementation ofhot water and percussion scarification A2a Hot water bath A2b Paint-shaker set-up A2c Placing soil tin containing seeds at end ofpaint shaker ann
52
Las Cruces NM elevation of 1300 meters) Each 100-seed replicate was placed in a
test tube and each test tube was immediately removed to cool at room temperature
Percussion scarification was implemented using a pneumatic paint shaker
(Central Pneumatic model 00422 Camarillo CA) (Figure A2b) This allowed
standardization of treatment intensity Compressed air pressure was maintained at
530kPa plusmn 30kPa (80psi plusmn 5psi) resulting in approximately 350 oscillations per
minute Each lOO-seed sample was placed in a 118 ml (4 oz) soil tin for shaking To
maximize lateral movement of paint shaker a paint can was used as a spacer to place
soil tins at the end of the shaking arm (Figure A2c) This allowed shaking to occur at
the greatest distance from the pivot point (fulcrum) Following all treatments
including the control seeds were soaked for twenty-four hours in distilled water prior
to plating out
Gennination was evaluated on lab benches at room temperature Each
replicate of seeds was placed in a 100 cm petri dish on moistened filter paper with
humidity maintained by enclosing the petri dishes in ziplock bags Gennination was
monitored daily for 14 days in accordance with International Seed Testing
Association standards for Robinia species (ISTA 1999) Gennination was defined as
protrusion of the radicle from the embryo by at least 1mm as viewed by the naked
eye
Categorical Analysis of Variance using Prpc Catmod (SAS Institute 1989)
was used to detennine treatment differences in germination percentages for each
source This procedure is an extension of a Chi-square test of homogeneity using the
53
natural log of the ratio of germinated to non-germinated seeds for each treatment Pshy
values less than 005 were considered significant A limited set of pairwiseshy
comparisons was conducted to compare treatment mean percents using a
conservative alpha value of 005 divided by the number of comparisons
Results
Percussion maximized germination for both New Mexico locust and black
locust There were three germination response levels for New Mexico locust (Figure
A3) First there was a low response in the control indicating a dormant seed lot
The hot water treatment and I-minute duration of percussion share a second
intermediate response Germination was highest at percussion levels from 2 to 10
minutes
A selected percussion treatment intermediate in time (5 minutes) produced the
steepest germination curve indicating the fastest germination rate (Figure A4) The
hot water treatment produced an intermediate curve or relatively slower germination
rate The control curve was very flat indicating the slowest germination rate for New
Mexico locust
Control seeds germinated poorly for black locust as well (Figure A5) Hot
water scarification improved germination over the control but all levels of percussion
improved germination over hot water The optimal durations of percussion treatment
were bracketed with a significant increase from the 1 to 2 minute percussion level a
high germination response from 2 to 5 minutes of percussion and finally a significant
54
~ d 25 CL)
p t1 0 ~ t1
g Vl ~ Vl
100~---------------------------------------------------------------
80
60
40
20
o Control Hot Water Percussion
I min Percussion
2 min Percussion
3 min
Scarification
Percussion 4 min
Figure A3 Effect of scarification on germination response for New Mexico locust
Percussion 5 min
=significant difference between adjacent treatment bars ns = no significant difference
Percussion 10 min
~ ~ () u agt
Il-t d o ~
~ ~ c3
100middotlr~~~------------------------------------e- Percussion 5min middot 0middot Hot Water -- Control
80-J=middot======
6b-middot-lmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middot--middotmiddot-middot~-middotmiddot -middotmiddot middot middotmiddot-middot 1
40
20
-----~-----------
0 0 0 middotmiddotmiddotmiddotmiddot0middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot0
0 --~-----y-----~------_---~--- T-----~
o shy -TDay 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A4 Effect of selected scarification treatments on germination speed for New Mexico locust First seven days of fourteen-day experiment are shown
Day 7
80
~ cd
5 60 ()
[) Cl d 0 1t1 d
40sect dUl
-J
20
o
ns
Scarification
Figure A5 Effect of scarification on germination response for black locust = significant difference between adjacent treatment bars ns = no significant difference
decrease at the 10-minute level This drop-off suggests that 10 minutes of percussion
over-treats seeds from this lot of-black locust
A selected percussion treatment intermediate in time (5 minutes) also had the
fastest germination rate for black locust (Figure A6)_ The hot water curve is
somewhat flatter indicating a slower germination rate The control curve is very flat
indicating a slow germination rate
A qualitative difference between hot water and percussion-treated seeds was
also observed Along with healthy germinants the hot water treatment produced
over-treated seeds (Figure A 7a) Examples of damaged germinants include seeds
where the radicle did not elongate (Figure A 7b) seeds where the radicle elongated
but was delayed in freeing itself from the seed coat (Figure A7c) and seeds where the
radicle broke off completely from the embryo (Figure A7d) In addition to these
examples of over-treatment there were numerous small dark seeds that failed to
imbibe water For percussion treatment nearly all seeds took up water (Figure A8a)
Germination was rapid and uniform with little damage evident (Figure A8b)
To summarize both New Mexico locust and black locust seed lots were
dormant The standard hot water scarification treatment improved germination but
only to 56 and 41 for New Mexico locust and black locust respectively Nearly
all percussion durations improved total germination as well as germination rate over
the hot water treatment Germination was greater than 90 for percussion durations
of 4 5 and 10 minutes for New Mexico locust and 3 4 and 5 minutes for black locust
58
CI)
sgt t CI) 0
igt 0
sect ttl t E
Vl 0
c3
100r==~==~~=r------------------------------------~ ___ Percussion 5 min
middotmiddotmiddot0middotmiddotmiddot Hot Water -lIf- Control
80r=========~--~~----------------------------~
60
40
20 -~-~---~---------- ---~------------I
omiddot
O
_~_----------lIf-----~bull -----_ shy---shy I
Day 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A6 Effect of selected scarification treatments on germination speed for black locust First seven days of fourteen-day experiment are shown
Day 7
a
b c d
Figure A7 Damage to New Mexico locust seeds observed during germination counts following hot water scarification A7a Hot water replicate including damaged seedlings and numerous unimbibed seeds A7b Close-up ofseed where radicle failed to elongate A7c Close-up ofseed where radicle elongated but was delayed in freeing itself from the seed coat A7d Close-up ofseed where radicle completely broke offfrom embryo
60
a
b
Figure A8 New Mexico locust seeds following percussion treatment at time of germination counts A8a Percussion replicate with uniformly healthy seedlings and near complete imbibition of seeds A8b Close-up ofpercussion-treated seedling
61
Discussion
Papilionoid seeds have a specific anatomical feature known as the strophiole
or lens-the natural site of water entry to the seed (Hamly 1932) The strophiole is
located on the cotyledonary lobe of a locust seed (Figure A9) When a papilionoid
legume seed is percussed for the appropriate amount of time repeated hits on the
integrated seed coat loosen the constrained cells of the strophiolar region without
excessively damaging the rest of the seed coat (Ballard 1976)
When a percussed seed is soaked water enters exclusively through the
strophiole in a controlled manner (Kelly and Staden 1987) This regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues This
contrasts with seeds that have been hot water or acid scarified treatments that can
randomly degrade the seed coat Unlocalized cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rehman et al 1998 Marunda 1990)
Papilionoids are the largest subfamily of legumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust and black locust these legumes often play an integral role in the
revegetation ofdisturbed lands Developing superior scarification methods to the
standard hot water and acid treatments should fadlitate the use ofthese legumes in
restoration projects
62
Figure A9 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (bc)
63
APPENDIXB
GERMINATION PERCENTS AND STANDARD ERRORS FOR SOURCE
BLOCK AND TREATMENT FOR NEW MEXICO LOCUST
Table B 1 Gennination percents and standard errors for source block and treatment for New Mexico locust
Source Block Treatment Germination Percentage Standard Error
Capitan 1 Immersion 74 437 Capitan 2 Immersion 69 462 Capitan 3 Immersion 71 454 Capitan 4 Immersion 67 470 Capitan Boiling 30 sec 79 407 Capitan 2 Boiling 30 sec 76 427 Capitan 3 Boiling 30 sec 72 449 Capitan 4 Boiling 30 sec 76 427 Capitan Boiling 60 sec 71 454 Capitan 2 Boiling 60 sec 54 498 Capitan 3 Boiling 60 sec 67 470 Capitan 4 Boiling 60 sec 76 427 Capitan I Control 5 218 Capitan 2 Control 7 255 Capitan 3 Control 4 196 Capitan 4 Control 5 218 Capitan 1 Percussion I min 47 499 Capitan 2 Percussion I min
470
Capitan Percussion I min 39 488
Capitan 4 Percussion I min 38 485 Capitan I Percussion 2 min 55 497 Capitan Capitan
2
Percussion 2 min Percussion 2 min
71 65
454 477
Capitan 4 Percussion 2 min 56 496 Capitan I Percussion 4 min 86 347 Capitan 2 Percussion 4 min 79 407 Capitan 3 Percussion 4 min 81 392 Capitan 4 Percussion 4 min 82 384 Capitan I Percussion 8 min 90 300 Capitan 2 Percussion 8 min 88 325 Capitan 3 Percussion 8 min 90 300 Capitan 4 Percussion 8 min 90 300 Gila I Immersion 71 454 Gila 2 Immersion 77 421 Gila
Immersion 75 433
Gila 4 Immersion 72 449
Gila I Boiling 30 sec 68 466
Gila 2 Boiling 30 sec 81 392
Gila 3 Boiling 30 sec 54 498
Gila 4 Boiling 30 sec 63 483
Gila I Boiling 60 sec 55 497
Gila 2 Boiling 60 sec 68 466
Gila 3 Boiling 60 sec 57 495
Gila 4 Boiling 60 sec 70 458
65
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Gila 2 Control 13 336 Gila 3 Control 13 336 Gila 4 Control 20 400 Gila 1 Percussion 1 min 66 474 Gila 2 Percussion 1 min 57 495 Gila 3 Percussion 1 min 73 444 Gila 4 Percussion 1 min 82 384 Gila 1 Percussion 2 min 81 392 Gila 2 Percussion 2 min 86 347 Gila 3 Percussion 2 min 85 357 Gila 4 Percussion 2 min 54 498 Gila 1 Percussion 4 min 79 407 Gila 2 Percussion 4 min 83 376 Gila 3 Percussion 4 min 84 367 Gila 4 Percussion 4 min 79 407 Gila 1 Percussion 8 min 51 500 Gila 2 Percussion 8 min 76 427 Gila 3 Percussion 8 min 52 500 Gila 4 Percussion 8 min 74 439 Holman 1 Immersion 83 376 Holman 2 Immersion 86 347 Holman 3 Immersion 81 392 Holman 4 Immersion 90 300 Holman 1 Boiling 30 sec 90 300 Holman 2 Boiling 30 sec 85 357 Holman 3 Boiling 30 sec 87 336 Holman 4 Boiling 30 sec 83 376 Holman 1 Boiling 60 sec 79 407 Holman 2 Boiling 60 sec 80 400 Holman 3 Boiling 60 sec 86 347 Holman 4 Boiling 60 sec 93 255 Holman Control 25 433 Holman 2 Control 19 392 Holman Control 21 407
Holman 4 Control 24 427
Holman Percussion 1 min 77 421
Holman 2 Percussion 1 min 85 357
Holman 3 Percussion 1 min 80 400
Holman 4 Percussion 1 min 89 313
Holman 1 Percussion 2 min 88 325
Holman 2 Percussion 2 min 96 196
Holman 3 Percussion 2 min 93 255
Holman 4 Percussion 2 min 96 196
Holman 1 Percussion 4 min 92 271
Holman 2 Percussion 4 min 97 171
66
I
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Holman 3 Percussion 4 min 91 286 Holman 4 Percussion 4 min 96 196 Holman 1 Percussion 8 min 88 325 Holman 2 Percussion 8 min 85 357 Holman 3 Percussion 8 min 83 376 Holman 4 Percussion 8 min 82 384 Jemez 1 Immersion 53 499 Jemez 2 Immersion 44 496 Jemez 3 Immersion 52 500 Jemez 4 Immersion 70 458 Jemez 1 Boiling 30 sec 58 494 Jemez 2 Boiling 30 sec 55 497 Jemez 3 Boiling 30 sec 48 500 Jemez 4 Boiling 30 sec 65 477 Jemez 1 Boiling 60 sec 68 466 Jemez 2 Boiling 60 sec 63 483 Jemez 3 Boiling 60 sec 73 444 Jemez 4 Boiling 60 sec 89 313 Jemez 1 Control 2 140 Jemez 2 Control 9 286 Jemez 3 Control 6 237 Jemez 4 Control 8 271 Jemez 1 Percussion 1 min 58 494 Jemez 2 Percussion 1 min 63 483 Jemez 3 Percussion 1 min 77 421 Jemez 4 Percussion 1 min 89 313 Jemez 1 Percussion 2 min 68 466 Jemez 2 Percussion 2 min 64 480 Jemez 3 Percussion 2 min 80 400 Jemez 4 Percussion 2 min 83 376 Jemez Percussion 4 min 68 466 Jemez 2 Percussion 4 min 79 407 Jemez 3 Percussion 4 min 70 458 Jemez 4 Percussion 4 min 83 376 Jemez 1 Percussion 8 min 52 500 Jemez 2 Percussion 8 min 59 492 Jemez 3 Percussion 8 min 61 488 Jemez 4 Percussion 8 min 70 458 Magdalena 1 Immersion 69 462 Magdalena 2 Immersion 69 462 Magdalena J Immersion 70 458 Magdalena 4 Immersion 85 357 Magdalena 1 Boiling 30 sec 65 477 Magdalena 2 Boiling 30 sec 71 454 Magdalena 3 Boiling 30 sec 68 466
67
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Magdalena 4 Boiling 30 sec 83 376 Magdalena 1 Boiling 60 sec 63 483 Magdalena 2 Boiling 60 sec 68 466 Magdalena 3 Boiling 60 sec 63 483 Magdalena 4 Boiling 60 sec 82 384 Magdalena 1 Control 11 313 Magdalena 2 Control 6 237 Magdalena 3 Control 5 218 Magdalena 4 Control 4 196 Magdalena Percussion 1 min 32 466 Magdalena 2 Percussion 1 min 45 497 Magdalena 3 Percussion 1 min 46 498 Magdalena 4 Percussion I min 36 480 Magdalena 1 Percussion 2 min 67 470 Magdalena 2 Percussion 2 min 68 466 Magdalena 3 Percussion 2 min 65 477 Magdalena 4 Percussion 2 min 81 392 Magdalena 1 Percussion 4 min 87 336 Magdalena 2 Percussion 4 min 97 171 Magdalena 3 Percussion 4 min 83 376 Magdalena 4 Percussion 4 min 97 171 Magdalena 1 Percussion 8 min 98 lAO Magdalena 2 Percussion 8 min 100 000 Magdalena 3 Percussion 8 min 96 196 Magdalena 4 Percussion 8 min 98 lAO Manzano 1 Immersion 67 470 Manzano 2 Immersion 57 495 Manzano 3 Immersion 72 4049 Manzano 4 Immersion 73 4044 Manzano 1 Boiling 30 sec 70 458 Manzano 2 Boiling 30 sec 64 480 Manzano 3 Boiling 30 sec 71 454 Manzano 4 Boiling 30 sec 80 400 Manzano I Boiling 60 sec 50 500 Manzano 2 Boiling 60 sec 53 499 Manzano 3 Boiling 60 sec 59 492 Manzano 4 Boiling 60 sec 74 439 Manzano I Control 4 196 Manzano 2 Control 8 271 Manzano 3 Control 1 099 Manzano 4 Control 6 237 Manzano 1 Percussion 1 min 52 500 Manzano 2 Percussion 1 min 39 488 Manzano 3 Percussion 1 min 36 480 Manzano 4 Percussion 1 min 51 500
68
Table B 1 Continued
Source Block Treatment Gerrni~ation Percentage Standard Error
Manzano Percussion 2 min 53 499 Manzano 2 Percussion 2 min 58 494 Manzano 3 Percussion 2 min 61 488 Manzano 4 Percussion 2 min 62 485 Manzano 1 Percussion 4 min 76 427 Manzano 2 Percussion 4 min 85 357 Manzano 3 Percussion 4 min 79 407 Manzano 4 Percussion 4 min 83 376 Manzano 1 Percussion 8 min 87 336 Manzano 2 Percussion 8 min 83 376 Manzano 3 Percussion 8 min 83 376 Manzano 4 Percussion 8 min 75 433 Raton 1 Immersion 54 498 Raton 2 Immersion 37 483 Raton 3 Immersion 39 488 Raton 4 Immersion 69 462 Raton 1 Boiling 30 sec 36 480 Raton 2 Boiling 30 sec 20 400 Raton 3 Boiling 30 sec 28 449 Raton 4 Boiling 30 sec 60 490 Raton 1 Boiling 60 sec 15 357 Raton 2 Boiling 60 sec 171 Raton 3 Boiling 60 sec 4 196 Raton 4 Boiling 60 sec 35 477 Raton 1 Control 4 196 Raton 2 Control 12 325 Raton 3 Control 2 140 Raton 4 Control 6 237 Raton 1 Percussion 1 min 52 500 Raton 2 Percussion I min 29 454 Raton 3 Percussion I min 35 477 Raton 4 Percussion 1 min 31 462 Raton 1 Percussion 2 min 55 497 Raton 2 Percussion 2 min 35 477 Raton 3 Percussion 2 min 33 470 Raton 4 Percussion 2 min 42 494 Raton 1 Percussion 4 min 65 477 Raton 2 Percussion 4 min 52 500 Raton Percussion 4 min 51 500 Raton 4 Percussion 4 min 53 499 Raton 1 Percussion 8 min 88 325 Raton 2 Percussion 8 min 80 400 Raton 3 Percussion 8 min 73 444 Raton 4 Percussion 8 min 74 439 Sacramento 1 Immersion 43 495
69
Table B 1 Continued
-Source Block Treatment Germination Percentage Standard Error
Sacramento 2 Immersion 30 458 Sacramento 3 Immersion 38 485 Sacramento 4 Immersion 56 496 Sacramento 1 Boiling 30 sec 47 499 Sacramento 2 Boiling 30 sec 48 500 Sacramento 3 Boiling 30 sec 48 500 Sacramento 4 Boiling 30 sec 56 496 Sacramento 1 Boiling 60 sec 52 500 Sacramento 2 Boiling 60 sec 67 470 Sacramento 3 Boiling 60 sec 43 495 Sacramento 4 Boiling 60 sec 48 500 Sacramento 1 Control 0 000 Sacramento 2 Control 3 171 Sacramento 3 Control 3 171 Sacramento 4 Control 099 Sacramento 1 Percussion 1 min 57 495 Sacramento 2 Percussion 1 min 61 488 Sacramento 3 Percussion 1 min 62 485 Sacramento 4 Percussion 1 min 55 497 Sacramento 1 Percussion 2 min 84 367 Sacramento 2 Percussion 2 min 70 458 Sacramento 3 Percussion 2 min 81 392 Sacramento 4 Percussion 2 min 69 462 Sacramento 1 Percussion 4 min 91 286 Sacramento 2 Percussion 4 min 84 367 Sacramento 3 Percussion 4 min 84 367 Sacramento 4 Percussion 4 min 94 237 Sacramento 1 Percussion 8 min 82 384 Sacramento 2 Percussion 8 min 81 392 Sacramento 3 Percussion 8 min 82 384 Sacramento 4 Percussion 8 min 81 392 Sandia 1 Immersion 76 427 Sandia 2 Immersion 70 458 Sandia 3 Immersion 70 458 Sandia 4 Immersion 81 392 Sandia 1 Boiling 30 sec 78 414 Sandia 2 Boiling 30 sec 71 454 Sandia 3 Boiling 30 sec 63 483 Sandia 4 Boiling 30 sec 78 414 Sandia 1 Boiling 60 sec 67 470 Sandia 2 Boiling 60 sec 65 477 Sandia 3 Boiling 60 sec 74 439 Sandia 4 Boiling 60 sec 66 474 Sandia 1 Control 16 367 Sandia 2 Control 7 255
70
Table B 1 Continued
Source Block Treatment Gennination Percentage Standard Error
Sandia 3 Control 8 271 Sandia 4 Control 16 367 Sandia 1 Percussion 1 min 67 470 Sandia 2 Percussion 1 min 61 488 Sandia 3 Percussion 1 min 66 474 Sandia 4 Percussion 1 min 59 492 Sandia I Percussion 2 min 82 384 Sandia 2 Percussion 2 min 83 376 Sandia 3 Percussion 2 min 81 392 Sandia 4 Percussion 2 min 84 367 Sandia 1 Percussion 4 min 89 313 Sandia 2 Percussion 4 min 86 347 Sandia 3 Percussion 4 min 89 313 Sandia 4 Percussion 4 min 85 357 Sandia 1 Percussion 8 min 80 400 Sandia 2 Percussion 8 min 77 421 Sandia 3 Percussion 8 min 69 462 Sandia 4 Percussion 8 min 66 474
LITERATURE CITED
Ashby WC Vogel WG Rogers NF 1985 Black locust in the reclamation equation General Technical Report Northeastern Forest Experiment Station USDA Forest Service NE-I05 12 p
Ballard LAT 1973 Physical barriers to gennination Seed Sci Technoll 285-303
___ 1976 Strophiolar water conduction in the seeds ofTrifolieae induced by action on the testa at nonstrophiolar sites Austr J Plant Physiol3 465-469
Barton LV 1947 Special studies on seed coat impenneability Contrib Boyce Thompson Inst 14 355-362
Baskin CC Baskin JM 1998 Seeds Ecology biogeography and evolution of donnancy and gennination Academic Press London UK 666 p
Bewley JD 1997 Seed germination and donnancy Plant Ce119 1055-1066
Bewley JD Black M 1994 Seeds physiology of development and germination 2nd ed Plenum Press New York NY 445 p
Briscoe CB 1996 Growing Nitrogen Fixing Trees in Acid Soils In MH Powell (ed) Nitrogen Fixing Trees for Acid Soils- A Field ManuaL Winrock International Morrilton AR 22 p
Copeland LO McDonald MB 1995 Principles of Seed Science and Technology 3rded Chapman amp Hall New York NY 409 p
Cox RE Klett JE 1984 Seed gennination requirements of native Colorado plants for use in the landscape Plant Propag 30(2) 6-10
Dell B 1980 Structure and function ofthe strophiolar plug in seeds ofAlbizia Lophantha Am J Bot 67 556-563
Dick-Peddie WA 1993 New Mexico Vegetation Past Present and Future University ofNew Mexico Press Albuquerque NM 244 p
Dreesen DR 2001 Propagation protocol for production ofcontainer Robinia neomexicana plants Los Lunas Plant Materials Center Los Lunas New Mexico In Native Plant Network URL httpwwwnativeplantnetworkorg (accessed 9 January 2002) Moscow (ID) University ofIdaho CoIl Natural Resources For Res Nursery
72
Dreesen DR Harrington JT 1997 Propagation of native plants for restoration projects in the Southwestern US- Preliminary Investigations In Landis TD Thompson JR tech coords National Proceedings Forest and Conservation Nursery Assocs Gen Tech Rep PNW-GTR-419 Portland OR USDA For Ser PNW Res Sta 77-88
Gottfried GJ 1980 Control ofNew Mexican locust and the effect on planted ponderosa pine in central Arizona US For Ser Res Note RM-386 Rocky Mountain For Range ExpStn Fort Collins CO
Gosling PG Samuel YK Jones SK 1995 A systematic examination of germination temperature chipping and water temperaturesoak duration pretreatments on the seeds ofLeucaena leucocephala Seed Sci amp Technol 23521-532
Hamly DH 1932 Softening of the seeds of Melilotus alba Bot Gazette 93 345shy375
Hanna PJ 1984 Anatomical features of the seed coat ofAcacia kempeana (Mueller) which relate to increased germination rate induced by heat treatment New Phytol 96 23-29
Hanks JP Dick-Peddie WA 1974 Vegetation patterns of the White Mountains New Mexico Southwest Naturalist 18(4) 371-382
Hine S Harrington JT Dreesen DR 1997 Seed scarification requirements for Robinia neomexicana In Harrington JTtech coord Proc Third SouthwestemContainer Growers Conf Feb 12-13 1997 Ft Collins CO
Hopkinson JM 1993 The strophiole of leguminous seeds Newsletter- IntI Herbage Seed Production Res Grp 18 7-8
---- 1997 Differences in hardseededness between Cunningham and Tarramba leucaena In Leucnet News The Newsletter of the International LeucaenaRes and Devlt Netwk No4
ISTA (International Seed Testing Association) 1999 International rules for seed testing rules Seed Sci amp Technol27 Supplement
Kelly KM van Staden J 1987 The lens as the site of permeability in the papilionoid seed Aspalathus linearis J Plant Physiol 128(45) 395-404
73
Keresztesi B 1988 Black locust the tree of agriculture Outlook on Agr 17(2) 77~85
Kigel 1 and Galili G 1995 Seed development and germination Marcel Dekker New York 853 p
Klemmedson 10 1994 New Mexico locust and parent material Influence on macronutrients of forest floor and soil Soil Sci Soc Am 1 58 974~980
Kuo WH Tam AW 1988 The pathway of water absorption of mung bean seeds Seed Sci amp Technol16(1) 139~144
Leadem CL 1997 Dormancy--unlocking seed secrets In Landis TD Thompson lR tech coords National Proceedings Forestand Conservation Nursery Assocs Gen Tech Rep PNW-G TR-419 Portland OR USDA For Ser PNW Res Sta 43-52
Lin Y Wagner MR Seweyestewa A Honahni MD DeGomez M McMillin JD 1996 Influence of cultural treatments on seed germination and seedling growth of Robinia neomexicana Forest Farm and Community Tree Res Reports Volume 1
Marunda CT 1990 Effects of seed pretreatment on the development ofAcacia auriculforinis and A holosericea seedlings ACIAR Proceedings Series No 28
Meginnis HG 1937 Sulphuric acid treatment to increase germination of black locust seed USDA Circ 453 34 p
Miklas PN Towensend CE Ladd SL 1987 Seedcoat anatomy and the scarification ofcicer milkvetch seed Crop Sci 27 766-772
Monsen SB 1984 Use of shrubs on mine spoils In The Challenge ofProducing Native Plants for the Intermountain Area Proceedings Intermountain Nurserymans Association 1983 conference Las Vegas NV compo PM Murphy 26-31 Ogden UT USDA For Ser Gen Tech Report INT-l68
Olson DF 1974 Robinia L Locust In Seeds of Woody Plants in the US tech coord CS Schopmeyer 728-731 USDA For Ser Agr Handbk No450
Rehman S Loescher RNl Harris P1C 1998 Dormancy breaking and germination ofAcacia salicina Lindl seeds Seed Sci amp Techno127 553-557
Rolston MP 1978 Water impermeable seed dormancy Bot Rev 44 365-396
74
SAS Institute Inc 1989 SASSTAT Users Guide Version 6 4th Edition Volume 1 Cary NC SAS Institute Inc 943 p
SerratoValenti G Conara L Ferrando M Modenesi P 1993 Structural and histochemical features ofStylosanthes scabra (Leguminosae Papilionoidae) seed coat as related to water entry Can J Bot 71 834-840
Simpson BJ 1988 A field guide to Texas trees Austin TX Texas Monthly Press 372p
Tran VN Cavanagh AK 1984 Structural aspects ofdormancy In Seed Physiology Vol 2 Murray DR (ed) p1-44 Academic Press Australia
USDA Forest Service 1948 Woody-plant seed manual USDA Misc Pub1654 416p
Vines RA 1960 Trees Shrubs and Woody Vines of the Southwest Austin Univ ofTexas Press 1104 p
Wagner MR Larson RE Craig TP 1992 Ecology and silvics ofNew Mexico locust (Robinia neomexicana) with an emphasis on the arboreal form p53-57 In JW Hanover K Miller and S Plesko (eds) Proc Intemat Confon Black Locust Biology Culture and Utilization Mich State Univ USA
Wilson JK 1944 Immersing seeds of species of Robinia in boiling water hastens germination J For 42 453-454
Zimmerman RW Carpenter SB 1980 First-year coppice production from a 5shyyear old black locust stand on surface mine spoil p309-314 Centr Hardwd For ConfProc University ofMissouri Columbia
Untreated Treated
Figure 9 SEM images ofuntreated vs 4-minute percussion-treated strophiolar cells ofNew Mexico locust seeds Treated cells are loosened separated and raised
DISCUSSION
Determining the Optimal Treatment
Averaging results of scarification treatmems across seed sources is a useful
tool to predict which treatment is most likely to be successful for an unknown seed
source Averaged over all sources four- and eight-minute percussion treatments
resulted in the highest germination 815 and 790 respectively and produced
germination that was as fast or faster than all other treatments Although there was no
difference between these two treatments on average an argument for 4 minutes of
percussion as a standard protocol for treating New Mexico locust seeds can be made
Descriptively 4 minutes of percussion improved germination over 8 minutes
ofpercussion for a majority of sources (five out of nine) 8 minutes of percussion
improved germination for three ofnine sources with no difference for a fmal source
For individual sources 4 minutes ofpercussion was always the best or secondmiddot best
treatment and was always equal to or better than all hot water treatments
Germination at 8 minutes ofpercussion was lower than all other percussion durations
for the Jemez and Gila sources and was lower than at least one hot water treatment for
these sources as well
The x-ray radiographs of the Sacramento seed source illustrate the excessive
internal and external seed damage that can occur with 8 minutes of percussion
germination counts 8-minute percussed seeds from Holman and Capitan
ClUll signs of damage that were visible to the naked eye (see below)
r~evertheless some of these seeds still fit the criteria for germination Neither x-ray
36
photography nor visible observations during seed counts revealed seed damage at the
4-minute percussion level In operational trials seeds percussed for 4 minutes
developed into healthy seedlings (Khadduri and Harrington unpublished data)
Strophiole and Seed Coat Behavior
Four minutes ofpercussion successfully breaks seed dormancy in a large
percentage ofNew Mexico locust seeds without excessively damaging the
surrounding seed coat or internal seed coat structures Most papilionoid legume
seeds including seeds ofNew Mexico locust have a specific region of the seed coat
known as the strophiole or lens (Hamly 1932) The strophiole is located on the
cotyledonary lobe of a locust seed (Figure 10 a-c) If seeds are not indiscriminately
damaged the strophiole is the first point of water entry to the embryo (Hopkinson
1997) When a papilionoid seed is percussed for the appropriate amount of time
repeated hits on the integrated seed coat loosen the constrained cells of the strophiolar
region without excessively damaging the rest of the seed coat (Ballard 1976) SEM
photos illuStrate that 4 minutes of percussion loosened and separated strophiolar cells
ofNew Mexico locust seeds when compared with constrained organized strophiolar
cells of untreated seeds (Figure 9) Research-has shown that water passes to the
embryo through the treated strophiole in a controlled manner and regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues
(Kelly and van Staden 1987)
37
10 Strophiole ofunimbibed New Mexico locust seed (a) and following uptake (bc)
38
In contrast hot-water treatments can excessively degrade the seed coat
(Rebman et al 1998 Marunda 1990) Random cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rebman et aI 1998 Marunda 1990) Along with a
proportionately large amount ofun-imbibed (thus under-treated) seeds (Figure l1a)
damage was observed in hot-water treated seeds from six sources during germination
counts Damage included seeds where the radicle failed to elongate (Figure 11 b)
seeds where the radicle elongated but was delayed in freeing itself from the seed coat
(Figure 11c) and seeds where the radicle completely broke off from the embryo
(Figure lId) In addition some seeds germinated in reverse (the cotyledon emerged
fIrst) If the radicle emerged completely from the seed coat they were counted as
germinated
While nearly all seeds treated for 8 minutes ofpercussion imbibed water
damage was evident in x-ray evaluations (Sacramento seed source) and in
germination counts (Holman and Capitan) at this treatment level Seed damage
during germination counts was similar to levels found in hot-water treated seeds
suggesting that over-treatment by both hot water and longer durations of percussion
may result in similar damage to seed structure
The Sacramento and Holman seed sources were two of the fIve sources that
opcrplltlpn in germination from the 4- to 8-minute durations of percussion
the benefIts ofloosening the strophiole as evidenced by imbibition of
all 8-minute treated seeds were outweighed by external and internal seed coat
39
a
b c d
11 Damage to New Mexico locust seeds observed during germination cotmts following hot water scarification 9a Hot water replicate including ~t seedlings and numerous unimbibed seeds 9b Close-up of seed where raallCle failed to elongate 9c Close-up ofseed where radicle elongated but was
-gtIn in freeing itself from the seed coat 9d Close-up ofseed where radicle broke off from embryo
40
damage (which also may be contributing to high imbibition) For the three sources
for which 8 minutes of percussion resulted in maximum germination extended
percussion of seeds necessary to loosen the strophiole may not have excessively
damaged the seed anatomy The Manzano source did not significantly change in
germination from 4 to 8 minutes of percussion suggesting that an increase in
permeable seeds at the 8-minute treatment duration was balanced by the number of
seeds that failed to germinate due to seed damage To summarize the 4-minute
percussion treatment improved germination over 8 minutes ofpercussion for a
majority of sources (five ofnine) the 8-minute percussion treatment improved
germination over 4-minutes ofpercussion for three sources and there was no
difference with the Manzano source Notably for the Raton source 8 minutes of
percussion increased germination by 235 percentage points over 4-minutes of
percussion (7875 vs 5525 respectively) the next best treatment Raton was the
only source to result in germination less than 75 for 4 minutes ofpercussion so a
grower may wish to apply longer percussion durations to lots that do not respond well
to this treatment level
Finally there was an interesting trend in the Jemez and Raton sources seed
that significantly contributed to the source by treatment interaction (Figure 12) A
~u test showed that the Jemez source had a significantly lighter seed weight than
other sources while the Raton source had an average seed weight For the Jemez
the longest duration of boiling increased germination Conversely only short
U4UVll) of percussion were needed to achieve high germination and the longest
41
80 _ Jemez a Raton
iD
~ C iD e iD
Q c 0 ~
c ~ iD 0
20
0------shy
Immersion Boiling 30 sec Boiling 60 sec
Hot Water Scarification
10degTr========~--------~------------------------------
80
60
40
20
_ Jemez Raton
0------shy
1 min 2 min 4 min 8 min
Percussion Scarification
Figure 12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources
42
b
--~----~~~~========~---- duration ofpercussion reduced germination For the Raton source longer durations
ofboiling consistently decreased germination whereas longer percussion treatments
consistently increased germination
A possible explanation is that a thick seed coat is associated with a loose
strophiole structure and that a thin seed coat is associated with a constrained
strophiole This contrast merits further study and may suggest an ecological
adaptation Further investigation ofthis phenomenon may contribute to successful
pre-screening ofunknown seed sources to determine optimal treatment leveL
43
CONCLUSION
Based on results obtained in pilot study (see Appendix) and thesis
experiments 4 minutes ofpercussion is recommended as a standard protocol for
treating an unknown source ofNew Mexico locust seed In addition to laboratory
results the 4-minute percussion treatment has resulted in germination greater than
80 and healthy seedling grow-outs for production purposes (unpublished data) In
laboratory experiments only one hundred seeds per 118 ml (40z) soil tin were
percussed at a time whereas for production we have treated up to five hundred seeds
per tin For large-scale production ofNew Mexico locust seedlings growers may
wish to experiment with larger quantities of seed and larger-size soil tins
Seed storage trials following percussion treatments were not conducted
However germination greater than 80 and healthy seedlings were obtained when
seeds were percussion-treated and subsequently stored at room-temperature (22degC)
for one week (unpublished data) Seed storage trials merit further study as the
standard hot water treatment leaves seeds in a softened condition that is not
conducive to storage
Hydroseeding is an effective method for direct seeding in reclamation
situations Percussion treatment may be conducive to hydro seeding as seeds remain
a dry relatively undamaged yet permeable condition following treatment Sulfuric
seeds also remain dry following treatment but extensive damage to seed coat
poor seedling vigor have been reported with this treatment (Kelly and van Staden
A grow-out study using a hydro seeder on a reclamation site testing percussion
44
against other treatments such as sulfuric acid and mechanical scarification also
merits further study
Papilionoids are the largest subfamily oflegumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust these legumes often play an integral role in the revegetation of
disturbed lands The pilot study (Appendix) indicates that percussion scarification
successfully treats the widely used black locust as well Developing superior
scarification methods to the standard hot water mechanical scarification and acid
treatments may facilitate the use ofpapilionoid legumes in reclamation projects
45
APPENDICES
APPENDIX A
PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR
NEW MEXICO LOCUST AND BLACK LOCUST SEEDS
Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4
Hot water and sulfuric acid soaks are traditional treatments for seeds of many
temperate woody legwnes including locusts However these scarification techniques
often produce inconsistent germination Percussion scarification where seeds are
repeatedly propelled against a hard surface was compared with hot water
scarification to evaluate treatment efficacy for New Mexico locust (Robinia
neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment
seeds were placed in a 98degC water bath which was immediately removed from the
heat source For percussion scarification seeds were placed in a soil sample tin and
agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the
control were followed by 24-hour water soaks Hot water baths resulted in 56 and
41 germination for New Mexico locust and black locust respectively For both
species nearly all durations ofpercussion increased germination over the hot water
treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3
4 and 5 minutes for black locust resulted in at least 90 germination Traditional
scarification treatments randomly degrade the entire seed coat which can lead to
tissue damage during water uptake Percussion scarification specifically weakens the
strophiole the natural source ofwater entry to the seed in papilionoid legwnes
INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom
2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031
48
Following percussion imbibition is controlled through the strophiole and underlying
tissue is protected
Keywords New Mexico locust black locust scarification percussion germination
Introduction
Locust trees (Robinia spp) are aggressive pioneer species that quickly
colonize disturbed land fix atmospheric nitrogen in the soil and stabilize slopes that
are prone to erosion (Klemmedson 1994 Ashby et al 1985) (Figure A1ab)
Recognition of these attributes has contributed to increased demand for locusts in
reclamation projects Locust seeds however exhibit physical dormancy where a
thick seed coat prevents the movement of water and gasses to the embryo (Leadem
1997)
Sulfuric acid or hot water soaks have traditionally been used to break seed
dormancy in macro-propagation of locusts Many growers are moving away from the
use of su1furic acid in treating locust seeds (Dreesen and Harrington 1997) In
addition to being dangerous acid soak durations must be specifically correlated to
seed lot (Olson 1974) Collections of native seed can vary tremendously in seed size
seed weight and hard seededness etc This variability limits the utility of sulfuric
acid soaks as even within a given lot some seeds may be damaged whereas others
are under-treated and fail to imbibe (take up water) A survey of Southwestern
container growers indicates that hot water baths are currently the most common
scarification method for locust seeds (Hine et al 1997) However hot water baths
also produce inconsistent germination (Lin et al 1996)
49
b
Figure AI New Mexico locust a primary invader following disturbance Ala Crown-sprouting at edge ofburn near Los Alamos NM--one year after Cerro Grande fire Alb Colonizing road cut Gila National Forest NM
50
Percussion scarification where seeds are repeatedly propelled against a hard
surface is an alternative dormancy-breaking method that has proven successful in
legumes related to locusts (Hamly 1932 Barton 1947) We compared this alternative
treatment to hot water scarification to evaluate treatment efficacy for New Mexico
locust and black locust seeds
Materials and Methods
Commercial New Mexico locust seeds (Western Native Seed Coaldale CO)
were collected fall 2000 in Huerfano County CO Black locust seeds were collected
September 2000 from Taos County NM by harvesting ripe pods from several trees at
a distance of up to 3 m from ground level Black locust seeds were allowed to air-dry
for 3 weeks and were threshed and separated from large chaffusing a greenhouse
fan A South Dakota Seed Blower (Chicago Illinltois) was used to remove fine chaff
from both seed lots Seeds were storedmiddotat 2-4C until the germination study was
conducted in March 2001
This study compared hot water scarification with percussion scarification
using treatment and species as experimental factors Along with a control
scarification treatments included immersion of seed in boiling water as well as
percussion durations of 1 2 3 45 or 10 minutes Four 100-seed samples were used
to test each species by scarification treatment combination
For the hot water treatment test tubes were filled with 30 mL water and
placed in a water-filled beaker (Figure A2a) The bath was raised to boiling (-98C at
51
b
c
Figure A2 Implementation ofhot water and percussion scarification A2a Hot water bath A2b Paint-shaker set-up A2c Placing soil tin containing seeds at end ofpaint shaker ann
52
Las Cruces NM elevation of 1300 meters) Each 100-seed replicate was placed in a
test tube and each test tube was immediately removed to cool at room temperature
Percussion scarification was implemented using a pneumatic paint shaker
(Central Pneumatic model 00422 Camarillo CA) (Figure A2b) This allowed
standardization of treatment intensity Compressed air pressure was maintained at
530kPa plusmn 30kPa (80psi plusmn 5psi) resulting in approximately 350 oscillations per
minute Each lOO-seed sample was placed in a 118 ml (4 oz) soil tin for shaking To
maximize lateral movement of paint shaker a paint can was used as a spacer to place
soil tins at the end of the shaking arm (Figure A2c) This allowed shaking to occur at
the greatest distance from the pivot point (fulcrum) Following all treatments
including the control seeds were soaked for twenty-four hours in distilled water prior
to plating out
Gennination was evaluated on lab benches at room temperature Each
replicate of seeds was placed in a 100 cm petri dish on moistened filter paper with
humidity maintained by enclosing the petri dishes in ziplock bags Gennination was
monitored daily for 14 days in accordance with International Seed Testing
Association standards for Robinia species (ISTA 1999) Gennination was defined as
protrusion of the radicle from the embryo by at least 1mm as viewed by the naked
eye
Categorical Analysis of Variance using Prpc Catmod (SAS Institute 1989)
was used to detennine treatment differences in germination percentages for each
source This procedure is an extension of a Chi-square test of homogeneity using the
53
natural log of the ratio of germinated to non-germinated seeds for each treatment Pshy
values less than 005 were considered significant A limited set of pairwiseshy
comparisons was conducted to compare treatment mean percents using a
conservative alpha value of 005 divided by the number of comparisons
Results
Percussion maximized germination for both New Mexico locust and black
locust There were three germination response levels for New Mexico locust (Figure
A3) First there was a low response in the control indicating a dormant seed lot
The hot water treatment and I-minute duration of percussion share a second
intermediate response Germination was highest at percussion levels from 2 to 10
minutes
A selected percussion treatment intermediate in time (5 minutes) produced the
steepest germination curve indicating the fastest germination rate (Figure A4) The
hot water treatment produced an intermediate curve or relatively slower germination
rate The control curve was very flat indicating the slowest germination rate for New
Mexico locust
Control seeds germinated poorly for black locust as well (Figure A5) Hot
water scarification improved germination over the control but all levels of percussion
improved germination over hot water The optimal durations of percussion treatment
were bracketed with a significant increase from the 1 to 2 minute percussion level a
high germination response from 2 to 5 minutes of percussion and finally a significant
54
~ d 25 CL)
p t1 0 ~ t1
g Vl ~ Vl
100~---------------------------------------------------------------
80
60
40
20
o Control Hot Water Percussion
I min Percussion
2 min Percussion
3 min
Scarification
Percussion 4 min
Figure A3 Effect of scarification on germination response for New Mexico locust
Percussion 5 min
=significant difference between adjacent treatment bars ns = no significant difference
Percussion 10 min
~ ~ () u agt
Il-t d o ~
~ ~ c3
100middotlr~~~------------------------------------e- Percussion 5min middot 0middot Hot Water -- Control
80-J=middot======
6b-middot-lmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middot--middotmiddot-middot~-middotmiddot -middotmiddot middot middotmiddot-middot 1
40
20
-----~-----------
0 0 0 middotmiddotmiddotmiddotmiddot0middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot0
0 --~-----y-----~------_---~--- T-----~
o shy -TDay 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A4 Effect of selected scarification treatments on germination speed for New Mexico locust First seven days of fourteen-day experiment are shown
Day 7
80
~ cd
5 60 ()
[) Cl d 0 1t1 d
40sect dUl
-J
20
o
ns
Scarification
Figure A5 Effect of scarification on germination response for black locust = significant difference between adjacent treatment bars ns = no significant difference
decrease at the 10-minute level This drop-off suggests that 10 minutes of percussion
over-treats seeds from this lot of-black locust
A selected percussion treatment intermediate in time (5 minutes) also had the
fastest germination rate for black locust (Figure A6)_ The hot water curve is
somewhat flatter indicating a slower germination rate The control curve is very flat
indicating a slow germination rate
A qualitative difference between hot water and percussion-treated seeds was
also observed Along with healthy germinants the hot water treatment produced
over-treated seeds (Figure A 7a) Examples of damaged germinants include seeds
where the radicle did not elongate (Figure A 7b) seeds where the radicle elongated
but was delayed in freeing itself from the seed coat (Figure A7c) and seeds where the
radicle broke off completely from the embryo (Figure A7d) In addition to these
examples of over-treatment there were numerous small dark seeds that failed to
imbibe water For percussion treatment nearly all seeds took up water (Figure A8a)
Germination was rapid and uniform with little damage evident (Figure A8b)
To summarize both New Mexico locust and black locust seed lots were
dormant The standard hot water scarification treatment improved germination but
only to 56 and 41 for New Mexico locust and black locust respectively Nearly
all percussion durations improved total germination as well as germination rate over
the hot water treatment Germination was greater than 90 for percussion durations
of 4 5 and 10 minutes for New Mexico locust and 3 4 and 5 minutes for black locust
58
CI)
sgt t CI) 0
igt 0
sect ttl t E
Vl 0
c3
100r==~==~~=r------------------------------------~ ___ Percussion 5 min
middotmiddotmiddot0middotmiddotmiddot Hot Water -lIf- Control
80r=========~--~~----------------------------~
60
40
20 -~-~---~---------- ---~------------I
omiddot
O
_~_----------lIf-----~bull -----_ shy---shy I
Day 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A6 Effect of selected scarification treatments on germination speed for black locust First seven days of fourteen-day experiment are shown
Day 7
a
b c d
Figure A7 Damage to New Mexico locust seeds observed during germination counts following hot water scarification A7a Hot water replicate including damaged seedlings and numerous unimbibed seeds A7b Close-up ofseed where radicle failed to elongate A7c Close-up ofseed where radicle elongated but was delayed in freeing itself from the seed coat A7d Close-up ofseed where radicle completely broke offfrom embryo
60
a
b
Figure A8 New Mexico locust seeds following percussion treatment at time of germination counts A8a Percussion replicate with uniformly healthy seedlings and near complete imbibition of seeds A8b Close-up ofpercussion-treated seedling
61
Discussion
Papilionoid seeds have a specific anatomical feature known as the strophiole
or lens-the natural site of water entry to the seed (Hamly 1932) The strophiole is
located on the cotyledonary lobe of a locust seed (Figure A9) When a papilionoid
legume seed is percussed for the appropriate amount of time repeated hits on the
integrated seed coat loosen the constrained cells of the strophiolar region without
excessively damaging the rest of the seed coat (Ballard 1976)
When a percussed seed is soaked water enters exclusively through the
strophiole in a controlled manner (Kelly and Staden 1987) This regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues This
contrasts with seeds that have been hot water or acid scarified treatments that can
randomly degrade the seed coat Unlocalized cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rehman et al 1998 Marunda 1990)
Papilionoids are the largest subfamily of legumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust and black locust these legumes often play an integral role in the
revegetation ofdisturbed lands Developing superior scarification methods to the
standard hot water and acid treatments should fadlitate the use ofthese legumes in
restoration projects
62
Figure A9 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (bc)
63
APPENDIXB
GERMINATION PERCENTS AND STANDARD ERRORS FOR SOURCE
BLOCK AND TREATMENT FOR NEW MEXICO LOCUST
Table B 1 Gennination percents and standard errors for source block and treatment for New Mexico locust
Source Block Treatment Germination Percentage Standard Error
Capitan 1 Immersion 74 437 Capitan 2 Immersion 69 462 Capitan 3 Immersion 71 454 Capitan 4 Immersion 67 470 Capitan Boiling 30 sec 79 407 Capitan 2 Boiling 30 sec 76 427 Capitan 3 Boiling 30 sec 72 449 Capitan 4 Boiling 30 sec 76 427 Capitan Boiling 60 sec 71 454 Capitan 2 Boiling 60 sec 54 498 Capitan 3 Boiling 60 sec 67 470 Capitan 4 Boiling 60 sec 76 427 Capitan I Control 5 218 Capitan 2 Control 7 255 Capitan 3 Control 4 196 Capitan 4 Control 5 218 Capitan 1 Percussion I min 47 499 Capitan 2 Percussion I min
470
Capitan Percussion I min 39 488
Capitan 4 Percussion I min 38 485 Capitan I Percussion 2 min 55 497 Capitan Capitan
2
Percussion 2 min Percussion 2 min
71 65
454 477
Capitan 4 Percussion 2 min 56 496 Capitan I Percussion 4 min 86 347 Capitan 2 Percussion 4 min 79 407 Capitan 3 Percussion 4 min 81 392 Capitan 4 Percussion 4 min 82 384 Capitan I Percussion 8 min 90 300 Capitan 2 Percussion 8 min 88 325 Capitan 3 Percussion 8 min 90 300 Capitan 4 Percussion 8 min 90 300 Gila I Immersion 71 454 Gila 2 Immersion 77 421 Gila
Immersion 75 433
Gila 4 Immersion 72 449
Gila I Boiling 30 sec 68 466
Gila 2 Boiling 30 sec 81 392
Gila 3 Boiling 30 sec 54 498
Gila 4 Boiling 30 sec 63 483
Gila I Boiling 60 sec 55 497
Gila 2 Boiling 60 sec 68 466
Gila 3 Boiling 60 sec 57 495
Gila 4 Boiling 60 sec 70 458
65
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Gila 2 Control 13 336 Gila 3 Control 13 336 Gila 4 Control 20 400 Gila 1 Percussion 1 min 66 474 Gila 2 Percussion 1 min 57 495 Gila 3 Percussion 1 min 73 444 Gila 4 Percussion 1 min 82 384 Gila 1 Percussion 2 min 81 392 Gila 2 Percussion 2 min 86 347 Gila 3 Percussion 2 min 85 357 Gila 4 Percussion 2 min 54 498 Gila 1 Percussion 4 min 79 407 Gila 2 Percussion 4 min 83 376 Gila 3 Percussion 4 min 84 367 Gila 4 Percussion 4 min 79 407 Gila 1 Percussion 8 min 51 500 Gila 2 Percussion 8 min 76 427 Gila 3 Percussion 8 min 52 500 Gila 4 Percussion 8 min 74 439 Holman 1 Immersion 83 376 Holman 2 Immersion 86 347 Holman 3 Immersion 81 392 Holman 4 Immersion 90 300 Holman 1 Boiling 30 sec 90 300 Holman 2 Boiling 30 sec 85 357 Holman 3 Boiling 30 sec 87 336 Holman 4 Boiling 30 sec 83 376 Holman 1 Boiling 60 sec 79 407 Holman 2 Boiling 60 sec 80 400 Holman 3 Boiling 60 sec 86 347 Holman 4 Boiling 60 sec 93 255 Holman Control 25 433 Holman 2 Control 19 392 Holman Control 21 407
Holman 4 Control 24 427
Holman Percussion 1 min 77 421
Holman 2 Percussion 1 min 85 357
Holman 3 Percussion 1 min 80 400
Holman 4 Percussion 1 min 89 313
Holman 1 Percussion 2 min 88 325
Holman 2 Percussion 2 min 96 196
Holman 3 Percussion 2 min 93 255
Holman 4 Percussion 2 min 96 196
Holman 1 Percussion 4 min 92 271
Holman 2 Percussion 4 min 97 171
66
I
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Holman 3 Percussion 4 min 91 286 Holman 4 Percussion 4 min 96 196 Holman 1 Percussion 8 min 88 325 Holman 2 Percussion 8 min 85 357 Holman 3 Percussion 8 min 83 376 Holman 4 Percussion 8 min 82 384 Jemez 1 Immersion 53 499 Jemez 2 Immersion 44 496 Jemez 3 Immersion 52 500 Jemez 4 Immersion 70 458 Jemez 1 Boiling 30 sec 58 494 Jemez 2 Boiling 30 sec 55 497 Jemez 3 Boiling 30 sec 48 500 Jemez 4 Boiling 30 sec 65 477 Jemez 1 Boiling 60 sec 68 466 Jemez 2 Boiling 60 sec 63 483 Jemez 3 Boiling 60 sec 73 444 Jemez 4 Boiling 60 sec 89 313 Jemez 1 Control 2 140 Jemez 2 Control 9 286 Jemez 3 Control 6 237 Jemez 4 Control 8 271 Jemez 1 Percussion 1 min 58 494 Jemez 2 Percussion 1 min 63 483 Jemez 3 Percussion 1 min 77 421 Jemez 4 Percussion 1 min 89 313 Jemez 1 Percussion 2 min 68 466 Jemez 2 Percussion 2 min 64 480 Jemez 3 Percussion 2 min 80 400 Jemez 4 Percussion 2 min 83 376 Jemez Percussion 4 min 68 466 Jemez 2 Percussion 4 min 79 407 Jemez 3 Percussion 4 min 70 458 Jemez 4 Percussion 4 min 83 376 Jemez 1 Percussion 8 min 52 500 Jemez 2 Percussion 8 min 59 492 Jemez 3 Percussion 8 min 61 488 Jemez 4 Percussion 8 min 70 458 Magdalena 1 Immersion 69 462 Magdalena 2 Immersion 69 462 Magdalena J Immersion 70 458 Magdalena 4 Immersion 85 357 Magdalena 1 Boiling 30 sec 65 477 Magdalena 2 Boiling 30 sec 71 454 Magdalena 3 Boiling 30 sec 68 466
67
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Magdalena 4 Boiling 30 sec 83 376 Magdalena 1 Boiling 60 sec 63 483 Magdalena 2 Boiling 60 sec 68 466 Magdalena 3 Boiling 60 sec 63 483 Magdalena 4 Boiling 60 sec 82 384 Magdalena 1 Control 11 313 Magdalena 2 Control 6 237 Magdalena 3 Control 5 218 Magdalena 4 Control 4 196 Magdalena Percussion 1 min 32 466 Magdalena 2 Percussion 1 min 45 497 Magdalena 3 Percussion 1 min 46 498 Magdalena 4 Percussion I min 36 480 Magdalena 1 Percussion 2 min 67 470 Magdalena 2 Percussion 2 min 68 466 Magdalena 3 Percussion 2 min 65 477 Magdalena 4 Percussion 2 min 81 392 Magdalena 1 Percussion 4 min 87 336 Magdalena 2 Percussion 4 min 97 171 Magdalena 3 Percussion 4 min 83 376 Magdalena 4 Percussion 4 min 97 171 Magdalena 1 Percussion 8 min 98 lAO Magdalena 2 Percussion 8 min 100 000 Magdalena 3 Percussion 8 min 96 196 Magdalena 4 Percussion 8 min 98 lAO Manzano 1 Immersion 67 470 Manzano 2 Immersion 57 495 Manzano 3 Immersion 72 4049 Manzano 4 Immersion 73 4044 Manzano 1 Boiling 30 sec 70 458 Manzano 2 Boiling 30 sec 64 480 Manzano 3 Boiling 30 sec 71 454 Manzano 4 Boiling 30 sec 80 400 Manzano I Boiling 60 sec 50 500 Manzano 2 Boiling 60 sec 53 499 Manzano 3 Boiling 60 sec 59 492 Manzano 4 Boiling 60 sec 74 439 Manzano I Control 4 196 Manzano 2 Control 8 271 Manzano 3 Control 1 099 Manzano 4 Control 6 237 Manzano 1 Percussion 1 min 52 500 Manzano 2 Percussion 1 min 39 488 Manzano 3 Percussion 1 min 36 480 Manzano 4 Percussion 1 min 51 500
68
Table B 1 Continued
Source Block Treatment Gerrni~ation Percentage Standard Error
Manzano Percussion 2 min 53 499 Manzano 2 Percussion 2 min 58 494 Manzano 3 Percussion 2 min 61 488 Manzano 4 Percussion 2 min 62 485 Manzano 1 Percussion 4 min 76 427 Manzano 2 Percussion 4 min 85 357 Manzano 3 Percussion 4 min 79 407 Manzano 4 Percussion 4 min 83 376 Manzano 1 Percussion 8 min 87 336 Manzano 2 Percussion 8 min 83 376 Manzano 3 Percussion 8 min 83 376 Manzano 4 Percussion 8 min 75 433 Raton 1 Immersion 54 498 Raton 2 Immersion 37 483 Raton 3 Immersion 39 488 Raton 4 Immersion 69 462 Raton 1 Boiling 30 sec 36 480 Raton 2 Boiling 30 sec 20 400 Raton 3 Boiling 30 sec 28 449 Raton 4 Boiling 30 sec 60 490 Raton 1 Boiling 60 sec 15 357 Raton 2 Boiling 60 sec 171 Raton 3 Boiling 60 sec 4 196 Raton 4 Boiling 60 sec 35 477 Raton 1 Control 4 196 Raton 2 Control 12 325 Raton 3 Control 2 140 Raton 4 Control 6 237 Raton 1 Percussion 1 min 52 500 Raton 2 Percussion I min 29 454 Raton 3 Percussion I min 35 477 Raton 4 Percussion 1 min 31 462 Raton 1 Percussion 2 min 55 497 Raton 2 Percussion 2 min 35 477 Raton 3 Percussion 2 min 33 470 Raton 4 Percussion 2 min 42 494 Raton 1 Percussion 4 min 65 477 Raton 2 Percussion 4 min 52 500 Raton Percussion 4 min 51 500 Raton 4 Percussion 4 min 53 499 Raton 1 Percussion 8 min 88 325 Raton 2 Percussion 8 min 80 400 Raton 3 Percussion 8 min 73 444 Raton 4 Percussion 8 min 74 439 Sacramento 1 Immersion 43 495
69
Table B 1 Continued
-Source Block Treatment Germination Percentage Standard Error
Sacramento 2 Immersion 30 458 Sacramento 3 Immersion 38 485 Sacramento 4 Immersion 56 496 Sacramento 1 Boiling 30 sec 47 499 Sacramento 2 Boiling 30 sec 48 500 Sacramento 3 Boiling 30 sec 48 500 Sacramento 4 Boiling 30 sec 56 496 Sacramento 1 Boiling 60 sec 52 500 Sacramento 2 Boiling 60 sec 67 470 Sacramento 3 Boiling 60 sec 43 495 Sacramento 4 Boiling 60 sec 48 500 Sacramento 1 Control 0 000 Sacramento 2 Control 3 171 Sacramento 3 Control 3 171 Sacramento 4 Control 099 Sacramento 1 Percussion 1 min 57 495 Sacramento 2 Percussion 1 min 61 488 Sacramento 3 Percussion 1 min 62 485 Sacramento 4 Percussion 1 min 55 497 Sacramento 1 Percussion 2 min 84 367 Sacramento 2 Percussion 2 min 70 458 Sacramento 3 Percussion 2 min 81 392 Sacramento 4 Percussion 2 min 69 462 Sacramento 1 Percussion 4 min 91 286 Sacramento 2 Percussion 4 min 84 367 Sacramento 3 Percussion 4 min 84 367 Sacramento 4 Percussion 4 min 94 237 Sacramento 1 Percussion 8 min 82 384 Sacramento 2 Percussion 8 min 81 392 Sacramento 3 Percussion 8 min 82 384 Sacramento 4 Percussion 8 min 81 392 Sandia 1 Immersion 76 427 Sandia 2 Immersion 70 458 Sandia 3 Immersion 70 458 Sandia 4 Immersion 81 392 Sandia 1 Boiling 30 sec 78 414 Sandia 2 Boiling 30 sec 71 454 Sandia 3 Boiling 30 sec 63 483 Sandia 4 Boiling 30 sec 78 414 Sandia 1 Boiling 60 sec 67 470 Sandia 2 Boiling 60 sec 65 477 Sandia 3 Boiling 60 sec 74 439 Sandia 4 Boiling 60 sec 66 474 Sandia 1 Control 16 367 Sandia 2 Control 7 255
70
Table B 1 Continued
Source Block Treatment Gennination Percentage Standard Error
Sandia 3 Control 8 271 Sandia 4 Control 16 367 Sandia 1 Percussion 1 min 67 470 Sandia 2 Percussion 1 min 61 488 Sandia 3 Percussion 1 min 66 474 Sandia 4 Percussion 1 min 59 492 Sandia I Percussion 2 min 82 384 Sandia 2 Percussion 2 min 83 376 Sandia 3 Percussion 2 min 81 392 Sandia 4 Percussion 2 min 84 367 Sandia 1 Percussion 4 min 89 313 Sandia 2 Percussion 4 min 86 347 Sandia 3 Percussion 4 min 89 313 Sandia 4 Percussion 4 min 85 357 Sandia 1 Percussion 8 min 80 400 Sandia 2 Percussion 8 min 77 421 Sandia 3 Percussion 8 min 69 462 Sandia 4 Percussion 8 min 66 474
LITERATURE CITED
Ashby WC Vogel WG Rogers NF 1985 Black locust in the reclamation equation General Technical Report Northeastern Forest Experiment Station USDA Forest Service NE-I05 12 p
Ballard LAT 1973 Physical barriers to gennination Seed Sci Technoll 285-303
___ 1976 Strophiolar water conduction in the seeds ofTrifolieae induced by action on the testa at nonstrophiolar sites Austr J Plant Physiol3 465-469
Barton LV 1947 Special studies on seed coat impenneability Contrib Boyce Thompson Inst 14 355-362
Baskin CC Baskin JM 1998 Seeds Ecology biogeography and evolution of donnancy and gennination Academic Press London UK 666 p
Bewley JD 1997 Seed germination and donnancy Plant Ce119 1055-1066
Bewley JD Black M 1994 Seeds physiology of development and germination 2nd ed Plenum Press New York NY 445 p
Briscoe CB 1996 Growing Nitrogen Fixing Trees in Acid Soils In MH Powell (ed) Nitrogen Fixing Trees for Acid Soils- A Field ManuaL Winrock International Morrilton AR 22 p
Copeland LO McDonald MB 1995 Principles of Seed Science and Technology 3rded Chapman amp Hall New York NY 409 p
Cox RE Klett JE 1984 Seed gennination requirements of native Colorado plants for use in the landscape Plant Propag 30(2) 6-10
Dell B 1980 Structure and function ofthe strophiolar plug in seeds ofAlbizia Lophantha Am J Bot 67 556-563
Dick-Peddie WA 1993 New Mexico Vegetation Past Present and Future University ofNew Mexico Press Albuquerque NM 244 p
Dreesen DR 2001 Propagation protocol for production ofcontainer Robinia neomexicana plants Los Lunas Plant Materials Center Los Lunas New Mexico In Native Plant Network URL httpwwwnativeplantnetworkorg (accessed 9 January 2002) Moscow (ID) University ofIdaho CoIl Natural Resources For Res Nursery
72
Dreesen DR Harrington JT 1997 Propagation of native plants for restoration projects in the Southwestern US- Preliminary Investigations In Landis TD Thompson JR tech coords National Proceedings Forest and Conservation Nursery Assocs Gen Tech Rep PNW-GTR-419 Portland OR USDA For Ser PNW Res Sta 77-88
Gottfried GJ 1980 Control ofNew Mexican locust and the effect on planted ponderosa pine in central Arizona US For Ser Res Note RM-386 Rocky Mountain For Range ExpStn Fort Collins CO
Gosling PG Samuel YK Jones SK 1995 A systematic examination of germination temperature chipping and water temperaturesoak duration pretreatments on the seeds ofLeucaena leucocephala Seed Sci amp Technol 23521-532
Hamly DH 1932 Softening of the seeds of Melilotus alba Bot Gazette 93 345shy375
Hanna PJ 1984 Anatomical features of the seed coat ofAcacia kempeana (Mueller) which relate to increased germination rate induced by heat treatment New Phytol 96 23-29
Hanks JP Dick-Peddie WA 1974 Vegetation patterns of the White Mountains New Mexico Southwest Naturalist 18(4) 371-382
Hine S Harrington JT Dreesen DR 1997 Seed scarification requirements for Robinia neomexicana In Harrington JTtech coord Proc Third SouthwestemContainer Growers Conf Feb 12-13 1997 Ft Collins CO
Hopkinson JM 1993 The strophiole of leguminous seeds Newsletter- IntI Herbage Seed Production Res Grp 18 7-8
---- 1997 Differences in hardseededness between Cunningham and Tarramba leucaena In Leucnet News The Newsletter of the International LeucaenaRes and Devlt Netwk No4
ISTA (International Seed Testing Association) 1999 International rules for seed testing rules Seed Sci amp Technol27 Supplement
Kelly KM van Staden J 1987 The lens as the site of permeability in the papilionoid seed Aspalathus linearis J Plant Physiol 128(45) 395-404
73
Keresztesi B 1988 Black locust the tree of agriculture Outlook on Agr 17(2) 77~85
Kigel 1 and Galili G 1995 Seed development and germination Marcel Dekker New York 853 p
Klemmedson 10 1994 New Mexico locust and parent material Influence on macronutrients of forest floor and soil Soil Sci Soc Am 1 58 974~980
Kuo WH Tam AW 1988 The pathway of water absorption of mung bean seeds Seed Sci amp Technol16(1) 139~144
Leadem CL 1997 Dormancy--unlocking seed secrets In Landis TD Thompson lR tech coords National Proceedings Forestand Conservation Nursery Assocs Gen Tech Rep PNW-G TR-419 Portland OR USDA For Ser PNW Res Sta 43-52
Lin Y Wagner MR Seweyestewa A Honahni MD DeGomez M McMillin JD 1996 Influence of cultural treatments on seed germination and seedling growth of Robinia neomexicana Forest Farm and Community Tree Res Reports Volume 1
Marunda CT 1990 Effects of seed pretreatment on the development ofAcacia auriculforinis and A holosericea seedlings ACIAR Proceedings Series No 28
Meginnis HG 1937 Sulphuric acid treatment to increase germination of black locust seed USDA Circ 453 34 p
Miklas PN Towensend CE Ladd SL 1987 Seedcoat anatomy and the scarification ofcicer milkvetch seed Crop Sci 27 766-772
Monsen SB 1984 Use of shrubs on mine spoils In The Challenge ofProducing Native Plants for the Intermountain Area Proceedings Intermountain Nurserymans Association 1983 conference Las Vegas NV compo PM Murphy 26-31 Ogden UT USDA For Ser Gen Tech Report INT-l68
Olson DF 1974 Robinia L Locust In Seeds of Woody Plants in the US tech coord CS Schopmeyer 728-731 USDA For Ser Agr Handbk No450
Rehman S Loescher RNl Harris P1C 1998 Dormancy breaking and germination ofAcacia salicina Lindl seeds Seed Sci amp Techno127 553-557
Rolston MP 1978 Water impermeable seed dormancy Bot Rev 44 365-396
74
SAS Institute Inc 1989 SASSTAT Users Guide Version 6 4th Edition Volume 1 Cary NC SAS Institute Inc 943 p
SerratoValenti G Conara L Ferrando M Modenesi P 1993 Structural and histochemical features ofStylosanthes scabra (Leguminosae Papilionoidae) seed coat as related to water entry Can J Bot 71 834-840
Simpson BJ 1988 A field guide to Texas trees Austin TX Texas Monthly Press 372p
Tran VN Cavanagh AK 1984 Structural aspects ofdormancy In Seed Physiology Vol 2 Murray DR (ed) p1-44 Academic Press Australia
USDA Forest Service 1948 Woody-plant seed manual USDA Misc Pub1654 416p
Vines RA 1960 Trees Shrubs and Woody Vines of the Southwest Austin Univ ofTexas Press 1104 p
Wagner MR Larson RE Craig TP 1992 Ecology and silvics ofNew Mexico locust (Robinia neomexicana) with an emphasis on the arboreal form p53-57 In JW Hanover K Miller and S Plesko (eds) Proc Intemat Confon Black Locust Biology Culture and Utilization Mich State Univ USA
Wilson JK 1944 Immersing seeds of species of Robinia in boiling water hastens germination J For 42 453-454
Zimmerman RW Carpenter SB 1980 First-year coppice production from a 5shyyear old black locust stand on surface mine spoil p309-314 Centr Hardwd For ConfProc University ofMissouri Columbia
DISCUSSION
Determining the Optimal Treatment
Averaging results of scarification treatmems across seed sources is a useful
tool to predict which treatment is most likely to be successful for an unknown seed
source Averaged over all sources four- and eight-minute percussion treatments
resulted in the highest germination 815 and 790 respectively and produced
germination that was as fast or faster than all other treatments Although there was no
difference between these two treatments on average an argument for 4 minutes of
percussion as a standard protocol for treating New Mexico locust seeds can be made
Descriptively 4 minutes of percussion improved germination over 8 minutes
ofpercussion for a majority of sources (five out of nine) 8 minutes of percussion
improved germination for three ofnine sources with no difference for a fmal source
For individual sources 4 minutes ofpercussion was always the best or secondmiddot best
treatment and was always equal to or better than all hot water treatments
Germination at 8 minutes ofpercussion was lower than all other percussion durations
for the Jemez and Gila sources and was lower than at least one hot water treatment for
these sources as well
The x-ray radiographs of the Sacramento seed source illustrate the excessive
internal and external seed damage that can occur with 8 minutes of percussion
germination counts 8-minute percussed seeds from Holman and Capitan
ClUll signs of damage that were visible to the naked eye (see below)
r~evertheless some of these seeds still fit the criteria for germination Neither x-ray
36
photography nor visible observations during seed counts revealed seed damage at the
4-minute percussion level In operational trials seeds percussed for 4 minutes
developed into healthy seedlings (Khadduri and Harrington unpublished data)
Strophiole and Seed Coat Behavior
Four minutes ofpercussion successfully breaks seed dormancy in a large
percentage ofNew Mexico locust seeds without excessively damaging the
surrounding seed coat or internal seed coat structures Most papilionoid legume
seeds including seeds ofNew Mexico locust have a specific region of the seed coat
known as the strophiole or lens (Hamly 1932) The strophiole is located on the
cotyledonary lobe of a locust seed (Figure 10 a-c) If seeds are not indiscriminately
damaged the strophiole is the first point of water entry to the embryo (Hopkinson
1997) When a papilionoid seed is percussed for the appropriate amount of time
repeated hits on the integrated seed coat loosen the constrained cells of the strophiolar
region without excessively damaging the rest of the seed coat (Ballard 1976) SEM
photos illuStrate that 4 minutes of percussion loosened and separated strophiolar cells
ofNew Mexico locust seeds when compared with constrained organized strophiolar
cells of untreated seeds (Figure 9) Research-has shown that water passes to the
embryo through the treated strophiole in a controlled manner and regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues
(Kelly and van Staden 1987)
37
10 Strophiole ofunimbibed New Mexico locust seed (a) and following uptake (bc)
38
In contrast hot-water treatments can excessively degrade the seed coat
(Rebman et al 1998 Marunda 1990) Random cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rebman et aI 1998 Marunda 1990) Along with a
proportionately large amount ofun-imbibed (thus under-treated) seeds (Figure l1a)
damage was observed in hot-water treated seeds from six sources during germination
counts Damage included seeds where the radicle failed to elongate (Figure 11 b)
seeds where the radicle elongated but was delayed in freeing itself from the seed coat
(Figure 11c) and seeds where the radicle completely broke off from the embryo
(Figure lId) In addition some seeds germinated in reverse (the cotyledon emerged
fIrst) If the radicle emerged completely from the seed coat they were counted as
germinated
While nearly all seeds treated for 8 minutes ofpercussion imbibed water
damage was evident in x-ray evaluations (Sacramento seed source) and in
germination counts (Holman and Capitan) at this treatment level Seed damage
during germination counts was similar to levels found in hot-water treated seeds
suggesting that over-treatment by both hot water and longer durations of percussion
may result in similar damage to seed structure
The Sacramento and Holman seed sources were two of the fIve sources that
opcrplltlpn in germination from the 4- to 8-minute durations of percussion
the benefIts ofloosening the strophiole as evidenced by imbibition of
all 8-minute treated seeds were outweighed by external and internal seed coat
39
a
b c d
11 Damage to New Mexico locust seeds observed during germination cotmts following hot water scarification 9a Hot water replicate including ~t seedlings and numerous unimbibed seeds 9b Close-up of seed where raallCle failed to elongate 9c Close-up ofseed where radicle elongated but was
-gtIn in freeing itself from the seed coat 9d Close-up ofseed where radicle broke off from embryo
40
damage (which also may be contributing to high imbibition) For the three sources
for which 8 minutes of percussion resulted in maximum germination extended
percussion of seeds necessary to loosen the strophiole may not have excessively
damaged the seed anatomy The Manzano source did not significantly change in
germination from 4 to 8 minutes of percussion suggesting that an increase in
permeable seeds at the 8-minute treatment duration was balanced by the number of
seeds that failed to germinate due to seed damage To summarize the 4-minute
percussion treatment improved germination over 8 minutes ofpercussion for a
majority of sources (five ofnine) the 8-minute percussion treatment improved
germination over 4-minutes ofpercussion for three sources and there was no
difference with the Manzano source Notably for the Raton source 8 minutes of
percussion increased germination by 235 percentage points over 4-minutes of
percussion (7875 vs 5525 respectively) the next best treatment Raton was the
only source to result in germination less than 75 for 4 minutes ofpercussion so a
grower may wish to apply longer percussion durations to lots that do not respond well
to this treatment level
Finally there was an interesting trend in the Jemez and Raton sources seed
that significantly contributed to the source by treatment interaction (Figure 12) A
~u test showed that the Jemez source had a significantly lighter seed weight than
other sources while the Raton source had an average seed weight For the Jemez
the longest duration of boiling increased germination Conversely only short
U4UVll) of percussion were needed to achieve high germination and the longest
41
80 _ Jemez a Raton
iD
~ C iD e iD
Q c 0 ~
c ~ iD 0
20
0------shy
Immersion Boiling 30 sec Boiling 60 sec
Hot Water Scarification
10degTr========~--------~------------------------------
80
60
40
20
_ Jemez Raton
0------shy
1 min 2 min 4 min 8 min
Percussion Scarification
Figure 12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources
42
b
--~----~~~~========~---- duration ofpercussion reduced germination For the Raton source longer durations
ofboiling consistently decreased germination whereas longer percussion treatments
consistently increased germination
A possible explanation is that a thick seed coat is associated with a loose
strophiole structure and that a thin seed coat is associated with a constrained
strophiole This contrast merits further study and may suggest an ecological
adaptation Further investigation ofthis phenomenon may contribute to successful
pre-screening ofunknown seed sources to determine optimal treatment leveL
43
CONCLUSION
Based on results obtained in pilot study (see Appendix) and thesis
experiments 4 minutes ofpercussion is recommended as a standard protocol for
treating an unknown source ofNew Mexico locust seed In addition to laboratory
results the 4-minute percussion treatment has resulted in germination greater than
80 and healthy seedling grow-outs for production purposes (unpublished data) In
laboratory experiments only one hundred seeds per 118 ml (40z) soil tin were
percussed at a time whereas for production we have treated up to five hundred seeds
per tin For large-scale production ofNew Mexico locust seedlings growers may
wish to experiment with larger quantities of seed and larger-size soil tins
Seed storage trials following percussion treatments were not conducted
However germination greater than 80 and healthy seedlings were obtained when
seeds were percussion-treated and subsequently stored at room-temperature (22degC)
for one week (unpublished data) Seed storage trials merit further study as the
standard hot water treatment leaves seeds in a softened condition that is not
conducive to storage
Hydroseeding is an effective method for direct seeding in reclamation
situations Percussion treatment may be conducive to hydro seeding as seeds remain
a dry relatively undamaged yet permeable condition following treatment Sulfuric
seeds also remain dry following treatment but extensive damage to seed coat
poor seedling vigor have been reported with this treatment (Kelly and van Staden
A grow-out study using a hydro seeder on a reclamation site testing percussion
44
against other treatments such as sulfuric acid and mechanical scarification also
merits further study
Papilionoids are the largest subfamily oflegumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust these legumes often play an integral role in the revegetation of
disturbed lands The pilot study (Appendix) indicates that percussion scarification
successfully treats the widely used black locust as well Developing superior
scarification methods to the standard hot water mechanical scarification and acid
treatments may facilitate the use ofpapilionoid legumes in reclamation projects
45
APPENDICES
APPENDIX A
PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR
NEW MEXICO LOCUST AND BLACK LOCUST SEEDS
Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4
Hot water and sulfuric acid soaks are traditional treatments for seeds of many
temperate woody legwnes including locusts However these scarification techniques
often produce inconsistent germination Percussion scarification where seeds are
repeatedly propelled against a hard surface was compared with hot water
scarification to evaluate treatment efficacy for New Mexico locust (Robinia
neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment
seeds were placed in a 98degC water bath which was immediately removed from the
heat source For percussion scarification seeds were placed in a soil sample tin and
agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the
control were followed by 24-hour water soaks Hot water baths resulted in 56 and
41 germination for New Mexico locust and black locust respectively For both
species nearly all durations ofpercussion increased germination over the hot water
treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3
4 and 5 minutes for black locust resulted in at least 90 germination Traditional
scarification treatments randomly degrade the entire seed coat which can lead to
tissue damage during water uptake Percussion scarification specifically weakens the
strophiole the natural source ofwater entry to the seed in papilionoid legwnes
INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom
2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031
48
Following percussion imbibition is controlled through the strophiole and underlying
tissue is protected
Keywords New Mexico locust black locust scarification percussion germination
Introduction
Locust trees (Robinia spp) are aggressive pioneer species that quickly
colonize disturbed land fix atmospheric nitrogen in the soil and stabilize slopes that
are prone to erosion (Klemmedson 1994 Ashby et al 1985) (Figure A1ab)
Recognition of these attributes has contributed to increased demand for locusts in
reclamation projects Locust seeds however exhibit physical dormancy where a
thick seed coat prevents the movement of water and gasses to the embryo (Leadem
1997)
Sulfuric acid or hot water soaks have traditionally been used to break seed
dormancy in macro-propagation of locusts Many growers are moving away from the
use of su1furic acid in treating locust seeds (Dreesen and Harrington 1997) In
addition to being dangerous acid soak durations must be specifically correlated to
seed lot (Olson 1974) Collections of native seed can vary tremendously in seed size
seed weight and hard seededness etc This variability limits the utility of sulfuric
acid soaks as even within a given lot some seeds may be damaged whereas others
are under-treated and fail to imbibe (take up water) A survey of Southwestern
container growers indicates that hot water baths are currently the most common
scarification method for locust seeds (Hine et al 1997) However hot water baths
also produce inconsistent germination (Lin et al 1996)
49
b
Figure AI New Mexico locust a primary invader following disturbance Ala Crown-sprouting at edge ofburn near Los Alamos NM--one year after Cerro Grande fire Alb Colonizing road cut Gila National Forest NM
50
Percussion scarification where seeds are repeatedly propelled against a hard
surface is an alternative dormancy-breaking method that has proven successful in
legumes related to locusts (Hamly 1932 Barton 1947) We compared this alternative
treatment to hot water scarification to evaluate treatment efficacy for New Mexico
locust and black locust seeds
Materials and Methods
Commercial New Mexico locust seeds (Western Native Seed Coaldale CO)
were collected fall 2000 in Huerfano County CO Black locust seeds were collected
September 2000 from Taos County NM by harvesting ripe pods from several trees at
a distance of up to 3 m from ground level Black locust seeds were allowed to air-dry
for 3 weeks and were threshed and separated from large chaffusing a greenhouse
fan A South Dakota Seed Blower (Chicago Illinltois) was used to remove fine chaff
from both seed lots Seeds were storedmiddotat 2-4C until the germination study was
conducted in March 2001
This study compared hot water scarification with percussion scarification
using treatment and species as experimental factors Along with a control
scarification treatments included immersion of seed in boiling water as well as
percussion durations of 1 2 3 45 or 10 minutes Four 100-seed samples were used
to test each species by scarification treatment combination
For the hot water treatment test tubes were filled with 30 mL water and
placed in a water-filled beaker (Figure A2a) The bath was raised to boiling (-98C at
51
b
c
Figure A2 Implementation ofhot water and percussion scarification A2a Hot water bath A2b Paint-shaker set-up A2c Placing soil tin containing seeds at end ofpaint shaker ann
52
Las Cruces NM elevation of 1300 meters) Each 100-seed replicate was placed in a
test tube and each test tube was immediately removed to cool at room temperature
Percussion scarification was implemented using a pneumatic paint shaker
(Central Pneumatic model 00422 Camarillo CA) (Figure A2b) This allowed
standardization of treatment intensity Compressed air pressure was maintained at
530kPa plusmn 30kPa (80psi plusmn 5psi) resulting in approximately 350 oscillations per
minute Each lOO-seed sample was placed in a 118 ml (4 oz) soil tin for shaking To
maximize lateral movement of paint shaker a paint can was used as a spacer to place
soil tins at the end of the shaking arm (Figure A2c) This allowed shaking to occur at
the greatest distance from the pivot point (fulcrum) Following all treatments
including the control seeds were soaked for twenty-four hours in distilled water prior
to plating out
Gennination was evaluated on lab benches at room temperature Each
replicate of seeds was placed in a 100 cm petri dish on moistened filter paper with
humidity maintained by enclosing the petri dishes in ziplock bags Gennination was
monitored daily for 14 days in accordance with International Seed Testing
Association standards for Robinia species (ISTA 1999) Gennination was defined as
protrusion of the radicle from the embryo by at least 1mm as viewed by the naked
eye
Categorical Analysis of Variance using Prpc Catmod (SAS Institute 1989)
was used to detennine treatment differences in germination percentages for each
source This procedure is an extension of a Chi-square test of homogeneity using the
53
natural log of the ratio of germinated to non-germinated seeds for each treatment Pshy
values less than 005 were considered significant A limited set of pairwiseshy
comparisons was conducted to compare treatment mean percents using a
conservative alpha value of 005 divided by the number of comparisons
Results
Percussion maximized germination for both New Mexico locust and black
locust There were three germination response levels for New Mexico locust (Figure
A3) First there was a low response in the control indicating a dormant seed lot
The hot water treatment and I-minute duration of percussion share a second
intermediate response Germination was highest at percussion levels from 2 to 10
minutes
A selected percussion treatment intermediate in time (5 minutes) produced the
steepest germination curve indicating the fastest germination rate (Figure A4) The
hot water treatment produced an intermediate curve or relatively slower germination
rate The control curve was very flat indicating the slowest germination rate for New
Mexico locust
Control seeds germinated poorly for black locust as well (Figure A5) Hot
water scarification improved germination over the control but all levels of percussion
improved germination over hot water The optimal durations of percussion treatment
were bracketed with a significant increase from the 1 to 2 minute percussion level a
high germination response from 2 to 5 minutes of percussion and finally a significant
54
~ d 25 CL)
p t1 0 ~ t1
g Vl ~ Vl
100~---------------------------------------------------------------
80
60
40
20
o Control Hot Water Percussion
I min Percussion
2 min Percussion
3 min
Scarification
Percussion 4 min
Figure A3 Effect of scarification on germination response for New Mexico locust
Percussion 5 min
=significant difference between adjacent treatment bars ns = no significant difference
Percussion 10 min
~ ~ () u agt
Il-t d o ~
~ ~ c3
100middotlr~~~------------------------------------e- Percussion 5min middot 0middot Hot Water -- Control
80-J=middot======
6b-middot-lmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middot--middotmiddot-middot~-middotmiddot -middotmiddot middot middotmiddot-middot 1
40
20
-----~-----------
0 0 0 middotmiddotmiddotmiddotmiddot0middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot0
0 --~-----y-----~------_---~--- T-----~
o shy -TDay 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A4 Effect of selected scarification treatments on germination speed for New Mexico locust First seven days of fourteen-day experiment are shown
Day 7
80
~ cd
5 60 ()
[) Cl d 0 1t1 d
40sect dUl
-J
20
o
ns
Scarification
Figure A5 Effect of scarification on germination response for black locust = significant difference between adjacent treatment bars ns = no significant difference
decrease at the 10-minute level This drop-off suggests that 10 minutes of percussion
over-treats seeds from this lot of-black locust
A selected percussion treatment intermediate in time (5 minutes) also had the
fastest germination rate for black locust (Figure A6)_ The hot water curve is
somewhat flatter indicating a slower germination rate The control curve is very flat
indicating a slow germination rate
A qualitative difference between hot water and percussion-treated seeds was
also observed Along with healthy germinants the hot water treatment produced
over-treated seeds (Figure A 7a) Examples of damaged germinants include seeds
where the radicle did not elongate (Figure A 7b) seeds where the radicle elongated
but was delayed in freeing itself from the seed coat (Figure A7c) and seeds where the
radicle broke off completely from the embryo (Figure A7d) In addition to these
examples of over-treatment there were numerous small dark seeds that failed to
imbibe water For percussion treatment nearly all seeds took up water (Figure A8a)
Germination was rapid and uniform with little damage evident (Figure A8b)
To summarize both New Mexico locust and black locust seed lots were
dormant The standard hot water scarification treatment improved germination but
only to 56 and 41 for New Mexico locust and black locust respectively Nearly
all percussion durations improved total germination as well as germination rate over
the hot water treatment Germination was greater than 90 for percussion durations
of 4 5 and 10 minutes for New Mexico locust and 3 4 and 5 minutes for black locust
58
CI)
sgt t CI) 0
igt 0
sect ttl t E
Vl 0
c3
100r==~==~~=r------------------------------------~ ___ Percussion 5 min
middotmiddotmiddot0middotmiddotmiddot Hot Water -lIf- Control
80r=========~--~~----------------------------~
60
40
20 -~-~---~---------- ---~------------I
omiddot
O
_~_----------lIf-----~bull -----_ shy---shy I
Day 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A6 Effect of selected scarification treatments on germination speed for black locust First seven days of fourteen-day experiment are shown
Day 7
a
b c d
Figure A7 Damage to New Mexico locust seeds observed during germination counts following hot water scarification A7a Hot water replicate including damaged seedlings and numerous unimbibed seeds A7b Close-up ofseed where radicle failed to elongate A7c Close-up ofseed where radicle elongated but was delayed in freeing itself from the seed coat A7d Close-up ofseed where radicle completely broke offfrom embryo
60
a
b
Figure A8 New Mexico locust seeds following percussion treatment at time of germination counts A8a Percussion replicate with uniformly healthy seedlings and near complete imbibition of seeds A8b Close-up ofpercussion-treated seedling
61
Discussion
Papilionoid seeds have a specific anatomical feature known as the strophiole
or lens-the natural site of water entry to the seed (Hamly 1932) The strophiole is
located on the cotyledonary lobe of a locust seed (Figure A9) When a papilionoid
legume seed is percussed for the appropriate amount of time repeated hits on the
integrated seed coat loosen the constrained cells of the strophiolar region without
excessively damaging the rest of the seed coat (Ballard 1976)
When a percussed seed is soaked water enters exclusively through the
strophiole in a controlled manner (Kelly and Staden 1987) This regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues This
contrasts with seeds that have been hot water or acid scarified treatments that can
randomly degrade the seed coat Unlocalized cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rehman et al 1998 Marunda 1990)
Papilionoids are the largest subfamily of legumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust and black locust these legumes often play an integral role in the
revegetation ofdisturbed lands Developing superior scarification methods to the
standard hot water and acid treatments should fadlitate the use ofthese legumes in
restoration projects
62
Figure A9 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (bc)
63
APPENDIXB
GERMINATION PERCENTS AND STANDARD ERRORS FOR SOURCE
BLOCK AND TREATMENT FOR NEW MEXICO LOCUST
Table B 1 Gennination percents and standard errors for source block and treatment for New Mexico locust
Source Block Treatment Germination Percentage Standard Error
Capitan 1 Immersion 74 437 Capitan 2 Immersion 69 462 Capitan 3 Immersion 71 454 Capitan 4 Immersion 67 470 Capitan Boiling 30 sec 79 407 Capitan 2 Boiling 30 sec 76 427 Capitan 3 Boiling 30 sec 72 449 Capitan 4 Boiling 30 sec 76 427 Capitan Boiling 60 sec 71 454 Capitan 2 Boiling 60 sec 54 498 Capitan 3 Boiling 60 sec 67 470 Capitan 4 Boiling 60 sec 76 427 Capitan I Control 5 218 Capitan 2 Control 7 255 Capitan 3 Control 4 196 Capitan 4 Control 5 218 Capitan 1 Percussion I min 47 499 Capitan 2 Percussion I min
470
Capitan Percussion I min 39 488
Capitan 4 Percussion I min 38 485 Capitan I Percussion 2 min 55 497 Capitan Capitan
2
Percussion 2 min Percussion 2 min
71 65
454 477
Capitan 4 Percussion 2 min 56 496 Capitan I Percussion 4 min 86 347 Capitan 2 Percussion 4 min 79 407 Capitan 3 Percussion 4 min 81 392 Capitan 4 Percussion 4 min 82 384 Capitan I Percussion 8 min 90 300 Capitan 2 Percussion 8 min 88 325 Capitan 3 Percussion 8 min 90 300 Capitan 4 Percussion 8 min 90 300 Gila I Immersion 71 454 Gila 2 Immersion 77 421 Gila
Immersion 75 433
Gila 4 Immersion 72 449
Gila I Boiling 30 sec 68 466
Gila 2 Boiling 30 sec 81 392
Gila 3 Boiling 30 sec 54 498
Gila 4 Boiling 30 sec 63 483
Gila I Boiling 60 sec 55 497
Gila 2 Boiling 60 sec 68 466
Gila 3 Boiling 60 sec 57 495
Gila 4 Boiling 60 sec 70 458
65
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Gila 2 Control 13 336 Gila 3 Control 13 336 Gila 4 Control 20 400 Gila 1 Percussion 1 min 66 474 Gila 2 Percussion 1 min 57 495 Gila 3 Percussion 1 min 73 444 Gila 4 Percussion 1 min 82 384 Gila 1 Percussion 2 min 81 392 Gila 2 Percussion 2 min 86 347 Gila 3 Percussion 2 min 85 357 Gila 4 Percussion 2 min 54 498 Gila 1 Percussion 4 min 79 407 Gila 2 Percussion 4 min 83 376 Gila 3 Percussion 4 min 84 367 Gila 4 Percussion 4 min 79 407 Gila 1 Percussion 8 min 51 500 Gila 2 Percussion 8 min 76 427 Gila 3 Percussion 8 min 52 500 Gila 4 Percussion 8 min 74 439 Holman 1 Immersion 83 376 Holman 2 Immersion 86 347 Holman 3 Immersion 81 392 Holman 4 Immersion 90 300 Holman 1 Boiling 30 sec 90 300 Holman 2 Boiling 30 sec 85 357 Holman 3 Boiling 30 sec 87 336 Holman 4 Boiling 30 sec 83 376 Holman 1 Boiling 60 sec 79 407 Holman 2 Boiling 60 sec 80 400 Holman 3 Boiling 60 sec 86 347 Holman 4 Boiling 60 sec 93 255 Holman Control 25 433 Holman 2 Control 19 392 Holman Control 21 407
Holman 4 Control 24 427
Holman Percussion 1 min 77 421
Holman 2 Percussion 1 min 85 357
Holman 3 Percussion 1 min 80 400
Holman 4 Percussion 1 min 89 313
Holman 1 Percussion 2 min 88 325
Holman 2 Percussion 2 min 96 196
Holman 3 Percussion 2 min 93 255
Holman 4 Percussion 2 min 96 196
Holman 1 Percussion 4 min 92 271
Holman 2 Percussion 4 min 97 171
66
I
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Holman 3 Percussion 4 min 91 286 Holman 4 Percussion 4 min 96 196 Holman 1 Percussion 8 min 88 325 Holman 2 Percussion 8 min 85 357 Holman 3 Percussion 8 min 83 376 Holman 4 Percussion 8 min 82 384 Jemez 1 Immersion 53 499 Jemez 2 Immersion 44 496 Jemez 3 Immersion 52 500 Jemez 4 Immersion 70 458 Jemez 1 Boiling 30 sec 58 494 Jemez 2 Boiling 30 sec 55 497 Jemez 3 Boiling 30 sec 48 500 Jemez 4 Boiling 30 sec 65 477 Jemez 1 Boiling 60 sec 68 466 Jemez 2 Boiling 60 sec 63 483 Jemez 3 Boiling 60 sec 73 444 Jemez 4 Boiling 60 sec 89 313 Jemez 1 Control 2 140 Jemez 2 Control 9 286 Jemez 3 Control 6 237 Jemez 4 Control 8 271 Jemez 1 Percussion 1 min 58 494 Jemez 2 Percussion 1 min 63 483 Jemez 3 Percussion 1 min 77 421 Jemez 4 Percussion 1 min 89 313 Jemez 1 Percussion 2 min 68 466 Jemez 2 Percussion 2 min 64 480 Jemez 3 Percussion 2 min 80 400 Jemez 4 Percussion 2 min 83 376 Jemez Percussion 4 min 68 466 Jemez 2 Percussion 4 min 79 407 Jemez 3 Percussion 4 min 70 458 Jemez 4 Percussion 4 min 83 376 Jemez 1 Percussion 8 min 52 500 Jemez 2 Percussion 8 min 59 492 Jemez 3 Percussion 8 min 61 488 Jemez 4 Percussion 8 min 70 458 Magdalena 1 Immersion 69 462 Magdalena 2 Immersion 69 462 Magdalena J Immersion 70 458 Magdalena 4 Immersion 85 357 Magdalena 1 Boiling 30 sec 65 477 Magdalena 2 Boiling 30 sec 71 454 Magdalena 3 Boiling 30 sec 68 466
67
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Magdalena 4 Boiling 30 sec 83 376 Magdalena 1 Boiling 60 sec 63 483 Magdalena 2 Boiling 60 sec 68 466 Magdalena 3 Boiling 60 sec 63 483 Magdalena 4 Boiling 60 sec 82 384 Magdalena 1 Control 11 313 Magdalena 2 Control 6 237 Magdalena 3 Control 5 218 Magdalena 4 Control 4 196 Magdalena Percussion 1 min 32 466 Magdalena 2 Percussion 1 min 45 497 Magdalena 3 Percussion 1 min 46 498 Magdalena 4 Percussion I min 36 480 Magdalena 1 Percussion 2 min 67 470 Magdalena 2 Percussion 2 min 68 466 Magdalena 3 Percussion 2 min 65 477 Magdalena 4 Percussion 2 min 81 392 Magdalena 1 Percussion 4 min 87 336 Magdalena 2 Percussion 4 min 97 171 Magdalena 3 Percussion 4 min 83 376 Magdalena 4 Percussion 4 min 97 171 Magdalena 1 Percussion 8 min 98 lAO Magdalena 2 Percussion 8 min 100 000 Magdalena 3 Percussion 8 min 96 196 Magdalena 4 Percussion 8 min 98 lAO Manzano 1 Immersion 67 470 Manzano 2 Immersion 57 495 Manzano 3 Immersion 72 4049 Manzano 4 Immersion 73 4044 Manzano 1 Boiling 30 sec 70 458 Manzano 2 Boiling 30 sec 64 480 Manzano 3 Boiling 30 sec 71 454 Manzano 4 Boiling 30 sec 80 400 Manzano I Boiling 60 sec 50 500 Manzano 2 Boiling 60 sec 53 499 Manzano 3 Boiling 60 sec 59 492 Manzano 4 Boiling 60 sec 74 439 Manzano I Control 4 196 Manzano 2 Control 8 271 Manzano 3 Control 1 099 Manzano 4 Control 6 237 Manzano 1 Percussion 1 min 52 500 Manzano 2 Percussion 1 min 39 488 Manzano 3 Percussion 1 min 36 480 Manzano 4 Percussion 1 min 51 500
68
Table B 1 Continued
Source Block Treatment Gerrni~ation Percentage Standard Error
Manzano Percussion 2 min 53 499 Manzano 2 Percussion 2 min 58 494 Manzano 3 Percussion 2 min 61 488 Manzano 4 Percussion 2 min 62 485 Manzano 1 Percussion 4 min 76 427 Manzano 2 Percussion 4 min 85 357 Manzano 3 Percussion 4 min 79 407 Manzano 4 Percussion 4 min 83 376 Manzano 1 Percussion 8 min 87 336 Manzano 2 Percussion 8 min 83 376 Manzano 3 Percussion 8 min 83 376 Manzano 4 Percussion 8 min 75 433 Raton 1 Immersion 54 498 Raton 2 Immersion 37 483 Raton 3 Immersion 39 488 Raton 4 Immersion 69 462 Raton 1 Boiling 30 sec 36 480 Raton 2 Boiling 30 sec 20 400 Raton 3 Boiling 30 sec 28 449 Raton 4 Boiling 30 sec 60 490 Raton 1 Boiling 60 sec 15 357 Raton 2 Boiling 60 sec 171 Raton 3 Boiling 60 sec 4 196 Raton 4 Boiling 60 sec 35 477 Raton 1 Control 4 196 Raton 2 Control 12 325 Raton 3 Control 2 140 Raton 4 Control 6 237 Raton 1 Percussion 1 min 52 500 Raton 2 Percussion I min 29 454 Raton 3 Percussion I min 35 477 Raton 4 Percussion 1 min 31 462 Raton 1 Percussion 2 min 55 497 Raton 2 Percussion 2 min 35 477 Raton 3 Percussion 2 min 33 470 Raton 4 Percussion 2 min 42 494 Raton 1 Percussion 4 min 65 477 Raton 2 Percussion 4 min 52 500 Raton Percussion 4 min 51 500 Raton 4 Percussion 4 min 53 499 Raton 1 Percussion 8 min 88 325 Raton 2 Percussion 8 min 80 400 Raton 3 Percussion 8 min 73 444 Raton 4 Percussion 8 min 74 439 Sacramento 1 Immersion 43 495
69
Table B 1 Continued
-Source Block Treatment Germination Percentage Standard Error
Sacramento 2 Immersion 30 458 Sacramento 3 Immersion 38 485 Sacramento 4 Immersion 56 496 Sacramento 1 Boiling 30 sec 47 499 Sacramento 2 Boiling 30 sec 48 500 Sacramento 3 Boiling 30 sec 48 500 Sacramento 4 Boiling 30 sec 56 496 Sacramento 1 Boiling 60 sec 52 500 Sacramento 2 Boiling 60 sec 67 470 Sacramento 3 Boiling 60 sec 43 495 Sacramento 4 Boiling 60 sec 48 500 Sacramento 1 Control 0 000 Sacramento 2 Control 3 171 Sacramento 3 Control 3 171 Sacramento 4 Control 099 Sacramento 1 Percussion 1 min 57 495 Sacramento 2 Percussion 1 min 61 488 Sacramento 3 Percussion 1 min 62 485 Sacramento 4 Percussion 1 min 55 497 Sacramento 1 Percussion 2 min 84 367 Sacramento 2 Percussion 2 min 70 458 Sacramento 3 Percussion 2 min 81 392 Sacramento 4 Percussion 2 min 69 462 Sacramento 1 Percussion 4 min 91 286 Sacramento 2 Percussion 4 min 84 367 Sacramento 3 Percussion 4 min 84 367 Sacramento 4 Percussion 4 min 94 237 Sacramento 1 Percussion 8 min 82 384 Sacramento 2 Percussion 8 min 81 392 Sacramento 3 Percussion 8 min 82 384 Sacramento 4 Percussion 8 min 81 392 Sandia 1 Immersion 76 427 Sandia 2 Immersion 70 458 Sandia 3 Immersion 70 458 Sandia 4 Immersion 81 392 Sandia 1 Boiling 30 sec 78 414 Sandia 2 Boiling 30 sec 71 454 Sandia 3 Boiling 30 sec 63 483 Sandia 4 Boiling 30 sec 78 414 Sandia 1 Boiling 60 sec 67 470 Sandia 2 Boiling 60 sec 65 477 Sandia 3 Boiling 60 sec 74 439 Sandia 4 Boiling 60 sec 66 474 Sandia 1 Control 16 367 Sandia 2 Control 7 255
70
Table B 1 Continued
Source Block Treatment Gennination Percentage Standard Error
Sandia 3 Control 8 271 Sandia 4 Control 16 367 Sandia 1 Percussion 1 min 67 470 Sandia 2 Percussion 1 min 61 488 Sandia 3 Percussion 1 min 66 474 Sandia 4 Percussion 1 min 59 492 Sandia I Percussion 2 min 82 384 Sandia 2 Percussion 2 min 83 376 Sandia 3 Percussion 2 min 81 392 Sandia 4 Percussion 2 min 84 367 Sandia 1 Percussion 4 min 89 313 Sandia 2 Percussion 4 min 86 347 Sandia 3 Percussion 4 min 89 313 Sandia 4 Percussion 4 min 85 357 Sandia 1 Percussion 8 min 80 400 Sandia 2 Percussion 8 min 77 421 Sandia 3 Percussion 8 min 69 462 Sandia 4 Percussion 8 min 66 474
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photography nor visible observations during seed counts revealed seed damage at the
4-minute percussion level In operational trials seeds percussed for 4 minutes
developed into healthy seedlings (Khadduri and Harrington unpublished data)
Strophiole and Seed Coat Behavior
Four minutes ofpercussion successfully breaks seed dormancy in a large
percentage ofNew Mexico locust seeds without excessively damaging the
surrounding seed coat or internal seed coat structures Most papilionoid legume
seeds including seeds ofNew Mexico locust have a specific region of the seed coat
known as the strophiole or lens (Hamly 1932) The strophiole is located on the
cotyledonary lobe of a locust seed (Figure 10 a-c) If seeds are not indiscriminately
damaged the strophiole is the first point of water entry to the embryo (Hopkinson
1997) When a papilionoid seed is percussed for the appropriate amount of time
repeated hits on the integrated seed coat loosen the constrained cells of the strophiolar
region without excessively damaging the rest of the seed coat (Ballard 1976) SEM
photos illuStrate that 4 minutes of percussion loosened and separated strophiolar cells
ofNew Mexico locust seeds when compared with constrained organized strophiolar
cells of untreated seeds (Figure 9) Research-has shown that water passes to the
embryo through the treated strophiole in a controlled manner and regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues
(Kelly and van Staden 1987)
37
10 Strophiole ofunimbibed New Mexico locust seed (a) and following uptake (bc)
38
In contrast hot-water treatments can excessively degrade the seed coat
(Rebman et al 1998 Marunda 1990) Random cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rebman et aI 1998 Marunda 1990) Along with a
proportionately large amount ofun-imbibed (thus under-treated) seeds (Figure l1a)
damage was observed in hot-water treated seeds from six sources during germination
counts Damage included seeds where the radicle failed to elongate (Figure 11 b)
seeds where the radicle elongated but was delayed in freeing itself from the seed coat
(Figure 11c) and seeds where the radicle completely broke off from the embryo
(Figure lId) In addition some seeds germinated in reverse (the cotyledon emerged
fIrst) If the radicle emerged completely from the seed coat they were counted as
germinated
While nearly all seeds treated for 8 minutes ofpercussion imbibed water
damage was evident in x-ray evaluations (Sacramento seed source) and in
germination counts (Holman and Capitan) at this treatment level Seed damage
during germination counts was similar to levels found in hot-water treated seeds
suggesting that over-treatment by both hot water and longer durations of percussion
may result in similar damage to seed structure
The Sacramento and Holman seed sources were two of the fIve sources that
opcrplltlpn in germination from the 4- to 8-minute durations of percussion
the benefIts ofloosening the strophiole as evidenced by imbibition of
all 8-minute treated seeds were outweighed by external and internal seed coat
39
a
b c d
11 Damage to New Mexico locust seeds observed during germination cotmts following hot water scarification 9a Hot water replicate including ~t seedlings and numerous unimbibed seeds 9b Close-up of seed where raallCle failed to elongate 9c Close-up ofseed where radicle elongated but was
-gtIn in freeing itself from the seed coat 9d Close-up ofseed where radicle broke off from embryo
40
damage (which also may be contributing to high imbibition) For the three sources
for which 8 minutes of percussion resulted in maximum germination extended
percussion of seeds necessary to loosen the strophiole may not have excessively
damaged the seed anatomy The Manzano source did not significantly change in
germination from 4 to 8 minutes of percussion suggesting that an increase in
permeable seeds at the 8-minute treatment duration was balanced by the number of
seeds that failed to germinate due to seed damage To summarize the 4-minute
percussion treatment improved germination over 8 minutes ofpercussion for a
majority of sources (five ofnine) the 8-minute percussion treatment improved
germination over 4-minutes ofpercussion for three sources and there was no
difference with the Manzano source Notably for the Raton source 8 minutes of
percussion increased germination by 235 percentage points over 4-minutes of
percussion (7875 vs 5525 respectively) the next best treatment Raton was the
only source to result in germination less than 75 for 4 minutes ofpercussion so a
grower may wish to apply longer percussion durations to lots that do not respond well
to this treatment level
Finally there was an interesting trend in the Jemez and Raton sources seed
that significantly contributed to the source by treatment interaction (Figure 12) A
~u test showed that the Jemez source had a significantly lighter seed weight than
other sources while the Raton source had an average seed weight For the Jemez
the longest duration of boiling increased germination Conversely only short
U4UVll) of percussion were needed to achieve high germination and the longest
41
80 _ Jemez a Raton
iD
~ C iD e iD
Q c 0 ~
c ~ iD 0
20
0------shy
Immersion Boiling 30 sec Boiling 60 sec
Hot Water Scarification
10degTr========~--------~------------------------------
80
60
40
20
_ Jemez Raton
0------shy
1 min 2 min 4 min 8 min
Percussion Scarification
Figure 12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources
42
b
--~----~~~~========~---- duration ofpercussion reduced germination For the Raton source longer durations
ofboiling consistently decreased germination whereas longer percussion treatments
consistently increased germination
A possible explanation is that a thick seed coat is associated with a loose
strophiole structure and that a thin seed coat is associated with a constrained
strophiole This contrast merits further study and may suggest an ecological
adaptation Further investigation ofthis phenomenon may contribute to successful
pre-screening ofunknown seed sources to determine optimal treatment leveL
43
CONCLUSION
Based on results obtained in pilot study (see Appendix) and thesis
experiments 4 minutes ofpercussion is recommended as a standard protocol for
treating an unknown source ofNew Mexico locust seed In addition to laboratory
results the 4-minute percussion treatment has resulted in germination greater than
80 and healthy seedling grow-outs for production purposes (unpublished data) In
laboratory experiments only one hundred seeds per 118 ml (40z) soil tin were
percussed at a time whereas for production we have treated up to five hundred seeds
per tin For large-scale production ofNew Mexico locust seedlings growers may
wish to experiment with larger quantities of seed and larger-size soil tins
Seed storage trials following percussion treatments were not conducted
However germination greater than 80 and healthy seedlings were obtained when
seeds were percussion-treated and subsequently stored at room-temperature (22degC)
for one week (unpublished data) Seed storage trials merit further study as the
standard hot water treatment leaves seeds in a softened condition that is not
conducive to storage
Hydroseeding is an effective method for direct seeding in reclamation
situations Percussion treatment may be conducive to hydro seeding as seeds remain
a dry relatively undamaged yet permeable condition following treatment Sulfuric
seeds also remain dry following treatment but extensive damage to seed coat
poor seedling vigor have been reported with this treatment (Kelly and van Staden
A grow-out study using a hydro seeder on a reclamation site testing percussion
44
against other treatments such as sulfuric acid and mechanical scarification also
merits further study
Papilionoids are the largest subfamily oflegumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust these legumes often play an integral role in the revegetation of
disturbed lands The pilot study (Appendix) indicates that percussion scarification
successfully treats the widely used black locust as well Developing superior
scarification methods to the standard hot water mechanical scarification and acid
treatments may facilitate the use ofpapilionoid legumes in reclamation projects
45
APPENDICES
APPENDIX A
PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR
NEW MEXICO LOCUST AND BLACK LOCUST SEEDS
Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4
Hot water and sulfuric acid soaks are traditional treatments for seeds of many
temperate woody legwnes including locusts However these scarification techniques
often produce inconsistent germination Percussion scarification where seeds are
repeatedly propelled against a hard surface was compared with hot water
scarification to evaluate treatment efficacy for New Mexico locust (Robinia
neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment
seeds were placed in a 98degC water bath which was immediately removed from the
heat source For percussion scarification seeds were placed in a soil sample tin and
agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the
control were followed by 24-hour water soaks Hot water baths resulted in 56 and
41 germination for New Mexico locust and black locust respectively For both
species nearly all durations ofpercussion increased germination over the hot water
treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3
4 and 5 minutes for black locust resulted in at least 90 germination Traditional
scarification treatments randomly degrade the entire seed coat which can lead to
tissue damage during water uptake Percussion scarification specifically weakens the
strophiole the natural source ofwater entry to the seed in papilionoid legwnes
INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom
2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031
48
Following percussion imbibition is controlled through the strophiole and underlying
tissue is protected
Keywords New Mexico locust black locust scarification percussion germination
Introduction
Locust trees (Robinia spp) are aggressive pioneer species that quickly
colonize disturbed land fix atmospheric nitrogen in the soil and stabilize slopes that
are prone to erosion (Klemmedson 1994 Ashby et al 1985) (Figure A1ab)
Recognition of these attributes has contributed to increased demand for locusts in
reclamation projects Locust seeds however exhibit physical dormancy where a
thick seed coat prevents the movement of water and gasses to the embryo (Leadem
1997)
Sulfuric acid or hot water soaks have traditionally been used to break seed
dormancy in macro-propagation of locusts Many growers are moving away from the
use of su1furic acid in treating locust seeds (Dreesen and Harrington 1997) In
addition to being dangerous acid soak durations must be specifically correlated to
seed lot (Olson 1974) Collections of native seed can vary tremendously in seed size
seed weight and hard seededness etc This variability limits the utility of sulfuric
acid soaks as even within a given lot some seeds may be damaged whereas others
are under-treated and fail to imbibe (take up water) A survey of Southwestern
container growers indicates that hot water baths are currently the most common
scarification method for locust seeds (Hine et al 1997) However hot water baths
also produce inconsistent germination (Lin et al 1996)
49
b
Figure AI New Mexico locust a primary invader following disturbance Ala Crown-sprouting at edge ofburn near Los Alamos NM--one year after Cerro Grande fire Alb Colonizing road cut Gila National Forest NM
50
Percussion scarification where seeds are repeatedly propelled against a hard
surface is an alternative dormancy-breaking method that has proven successful in
legumes related to locusts (Hamly 1932 Barton 1947) We compared this alternative
treatment to hot water scarification to evaluate treatment efficacy for New Mexico
locust and black locust seeds
Materials and Methods
Commercial New Mexico locust seeds (Western Native Seed Coaldale CO)
were collected fall 2000 in Huerfano County CO Black locust seeds were collected
September 2000 from Taos County NM by harvesting ripe pods from several trees at
a distance of up to 3 m from ground level Black locust seeds were allowed to air-dry
for 3 weeks and were threshed and separated from large chaffusing a greenhouse
fan A South Dakota Seed Blower (Chicago Illinltois) was used to remove fine chaff
from both seed lots Seeds were storedmiddotat 2-4C until the germination study was
conducted in March 2001
This study compared hot water scarification with percussion scarification
using treatment and species as experimental factors Along with a control
scarification treatments included immersion of seed in boiling water as well as
percussion durations of 1 2 3 45 or 10 minutes Four 100-seed samples were used
to test each species by scarification treatment combination
For the hot water treatment test tubes were filled with 30 mL water and
placed in a water-filled beaker (Figure A2a) The bath was raised to boiling (-98C at
51
b
c
Figure A2 Implementation ofhot water and percussion scarification A2a Hot water bath A2b Paint-shaker set-up A2c Placing soil tin containing seeds at end ofpaint shaker ann
52
Las Cruces NM elevation of 1300 meters) Each 100-seed replicate was placed in a
test tube and each test tube was immediately removed to cool at room temperature
Percussion scarification was implemented using a pneumatic paint shaker
(Central Pneumatic model 00422 Camarillo CA) (Figure A2b) This allowed
standardization of treatment intensity Compressed air pressure was maintained at
530kPa plusmn 30kPa (80psi plusmn 5psi) resulting in approximately 350 oscillations per
minute Each lOO-seed sample was placed in a 118 ml (4 oz) soil tin for shaking To
maximize lateral movement of paint shaker a paint can was used as a spacer to place
soil tins at the end of the shaking arm (Figure A2c) This allowed shaking to occur at
the greatest distance from the pivot point (fulcrum) Following all treatments
including the control seeds were soaked for twenty-four hours in distilled water prior
to plating out
Gennination was evaluated on lab benches at room temperature Each
replicate of seeds was placed in a 100 cm petri dish on moistened filter paper with
humidity maintained by enclosing the petri dishes in ziplock bags Gennination was
monitored daily for 14 days in accordance with International Seed Testing
Association standards for Robinia species (ISTA 1999) Gennination was defined as
protrusion of the radicle from the embryo by at least 1mm as viewed by the naked
eye
Categorical Analysis of Variance using Prpc Catmod (SAS Institute 1989)
was used to detennine treatment differences in germination percentages for each
source This procedure is an extension of a Chi-square test of homogeneity using the
53
natural log of the ratio of germinated to non-germinated seeds for each treatment Pshy
values less than 005 were considered significant A limited set of pairwiseshy
comparisons was conducted to compare treatment mean percents using a
conservative alpha value of 005 divided by the number of comparisons
Results
Percussion maximized germination for both New Mexico locust and black
locust There were three germination response levels for New Mexico locust (Figure
A3) First there was a low response in the control indicating a dormant seed lot
The hot water treatment and I-minute duration of percussion share a second
intermediate response Germination was highest at percussion levels from 2 to 10
minutes
A selected percussion treatment intermediate in time (5 minutes) produced the
steepest germination curve indicating the fastest germination rate (Figure A4) The
hot water treatment produced an intermediate curve or relatively slower germination
rate The control curve was very flat indicating the slowest germination rate for New
Mexico locust
Control seeds germinated poorly for black locust as well (Figure A5) Hot
water scarification improved germination over the control but all levels of percussion
improved germination over hot water The optimal durations of percussion treatment
were bracketed with a significant increase from the 1 to 2 minute percussion level a
high germination response from 2 to 5 minutes of percussion and finally a significant
54
~ d 25 CL)
p t1 0 ~ t1
g Vl ~ Vl
100~---------------------------------------------------------------
80
60
40
20
o Control Hot Water Percussion
I min Percussion
2 min Percussion
3 min
Scarification
Percussion 4 min
Figure A3 Effect of scarification on germination response for New Mexico locust
Percussion 5 min
=significant difference between adjacent treatment bars ns = no significant difference
Percussion 10 min
~ ~ () u agt
Il-t d o ~
~ ~ c3
100middotlr~~~------------------------------------e- Percussion 5min middot 0middot Hot Water -- Control
80-J=middot======
6b-middot-lmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middot--middotmiddot-middot~-middotmiddot -middotmiddot middot middotmiddot-middot 1
40
20
-----~-----------
0 0 0 middotmiddotmiddotmiddotmiddot0middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot0
0 --~-----y-----~------_---~--- T-----~
o shy -TDay 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A4 Effect of selected scarification treatments on germination speed for New Mexico locust First seven days of fourteen-day experiment are shown
Day 7
80
~ cd
5 60 ()
[) Cl d 0 1t1 d
40sect dUl
-J
20
o
ns
Scarification
Figure A5 Effect of scarification on germination response for black locust = significant difference between adjacent treatment bars ns = no significant difference
decrease at the 10-minute level This drop-off suggests that 10 minutes of percussion
over-treats seeds from this lot of-black locust
A selected percussion treatment intermediate in time (5 minutes) also had the
fastest germination rate for black locust (Figure A6)_ The hot water curve is
somewhat flatter indicating a slower germination rate The control curve is very flat
indicating a slow germination rate
A qualitative difference between hot water and percussion-treated seeds was
also observed Along with healthy germinants the hot water treatment produced
over-treated seeds (Figure A 7a) Examples of damaged germinants include seeds
where the radicle did not elongate (Figure A 7b) seeds where the radicle elongated
but was delayed in freeing itself from the seed coat (Figure A7c) and seeds where the
radicle broke off completely from the embryo (Figure A7d) In addition to these
examples of over-treatment there were numerous small dark seeds that failed to
imbibe water For percussion treatment nearly all seeds took up water (Figure A8a)
Germination was rapid and uniform with little damage evident (Figure A8b)
To summarize both New Mexico locust and black locust seed lots were
dormant The standard hot water scarification treatment improved germination but
only to 56 and 41 for New Mexico locust and black locust respectively Nearly
all percussion durations improved total germination as well as germination rate over
the hot water treatment Germination was greater than 90 for percussion durations
of 4 5 and 10 minutes for New Mexico locust and 3 4 and 5 minutes for black locust
58
CI)
sgt t CI) 0
igt 0
sect ttl t E
Vl 0
c3
100r==~==~~=r------------------------------------~ ___ Percussion 5 min
middotmiddotmiddot0middotmiddotmiddot Hot Water -lIf- Control
80r=========~--~~----------------------------~
60
40
20 -~-~---~---------- ---~------------I
omiddot
O
_~_----------lIf-----~bull -----_ shy---shy I
Day 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A6 Effect of selected scarification treatments on germination speed for black locust First seven days of fourteen-day experiment are shown
Day 7
a
b c d
Figure A7 Damage to New Mexico locust seeds observed during germination counts following hot water scarification A7a Hot water replicate including damaged seedlings and numerous unimbibed seeds A7b Close-up ofseed where radicle failed to elongate A7c Close-up ofseed where radicle elongated but was delayed in freeing itself from the seed coat A7d Close-up ofseed where radicle completely broke offfrom embryo
60
a
b
Figure A8 New Mexico locust seeds following percussion treatment at time of germination counts A8a Percussion replicate with uniformly healthy seedlings and near complete imbibition of seeds A8b Close-up ofpercussion-treated seedling
61
Discussion
Papilionoid seeds have a specific anatomical feature known as the strophiole
or lens-the natural site of water entry to the seed (Hamly 1932) The strophiole is
located on the cotyledonary lobe of a locust seed (Figure A9) When a papilionoid
legume seed is percussed for the appropriate amount of time repeated hits on the
integrated seed coat loosen the constrained cells of the strophiolar region without
excessively damaging the rest of the seed coat (Ballard 1976)
When a percussed seed is soaked water enters exclusively through the
strophiole in a controlled manner (Kelly and Staden 1987) This regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues This
contrasts with seeds that have been hot water or acid scarified treatments that can
randomly degrade the seed coat Unlocalized cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rehman et al 1998 Marunda 1990)
Papilionoids are the largest subfamily of legumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust and black locust these legumes often play an integral role in the
revegetation ofdisturbed lands Developing superior scarification methods to the
standard hot water and acid treatments should fadlitate the use ofthese legumes in
restoration projects
62
Figure A9 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (bc)
63
APPENDIXB
GERMINATION PERCENTS AND STANDARD ERRORS FOR SOURCE
BLOCK AND TREATMENT FOR NEW MEXICO LOCUST
Table B 1 Gennination percents and standard errors for source block and treatment for New Mexico locust
Source Block Treatment Germination Percentage Standard Error
Capitan 1 Immersion 74 437 Capitan 2 Immersion 69 462 Capitan 3 Immersion 71 454 Capitan 4 Immersion 67 470 Capitan Boiling 30 sec 79 407 Capitan 2 Boiling 30 sec 76 427 Capitan 3 Boiling 30 sec 72 449 Capitan 4 Boiling 30 sec 76 427 Capitan Boiling 60 sec 71 454 Capitan 2 Boiling 60 sec 54 498 Capitan 3 Boiling 60 sec 67 470 Capitan 4 Boiling 60 sec 76 427 Capitan I Control 5 218 Capitan 2 Control 7 255 Capitan 3 Control 4 196 Capitan 4 Control 5 218 Capitan 1 Percussion I min 47 499 Capitan 2 Percussion I min
470
Capitan Percussion I min 39 488
Capitan 4 Percussion I min 38 485 Capitan I Percussion 2 min 55 497 Capitan Capitan
2
Percussion 2 min Percussion 2 min
71 65
454 477
Capitan 4 Percussion 2 min 56 496 Capitan I Percussion 4 min 86 347 Capitan 2 Percussion 4 min 79 407 Capitan 3 Percussion 4 min 81 392 Capitan 4 Percussion 4 min 82 384 Capitan I Percussion 8 min 90 300 Capitan 2 Percussion 8 min 88 325 Capitan 3 Percussion 8 min 90 300 Capitan 4 Percussion 8 min 90 300 Gila I Immersion 71 454 Gila 2 Immersion 77 421 Gila
Immersion 75 433
Gila 4 Immersion 72 449
Gila I Boiling 30 sec 68 466
Gila 2 Boiling 30 sec 81 392
Gila 3 Boiling 30 sec 54 498
Gila 4 Boiling 30 sec 63 483
Gila I Boiling 60 sec 55 497
Gila 2 Boiling 60 sec 68 466
Gila 3 Boiling 60 sec 57 495
Gila 4 Boiling 60 sec 70 458
65
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Gila 2 Control 13 336 Gila 3 Control 13 336 Gila 4 Control 20 400 Gila 1 Percussion 1 min 66 474 Gila 2 Percussion 1 min 57 495 Gila 3 Percussion 1 min 73 444 Gila 4 Percussion 1 min 82 384 Gila 1 Percussion 2 min 81 392 Gila 2 Percussion 2 min 86 347 Gila 3 Percussion 2 min 85 357 Gila 4 Percussion 2 min 54 498 Gila 1 Percussion 4 min 79 407 Gila 2 Percussion 4 min 83 376 Gila 3 Percussion 4 min 84 367 Gila 4 Percussion 4 min 79 407 Gila 1 Percussion 8 min 51 500 Gila 2 Percussion 8 min 76 427 Gila 3 Percussion 8 min 52 500 Gila 4 Percussion 8 min 74 439 Holman 1 Immersion 83 376 Holman 2 Immersion 86 347 Holman 3 Immersion 81 392 Holman 4 Immersion 90 300 Holman 1 Boiling 30 sec 90 300 Holman 2 Boiling 30 sec 85 357 Holman 3 Boiling 30 sec 87 336 Holman 4 Boiling 30 sec 83 376 Holman 1 Boiling 60 sec 79 407 Holman 2 Boiling 60 sec 80 400 Holman 3 Boiling 60 sec 86 347 Holman 4 Boiling 60 sec 93 255 Holman Control 25 433 Holman 2 Control 19 392 Holman Control 21 407
Holman 4 Control 24 427
Holman Percussion 1 min 77 421
Holman 2 Percussion 1 min 85 357
Holman 3 Percussion 1 min 80 400
Holman 4 Percussion 1 min 89 313
Holman 1 Percussion 2 min 88 325
Holman 2 Percussion 2 min 96 196
Holman 3 Percussion 2 min 93 255
Holman 4 Percussion 2 min 96 196
Holman 1 Percussion 4 min 92 271
Holman 2 Percussion 4 min 97 171
66
I
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Holman 3 Percussion 4 min 91 286 Holman 4 Percussion 4 min 96 196 Holman 1 Percussion 8 min 88 325 Holman 2 Percussion 8 min 85 357 Holman 3 Percussion 8 min 83 376 Holman 4 Percussion 8 min 82 384 Jemez 1 Immersion 53 499 Jemez 2 Immersion 44 496 Jemez 3 Immersion 52 500 Jemez 4 Immersion 70 458 Jemez 1 Boiling 30 sec 58 494 Jemez 2 Boiling 30 sec 55 497 Jemez 3 Boiling 30 sec 48 500 Jemez 4 Boiling 30 sec 65 477 Jemez 1 Boiling 60 sec 68 466 Jemez 2 Boiling 60 sec 63 483 Jemez 3 Boiling 60 sec 73 444 Jemez 4 Boiling 60 sec 89 313 Jemez 1 Control 2 140 Jemez 2 Control 9 286 Jemez 3 Control 6 237 Jemez 4 Control 8 271 Jemez 1 Percussion 1 min 58 494 Jemez 2 Percussion 1 min 63 483 Jemez 3 Percussion 1 min 77 421 Jemez 4 Percussion 1 min 89 313 Jemez 1 Percussion 2 min 68 466 Jemez 2 Percussion 2 min 64 480 Jemez 3 Percussion 2 min 80 400 Jemez 4 Percussion 2 min 83 376 Jemez Percussion 4 min 68 466 Jemez 2 Percussion 4 min 79 407 Jemez 3 Percussion 4 min 70 458 Jemez 4 Percussion 4 min 83 376 Jemez 1 Percussion 8 min 52 500 Jemez 2 Percussion 8 min 59 492 Jemez 3 Percussion 8 min 61 488 Jemez 4 Percussion 8 min 70 458 Magdalena 1 Immersion 69 462 Magdalena 2 Immersion 69 462 Magdalena J Immersion 70 458 Magdalena 4 Immersion 85 357 Magdalena 1 Boiling 30 sec 65 477 Magdalena 2 Boiling 30 sec 71 454 Magdalena 3 Boiling 30 sec 68 466
67
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Magdalena 4 Boiling 30 sec 83 376 Magdalena 1 Boiling 60 sec 63 483 Magdalena 2 Boiling 60 sec 68 466 Magdalena 3 Boiling 60 sec 63 483 Magdalena 4 Boiling 60 sec 82 384 Magdalena 1 Control 11 313 Magdalena 2 Control 6 237 Magdalena 3 Control 5 218 Magdalena 4 Control 4 196 Magdalena Percussion 1 min 32 466 Magdalena 2 Percussion 1 min 45 497 Magdalena 3 Percussion 1 min 46 498 Magdalena 4 Percussion I min 36 480 Magdalena 1 Percussion 2 min 67 470 Magdalena 2 Percussion 2 min 68 466 Magdalena 3 Percussion 2 min 65 477 Magdalena 4 Percussion 2 min 81 392 Magdalena 1 Percussion 4 min 87 336 Magdalena 2 Percussion 4 min 97 171 Magdalena 3 Percussion 4 min 83 376 Magdalena 4 Percussion 4 min 97 171 Magdalena 1 Percussion 8 min 98 lAO Magdalena 2 Percussion 8 min 100 000 Magdalena 3 Percussion 8 min 96 196 Magdalena 4 Percussion 8 min 98 lAO Manzano 1 Immersion 67 470 Manzano 2 Immersion 57 495 Manzano 3 Immersion 72 4049 Manzano 4 Immersion 73 4044 Manzano 1 Boiling 30 sec 70 458 Manzano 2 Boiling 30 sec 64 480 Manzano 3 Boiling 30 sec 71 454 Manzano 4 Boiling 30 sec 80 400 Manzano I Boiling 60 sec 50 500 Manzano 2 Boiling 60 sec 53 499 Manzano 3 Boiling 60 sec 59 492 Manzano 4 Boiling 60 sec 74 439 Manzano I Control 4 196 Manzano 2 Control 8 271 Manzano 3 Control 1 099 Manzano 4 Control 6 237 Manzano 1 Percussion 1 min 52 500 Manzano 2 Percussion 1 min 39 488 Manzano 3 Percussion 1 min 36 480 Manzano 4 Percussion 1 min 51 500
68
Table B 1 Continued
Source Block Treatment Gerrni~ation Percentage Standard Error
Manzano Percussion 2 min 53 499 Manzano 2 Percussion 2 min 58 494 Manzano 3 Percussion 2 min 61 488 Manzano 4 Percussion 2 min 62 485 Manzano 1 Percussion 4 min 76 427 Manzano 2 Percussion 4 min 85 357 Manzano 3 Percussion 4 min 79 407 Manzano 4 Percussion 4 min 83 376 Manzano 1 Percussion 8 min 87 336 Manzano 2 Percussion 8 min 83 376 Manzano 3 Percussion 8 min 83 376 Manzano 4 Percussion 8 min 75 433 Raton 1 Immersion 54 498 Raton 2 Immersion 37 483 Raton 3 Immersion 39 488 Raton 4 Immersion 69 462 Raton 1 Boiling 30 sec 36 480 Raton 2 Boiling 30 sec 20 400 Raton 3 Boiling 30 sec 28 449 Raton 4 Boiling 30 sec 60 490 Raton 1 Boiling 60 sec 15 357 Raton 2 Boiling 60 sec 171 Raton 3 Boiling 60 sec 4 196 Raton 4 Boiling 60 sec 35 477 Raton 1 Control 4 196 Raton 2 Control 12 325 Raton 3 Control 2 140 Raton 4 Control 6 237 Raton 1 Percussion 1 min 52 500 Raton 2 Percussion I min 29 454 Raton 3 Percussion I min 35 477 Raton 4 Percussion 1 min 31 462 Raton 1 Percussion 2 min 55 497 Raton 2 Percussion 2 min 35 477 Raton 3 Percussion 2 min 33 470 Raton 4 Percussion 2 min 42 494 Raton 1 Percussion 4 min 65 477 Raton 2 Percussion 4 min 52 500 Raton Percussion 4 min 51 500 Raton 4 Percussion 4 min 53 499 Raton 1 Percussion 8 min 88 325 Raton 2 Percussion 8 min 80 400 Raton 3 Percussion 8 min 73 444 Raton 4 Percussion 8 min 74 439 Sacramento 1 Immersion 43 495
69
Table B 1 Continued
-Source Block Treatment Germination Percentage Standard Error
Sacramento 2 Immersion 30 458 Sacramento 3 Immersion 38 485 Sacramento 4 Immersion 56 496 Sacramento 1 Boiling 30 sec 47 499 Sacramento 2 Boiling 30 sec 48 500 Sacramento 3 Boiling 30 sec 48 500 Sacramento 4 Boiling 30 sec 56 496 Sacramento 1 Boiling 60 sec 52 500 Sacramento 2 Boiling 60 sec 67 470 Sacramento 3 Boiling 60 sec 43 495 Sacramento 4 Boiling 60 sec 48 500 Sacramento 1 Control 0 000 Sacramento 2 Control 3 171 Sacramento 3 Control 3 171 Sacramento 4 Control 099 Sacramento 1 Percussion 1 min 57 495 Sacramento 2 Percussion 1 min 61 488 Sacramento 3 Percussion 1 min 62 485 Sacramento 4 Percussion 1 min 55 497 Sacramento 1 Percussion 2 min 84 367 Sacramento 2 Percussion 2 min 70 458 Sacramento 3 Percussion 2 min 81 392 Sacramento 4 Percussion 2 min 69 462 Sacramento 1 Percussion 4 min 91 286 Sacramento 2 Percussion 4 min 84 367 Sacramento 3 Percussion 4 min 84 367 Sacramento 4 Percussion 4 min 94 237 Sacramento 1 Percussion 8 min 82 384 Sacramento 2 Percussion 8 min 81 392 Sacramento 3 Percussion 8 min 82 384 Sacramento 4 Percussion 8 min 81 392 Sandia 1 Immersion 76 427 Sandia 2 Immersion 70 458 Sandia 3 Immersion 70 458 Sandia 4 Immersion 81 392 Sandia 1 Boiling 30 sec 78 414 Sandia 2 Boiling 30 sec 71 454 Sandia 3 Boiling 30 sec 63 483 Sandia 4 Boiling 30 sec 78 414 Sandia 1 Boiling 60 sec 67 470 Sandia 2 Boiling 60 sec 65 477 Sandia 3 Boiling 60 sec 74 439 Sandia 4 Boiling 60 sec 66 474 Sandia 1 Control 16 367 Sandia 2 Control 7 255
70
Table B 1 Continued
Source Block Treatment Gennination Percentage Standard Error
Sandia 3 Control 8 271 Sandia 4 Control 16 367 Sandia 1 Percussion 1 min 67 470 Sandia 2 Percussion 1 min 61 488 Sandia 3 Percussion 1 min 66 474 Sandia 4 Percussion 1 min 59 492 Sandia I Percussion 2 min 82 384 Sandia 2 Percussion 2 min 83 376 Sandia 3 Percussion 2 min 81 392 Sandia 4 Percussion 2 min 84 367 Sandia 1 Percussion 4 min 89 313 Sandia 2 Percussion 4 min 86 347 Sandia 3 Percussion 4 min 89 313 Sandia 4 Percussion 4 min 85 357 Sandia 1 Percussion 8 min 80 400 Sandia 2 Percussion 8 min 77 421 Sandia 3 Percussion 8 min 69 462 Sandia 4 Percussion 8 min 66 474
LITERATURE CITED
Ashby WC Vogel WG Rogers NF 1985 Black locust in the reclamation equation General Technical Report Northeastern Forest Experiment Station USDA Forest Service NE-I05 12 p
Ballard LAT 1973 Physical barriers to gennination Seed Sci Technoll 285-303
___ 1976 Strophiolar water conduction in the seeds ofTrifolieae induced by action on the testa at nonstrophiolar sites Austr J Plant Physiol3 465-469
Barton LV 1947 Special studies on seed coat impenneability Contrib Boyce Thompson Inst 14 355-362
Baskin CC Baskin JM 1998 Seeds Ecology biogeography and evolution of donnancy and gennination Academic Press London UK 666 p
Bewley JD 1997 Seed germination and donnancy Plant Ce119 1055-1066
Bewley JD Black M 1994 Seeds physiology of development and germination 2nd ed Plenum Press New York NY 445 p
Briscoe CB 1996 Growing Nitrogen Fixing Trees in Acid Soils In MH Powell (ed) Nitrogen Fixing Trees for Acid Soils- A Field ManuaL Winrock International Morrilton AR 22 p
Copeland LO McDonald MB 1995 Principles of Seed Science and Technology 3rded Chapman amp Hall New York NY 409 p
Cox RE Klett JE 1984 Seed gennination requirements of native Colorado plants for use in the landscape Plant Propag 30(2) 6-10
Dell B 1980 Structure and function ofthe strophiolar plug in seeds ofAlbizia Lophantha Am J Bot 67 556-563
Dick-Peddie WA 1993 New Mexico Vegetation Past Present and Future University ofNew Mexico Press Albuquerque NM 244 p
Dreesen DR 2001 Propagation protocol for production ofcontainer Robinia neomexicana plants Los Lunas Plant Materials Center Los Lunas New Mexico In Native Plant Network URL httpwwwnativeplantnetworkorg (accessed 9 January 2002) Moscow (ID) University ofIdaho CoIl Natural Resources For Res Nursery
72
Dreesen DR Harrington JT 1997 Propagation of native plants for restoration projects in the Southwestern US- Preliminary Investigations In Landis TD Thompson JR tech coords National Proceedings Forest and Conservation Nursery Assocs Gen Tech Rep PNW-GTR-419 Portland OR USDA For Ser PNW Res Sta 77-88
Gottfried GJ 1980 Control ofNew Mexican locust and the effect on planted ponderosa pine in central Arizona US For Ser Res Note RM-386 Rocky Mountain For Range ExpStn Fort Collins CO
Gosling PG Samuel YK Jones SK 1995 A systematic examination of germination temperature chipping and water temperaturesoak duration pretreatments on the seeds ofLeucaena leucocephala Seed Sci amp Technol 23521-532
Hamly DH 1932 Softening of the seeds of Melilotus alba Bot Gazette 93 345shy375
Hanna PJ 1984 Anatomical features of the seed coat ofAcacia kempeana (Mueller) which relate to increased germination rate induced by heat treatment New Phytol 96 23-29
Hanks JP Dick-Peddie WA 1974 Vegetation patterns of the White Mountains New Mexico Southwest Naturalist 18(4) 371-382
Hine S Harrington JT Dreesen DR 1997 Seed scarification requirements for Robinia neomexicana In Harrington JTtech coord Proc Third SouthwestemContainer Growers Conf Feb 12-13 1997 Ft Collins CO
Hopkinson JM 1993 The strophiole of leguminous seeds Newsletter- IntI Herbage Seed Production Res Grp 18 7-8
---- 1997 Differences in hardseededness between Cunningham and Tarramba leucaena In Leucnet News The Newsletter of the International LeucaenaRes and Devlt Netwk No4
ISTA (International Seed Testing Association) 1999 International rules for seed testing rules Seed Sci amp Technol27 Supplement
Kelly KM van Staden J 1987 The lens as the site of permeability in the papilionoid seed Aspalathus linearis J Plant Physiol 128(45) 395-404
73
Keresztesi B 1988 Black locust the tree of agriculture Outlook on Agr 17(2) 77~85
Kigel 1 and Galili G 1995 Seed development and germination Marcel Dekker New York 853 p
Klemmedson 10 1994 New Mexico locust and parent material Influence on macronutrients of forest floor and soil Soil Sci Soc Am 1 58 974~980
Kuo WH Tam AW 1988 The pathway of water absorption of mung bean seeds Seed Sci amp Technol16(1) 139~144
Leadem CL 1997 Dormancy--unlocking seed secrets In Landis TD Thompson lR tech coords National Proceedings Forestand Conservation Nursery Assocs Gen Tech Rep PNW-G TR-419 Portland OR USDA For Ser PNW Res Sta 43-52
Lin Y Wagner MR Seweyestewa A Honahni MD DeGomez M McMillin JD 1996 Influence of cultural treatments on seed germination and seedling growth of Robinia neomexicana Forest Farm and Community Tree Res Reports Volume 1
Marunda CT 1990 Effects of seed pretreatment on the development ofAcacia auriculforinis and A holosericea seedlings ACIAR Proceedings Series No 28
Meginnis HG 1937 Sulphuric acid treatment to increase germination of black locust seed USDA Circ 453 34 p
Miklas PN Towensend CE Ladd SL 1987 Seedcoat anatomy and the scarification ofcicer milkvetch seed Crop Sci 27 766-772
Monsen SB 1984 Use of shrubs on mine spoils In The Challenge ofProducing Native Plants for the Intermountain Area Proceedings Intermountain Nurserymans Association 1983 conference Las Vegas NV compo PM Murphy 26-31 Ogden UT USDA For Ser Gen Tech Report INT-l68
Olson DF 1974 Robinia L Locust In Seeds of Woody Plants in the US tech coord CS Schopmeyer 728-731 USDA For Ser Agr Handbk No450
Rehman S Loescher RNl Harris P1C 1998 Dormancy breaking and germination ofAcacia salicina Lindl seeds Seed Sci amp Techno127 553-557
Rolston MP 1978 Water impermeable seed dormancy Bot Rev 44 365-396
74
SAS Institute Inc 1989 SASSTAT Users Guide Version 6 4th Edition Volume 1 Cary NC SAS Institute Inc 943 p
SerratoValenti G Conara L Ferrando M Modenesi P 1993 Structural and histochemical features ofStylosanthes scabra (Leguminosae Papilionoidae) seed coat as related to water entry Can J Bot 71 834-840
Simpson BJ 1988 A field guide to Texas trees Austin TX Texas Monthly Press 372p
Tran VN Cavanagh AK 1984 Structural aspects ofdormancy In Seed Physiology Vol 2 Murray DR (ed) p1-44 Academic Press Australia
USDA Forest Service 1948 Woody-plant seed manual USDA Misc Pub1654 416p
Vines RA 1960 Trees Shrubs and Woody Vines of the Southwest Austin Univ ofTexas Press 1104 p
Wagner MR Larson RE Craig TP 1992 Ecology and silvics ofNew Mexico locust (Robinia neomexicana) with an emphasis on the arboreal form p53-57 In JW Hanover K Miller and S Plesko (eds) Proc Intemat Confon Black Locust Biology Culture and Utilization Mich State Univ USA
Wilson JK 1944 Immersing seeds of species of Robinia in boiling water hastens germination J For 42 453-454
Zimmerman RW Carpenter SB 1980 First-year coppice production from a 5shyyear old black locust stand on surface mine spoil p309-314 Centr Hardwd For ConfProc University ofMissouri Columbia
10 Strophiole ofunimbibed New Mexico locust seed (a) and following uptake (bc)
38
In contrast hot-water treatments can excessively degrade the seed coat
(Rebman et al 1998 Marunda 1990) Random cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rebman et aI 1998 Marunda 1990) Along with a
proportionately large amount ofun-imbibed (thus under-treated) seeds (Figure l1a)
damage was observed in hot-water treated seeds from six sources during germination
counts Damage included seeds where the radicle failed to elongate (Figure 11 b)
seeds where the radicle elongated but was delayed in freeing itself from the seed coat
(Figure 11c) and seeds where the radicle completely broke off from the embryo
(Figure lId) In addition some seeds germinated in reverse (the cotyledon emerged
fIrst) If the radicle emerged completely from the seed coat they were counted as
germinated
While nearly all seeds treated for 8 minutes ofpercussion imbibed water
damage was evident in x-ray evaluations (Sacramento seed source) and in
germination counts (Holman and Capitan) at this treatment level Seed damage
during germination counts was similar to levels found in hot-water treated seeds
suggesting that over-treatment by both hot water and longer durations of percussion
may result in similar damage to seed structure
The Sacramento and Holman seed sources were two of the fIve sources that
opcrplltlpn in germination from the 4- to 8-minute durations of percussion
the benefIts ofloosening the strophiole as evidenced by imbibition of
all 8-minute treated seeds were outweighed by external and internal seed coat
39
a
b c d
11 Damage to New Mexico locust seeds observed during germination cotmts following hot water scarification 9a Hot water replicate including ~t seedlings and numerous unimbibed seeds 9b Close-up of seed where raallCle failed to elongate 9c Close-up ofseed where radicle elongated but was
-gtIn in freeing itself from the seed coat 9d Close-up ofseed where radicle broke off from embryo
40
damage (which also may be contributing to high imbibition) For the three sources
for which 8 minutes of percussion resulted in maximum germination extended
percussion of seeds necessary to loosen the strophiole may not have excessively
damaged the seed anatomy The Manzano source did not significantly change in
germination from 4 to 8 minutes of percussion suggesting that an increase in
permeable seeds at the 8-minute treatment duration was balanced by the number of
seeds that failed to germinate due to seed damage To summarize the 4-minute
percussion treatment improved germination over 8 minutes ofpercussion for a
majority of sources (five ofnine) the 8-minute percussion treatment improved
germination over 4-minutes ofpercussion for three sources and there was no
difference with the Manzano source Notably for the Raton source 8 minutes of
percussion increased germination by 235 percentage points over 4-minutes of
percussion (7875 vs 5525 respectively) the next best treatment Raton was the
only source to result in germination less than 75 for 4 minutes ofpercussion so a
grower may wish to apply longer percussion durations to lots that do not respond well
to this treatment level
Finally there was an interesting trend in the Jemez and Raton sources seed
that significantly contributed to the source by treatment interaction (Figure 12) A
~u test showed that the Jemez source had a significantly lighter seed weight than
other sources while the Raton source had an average seed weight For the Jemez
the longest duration of boiling increased germination Conversely only short
U4UVll) of percussion were needed to achieve high germination and the longest
41
80 _ Jemez a Raton
iD
~ C iD e iD
Q c 0 ~
c ~ iD 0
20
0------shy
Immersion Boiling 30 sec Boiling 60 sec
Hot Water Scarification
10degTr========~--------~------------------------------
80
60
40
20
_ Jemez Raton
0------shy
1 min 2 min 4 min 8 min
Percussion Scarification
Figure 12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources
42
b
--~----~~~~========~---- duration ofpercussion reduced germination For the Raton source longer durations
ofboiling consistently decreased germination whereas longer percussion treatments
consistently increased germination
A possible explanation is that a thick seed coat is associated with a loose
strophiole structure and that a thin seed coat is associated with a constrained
strophiole This contrast merits further study and may suggest an ecological
adaptation Further investigation ofthis phenomenon may contribute to successful
pre-screening ofunknown seed sources to determine optimal treatment leveL
43
CONCLUSION
Based on results obtained in pilot study (see Appendix) and thesis
experiments 4 minutes ofpercussion is recommended as a standard protocol for
treating an unknown source ofNew Mexico locust seed In addition to laboratory
results the 4-minute percussion treatment has resulted in germination greater than
80 and healthy seedling grow-outs for production purposes (unpublished data) In
laboratory experiments only one hundred seeds per 118 ml (40z) soil tin were
percussed at a time whereas for production we have treated up to five hundred seeds
per tin For large-scale production ofNew Mexico locust seedlings growers may
wish to experiment with larger quantities of seed and larger-size soil tins
Seed storage trials following percussion treatments were not conducted
However germination greater than 80 and healthy seedlings were obtained when
seeds were percussion-treated and subsequently stored at room-temperature (22degC)
for one week (unpublished data) Seed storage trials merit further study as the
standard hot water treatment leaves seeds in a softened condition that is not
conducive to storage
Hydroseeding is an effective method for direct seeding in reclamation
situations Percussion treatment may be conducive to hydro seeding as seeds remain
a dry relatively undamaged yet permeable condition following treatment Sulfuric
seeds also remain dry following treatment but extensive damage to seed coat
poor seedling vigor have been reported with this treatment (Kelly and van Staden
A grow-out study using a hydro seeder on a reclamation site testing percussion
44
against other treatments such as sulfuric acid and mechanical scarification also
merits further study
Papilionoids are the largest subfamily oflegumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust these legumes often play an integral role in the revegetation of
disturbed lands The pilot study (Appendix) indicates that percussion scarification
successfully treats the widely used black locust as well Developing superior
scarification methods to the standard hot water mechanical scarification and acid
treatments may facilitate the use ofpapilionoid legumes in reclamation projects
45
APPENDICES
APPENDIX A
PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR
NEW MEXICO LOCUST AND BLACK LOCUST SEEDS
Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4
Hot water and sulfuric acid soaks are traditional treatments for seeds of many
temperate woody legwnes including locusts However these scarification techniques
often produce inconsistent germination Percussion scarification where seeds are
repeatedly propelled against a hard surface was compared with hot water
scarification to evaluate treatment efficacy for New Mexico locust (Robinia
neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment
seeds were placed in a 98degC water bath which was immediately removed from the
heat source For percussion scarification seeds were placed in a soil sample tin and
agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the
control were followed by 24-hour water soaks Hot water baths resulted in 56 and
41 germination for New Mexico locust and black locust respectively For both
species nearly all durations ofpercussion increased germination over the hot water
treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3
4 and 5 minutes for black locust resulted in at least 90 germination Traditional
scarification treatments randomly degrade the entire seed coat which can lead to
tissue damage during water uptake Percussion scarification specifically weakens the
strophiole the natural source ofwater entry to the seed in papilionoid legwnes
INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom
2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031
48
Following percussion imbibition is controlled through the strophiole and underlying
tissue is protected
Keywords New Mexico locust black locust scarification percussion germination
Introduction
Locust trees (Robinia spp) are aggressive pioneer species that quickly
colonize disturbed land fix atmospheric nitrogen in the soil and stabilize slopes that
are prone to erosion (Klemmedson 1994 Ashby et al 1985) (Figure A1ab)
Recognition of these attributes has contributed to increased demand for locusts in
reclamation projects Locust seeds however exhibit physical dormancy where a
thick seed coat prevents the movement of water and gasses to the embryo (Leadem
1997)
Sulfuric acid or hot water soaks have traditionally been used to break seed
dormancy in macro-propagation of locusts Many growers are moving away from the
use of su1furic acid in treating locust seeds (Dreesen and Harrington 1997) In
addition to being dangerous acid soak durations must be specifically correlated to
seed lot (Olson 1974) Collections of native seed can vary tremendously in seed size
seed weight and hard seededness etc This variability limits the utility of sulfuric
acid soaks as even within a given lot some seeds may be damaged whereas others
are under-treated and fail to imbibe (take up water) A survey of Southwestern
container growers indicates that hot water baths are currently the most common
scarification method for locust seeds (Hine et al 1997) However hot water baths
also produce inconsistent germination (Lin et al 1996)
49
b
Figure AI New Mexico locust a primary invader following disturbance Ala Crown-sprouting at edge ofburn near Los Alamos NM--one year after Cerro Grande fire Alb Colonizing road cut Gila National Forest NM
50
Percussion scarification where seeds are repeatedly propelled against a hard
surface is an alternative dormancy-breaking method that has proven successful in
legumes related to locusts (Hamly 1932 Barton 1947) We compared this alternative
treatment to hot water scarification to evaluate treatment efficacy for New Mexico
locust and black locust seeds
Materials and Methods
Commercial New Mexico locust seeds (Western Native Seed Coaldale CO)
were collected fall 2000 in Huerfano County CO Black locust seeds were collected
September 2000 from Taos County NM by harvesting ripe pods from several trees at
a distance of up to 3 m from ground level Black locust seeds were allowed to air-dry
for 3 weeks and were threshed and separated from large chaffusing a greenhouse
fan A South Dakota Seed Blower (Chicago Illinltois) was used to remove fine chaff
from both seed lots Seeds were storedmiddotat 2-4C until the germination study was
conducted in March 2001
This study compared hot water scarification with percussion scarification
using treatment and species as experimental factors Along with a control
scarification treatments included immersion of seed in boiling water as well as
percussion durations of 1 2 3 45 or 10 minutes Four 100-seed samples were used
to test each species by scarification treatment combination
For the hot water treatment test tubes were filled with 30 mL water and
placed in a water-filled beaker (Figure A2a) The bath was raised to boiling (-98C at
51
b
c
Figure A2 Implementation ofhot water and percussion scarification A2a Hot water bath A2b Paint-shaker set-up A2c Placing soil tin containing seeds at end ofpaint shaker ann
52
Las Cruces NM elevation of 1300 meters) Each 100-seed replicate was placed in a
test tube and each test tube was immediately removed to cool at room temperature
Percussion scarification was implemented using a pneumatic paint shaker
(Central Pneumatic model 00422 Camarillo CA) (Figure A2b) This allowed
standardization of treatment intensity Compressed air pressure was maintained at
530kPa plusmn 30kPa (80psi plusmn 5psi) resulting in approximately 350 oscillations per
minute Each lOO-seed sample was placed in a 118 ml (4 oz) soil tin for shaking To
maximize lateral movement of paint shaker a paint can was used as a spacer to place
soil tins at the end of the shaking arm (Figure A2c) This allowed shaking to occur at
the greatest distance from the pivot point (fulcrum) Following all treatments
including the control seeds were soaked for twenty-four hours in distilled water prior
to plating out
Gennination was evaluated on lab benches at room temperature Each
replicate of seeds was placed in a 100 cm petri dish on moistened filter paper with
humidity maintained by enclosing the petri dishes in ziplock bags Gennination was
monitored daily for 14 days in accordance with International Seed Testing
Association standards for Robinia species (ISTA 1999) Gennination was defined as
protrusion of the radicle from the embryo by at least 1mm as viewed by the naked
eye
Categorical Analysis of Variance using Prpc Catmod (SAS Institute 1989)
was used to detennine treatment differences in germination percentages for each
source This procedure is an extension of a Chi-square test of homogeneity using the
53
natural log of the ratio of germinated to non-germinated seeds for each treatment Pshy
values less than 005 were considered significant A limited set of pairwiseshy
comparisons was conducted to compare treatment mean percents using a
conservative alpha value of 005 divided by the number of comparisons
Results
Percussion maximized germination for both New Mexico locust and black
locust There were three germination response levels for New Mexico locust (Figure
A3) First there was a low response in the control indicating a dormant seed lot
The hot water treatment and I-minute duration of percussion share a second
intermediate response Germination was highest at percussion levels from 2 to 10
minutes
A selected percussion treatment intermediate in time (5 minutes) produced the
steepest germination curve indicating the fastest germination rate (Figure A4) The
hot water treatment produced an intermediate curve or relatively slower germination
rate The control curve was very flat indicating the slowest germination rate for New
Mexico locust
Control seeds germinated poorly for black locust as well (Figure A5) Hot
water scarification improved germination over the control but all levels of percussion
improved germination over hot water The optimal durations of percussion treatment
were bracketed with a significant increase from the 1 to 2 minute percussion level a
high germination response from 2 to 5 minutes of percussion and finally a significant
54
~ d 25 CL)
p t1 0 ~ t1
g Vl ~ Vl
100~---------------------------------------------------------------
80
60
40
20
o Control Hot Water Percussion
I min Percussion
2 min Percussion
3 min
Scarification
Percussion 4 min
Figure A3 Effect of scarification on germination response for New Mexico locust
Percussion 5 min
=significant difference between adjacent treatment bars ns = no significant difference
Percussion 10 min
~ ~ () u agt
Il-t d o ~
~ ~ c3
100middotlr~~~------------------------------------e- Percussion 5min middot 0middot Hot Water -- Control
80-J=middot======
6b-middot-lmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middot--middotmiddot-middot~-middotmiddot -middotmiddot middot middotmiddot-middot 1
40
20
-----~-----------
0 0 0 middotmiddotmiddotmiddotmiddot0middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot0
0 --~-----y-----~------_---~--- T-----~
o shy -TDay 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A4 Effect of selected scarification treatments on germination speed for New Mexico locust First seven days of fourteen-day experiment are shown
Day 7
80
~ cd
5 60 ()
[) Cl d 0 1t1 d
40sect dUl
-J
20
o
ns
Scarification
Figure A5 Effect of scarification on germination response for black locust = significant difference between adjacent treatment bars ns = no significant difference
decrease at the 10-minute level This drop-off suggests that 10 minutes of percussion
over-treats seeds from this lot of-black locust
A selected percussion treatment intermediate in time (5 minutes) also had the
fastest germination rate for black locust (Figure A6)_ The hot water curve is
somewhat flatter indicating a slower germination rate The control curve is very flat
indicating a slow germination rate
A qualitative difference between hot water and percussion-treated seeds was
also observed Along with healthy germinants the hot water treatment produced
over-treated seeds (Figure A 7a) Examples of damaged germinants include seeds
where the radicle did not elongate (Figure A 7b) seeds where the radicle elongated
but was delayed in freeing itself from the seed coat (Figure A7c) and seeds where the
radicle broke off completely from the embryo (Figure A7d) In addition to these
examples of over-treatment there were numerous small dark seeds that failed to
imbibe water For percussion treatment nearly all seeds took up water (Figure A8a)
Germination was rapid and uniform with little damage evident (Figure A8b)
To summarize both New Mexico locust and black locust seed lots were
dormant The standard hot water scarification treatment improved germination but
only to 56 and 41 for New Mexico locust and black locust respectively Nearly
all percussion durations improved total germination as well as germination rate over
the hot water treatment Germination was greater than 90 for percussion durations
of 4 5 and 10 minutes for New Mexico locust and 3 4 and 5 minutes for black locust
58
CI)
sgt t CI) 0
igt 0
sect ttl t E
Vl 0
c3
100r==~==~~=r------------------------------------~ ___ Percussion 5 min
middotmiddotmiddot0middotmiddotmiddot Hot Water -lIf- Control
80r=========~--~~----------------------------~
60
40
20 -~-~---~---------- ---~------------I
omiddot
O
_~_----------lIf-----~bull -----_ shy---shy I
Day 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A6 Effect of selected scarification treatments on germination speed for black locust First seven days of fourteen-day experiment are shown
Day 7
a
b c d
Figure A7 Damage to New Mexico locust seeds observed during germination counts following hot water scarification A7a Hot water replicate including damaged seedlings and numerous unimbibed seeds A7b Close-up ofseed where radicle failed to elongate A7c Close-up ofseed where radicle elongated but was delayed in freeing itself from the seed coat A7d Close-up ofseed where radicle completely broke offfrom embryo
60
a
b
Figure A8 New Mexico locust seeds following percussion treatment at time of germination counts A8a Percussion replicate with uniformly healthy seedlings and near complete imbibition of seeds A8b Close-up ofpercussion-treated seedling
61
Discussion
Papilionoid seeds have a specific anatomical feature known as the strophiole
or lens-the natural site of water entry to the seed (Hamly 1932) The strophiole is
located on the cotyledonary lobe of a locust seed (Figure A9) When a papilionoid
legume seed is percussed for the appropriate amount of time repeated hits on the
integrated seed coat loosen the constrained cells of the strophiolar region without
excessively damaging the rest of the seed coat (Ballard 1976)
When a percussed seed is soaked water enters exclusively through the
strophiole in a controlled manner (Kelly and Staden 1987) This regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues This
contrasts with seeds that have been hot water or acid scarified treatments that can
randomly degrade the seed coat Unlocalized cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rehman et al 1998 Marunda 1990)
Papilionoids are the largest subfamily of legumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust and black locust these legumes often play an integral role in the
revegetation ofdisturbed lands Developing superior scarification methods to the
standard hot water and acid treatments should fadlitate the use ofthese legumes in
restoration projects
62
Figure A9 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (bc)
63
APPENDIXB
GERMINATION PERCENTS AND STANDARD ERRORS FOR SOURCE
BLOCK AND TREATMENT FOR NEW MEXICO LOCUST
Table B 1 Gennination percents and standard errors for source block and treatment for New Mexico locust
Source Block Treatment Germination Percentage Standard Error
Capitan 1 Immersion 74 437 Capitan 2 Immersion 69 462 Capitan 3 Immersion 71 454 Capitan 4 Immersion 67 470 Capitan Boiling 30 sec 79 407 Capitan 2 Boiling 30 sec 76 427 Capitan 3 Boiling 30 sec 72 449 Capitan 4 Boiling 30 sec 76 427 Capitan Boiling 60 sec 71 454 Capitan 2 Boiling 60 sec 54 498 Capitan 3 Boiling 60 sec 67 470 Capitan 4 Boiling 60 sec 76 427 Capitan I Control 5 218 Capitan 2 Control 7 255 Capitan 3 Control 4 196 Capitan 4 Control 5 218 Capitan 1 Percussion I min 47 499 Capitan 2 Percussion I min
470
Capitan Percussion I min 39 488
Capitan 4 Percussion I min 38 485 Capitan I Percussion 2 min 55 497 Capitan Capitan
2
Percussion 2 min Percussion 2 min
71 65
454 477
Capitan 4 Percussion 2 min 56 496 Capitan I Percussion 4 min 86 347 Capitan 2 Percussion 4 min 79 407 Capitan 3 Percussion 4 min 81 392 Capitan 4 Percussion 4 min 82 384 Capitan I Percussion 8 min 90 300 Capitan 2 Percussion 8 min 88 325 Capitan 3 Percussion 8 min 90 300 Capitan 4 Percussion 8 min 90 300 Gila I Immersion 71 454 Gila 2 Immersion 77 421 Gila
Immersion 75 433
Gila 4 Immersion 72 449
Gila I Boiling 30 sec 68 466
Gila 2 Boiling 30 sec 81 392
Gila 3 Boiling 30 sec 54 498
Gila 4 Boiling 30 sec 63 483
Gila I Boiling 60 sec 55 497
Gila 2 Boiling 60 sec 68 466
Gila 3 Boiling 60 sec 57 495
Gila 4 Boiling 60 sec 70 458
65
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Gila 2 Control 13 336 Gila 3 Control 13 336 Gila 4 Control 20 400 Gila 1 Percussion 1 min 66 474 Gila 2 Percussion 1 min 57 495 Gila 3 Percussion 1 min 73 444 Gila 4 Percussion 1 min 82 384 Gila 1 Percussion 2 min 81 392 Gila 2 Percussion 2 min 86 347 Gila 3 Percussion 2 min 85 357 Gila 4 Percussion 2 min 54 498 Gila 1 Percussion 4 min 79 407 Gila 2 Percussion 4 min 83 376 Gila 3 Percussion 4 min 84 367 Gila 4 Percussion 4 min 79 407 Gila 1 Percussion 8 min 51 500 Gila 2 Percussion 8 min 76 427 Gila 3 Percussion 8 min 52 500 Gila 4 Percussion 8 min 74 439 Holman 1 Immersion 83 376 Holman 2 Immersion 86 347 Holman 3 Immersion 81 392 Holman 4 Immersion 90 300 Holman 1 Boiling 30 sec 90 300 Holman 2 Boiling 30 sec 85 357 Holman 3 Boiling 30 sec 87 336 Holman 4 Boiling 30 sec 83 376 Holman 1 Boiling 60 sec 79 407 Holman 2 Boiling 60 sec 80 400 Holman 3 Boiling 60 sec 86 347 Holman 4 Boiling 60 sec 93 255 Holman Control 25 433 Holman 2 Control 19 392 Holman Control 21 407
Holman 4 Control 24 427
Holman Percussion 1 min 77 421
Holman 2 Percussion 1 min 85 357
Holman 3 Percussion 1 min 80 400
Holman 4 Percussion 1 min 89 313
Holman 1 Percussion 2 min 88 325
Holman 2 Percussion 2 min 96 196
Holman 3 Percussion 2 min 93 255
Holman 4 Percussion 2 min 96 196
Holman 1 Percussion 4 min 92 271
Holman 2 Percussion 4 min 97 171
66
I
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Holman 3 Percussion 4 min 91 286 Holman 4 Percussion 4 min 96 196 Holman 1 Percussion 8 min 88 325 Holman 2 Percussion 8 min 85 357 Holman 3 Percussion 8 min 83 376 Holman 4 Percussion 8 min 82 384 Jemez 1 Immersion 53 499 Jemez 2 Immersion 44 496 Jemez 3 Immersion 52 500 Jemez 4 Immersion 70 458 Jemez 1 Boiling 30 sec 58 494 Jemez 2 Boiling 30 sec 55 497 Jemez 3 Boiling 30 sec 48 500 Jemez 4 Boiling 30 sec 65 477 Jemez 1 Boiling 60 sec 68 466 Jemez 2 Boiling 60 sec 63 483 Jemez 3 Boiling 60 sec 73 444 Jemez 4 Boiling 60 sec 89 313 Jemez 1 Control 2 140 Jemez 2 Control 9 286 Jemez 3 Control 6 237 Jemez 4 Control 8 271 Jemez 1 Percussion 1 min 58 494 Jemez 2 Percussion 1 min 63 483 Jemez 3 Percussion 1 min 77 421 Jemez 4 Percussion 1 min 89 313 Jemez 1 Percussion 2 min 68 466 Jemez 2 Percussion 2 min 64 480 Jemez 3 Percussion 2 min 80 400 Jemez 4 Percussion 2 min 83 376 Jemez Percussion 4 min 68 466 Jemez 2 Percussion 4 min 79 407 Jemez 3 Percussion 4 min 70 458 Jemez 4 Percussion 4 min 83 376 Jemez 1 Percussion 8 min 52 500 Jemez 2 Percussion 8 min 59 492 Jemez 3 Percussion 8 min 61 488 Jemez 4 Percussion 8 min 70 458 Magdalena 1 Immersion 69 462 Magdalena 2 Immersion 69 462 Magdalena J Immersion 70 458 Magdalena 4 Immersion 85 357 Magdalena 1 Boiling 30 sec 65 477 Magdalena 2 Boiling 30 sec 71 454 Magdalena 3 Boiling 30 sec 68 466
67
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Magdalena 4 Boiling 30 sec 83 376 Magdalena 1 Boiling 60 sec 63 483 Magdalena 2 Boiling 60 sec 68 466 Magdalena 3 Boiling 60 sec 63 483 Magdalena 4 Boiling 60 sec 82 384 Magdalena 1 Control 11 313 Magdalena 2 Control 6 237 Magdalena 3 Control 5 218 Magdalena 4 Control 4 196 Magdalena Percussion 1 min 32 466 Magdalena 2 Percussion 1 min 45 497 Magdalena 3 Percussion 1 min 46 498 Magdalena 4 Percussion I min 36 480 Magdalena 1 Percussion 2 min 67 470 Magdalena 2 Percussion 2 min 68 466 Magdalena 3 Percussion 2 min 65 477 Magdalena 4 Percussion 2 min 81 392 Magdalena 1 Percussion 4 min 87 336 Magdalena 2 Percussion 4 min 97 171 Magdalena 3 Percussion 4 min 83 376 Magdalena 4 Percussion 4 min 97 171 Magdalena 1 Percussion 8 min 98 lAO Magdalena 2 Percussion 8 min 100 000 Magdalena 3 Percussion 8 min 96 196 Magdalena 4 Percussion 8 min 98 lAO Manzano 1 Immersion 67 470 Manzano 2 Immersion 57 495 Manzano 3 Immersion 72 4049 Manzano 4 Immersion 73 4044 Manzano 1 Boiling 30 sec 70 458 Manzano 2 Boiling 30 sec 64 480 Manzano 3 Boiling 30 sec 71 454 Manzano 4 Boiling 30 sec 80 400 Manzano I Boiling 60 sec 50 500 Manzano 2 Boiling 60 sec 53 499 Manzano 3 Boiling 60 sec 59 492 Manzano 4 Boiling 60 sec 74 439 Manzano I Control 4 196 Manzano 2 Control 8 271 Manzano 3 Control 1 099 Manzano 4 Control 6 237 Manzano 1 Percussion 1 min 52 500 Manzano 2 Percussion 1 min 39 488 Manzano 3 Percussion 1 min 36 480 Manzano 4 Percussion 1 min 51 500
68
Table B 1 Continued
Source Block Treatment Gerrni~ation Percentage Standard Error
Manzano Percussion 2 min 53 499 Manzano 2 Percussion 2 min 58 494 Manzano 3 Percussion 2 min 61 488 Manzano 4 Percussion 2 min 62 485 Manzano 1 Percussion 4 min 76 427 Manzano 2 Percussion 4 min 85 357 Manzano 3 Percussion 4 min 79 407 Manzano 4 Percussion 4 min 83 376 Manzano 1 Percussion 8 min 87 336 Manzano 2 Percussion 8 min 83 376 Manzano 3 Percussion 8 min 83 376 Manzano 4 Percussion 8 min 75 433 Raton 1 Immersion 54 498 Raton 2 Immersion 37 483 Raton 3 Immersion 39 488 Raton 4 Immersion 69 462 Raton 1 Boiling 30 sec 36 480 Raton 2 Boiling 30 sec 20 400 Raton 3 Boiling 30 sec 28 449 Raton 4 Boiling 30 sec 60 490 Raton 1 Boiling 60 sec 15 357 Raton 2 Boiling 60 sec 171 Raton 3 Boiling 60 sec 4 196 Raton 4 Boiling 60 sec 35 477 Raton 1 Control 4 196 Raton 2 Control 12 325 Raton 3 Control 2 140 Raton 4 Control 6 237 Raton 1 Percussion 1 min 52 500 Raton 2 Percussion I min 29 454 Raton 3 Percussion I min 35 477 Raton 4 Percussion 1 min 31 462 Raton 1 Percussion 2 min 55 497 Raton 2 Percussion 2 min 35 477 Raton 3 Percussion 2 min 33 470 Raton 4 Percussion 2 min 42 494 Raton 1 Percussion 4 min 65 477 Raton 2 Percussion 4 min 52 500 Raton Percussion 4 min 51 500 Raton 4 Percussion 4 min 53 499 Raton 1 Percussion 8 min 88 325 Raton 2 Percussion 8 min 80 400 Raton 3 Percussion 8 min 73 444 Raton 4 Percussion 8 min 74 439 Sacramento 1 Immersion 43 495
69
Table B 1 Continued
-Source Block Treatment Germination Percentage Standard Error
Sacramento 2 Immersion 30 458 Sacramento 3 Immersion 38 485 Sacramento 4 Immersion 56 496 Sacramento 1 Boiling 30 sec 47 499 Sacramento 2 Boiling 30 sec 48 500 Sacramento 3 Boiling 30 sec 48 500 Sacramento 4 Boiling 30 sec 56 496 Sacramento 1 Boiling 60 sec 52 500 Sacramento 2 Boiling 60 sec 67 470 Sacramento 3 Boiling 60 sec 43 495 Sacramento 4 Boiling 60 sec 48 500 Sacramento 1 Control 0 000 Sacramento 2 Control 3 171 Sacramento 3 Control 3 171 Sacramento 4 Control 099 Sacramento 1 Percussion 1 min 57 495 Sacramento 2 Percussion 1 min 61 488 Sacramento 3 Percussion 1 min 62 485 Sacramento 4 Percussion 1 min 55 497 Sacramento 1 Percussion 2 min 84 367 Sacramento 2 Percussion 2 min 70 458 Sacramento 3 Percussion 2 min 81 392 Sacramento 4 Percussion 2 min 69 462 Sacramento 1 Percussion 4 min 91 286 Sacramento 2 Percussion 4 min 84 367 Sacramento 3 Percussion 4 min 84 367 Sacramento 4 Percussion 4 min 94 237 Sacramento 1 Percussion 8 min 82 384 Sacramento 2 Percussion 8 min 81 392 Sacramento 3 Percussion 8 min 82 384 Sacramento 4 Percussion 8 min 81 392 Sandia 1 Immersion 76 427 Sandia 2 Immersion 70 458 Sandia 3 Immersion 70 458 Sandia 4 Immersion 81 392 Sandia 1 Boiling 30 sec 78 414 Sandia 2 Boiling 30 sec 71 454 Sandia 3 Boiling 30 sec 63 483 Sandia 4 Boiling 30 sec 78 414 Sandia 1 Boiling 60 sec 67 470 Sandia 2 Boiling 60 sec 65 477 Sandia 3 Boiling 60 sec 74 439 Sandia 4 Boiling 60 sec 66 474 Sandia 1 Control 16 367 Sandia 2 Control 7 255
70
Table B 1 Continued
Source Block Treatment Gennination Percentage Standard Error
Sandia 3 Control 8 271 Sandia 4 Control 16 367 Sandia 1 Percussion 1 min 67 470 Sandia 2 Percussion 1 min 61 488 Sandia 3 Percussion 1 min 66 474 Sandia 4 Percussion 1 min 59 492 Sandia I Percussion 2 min 82 384 Sandia 2 Percussion 2 min 83 376 Sandia 3 Percussion 2 min 81 392 Sandia 4 Percussion 2 min 84 367 Sandia 1 Percussion 4 min 89 313 Sandia 2 Percussion 4 min 86 347 Sandia 3 Percussion 4 min 89 313 Sandia 4 Percussion 4 min 85 357 Sandia 1 Percussion 8 min 80 400 Sandia 2 Percussion 8 min 77 421 Sandia 3 Percussion 8 min 69 462 Sandia 4 Percussion 8 min 66 474
LITERATURE CITED
Ashby WC Vogel WG Rogers NF 1985 Black locust in the reclamation equation General Technical Report Northeastern Forest Experiment Station USDA Forest Service NE-I05 12 p
Ballard LAT 1973 Physical barriers to gennination Seed Sci Technoll 285-303
___ 1976 Strophiolar water conduction in the seeds ofTrifolieae induced by action on the testa at nonstrophiolar sites Austr J Plant Physiol3 465-469
Barton LV 1947 Special studies on seed coat impenneability Contrib Boyce Thompson Inst 14 355-362
Baskin CC Baskin JM 1998 Seeds Ecology biogeography and evolution of donnancy and gennination Academic Press London UK 666 p
Bewley JD 1997 Seed germination and donnancy Plant Ce119 1055-1066
Bewley JD Black M 1994 Seeds physiology of development and germination 2nd ed Plenum Press New York NY 445 p
Briscoe CB 1996 Growing Nitrogen Fixing Trees in Acid Soils In MH Powell (ed) Nitrogen Fixing Trees for Acid Soils- A Field ManuaL Winrock International Morrilton AR 22 p
Copeland LO McDonald MB 1995 Principles of Seed Science and Technology 3rded Chapman amp Hall New York NY 409 p
Cox RE Klett JE 1984 Seed gennination requirements of native Colorado plants for use in the landscape Plant Propag 30(2) 6-10
Dell B 1980 Structure and function ofthe strophiolar plug in seeds ofAlbizia Lophantha Am J Bot 67 556-563
Dick-Peddie WA 1993 New Mexico Vegetation Past Present and Future University ofNew Mexico Press Albuquerque NM 244 p
Dreesen DR 2001 Propagation protocol for production ofcontainer Robinia neomexicana plants Los Lunas Plant Materials Center Los Lunas New Mexico In Native Plant Network URL httpwwwnativeplantnetworkorg (accessed 9 January 2002) Moscow (ID) University ofIdaho CoIl Natural Resources For Res Nursery
72
Dreesen DR Harrington JT 1997 Propagation of native plants for restoration projects in the Southwestern US- Preliminary Investigations In Landis TD Thompson JR tech coords National Proceedings Forest and Conservation Nursery Assocs Gen Tech Rep PNW-GTR-419 Portland OR USDA For Ser PNW Res Sta 77-88
Gottfried GJ 1980 Control ofNew Mexican locust and the effect on planted ponderosa pine in central Arizona US For Ser Res Note RM-386 Rocky Mountain For Range ExpStn Fort Collins CO
Gosling PG Samuel YK Jones SK 1995 A systematic examination of germination temperature chipping and water temperaturesoak duration pretreatments on the seeds ofLeucaena leucocephala Seed Sci amp Technol 23521-532
Hamly DH 1932 Softening of the seeds of Melilotus alba Bot Gazette 93 345shy375
Hanna PJ 1984 Anatomical features of the seed coat ofAcacia kempeana (Mueller) which relate to increased germination rate induced by heat treatment New Phytol 96 23-29
Hanks JP Dick-Peddie WA 1974 Vegetation patterns of the White Mountains New Mexico Southwest Naturalist 18(4) 371-382
Hine S Harrington JT Dreesen DR 1997 Seed scarification requirements for Robinia neomexicana In Harrington JTtech coord Proc Third SouthwestemContainer Growers Conf Feb 12-13 1997 Ft Collins CO
Hopkinson JM 1993 The strophiole of leguminous seeds Newsletter- IntI Herbage Seed Production Res Grp 18 7-8
---- 1997 Differences in hardseededness between Cunningham and Tarramba leucaena In Leucnet News The Newsletter of the International LeucaenaRes and Devlt Netwk No4
ISTA (International Seed Testing Association) 1999 International rules for seed testing rules Seed Sci amp Technol27 Supplement
Kelly KM van Staden J 1987 The lens as the site of permeability in the papilionoid seed Aspalathus linearis J Plant Physiol 128(45) 395-404
73
Keresztesi B 1988 Black locust the tree of agriculture Outlook on Agr 17(2) 77~85
Kigel 1 and Galili G 1995 Seed development and germination Marcel Dekker New York 853 p
Klemmedson 10 1994 New Mexico locust and parent material Influence on macronutrients of forest floor and soil Soil Sci Soc Am 1 58 974~980
Kuo WH Tam AW 1988 The pathway of water absorption of mung bean seeds Seed Sci amp Technol16(1) 139~144
Leadem CL 1997 Dormancy--unlocking seed secrets In Landis TD Thompson lR tech coords National Proceedings Forestand Conservation Nursery Assocs Gen Tech Rep PNW-G TR-419 Portland OR USDA For Ser PNW Res Sta 43-52
Lin Y Wagner MR Seweyestewa A Honahni MD DeGomez M McMillin JD 1996 Influence of cultural treatments on seed germination and seedling growth of Robinia neomexicana Forest Farm and Community Tree Res Reports Volume 1
Marunda CT 1990 Effects of seed pretreatment on the development ofAcacia auriculforinis and A holosericea seedlings ACIAR Proceedings Series No 28
Meginnis HG 1937 Sulphuric acid treatment to increase germination of black locust seed USDA Circ 453 34 p
Miklas PN Towensend CE Ladd SL 1987 Seedcoat anatomy and the scarification ofcicer milkvetch seed Crop Sci 27 766-772
Monsen SB 1984 Use of shrubs on mine spoils In The Challenge ofProducing Native Plants for the Intermountain Area Proceedings Intermountain Nurserymans Association 1983 conference Las Vegas NV compo PM Murphy 26-31 Ogden UT USDA For Ser Gen Tech Report INT-l68
Olson DF 1974 Robinia L Locust In Seeds of Woody Plants in the US tech coord CS Schopmeyer 728-731 USDA For Ser Agr Handbk No450
Rehman S Loescher RNl Harris P1C 1998 Dormancy breaking and germination ofAcacia salicina Lindl seeds Seed Sci amp Techno127 553-557
Rolston MP 1978 Water impermeable seed dormancy Bot Rev 44 365-396
74
SAS Institute Inc 1989 SASSTAT Users Guide Version 6 4th Edition Volume 1 Cary NC SAS Institute Inc 943 p
SerratoValenti G Conara L Ferrando M Modenesi P 1993 Structural and histochemical features ofStylosanthes scabra (Leguminosae Papilionoidae) seed coat as related to water entry Can J Bot 71 834-840
Simpson BJ 1988 A field guide to Texas trees Austin TX Texas Monthly Press 372p
Tran VN Cavanagh AK 1984 Structural aspects ofdormancy In Seed Physiology Vol 2 Murray DR (ed) p1-44 Academic Press Australia
USDA Forest Service 1948 Woody-plant seed manual USDA Misc Pub1654 416p
Vines RA 1960 Trees Shrubs and Woody Vines of the Southwest Austin Univ ofTexas Press 1104 p
Wagner MR Larson RE Craig TP 1992 Ecology and silvics ofNew Mexico locust (Robinia neomexicana) with an emphasis on the arboreal form p53-57 In JW Hanover K Miller and S Plesko (eds) Proc Intemat Confon Black Locust Biology Culture and Utilization Mich State Univ USA
Wilson JK 1944 Immersing seeds of species of Robinia in boiling water hastens germination J For 42 453-454
Zimmerman RW Carpenter SB 1980 First-year coppice production from a 5shyyear old black locust stand on surface mine spoil p309-314 Centr Hardwd For ConfProc University ofMissouri Columbia
In contrast hot-water treatments can excessively degrade the seed coat
(Rebman et al 1998 Marunda 1990) Random cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rebman et aI 1998 Marunda 1990) Along with a
proportionately large amount ofun-imbibed (thus under-treated) seeds (Figure l1a)
damage was observed in hot-water treated seeds from six sources during germination
counts Damage included seeds where the radicle failed to elongate (Figure 11 b)
seeds where the radicle elongated but was delayed in freeing itself from the seed coat
(Figure 11c) and seeds where the radicle completely broke off from the embryo
(Figure lId) In addition some seeds germinated in reverse (the cotyledon emerged
fIrst) If the radicle emerged completely from the seed coat they were counted as
germinated
While nearly all seeds treated for 8 minutes ofpercussion imbibed water
damage was evident in x-ray evaluations (Sacramento seed source) and in
germination counts (Holman and Capitan) at this treatment level Seed damage
during germination counts was similar to levels found in hot-water treated seeds
suggesting that over-treatment by both hot water and longer durations of percussion
may result in similar damage to seed structure
The Sacramento and Holman seed sources were two of the fIve sources that
opcrplltlpn in germination from the 4- to 8-minute durations of percussion
the benefIts ofloosening the strophiole as evidenced by imbibition of
all 8-minute treated seeds were outweighed by external and internal seed coat
39
a
b c d
11 Damage to New Mexico locust seeds observed during germination cotmts following hot water scarification 9a Hot water replicate including ~t seedlings and numerous unimbibed seeds 9b Close-up of seed where raallCle failed to elongate 9c Close-up ofseed where radicle elongated but was
-gtIn in freeing itself from the seed coat 9d Close-up ofseed where radicle broke off from embryo
40
damage (which also may be contributing to high imbibition) For the three sources
for which 8 minutes of percussion resulted in maximum germination extended
percussion of seeds necessary to loosen the strophiole may not have excessively
damaged the seed anatomy The Manzano source did not significantly change in
germination from 4 to 8 minutes of percussion suggesting that an increase in
permeable seeds at the 8-minute treatment duration was balanced by the number of
seeds that failed to germinate due to seed damage To summarize the 4-minute
percussion treatment improved germination over 8 minutes ofpercussion for a
majority of sources (five ofnine) the 8-minute percussion treatment improved
germination over 4-minutes ofpercussion for three sources and there was no
difference with the Manzano source Notably for the Raton source 8 minutes of
percussion increased germination by 235 percentage points over 4-minutes of
percussion (7875 vs 5525 respectively) the next best treatment Raton was the
only source to result in germination less than 75 for 4 minutes ofpercussion so a
grower may wish to apply longer percussion durations to lots that do not respond well
to this treatment level
Finally there was an interesting trend in the Jemez and Raton sources seed
that significantly contributed to the source by treatment interaction (Figure 12) A
~u test showed that the Jemez source had a significantly lighter seed weight than
other sources while the Raton source had an average seed weight For the Jemez
the longest duration of boiling increased germination Conversely only short
U4UVll) of percussion were needed to achieve high germination and the longest
41
80 _ Jemez a Raton
iD
~ C iD e iD
Q c 0 ~
c ~ iD 0
20
0------shy
Immersion Boiling 30 sec Boiling 60 sec
Hot Water Scarification
10degTr========~--------~------------------------------
80
60
40
20
_ Jemez Raton
0------shy
1 min 2 min 4 min 8 min
Percussion Scarification
Figure 12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources
42
b
--~----~~~~========~---- duration ofpercussion reduced germination For the Raton source longer durations
ofboiling consistently decreased germination whereas longer percussion treatments
consistently increased germination
A possible explanation is that a thick seed coat is associated with a loose
strophiole structure and that a thin seed coat is associated with a constrained
strophiole This contrast merits further study and may suggest an ecological
adaptation Further investigation ofthis phenomenon may contribute to successful
pre-screening ofunknown seed sources to determine optimal treatment leveL
43
CONCLUSION
Based on results obtained in pilot study (see Appendix) and thesis
experiments 4 minutes ofpercussion is recommended as a standard protocol for
treating an unknown source ofNew Mexico locust seed In addition to laboratory
results the 4-minute percussion treatment has resulted in germination greater than
80 and healthy seedling grow-outs for production purposes (unpublished data) In
laboratory experiments only one hundred seeds per 118 ml (40z) soil tin were
percussed at a time whereas for production we have treated up to five hundred seeds
per tin For large-scale production ofNew Mexico locust seedlings growers may
wish to experiment with larger quantities of seed and larger-size soil tins
Seed storage trials following percussion treatments were not conducted
However germination greater than 80 and healthy seedlings were obtained when
seeds were percussion-treated and subsequently stored at room-temperature (22degC)
for one week (unpublished data) Seed storage trials merit further study as the
standard hot water treatment leaves seeds in a softened condition that is not
conducive to storage
Hydroseeding is an effective method for direct seeding in reclamation
situations Percussion treatment may be conducive to hydro seeding as seeds remain
a dry relatively undamaged yet permeable condition following treatment Sulfuric
seeds also remain dry following treatment but extensive damage to seed coat
poor seedling vigor have been reported with this treatment (Kelly and van Staden
A grow-out study using a hydro seeder on a reclamation site testing percussion
44
against other treatments such as sulfuric acid and mechanical scarification also
merits further study
Papilionoids are the largest subfamily oflegumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust these legumes often play an integral role in the revegetation of
disturbed lands The pilot study (Appendix) indicates that percussion scarification
successfully treats the widely used black locust as well Developing superior
scarification methods to the standard hot water mechanical scarification and acid
treatments may facilitate the use ofpapilionoid legumes in reclamation projects
45
APPENDICES
APPENDIX A
PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR
NEW MEXICO LOCUST AND BLACK LOCUST SEEDS
Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4
Hot water and sulfuric acid soaks are traditional treatments for seeds of many
temperate woody legwnes including locusts However these scarification techniques
often produce inconsistent germination Percussion scarification where seeds are
repeatedly propelled against a hard surface was compared with hot water
scarification to evaluate treatment efficacy for New Mexico locust (Robinia
neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment
seeds were placed in a 98degC water bath which was immediately removed from the
heat source For percussion scarification seeds were placed in a soil sample tin and
agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the
control were followed by 24-hour water soaks Hot water baths resulted in 56 and
41 germination for New Mexico locust and black locust respectively For both
species nearly all durations ofpercussion increased germination over the hot water
treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3
4 and 5 minutes for black locust resulted in at least 90 germination Traditional
scarification treatments randomly degrade the entire seed coat which can lead to
tissue damage during water uptake Percussion scarification specifically weakens the
strophiole the natural source ofwater entry to the seed in papilionoid legwnes
INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom
2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031
48
Following percussion imbibition is controlled through the strophiole and underlying
tissue is protected
Keywords New Mexico locust black locust scarification percussion germination
Introduction
Locust trees (Robinia spp) are aggressive pioneer species that quickly
colonize disturbed land fix atmospheric nitrogen in the soil and stabilize slopes that
are prone to erosion (Klemmedson 1994 Ashby et al 1985) (Figure A1ab)
Recognition of these attributes has contributed to increased demand for locusts in
reclamation projects Locust seeds however exhibit physical dormancy where a
thick seed coat prevents the movement of water and gasses to the embryo (Leadem
1997)
Sulfuric acid or hot water soaks have traditionally been used to break seed
dormancy in macro-propagation of locusts Many growers are moving away from the
use of su1furic acid in treating locust seeds (Dreesen and Harrington 1997) In
addition to being dangerous acid soak durations must be specifically correlated to
seed lot (Olson 1974) Collections of native seed can vary tremendously in seed size
seed weight and hard seededness etc This variability limits the utility of sulfuric
acid soaks as even within a given lot some seeds may be damaged whereas others
are under-treated and fail to imbibe (take up water) A survey of Southwestern
container growers indicates that hot water baths are currently the most common
scarification method for locust seeds (Hine et al 1997) However hot water baths
also produce inconsistent germination (Lin et al 1996)
49
b
Figure AI New Mexico locust a primary invader following disturbance Ala Crown-sprouting at edge ofburn near Los Alamos NM--one year after Cerro Grande fire Alb Colonizing road cut Gila National Forest NM
50
Percussion scarification where seeds are repeatedly propelled against a hard
surface is an alternative dormancy-breaking method that has proven successful in
legumes related to locusts (Hamly 1932 Barton 1947) We compared this alternative
treatment to hot water scarification to evaluate treatment efficacy for New Mexico
locust and black locust seeds
Materials and Methods
Commercial New Mexico locust seeds (Western Native Seed Coaldale CO)
were collected fall 2000 in Huerfano County CO Black locust seeds were collected
September 2000 from Taos County NM by harvesting ripe pods from several trees at
a distance of up to 3 m from ground level Black locust seeds were allowed to air-dry
for 3 weeks and were threshed and separated from large chaffusing a greenhouse
fan A South Dakota Seed Blower (Chicago Illinltois) was used to remove fine chaff
from both seed lots Seeds were storedmiddotat 2-4C until the germination study was
conducted in March 2001
This study compared hot water scarification with percussion scarification
using treatment and species as experimental factors Along with a control
scarification treatments included immersion of seed in boiling water as well as
percussion durations of 1 2 3 45 or 10 minutes Four 100-seed samples were used
to test each species by scarification treatment combination
For the hot water treatment test tubes were filled with 30 mL water and
placed in a water-filled beaker (Figure A2a) The bath was raised to boiling (-98C at
51
b
c
Figure A2 Implementation ofhot water and percussion scarification A2a Hot water bath A2b Paint-shaker set-up A2c Placing soil tin containing seeds at end ofpaint shaker ann
52
Las Cruces NM elevation of 1300 meters) Each 100-seed replicate was placed in a
test tube and each test tube was immediately removed to cool at room temperature
Percussion scarification was implemented using a pneumatic paint shaker
(Central Pneumatic model 00422 Camarillo CA) (Figure A2b) This allowed
standardization of treatment intensity Compressed air pressure was maintained at
530kPa plusmn 30kPa (80psi plusmn 5psi) resulting in approximately 350 oscillations per
minute Each lOO-seed sample was placed in a 118 ml (4 oz) soil tin for shaking To
maximize lateral movement of paint shaker a paint can was used as a spacer to place
soil tins at the end of the shaking arm (Figure A2c) This allowed shaking to occur at
the greatest distance from the pivot point (fulcrum) Following all treatments
including the control seeds were soaked for twenty-four hours in distilled water prior
to plating out
Gennination was evaluated on lab benches at room temperature Each
replicate of seeds was placed in a 100 cm petri dish on moistened filter paper with
humidity maintained by enclosing the petri dishes in ziplock bags Gennination was
monitored daily for 14 days in accordance with International Seed Testing
Association standards for Robinia species (ISTA 1999) Gennination was defined as
protrusion of the radicle from the embryo by at least 1mm as viewed by the naked
eye
Categorical Analysis of Variance using Prpc Catmod (SAS Institute 1989)
was used to detennine treatment differences in germination percentages for each
source This procedure is an extension of a Chi-square test of homogeneity using the
53
natural log of the ratio of germinated to non-germinated seeds for each treatment Pshy
values less than 005 were considered significant A limited set of pairwiseshy
comparisons was conducted to compare treatment mean percents using a
conservative alpha value of 005 divided by the number of comparisons
Results
Percussion maximized germination for both New Mexico locust and black
locust There were three germination response levels for New Mexico locust (Figure
A3) First there was a low response in the control indicating a dormant seed lot
The hot water treatment and I-minute duration of percussion share a second
intermediate response Germination was highest at percussion levels from 2 to 10
minutes
A selected percussion treatment intermediate in time (5 minutes) produced the
steepest germination curve indicating the fastest germination rate (Figure A4) The
hot water treatment produced an intermediate curve or relatively slower germination
rate The control curve was very flat indicating the slowest germination rate for New
Mexico locust
Control seeds germinated poorly for black locust as well (Figure A5) Hot
water scarification improved germination over the control but all levels of percussion
improved germination over hot water The optimal durations of percussion treatment
were bracketed with a significant increase from the 1 to 2 minute percussion level a
high germination response from 2 to 5 minutes of percussion and finally a significant
54
~ d 25 CL)
p t1 0 ~ t1
g Vl ~ Vl
100~---------------------------------------------------------------
80
60
40
20
o Control Hot Water Percussion
I min Percussion
2 min Percussion
3 min
Scarification
Percussion 4 min
Figure A3 Effect of scarification on germination response for New Mexico locust
Percussion 5 min
=significant difference between adjacent treatment bars ns = no significant difference
Percussion 10 min
~ ~ () u agt
Il-t d o ~
~ ~ c3
100middotlr~~~------------------------------------e- Percussion 5min middot 0middot Hot Water -- Control
80-J=middot======
6b-middot-lmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middot--middotmiddot-middot~-middotmiddot -middotmiddot middot middotmiddot-middot 1
40
20
-----~-----------
0 0 0 middotmiddotmiddotmiddotmiddot0middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot0
0 --~-----y-----~------_---~--- T-----~
o shy -TDay 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A4 Effect of selected scarification treatments on germination speed for New Mexico locust First seven days of fourteen-day experiment are shown
Day 7
80
~ cd
5 60 ()
[) Cl d 0 1t1 d
40sect dUl
-J
20
o
ns
Scarification
Figure A5 Effect of scarification on germination response for black locust = significant difference between adjacent treatment bars ns = no significant difference
decrease at the 10-minute level This drop-off suggests that 10 minutes of percussion
over-treats seeds from this lot of-black locust
A selected percussion treatment intermediate in time (5 minutes) also had the
fastest germination rate for black locust (Figure A6)_ The hot water curve is
somewhat flatter indicating a slower germination rate The control curve is very flat
indicating a slow germination rate
A qualitative difference between hot water and percussion-treated seeds was
also observed Along with healthy germinants the hot water treatment produced
over-treated seeds (Figure A 7a) Examples of damaged germinants include seeds
where the radicle did not elongate (Figure A 7b) seeds where the radicle elongated
but was delayed in freeing itself from the seed coat (Figure A7c) and seeds where the
radicle broke off completely from the embryo (Figure A7d) In addition to these
examples of over-treatment there were numerous small dark seeds that failed to
imbibe water For percussion treatment nearly all seeds took up water (Figure A8a)
Germination was rapid and uniform with little damage evident (Figure A8b)
To summarize both New Mexico locust and black locust seed lots were
dormant The standard hot water scarification treatment improved germination but
only to 56 and 41 for New Mexico locust and black locust respectively Nearly
all percussion durations improved total germination as well as germination rate over
the hot water treatment Germination was greater than 90 for percussion durations
of 4 5 and 10 minutes for New Mexico locust and 3 4 and 5 minutes for black locust
58
CI)
sgt t CI) 0
igt 0
sect ttl t E
Vl 0
c3
100r==~==~~=r------------------------------------~ ___ Percussion 5 min
middotmiddotmiddot0middotmiddotmiddot Hot Water -lIf- Control
80r=========~--~~----------------------------~
60
40
20 -~-~---~---------- ---~------------I
omiddot
O
_~_----------lIf-----~bull -----_ shy---shy I
Day 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A6 Effect of selected scarification treatments on germination speed for black locust First seven days of fourteen-day experiment are shown
Day 7
a
b c d
Figure A7 Damage to New Mexico locust seeds observed during germination counts following hot water scarification A7a Hot water replicate including damaged seedlings and numerous unimbibed seeds A7b Close-up ofseed where radicle failed to elongate A7c Close-up ofseed where radicle elongated but was delayed in freeing itself from the seed coat A7d Close-up ofseed where radicle completely broke offfrom embryo
60
a
b
Figure A8 New Mexico locust seeds following percussion treatment at time of germination counts A8a Percussion replicate with uniformly healthy seedlings and near complete imbibition of seeds A8b Close-up ofpercussion-treated seedling
61
Discussion
Papilionoid seeds have a specific anatomical feature known as the strophiole
or lens-the natural site of water entry to the seed (Hamly 1932) The strophiole is
located on the cotyledonary lobe of a locust seed (Figure A9) When a papilionoid
legume seed is percussed for the appropriate amount of time repeated hits on the
integrated seed coat loosen the constrained cells of the strophiolar region without
excessively damaging the rest of the seed coat (Ballard 1976)
When a percussed seed is soaked water enters exclusively through the
strophiole in a controlled manner (Kelly and Staden 1987) This regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues This
contrasts with seeds that have been hot water or acid scarified treatments that can
randomly degrade the seed coat Unlocalized cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rehman et al 1998 Marunda 1990)
Papilionoids are the largest subfamily of legumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust and black locust these legumes often play an integral role in the
revegetation ofdisturbed lands Developing superior scarification methods to the
standard hot water and acid treatments should fadlitate the use ofthese legumes in
restoration projects
62
Figure A9 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (bc)
63
APPENDIXB
GERMINATION PERCENTS AND STANDARD ERRORS FOR SOURCE
BLOCK AND TREATMENT FOR NEW MEXICO LOCUST
Table B 1 Gennination percents and standard errors for source block and treatment for New Mexico locust
Source Block Treatment Germination Percentage Standard Error
Capitan 1 Immersion 74 437 Capitan 2 Immersion 69 462 Capitan 3 Immersion 71 454 Capitan 4 Immersion 67 470 Capitan Boiling 30 sec 79 407 Capitan 2 Boiling 30 sec 76 427 Capitan 3 Boiling 30 sec 72 449 Capitan 4 Boiling 30 sec 76 427 Capitan Boiling 60 sec 71 454 Capitan 2 Boiling 60 sec 54 498 Capitan 3 Boiling 60 sec 67 470 Capitan 4 Boiling 60 sec 76 427 Capitan I Control 5 218 Capitan 2 Control 7 255 Capitan 3 Control 4 196 Capitan 4 Control 5 218 Capitan 1 Percussion I min 47 499 Capitan 2 Percussion I min
470
Capitan Percussion I min 39 488
Capitan 4 Percussion I min 38 485 Capitan I Percussion 2 min 55 497 Capitan Capitan
2
Percussion 2 min Percussion 2 min
71 65
454 477
Capitan 4 Percussion 2 min 56 496 Capitan I Percussion 4 min 86 347 Capitan 2 Percussion 4 min 79 407 Capitan 3 Percussion 4 min 81 392 Capitan 4 Percussion 4 min 82 384 Capitan I Percussion 8 min 90 300 Capitan 2 Percussion 8 min 88 325 Capitan 3 Percussion 8 min 90 300 Capitan 4 Percussion 8 min 90 300 Gila I Immersion 71 454 Gila 2 Immersion 77 421 Gila
Immersion 75 433
Gila 4 Immersion 72 449
Gila I Boiling 30 sec 68 466
Gila 2 Boiling 30 sec 81 392
Gila 3 Boiling 30 sec 54 498
Gila 4 Boiling 30 sec 63 483
Gila I Boiling 60 sec 55 497
Gila 2 Boiling 60 sec 68 466
Gila 3 Boiling 60 sec 57 495
Gila 4 Boiling 60 sec 70 458
65
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Gila 2 Control 13 336 Gila 3 Control 13 336 Gila 4 Control 20 400 Gila 1 Percussion 1 min 66 474 Gila 2 Percussion 1 min 57 495 Gila 3 Percussion 1 min 73 444 Gila 4 Percussion 1 min 82 384 Gila 1 Percussion 2 min 81 392 Gila 2 Percussion 2 min 86 347 Gila 3 Percussion 2 min 85 357 Gila 4 Percussion 2 min 54 498 Gila 1 Percussion 4 min 79 407 Gila 2 Percussion 4 min 83 376 Gila 3 Percussion 4 min 84 367 Gila 4 Percussion 4 min 79 407 Gila 1 Percussion 8 min 51 500 Gila 2 Percussion 8 min 76 427 Gila 3 Percussion 8 min 52 500 Gila 4 Percussion 8 min 74 439 Holman 1 Immersion 83 376 Holman 2 Immersion 86 347 Holman 3 Immersion 81 392 Holman 4 Immersion 90 300 Holman 1 Boiling 30 sec 90 300 Holman 2 Boiling 30 sec 85 357 Holman 3 Boiling 30 sec 87 336 Holman 4 Boiling 30 sec 83 376 Holman 1 Boiling 60 sec 79 407 Holman 2 Boiling 60 sec 80 400 Holman 3 Boiling 60 sec 86 347 Holman 4 Boiling 60 sec 93 255 Holman Control 25 433 Holman 2 Control 19 392 Holman Control 21 407
Holman 4 Control 24 427
Holman Percussion 1 min 77 421
Holman 2 Percussion 1 min 85 357
Holman 3 Percussion 1 min 80 400
Holman 4 Percussion 1 min 89 313
Holman 1 Percussion 2 min 88 325
Holman 2 Percussion 2 min 96 196
Holman 3 Percussion 2 min 93 255
Holman 4 Percussion 2 min 96 196
Holman 1 Percussion 4 min 92 271
Holman 2 Percussion 4 min 97 171
66
I
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Holman 3 Percussion 4 min 91 286 Holman 4 Percussion 4 min 96 196 Holman 1 Percussion 8 min 88 325 Holman 2 Percussion 8 min 85 357 Holman 3 Percussion 8 min 83 376 Holman 4 Percussion 8 min 82 384 Jemez 1 Immersion 53 499 Jemez 2 Immersion 44 496 Jemez 3 Immersion 52 500 Jemez 4 Immersion 70 458 Jemez 1 Boiling 30 sec 58 494 Jemez 2 Boiling 30 sec 55 497 Jemez 3 Boiling 30 sec 48 500 Jemez 4 Boiling 30 sec 65 477 Jemez 1 Boiling 60 sec 68 466 Jemez 2 Boiling 60 sec 63 483 Jemez 3 Boiling 60 sec 73 444 Jemez 4 Boiling 60 sec 89 313 Jemez 1 Control 2 140 Jemez 2 Control 9 286 Jemez 3 Control 6 237 Jemez 4 Control 8 271 Jemez 1 Percussion 1 min 58 494 Jemez 2 Percussion 1 min 63 483 Jemez 3 Percussion 1 min 77 421 Jemez 4 Percussion 1 min 89 313 Jemez 1 Percussion 2 min 68 466 Jemez 2 Percussion 2 min 64 480 Jemez 3 Percussion 2 min 80 400 Jemez 4 Percussion 2 min 83 376 Jemez Percussion 4 min 68 466 Jemez 2 Percussion 4 min 79 407 Jemez 3 Percussion 4 min 70 458 Jemez 4 Percussion 4 min 83 376 Jemez 1 Percussion 8 min 52 500 Jemez 2 Percussion 8 min 59 492 Jemez 3 Percussion 8 min 61 488 Jemez 4 Percussion 8 min 70 458 Magdalena 1 Immersion 69 462 Magdalena 2 Immersion 69 462 Magdalena J Immersion 70 458 Magdalena 4 Immersion 85 357 Magdalena 1 Boiling 30 sec 65 477 Magdalena 2 Boiling 30 sec 71 454 Magdalena 3 Boiling 30 sec 68 466
67
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Magdalena 4 Boiling 30 sec 83 376 Magdalena 1 Boiling 60 sec 63 483 Magdalena 2 Boiling 60 sec 68 466 Magdalena 3 Boiling 60 sec 63 483 Magdalena 4 Boiling 60 sec 82 384 Magdalena 1 Control 11 313 Magdalena 2 Control 6 237 Magdalena 3 Control 5 218 Magdalena 4 Control 4 196 Magdalena Percussion 1 min 32 466 Magdalena 2 Percussion 1 min 45 497 Magdalena 3 Percussion 1 min 46 498 Magdalena 4 Percussion I min 36 480 Magdalena 1 Percussion 2 min 67 470 Magdalena 2 Percussion 2 min 68 466 Magdalena 3 Percussion 2 min 65 477 Magdalena 4 Percussion 2 min 81 392 Magdalena 1 Percussion 4 min 87 336 Magdalena 2 Percussion 4 min 97 171 Magdalena 3 Percussion 4 min 83 376 Magdalena 4 Percussion 4 min 97 171 Magdalena 1 Percussion 8 min 98 lAO Magdalena 2 Percussion 8 min 100 000 Magdalena 3 Percussion 8 min 96 196 Magdalena 4 Percussion 8 min 98 lAO Manzano 1 Immersion 67 470 Manzano 2 Immersion 57 495 Manzano 3 Immersion 72 4049 Manzano 4 Immersion 73 4044 Manzano 1 Boiling 30 sec 70 458 Manzano 2 Boiling 30 sec 64 480 Manzano 3 Boiling 30 sec 71 454 Manzano 4 Boiling 30 sec 80 400 Manzano I Boiling 60 sec 50 500 Manzano 2 Boiling 60 sec 53 499 Manzano 3 Boiling 60 sec 59 492 Manzano 4 Boiling 60 sec 74 439 Manzano I Control 4 196 Manzano 2 Control 8 271 Manzano 3 Control 1 099 Manzano 4 Control 6 237 Manzano 1 Percussion 1 min 52 500 Manzano 2 Percussion 1 min 39 488 Manzano 3 Percussion 1 min 36 480 Manzano 4 Percussion 1 min 51 500
68
Table B 1 Continued
Source Block Treatment Gerrni~ation Percentage Standard Error
Manzano Percussion 2 min 53 499 Manzano 2 Percussion 2 min 58 494 Manzano 3 Percussion 2 min 61 488 Manzano 4 Percussion 2 min 62 485 Manzano 1 Percussion 4 min 76 427 Manzano 2 Percussion 4 min 85 357 Manzano 3 Percussion 4 min 79 407 Manzano 4 Percussion 4 min 83 376 Manzano 1 Percussion 8 min 87 336 Manzano 2 Percussion 8 min 83 376 Manzano 3 Percussion 8 min 83 376 Manzano 4 Percussion 8 min 75 433 Raton 1 Immersion 54 498 Raton 2 Immersion 37 483 Raton 3 Immersion 39 488 Raton 4 Immersion 69 462 Raton 1 Boiling 30 sec 36 480 Raton 2 Boiling 30 sec 20 400 Raton 3 Boiling 30 sec 28 449 Raton 4 Boiling 30 sec 60 490 Raton 1 Boiling 60 sec 15 357 Raton 2 Boiling 60 sec 171 Raton 3 Boiling 60 sec 4 196 Raton 4 Boiling 60 sec 35 477 Raton 1 Control 4 196 Raton 2 Control 12 325 Raton 3 Control 2 140 Raton 4 Control 6 237 Raton 1 Percussion 1 min 52 500 Raton 2 Percussion I min 29 454 Raton 3 Percussion I min 35 477 Raton 4 Percussion 1 min 31 462 Raton 1 Percussion 2 min 55 497 Raton 2 Percussion 2 min 35 477 Raton 3 Percussion 2 min 33 470 Raton 4 Percussion 2 min 42 494 Raton 1 Percussion 4 min 65 477 Raton 2 Percussion 4 min 52 500 Raton Percussion 4 min 51 500 Raton 4 Percussion 4 min 53 499 Raton 1 Percussion 8 min 88 325 Raton 2 Percussion 8 min 80 400 Raton 3 Percussion 8 min 73 444 Raton 4 Percussion 8 min 74 439 Sacramento 1 Immersion 43 495
69
Table B 1 Continued
-Source Block Treatment Germination Percentage Standard Error
Sacramento 2 Immersion 30 458 Sacramento 3 Immersion 38 485 Sacramento 4 Immersion 56 496 Sacramento 1 Boiling 30 sec 47 499 Sacramento 2 Boiling 30 sec 48 500 Sacramento 3 Boiling 30 sec 48 500 Sacramento 4 Boiling 30 sec 56 496 Sacramento 1 Boiling 60 sec 52 500 Sacramento 2 Boiling 60 sec 67 470 Sacramento 3 Boiling 60 sec 43 495 Sacramento 4 Boiling 60 sec 48 500 Sacramento 1 Control 0 000 Sacramento 2 Control 3 171 Sacramento 3 Control 3 171 Sacramento 4 Control 099 Sacramento 1 Percussion 1 min 57 495 Sacramento 2 Percussion 1 min 61 488 Sacramento 3 Percussion 1 min 62 485 Sacramento 4 Percussion 1 min 55 497 Sacramento 1 Percussion 2 min 84 367 Sacramento 2 Percussion 2 min 70 458 Sacramento 3 Percussion 2 min 81 392 Sacramento 4 Percussion 2 min 69 462 Sacramento 1 Percussion 4 min 91 286 Sacramento 2 Percussion 4 min 84 367 Sacramento 3 Percussion 4 min 84 367 Sacramento 4 Percussion 4 min 94 237 Sacramento 1 Percussion 8 min 82 384 Sacramento 2 Percussion 8 min 81 392 Sacramento 3 Percussion 8 min 82 384 Sacramento 4 Percussion 8 min 81 392 Sandia 1 Immersion 76 427 Sandia 2 Immersion 70 458 Sandia 3 Immersion 70 458 Sandia 4 Immersion 81 392 Sandia 1 Boiling 30 sec 78 414 Sandia 2 Boiling 30 sec 71 454 Sandia 3 Boiling 30 sec 63 483 Sandia 4 Boiling 30 sec 78 414 Sandia 1 Boiling 60 sec 67 470 Sandia 2 Boiling 60 sec 65 477 Sandia 3 Boiling 60 sec 74 439 Sandia 4 Boiling 60 sec 66 474 Sandia 1 Control 16 367 Sandia 2 Control 7 255
70
Table B 1 Continued
Source Block Treatment Gennination Percentage Standard Error
Sandia 3 Control 8 271 Sandia 4 Control 16 367 Sandia 1 Percussion 1 min 67 470 Sandia 2 Percussion 1 min 61 488 Sandia 3 Percussion 1 min 66 474 Sandia 4 Percussion 1 min 59 492 Sandia I Percussion 2 min 82 384 Sandia 2 Percussion 2 min 83 376 Sandia 3 Percussion 2 min 81 392 Sandia 4 Percussion 2 min 84 367 Sandia 1 Percussion 4 min 89 313 Sandia 2 Percussion 4 min 86 347 Sandia 3 Percussion 4 min 89 313 Sandia 4 Percussion 4 min 85 357 Sandia 1 Percussion 8 min 80 400 Sandia 2 Percussion 8 min 77 421 Sandia 3 Percussion 8 min 69 462 Sandia 4 Percussion 8 min 66 474
LITERATURE CITED
Ashby WC Vogel WG Rogers NF 1985 Black locust in the reclamation equation General Technical Report Northeastern Forest Experiment Station USDA Forest Service NE-I05 12 p
Ballard LAT 1973 Physical barriers to gennination Seed Sci Technoll 285-303
___ 1976 Strophiolar water conduction in the seeds ofTrifolieae induced by action on the testa at nonstrophiolar sites Austr J Plant Physiol3 465-469
Barton LV 1947 Special studies on seed coat impenneability Contrib Boyce Thompson Inst 14 355-362
Baskin CC Baskin JM 1998 Seeds Ecology biogeography and evolution of donnancy and gennination Academic Press London UK 666 p
Bewley JD 1997 Seed germination and donnancy Plant Ce119 1055-1066
Bewley JD Black M 1994 Seeds physiology of development and germination 2nd ed Plenum Press New York NY 445 p
Briscoe CB 1996 Growing Nitrogen Fixing Trees in Acid Soils In MH Powell (ed) Nitrogen Fixing Trees for Acid Soils- A Field ManuaL Winrock International Morrilton AR 22 p
Copeland LO McDonald MB 1995 Principles of Seed Science and Technology 3rded Chapman amp Hall New York NY 409 p
Cox RE Klett JE 1984 Seed gennination requirements of native Colorado plants for use in the landscape Plant Propag 30(2) 6-10
Dell B 1980 Structure and function ofthe strophiolar plug in seeds ofAlbizia Lophantha Am J Bot 67 556-563
Dick-Peddie WA 1993 New Mexico Vegetation Past Present and Future University ofNew Mexico Press Albuquerque NM 244 p
Dreesen DR 2001 Propagation protocol for production ofcontainer Robinia neomexicana plants Los Lunas Plant Materials Center Los Lunas New Mexico In Native Plant Network URL httpwwwnativeplantnetworkorg (accessed 9 January 2002) Moscow (ID) University ofIdaho CoIl Natural Resources For Res Nursery
72
Dreesen DR Harrington JT 1997 Propagation of native plants for restoration projects in the Southwestern US- Preliminary Investigations In Landis TD Thompson JR tech coords National Proceedings Forest and Conservation Nursery Assocs Gen Tech Rep PNW-GTR-419 Portland OR USDA For Ser PNW Res Sta 77-88
Gottfried GJ 1980 Control ofNew Mexican locust and the effect on planted ponderosa pine in central Arizona US For Ser Res Note RM-386 Rocky Mountain For Range ExpStn Fort Collins CO
Gosling PG Samuel YK Jones SK 1995 A systematic examination of germination temperature chipping and water temperaturesoak duration pretreatments on the seeds ofLeucaena leucocephala Seed Sci amp Technol 23521-532
Hamly DH 1932 Softening of the seeds of Melilotus alba Bot Gazette 93 345shy375
Hanna PJ 1984 Anatomical features of the seed coat ofAcacia kempeana (Mueller) which relate to increased germination rate induced by heat treatment New Phytol 96 23-29
Hanks JP Dick-Peddie WA 1974 Vegetation patterns of the White Mountains New Mexico Southwest Naturalist 18(4) 371-382
Hine S Harrington JT Dreesen DR 1997 Seed scarification requirements for Robinia neomexicana In Harrington JTtech coord Proc Third SouthwestemContainer Growers Conf Feb 12-13 1997 Ft Collins CO
Hopkinson JM 1993 The strophiole of leguminous seeds Newsletter- IntI Herbage Seed Production Res Grp 18 7-8
---- 1997 Differences in hardseededness between Cunningham and Tarramba leucaena In Leucnet News The Newsletter of the International LeucaenaRes and Devlt Netwk No4
ISTA (International Seed Testing Association) 1999 International rules for seed testing rules Seed Sci amp Technol27 Supplement
Kelly KM van Staden J 1987 The lens as the site of permeability in the papilionoid seed Aspalathus linearis J Plant Physiol 128(45) 395-404
73
Keresztesi B 1988 Black locust the tree of agriculture Outlook on Agr 17(2) 77~85
Kigel 1 and Galili G 1995 Seed development and germination Marcel Dekker New York 853 p
Klemmedson 10 1994 New Mexico locust and parent material Influence on macronutrients of forest floor and soil Soil Sci Soc Am 1 58 974~980
Kuo WH Tam AW 1988 The pathway of water absorption of mung bean seeds Seed Sci amp Technol16(1) 139~144
Leadem CL 1997 Dormancy--unlocking seed secrets In Landis TD Thompson lR tech coords National Proceedings Forestand Conservation Nursery Assocs Gen Tech Rep PNW-G TR-419 Portland OR USDA For Ser PNW Res Sta 43-52
Lin Y Wagner MR Seweyestewa A Honahni MD DeGomez M McMillin JD 1996 Influence of cultural treatments on seed germination and seedling growth of Robinia neomexicana Forest Farm and Community Tree Res Reports Volume 1
Marunda CT 1990 Effects of seed pretreatment on the development ofAcacia auriculforinis and A holosericea seedlings ACIAR Proceedings Series No 28
Meginnis HG 1937 Sulphuric acid treatment to increase germination of black locust seed USDA Circ 453 34 p
Miklas PN Towensend CE Ladd SL 1987 Seedcoat anatomy and the scarification ofcicer milkvetch seed Crop Sci 27 766-772
Monsen SB 1984 Use of shrubs on mine spoils In The Challenge ofProducing Native Plants for the Intermountain Area Proceedings Intermountain Nurserymans Association 1983 conference Las Vegas NV compo PM Murphy 26-31 Ogden UT USDA For Ser Gen Tech Report INT-l68
Olson DF 1974 Robinia L Locust In Seeds of Woody Plants in the US tech coord CS Schopmeyer 728-731 USDA For Ser Agr Handbk No450
Rehman S Loescher RNl Harris P1C 1998 Dormancy breaking and germination ofAcacia salicina Lindl seeds Seed Sci amp Techno127 553-557
Rolston MP 1978 Water impermeable seed dormancy Bot Rev 44 365-396
74
SAS Institute Inc 1989 SASSTAT Users Guide Version 6 4th Edition Volume 1 Cary NC SAS Institute Inc 943 p
SerratoValenti G Conara L Ferrando M Modenesi P 1993 Structural and histochemical features ofStylosanthes scabra (Leguminosae Papilionoidae) seed coat as related to water entry Can J Bot 71 834-840
Simpson BJ 1988 A field guide to Texas trees Austin TX Texas Monthly Press 372p
Tran VN Cavanagh AK 1984 Structural aspects ofdormancy In Seed Physiology Vol 2 Murray DR (ed) p1-44 Academic Press Australia
USDA Forest Service 1948 Woody-plant seed manual USDA Misc Pub1654 416p
Vines RA 1960 Trees Shrubs and Woody Vines of the Southwest Austin Univ ofTexas Press 1104 p
Wagner MR Larson RE Craig TP 1992 Ecology and silvics ofNew Mexico locust (Robinia neomexicana) with an emphasis on the arboreal form p53-57 In JW Hanover K Miller and S Plesko (eds) Proc Intemat Confon Black Locust Biology Culture and Utilization Mich State Univ USA
Wilson JK 1944 Immersing seeds of species of Robinia in boiling water hastens germination J For 42 453-454
Zimmerman RW Carpenter SB 1980 First-year coppice production from a 5shyyear old black locust stand on surface mine spoil p309-314 Centr Hardwd For ConfProc University ofMissouri Columbia
a
b c d
11 Damage to New Mexico locust seeds observed during germination cotmts following hot water scarification 9a Hot water replicate including ~t seedlings and numerous unimbibed seeds 9b Close-up of seed where raallCle failed to elongate 9c Close-up ofseed where radicle elongated but was
-gtIn in freeing itself from the seed coat 9d Close-up ofseed where radicle broke off from embryo
40
damage (which also may be contributing to high imbibition) For the three sources
for which 8 minutes of percussion resulted in maximum germination extended
percussion of seeds necessary to loosen the strophiole may not have excessively
damaged the seed anatomy The Manzano source did not significantly change in
germination from 4 to 8 minutes of percussion suggesting that an increase in
permeable seeds at the 8-minute treatment duration was balanced by the number of
seeds that failed to germinate due to seed damage To summarize the 4-minute
percussion treatment improved germination over 8 minutes ofpercussion for a
majority of sources (five ofnine) the 8-minute percussion treatment improved
germination over 4-minutes ofpercussion for three sources and there was no
difference with the Manzano source Notably for the Raton source 8 minutes of
percussion increased germination by 235 percentage points over 4-minutes of
percussion (7875 vs 5525 respectively) the next best treatment Raton was the
only source to result in germination less than 75 for 4 minutes ofpercussion so a
grower may wish to apply longer percussion durations to lots that do not respond well
to this treatment level
Finally there was an interesting trend in the Jemez and Raton sources seed
that significantly contributed to the source by treatment interaction (Figure 12) A
~u test showed that the Jemez source had a significantly lighter seed weight than
other sources while the Raton source had an average seed weight For the Jemez
the longest duration of boiling increased germination Conversely only short
U4UVll) of percussion were needed to achieve high germination and the longest
41
80 _ Jemez a Raton
iD
~ C iD e iD
Q c 0 ~
c ~ iD 0
20
0------shy
Immersion Boiling 30 sec Boiling 60 sec
Hot Water Scarification
10degTr========~--------~------------------------------
80
60
40
20
_ Jemez Raton
0------shy
1 min 2 min 4 min 8 min
Percussion Scarification
Figure 12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources
42
b
--~----~~~~========~---- duration ofpercussion reduced germination For the Raton source longer durations
ofboiling consistently decreased germination whereas longer percussion treatments
consistently increased germination
A possible explanation is that a thick seed coat is associated with a loose
strophiole structure and that a thin seed coat is associated with a constrained
strophiole This contrast merits further study and may suggest an ecological
adaptation Further investigation ofthis phenomenon may contribute to successful
pre-screening ofunknown seed sources to determine optimal treatment leveL
43
CONCLUSION
Based on results obtained in pilot study (see Appendix) and thesis
experiments 4 minutes ofpercussion is recommended as a standard protocol for
treating an unknown source ofNew Mexico locust seed In addition to laboratory
results the 4-minute percussion treatment has resulted in germination greater than
80 and healthy seedling grow-outs for production purposes (unpublished data) In
laboratory experiments only one hundred seeds per 118 ml (40z) soil tin were
percussed at a time whereas for production we have treated up to five hundred seeds
per tin For large-scale production ofNew Mexico locust seedlings growers may
wish to experiment with larger quantities of seed and larger-size soil tins
Seed storage trials following percussion treatments were not conducted
However germination greater than 80 and healthy seedlings were obtained when
seeds were percussion-treated and subsequently stored at room-temperature (22degC)
for one week (unpublished data) Seed storage trials merit further study as the
standard hot water treatment leaves seeds in a softened condition that is not
conducive to storage
Hydroseeding is an effective method for direct seeding in reclamation
situations Percussion treatment may be conducive to hydro seeding as seeds remain
a dry relatively undamaged yet permeable condition following treatment Sulfuric
seeds also remain dry following treatment but extensive damage to seed coat
poor seedling vigor have been reported with this treatment (Kelly and van Staden
A grow-out study using a hydro seeder on a reclamation site testing percussion
44
against other treatments such as sulfuric acid and mechanical scarification also
merits further study
Papilionoids are the largest subfamily oflegumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust these legumes often play an integral role in the revegetation of
disturbed lands The pilot study (Appendix) indicates that percussion scarification
successfully treats the widely used black locust as well Developing superior
scarification methods to the standard hot water mechanical scarification and acid
treatments may facilitate the use ofpapilionoid legumes in reclamation projects
45
APPENDICES
APPENDIX A
PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR
NEW MEXICO LOCUST AND BLACK LOCUST SEEDS
Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4
Hot water and sulfuric acid soaks are traditional treatments for seeds of many
temperate woody legwnes including locusts However these scarification techniques
often produce inconsistent germination Percussion scarification where seeds are
repeatedly propelled against a hard surface was compared with hot water
scarification to evaluate treatment efficacy for New Mexico locust (Robinia
neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment
seeds were placed in a 98degC water bath which was immediately removed from the
heat source For percussion scarification seeds were placed in a soil sample tin and
agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the
control were followed by 24-hour water soaks Hot water baths resulted in 56 and
41 germination for New Mexico locust and black locust respectively For both
species nearly all durations ofpercussion increased germination over the hot water
treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3
4 and 5 minutes for black locust resulted in at least 90 germination Traditional
scarification treatments randomly degrade the entire seed coat which can lead to
tissue damage during water uptake Percussion scarification specifically weakens the
strophiole the natural source ofwater entry to the seed in papilionoid legwnes
INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom
2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031
48
Following percussion imbibition is controlled through the strophiole and underlying
tissue is protected
Keywords New Mexico locust black locust scarification percussion germination
Introduction
Locust trees (Robinia spp) are aggressive pioneer species that quickly
colonize disturbed land fix atmospheric nitrogen in the soil and stabilize slopes that
are prone to erosion (Klemmedson 1994 Ashby et al 1985) (Figure A1ab)
Recognition of these attributes has contributed to increased demand for locusts in
reclamation projects Locust seeds however exhibit physical dormancy where a
thick seed coat prevents the movement of water and gasses to the embryo (Leadem
1997)
Sulfuric acid or hot water soaks have traditionally been used to break seed
dormancy in macro-propagation of locusts Many growers are moving away from the
use of su1furic acid in treating locust seeds (Dreesen and Harrington 1997) In
addition to being dangerous acid soak durations must be specifically correlated to
seed lot (Olson 1974) Collections of native seed can vary tremendously in seed size
seed weight and hard seededness etc This variability limits the utility of sulfuric
acid soaks as even within a given lot some seeds may be damaged whereas others
are under-treated and fail to imbibe (take up water) A survey of Southwestern
container growers indicates that hot water baths are currently the most common
scarification method for locust seeds (Hine et al 1997) However hot water baths
also produce inconsistent germination (Lin et al 1996)
49
b
Figure AI New Mexico locust a primary invader following disturbance Ala Crown-sprouting at edge ofburn near Los Alamos NM--one year after Cerro Grande fire Alb Colonizing road cut Gila National Forest NM
50
Percussion scarification where seeds are repeatedly propelled against a hard
surface is an alternative dormancy-breaking method that has proven successful in
legumes related to locusts (Hamly 1932 Barton 1947) We compared this alternative
treatment to hot water scarification to evaluate treatment efficacy for New Mexico
locust and black locust seeds
Materials and Methods
Commercial New Mexico locust seeds (Western Native Seed Coaldale CO)
were collected fall 2000 in Huerfano County CO Black locust seeds were collected
September 2000 from Taos County NM by harvesting ripe pods from several trees at
a distance of up to 3 m from ground level Black locust seeds were allowed to air-dry
for 3 weeks and were threshed and separated from large chaffusing a greenhouse
fan A South Dakota Seed Blower (Chicago Illinltois) was used to remove fine chaff
from both seed lots Seeds were storedmiddotat 2-4C until the germination study was
conducted in March 2001
This study compared hot water scarification with percussion scarification
using treatment and species as experimental factors Along with a control
scarification treatments included immersion of seed in boiling water as well as
percussion durations of 1 2 3 45 or 10 minutes Four 100-seed samples were used
to test each species by scarification treatment combination
For the hot water treatment test tubes were filled with 30 mL water and
placed in a water-filled beaker (Figure A2a) The bath was raised to boiling (-98C at
51
b
c
Figure A2 Implementation ofhot water and percussion scarification A2a Hot water bath A2b Paint-shaker set-up A2c Placing soil tin containing seeds at end ofpaint shaker ann
52
Las Cruces NM elevation of 1300 meters) Each 100-seed replicate was placed in a
test tube and each test tube was immediately removed to cool at room temperature
Percussion scarification was implemented using a pneumatic paint shaker
(Central Pneumatic model 00422 Camarillo CA) (Figure A2b) This allowed
standardization of treatment intensity Compressed air pressure was maintained at
530kPa plusmn 30kPa (80psi plusmn 5psi) resulting in approximately 350 oscillations per
minute Each lOO-seed sample was placed in a 118 ml (4 oz) soil tin for shaking To
maximize lateral movement of paint shaker a paint can was used as a spacer to place
soil tins at the end of the shaking arm (Figure A2c) This allowed shaking to occur at
the greatest distance from the pivot point (fulcrum) Following all treatments
including the control seeds were soaked for twenty-four hours in distilled water prior
to plating out
Gennination was evaluated on lab benches at room temperature Each
replicate of seeds was placed in a 100 cm petri dish on moistened filter paper with
humidity maintained by enclosing the petri dishes in ziplock bags Gennination was
monitored daily for 14 days in accordance with International Seed Testing
Association standards for Robinia species (ISTA 1999) Gennination was defined as
protrusion of the radicle from the embryo by at least 1mm as viewed by the naked
eye
Categorical Analysis of Variance using Prpc Catmod (SAS Institute 1989)
was used to detennine treatment differences in germination percentages for each
source This procedure is an extension of a Chi-square test of homogeneity using the
53
natural log of the ratio of germinated to non-germinated seeds for each treatment Pshy
values less than 005 were considered significant A limited set of pairwiseshy
comparisons was conducted to compare treatment mean percents using a
conservative alpha value of 005 divided by the number of comparisons
Results
Percussion maximized germination for both New Mexico locust and black
locust There were three germination response levels for New Mexico locust (Figure
A3) First there was a low response in the control indicating a dormant seed lot
The hot water treatment and I-minute duration of percussion share a second
intermediate response Germination was highest at percussion levels from 2 to 10
minutes
A selected percussion treatment intermediate in time (5 minutes) produced the
steepest germination curve indicating the fastest germination rate (Figure A4) The
hot water treatment produced an intermediate curve or relatively slower germination
rate The control curve was very flat indicating the slowest germination rate for New
Mexico locust
Control seeds germinated poorly for black locust as well (Figure A5) Hot
water scarification improved germination over the control but all levels of percussion
improved germination over hot water The optimal durations of percussion treatment
were bracketed with a significant increase from the 1 to 2 minute percussion level a
high germination response from 2 to 5 minutes of percussion and finally a significant
54
~ d 25 CL)
p t1 0 ~ t1
g Vl ~ Vl
100~---------------------------------------------------------------
80
60
40
20
o Control Hot Water Percussion
I min Percussion
2 min Percussion
3 min
Scarification
Percussion 4 min
Figure A3 Effect of scarification on germination response for New Mexico locust
Percussion 5 min
=significant difference between adjacent treatment bars ns = no significant difference
Percussion 10 min
~ ~ () u agt
Il-t d o ~
~ ~ c3
100middotlr~~~------------------------------------e- Percussion 5min middot 0middot Hot Water -- Control
80-J=middot======
6b-middot-lmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middot--middotmiddot-middot~-middotmiddot -middotmiddot middot middotmiddot-middot 1
40
20
-----~-----------
0 0 0 middotmiddotmiddotmiddotmiddot0middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot0
0 --~-----y-----~------_---~--- T-----~
o shy -TDay 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A4 Effect of selected scarification treatments on germination speed for New Mexico locust First seven days of fourteen-day experiment are shown
Day 7
80
~ cd
5 60 ()
[) Cl d 0 1t1 d
40sect dUl
-J
20
o
ns
Scarification
Figure A5 Effect of scarification on germination response for black locust = significant difference between adjacent treatment bars ns = no significant difference
decrease at the 10-minute level This drop-off suggests that 10 minutes of percussion
over-treats seeds from this lot of-black locust
A selected percussion treatment intermediate in time (5 minutes) also had the
fastest germination rate for black locust (Figure A6)_ The hot water curve is
somewhat flatter indicating a slower germination rate The control curve is very flat
indicating a slow germination rate
A qualitative difference between hot water and percussion-treated seeds was
also observed Along with healthy germinants the hot water treatment produced
over-treated seeds (Figure A 7a) Examples of damaged germinants include seeds
where the radicle did not elongate (Figure A 7b) seeds where the radicle elongated
but was delayed in freeing itself from the seed coat (Figure A7c) and seeds where the
radicle broke off completely from the embryo (Figure A7d) In addition to these
examples of over-treatment there were numerous small dark seeds that failed to
imbibe water For percussion treatment nearly all seeds took up water (Figure A8a)
Germination was rapid and uniform with little damage evident (Figure A8b)
To summarize both New Mexico locust and black locust seed lots were
dormant The standard hot water scarification treatment improved germination but
only to 56 and 41 for New Mexico locust and black locust respectively Nearly
all percussion durations improved total germination as well as germination rate over
the hot water treatment Germination was greater than 90 for percussion durations
of 4 5 and 10 minutes for New Mexico locust and 3 4 and 5 minutes for black locust
58
CI)
sgt t CI) 0
igt 0
sect ttl t E
Vl 0
c3
100r==~==~~=r------------------------------------~ ___ Percussion 5 min
middotmiddotmiddot0middotmiddotmiddot Hot Water -lIf- Control
80r=========~--~~----------------------------~
60
40
20 -~-~---~---------- ---~------------I
omiddot
O
_~_----------lIf-----~bull -----_ shy---shy I
Day 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A6 Effect of selected scarification treatments on germination speed for black locust First seven days of fourteen-day experiment are shown
Day 7
a
b c d
Figure A7 Damage to New Mexico locust seeds observed during germination counts following hot water scarification A7a Hot water replicate including damaged seedlings and numerous unimbibed seeds A7b Close-up ofseed where radicle failed to elongate A7c Close-up ofseed where radicle elongated but was delayed in freeing itself from the seed coat A7d Close-up ofseed where radicle completely broke offfrom embryo
60
a
b
Figure A8 New Mexico locust seeds following percussion treatment at time of germination counts A8a Percussion replicate with uniformly healthy seedlings and near complete imbibition of seeds A8b Close-up ofpercussion-treated seedling
61
Discussion
Papilionoid seeds have a specific anatomical feature known as the strophiole
or lens-the natural site of water entry to the seed (Hamly 1932) The strophiole is
located on the cotyledonary lobe of a locust seed (Figure A9) When a papilionoid
legume seed is percussed for the appropriate amount of time repeated hits on the
integrated seed coat loosen the constrained cells of the strophiolar region without
excessively damaging the rest of the seed coat (Ballard 1976)
When a percussed seed is soaked water enters exclusively through the
strophiole in a controlled manner (Kelly and Staden 1987) This regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues This
contrasts with seeds that have been hot water or acid scarified treatments that can
randomly degrade the seed coat Unlocalized cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rehman et al 1998 Marunda 1990)
Papilionoids are the largest subfamily of legumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust and black locust these legumes often play an integral role in the
revegetation ofdisturbed lands Developing superior scarification methods to the
standard hot water and acid treatments should fadlitate the use ofthese legumes in
restoration projects
62
Figure A9 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (bc)
63
APPENDIXB
GERMINATION PERCENTS AND STANDARD ERRORS FOR SOURCE
BLOCK AND TREATMENT FOR NEW MEXICO LOCUST
Table B 1 Gennination percents and standard errors for source block and treatment for New Mexico locust
Source Block Treatment Germination Percentage Standard Error
Capitan 1 Immersion 74 437 Capitan 2 Immersion 69 462 Capitan 3 Immersion 71 454 Capitan 4 Immersion 67 470 Capitan Boiling 30 sec 79 407 Capitan 2 Boiling 30 sec 76 427 Capitan 3 Boiling 30 sec 72 449 Capitan 4 Boiling 30 sec 76 427 Capitan Boiling 60 sec 71 454 Capitan 2 Boiling 60 sec 54 498 Capitan 3 Boiling 60 sec 67 470 Capitan 4 Boiling 60 sec 76 427 Capitan I Control 5 218 Capitan 2 Control 7 255 Capitan 3 Control 4 196 Capitan 4 Control 5 218 Capitan 1 Percussion I min 47 499 Capitan 2 Percussion I min
470
Capitan Percussion I min 39 488
Capitan 4 Percussion I min 38 485 Capitan I Percussion 2 min 55 497 Capitan Capitan
2
Percussion 2 min Percussion 2 min
71 65
454 477
Capitan 4 Percussion 2 min 56 496 Capitan I Percussion 4 min 86 347 Capitan 2 Percussion 4 min 79 407 Capitan 3 Percussion 4 min 81 392 Capitan 4 Percussion 4 min 82 384 Capitan I Percussion 8 min 90 300 Capitan 2 Percussion 8 min 88 325 Capitan 3 Percussion 8 min 90 300 Capitan 4 Percussion 8 min 90 300 Gila I Immersion 71 454 Gila 2 Immersion 77 421 Gila
Immersion 75 433
Gila 4 Immersion 72 449
Gila I Boiling 30 sec 68 466
Gila 2 Boiling 30 sec 81 392
Gila 3 Boiling 30 sec 54 498
Gila 4 Boiling 30 sec 63 483
Gila I Boiling 60 sec 55 497
Gila 2 Boiling 60 sec 68 466
Gila 3 Boiling 60 sec 57 495
Gila 4 Boiling 60 sec 70 458
65
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Gila 2 Control 13 336 Gila 3 Control 13 336 Gila 4 Control 20 400 Gila 1 Percussion 1 min 66 474 Gila 2 Percussion 1 min 57 495 Gila 3 Percussion 1 min 73 444 Gila 4 Percussion 1 min 82 384 Gila 1 Percussion 2 min 81 392 Gila 2 Percussion 2 min 86 347 Gila 3 Percussion 2 min 85 357 Gila 4 Percussion 2 min 54 498 Gila 1 Percussion 4 min 79 407 Gila 2 Percussion 4 min 83 376 Gila 3 Percussion 4 min 84 367 Gila 4 Percussion 4 min 79 407 Gila 1 Percussion 8 min 51 500 Gila 2 Percussion 8 min 76 427 Gila 3 Percussion 8 min 52 500 Gila 4 Percussion 8 min 74 439 Holman 1 Immersion 83 376 Holman 2 Immersion 86 347 Holman 3 Immersion 81 392 Holman 4 Immersion 90 300 Holman 1 Boiling 30 sec 90 300 Holman 2 Boiling 30 sec 85 357 Holman 3 Boiling 30 sec 87 336 Holman 4 Boiling 30 sec 83 376 Holman 1 Boiling 60 sec 79 407 Holman 2 Boiling 60 sec 80 400 Holman 3 Boiling 60 sec 86 347 Holman 4 Boiling 60 sec 93 255 Holman Control 25 433 Holman 2 Control 19 392 Holman Control 21 407
Holman 4 Control 24 427
Holman Percussion 1 min 77 421
Holman 2 Percussion 1 min 85 357
Holman 3 Percussion 1 min 80 400
Holman 4 Percussion 1 min 89 313
Holman 1 Percussion 2 min 88 325
Holman 2 Percussion 2 min 96 196
Holman 3 Percussion 2 min 93 255
Holman 4 Percussion 2 min 96 196
Holman 1 Percussion 4 min 92 271
Holman 2 Percussion 4 min 97 171
66
I
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Holman 3 Percussion 4 min 91 286 Holman 4 Percussion 4 min 96 196 Holman 1 Percussion 8 min 88 325 Holman 2 Percussion 8 min 85 357 Holman 3 Percussion 8 min 83 376 Holman 4 Percussion 8 min 82 384 Jemez 1 Immersion 53 499 Jemez 2 Immersion 44 496 Jemez 3 Immersion 52 500 Jemez 4 Immersion 70 458 Jemez 1 Boiling 30 sec 58 494 Jemez 2 Boiling 30 sec 55 497 Jemez 3 Boiling 30 sec 48 500 Jemez 4 Boiling 30 sec 65 477 Jemez 1 Boiling 60 sec 68 466 Jemez 2 Boiling 60 sec 63 483 Jemez 3 Boiling 60 sec 73 444 Jemez 4 Boiling 60 sec 89 313 Jemez 1 Control 2 140 Jemez 2 Control 9 286 Jemez 3 Control 6 237 Jemez 4 Control 8 271 Jemez 1 Percussion 1 min 58 494 Jemez 2 Percussion 1 min 63 483 Jemez 3 Percussion 1 min 77 421 Jemez 4 Percussion 1 min 89 313 Jemez 1 Percussion 2 min 68 466 Jemez 2 Percussion 2 min 64 480 Jemez 3 Percussion 2 min 80 400 Jemez 4 Percussion 2 min 83 376 Jemez Percussion 4 min 68 466 Jemez 2 Percussion 4 min 79 407 Jemez 3 Percussion 4 min 70 458 Jemez 4 Percussion 4 min 83 376 Jemez 1 Percussion 8 min 52 500 Jemez 2 Percussion 8 min 59 492 Jemez 3 Percussion 8 min 61 488 Jemez 4 Percussion 8 min 70 458 Magdalena 1 Immersion 69 462 Magdalena 2 Immersion 69 462 Magdalena J Immersion 70 458 Magdalena 4 Immersion 85 357 Magdalena 1 Boiling 30 sec 65 477 Magdalena 2 Boiling 30 sec 71 454 Magdalena 3 Boiling 30 sec 68 466
67
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Magdalena 4 Boiling 30 sec 83 376 Magdalena 1 Boiling 60 sec 63 483 Magdalena 2 Boiling 60 sec 68 466 Magdalena 3 Boiling 60 sec 63 483 Magdalena 4 Boiling 60 sec 82 384 Magdalena 1 Control 11 313 Magdalena 2 Control 6 237 Magdalena 3 Control 5 218 Magdalena 4 Control 4 196 Magdalena Percussion 1 min 32 466 Magdalena 2 Percussion 1 min 45 497 Magdalena 3 Percussion 1 min 46 498 Magdalena 4 Percussion I min 36 480 Magdalena 1 Percussion 2 min 67 470 Magdalena 2 Percussion 2 min 68 466 Magdalena 3 Percussion 2 min 65 477 Magdalena 4 Percussion 2 min 81 392 Magdalena 1 Percussion 4 min 87 336 Magdalena 2 Percussion 4 min 97 171 Magdalena 3 Percussion 4 min 83 376 Magdalena 4 Percussion 4 min 97 171 Magdalena 1 Percussion 8 min 98 lAO Magdalena 2 Percussion 8 min 100 000 Magdalena 3 Percussion 8 min 96 196 Magdalena 4 Percussion 8 min 98 lAO Manzano 1 Immersion 67 470 Manzano 2 Immersion 57 495 Manzano 3 Immersion 72 4049 Manzano 4 Immersion 73 4044 Manzano 1 Boiling 30 sec 70 458 Manzano 2 Boiling 30 sec 64 480 Manzano 3 Boiling 30 sec 71 454 Manzano 4 Boiling 30 sec 80 400 Manzano I Boiling 60 sec 50 500 Manzano 2 Boiling 60 sec 53 499 Manzano 3 Boiling 60 sec 59 492 Manzano 4 Boiling 60 sec 74 439 Manzano I Control 4 196 Manzano 2 Control 8 271 Manzano 3 Control 1 099 Manzano 4 Control 6 237 Manzano 1 Percussion 1 min 52 500 Manzano 2 Percussion 1 min 39 488 Manzano 3 Percussion 1 min 36 480 Manzano 4 Percussion 1 min 51 500
68
Table B 1 Continued
Source Block Treatment Gerrni~ation Percentage Standard Error
Manzano Percussion 2 min 53 499 Manzano 2 Percussion 2 min 58 494 Manzano 3 Percussion 2 min 61 488 Manzano 4 Percussion 2 min 62 485 Manzano 1 Percussion 4 min 76 427 Manzano 2 Percussion 4 min 85 357 Manzano 3 Percussion 4 min 79 407 Manzano 4 Percussion 4 min 83 376 Manzano 1 Percussion 8 min 87 336 Manzano 2 Percussion 8 min 83 376 Manzano 3 Percussion 8 min 83 376 Manzano 4 Percussion 8 min 75 433 Raton 1 Immersion 54 498 Raton 2 Immersion 37 483 Raton 3 Immersion 39 488 Raton 4 Immersion 69 462 Raton 1 Boiling 30 sec 36 480 Raton 2 Boiling 30 sec 20 400 Raton 3 Boiling 30 sec 28 449 Raton 4 Boiling 30 sec 60 490 Raton 1 Boiling 60 sec 15 357 Raton 2 Boiling 60 sec 171 Raton 3 Boiling 60 sec 4 196 Raton 4 Boiling 60 sec 35 477 Raton 1 Control 4 196 Raton 2 Control 12 325 Raton 3 Control 2 140 Raton 4 Control 6 237 Raton 1 Percussion 1 min 52 500 Raton 2 Percussion I min 29 454 Raton 3 Percussion I min 35 477 Raton 4 Percussion 1 min 31 462 Raton 1 Percussion 2 min 55 497 Raton 2 Percussion 2 min 35 477 Raton 3 Percussion 2 min 33 470 Raton 4 Percussion 2 min 42 494 Raton 1 Percussion 4 min 65 477 Raton 2 Percussion 4 min 52 500 Raton Percussion 4 min 51 500 Raton 4 Percussion 4 min 53 499 Raton 1 Percussion 8 min 88 325 Raton 2 Percussion 8 min 80 400 Raton 3 Percussion 8 min 73 444 Raton 4 Percussion 8 min 74 439 Sacramento 1 Immersion 43 495
69
Table B 1 Continued
-Source Block Treatment Germination Percentage Standard Error
Sacramento 2 Immersion 30 458 Sacramento 3 Immersion 38 485 Sacramento 4 Immersion 56 496 Sacramento 1 Boiling 30 sec 47 499 Sacramento 2 Boiling 30 sec 48 500 Sacramento 3 Boiling 30 sec 48 500 Sacramento 4 Boiling 30 sec 56 496 Sacramento 1 Boiling 60 sec 52 500 Sacramento 2 Boiling 60 sec 67 470 Sacramento 3 Boiling 60 sec 43 495 Sacramento 4 Boiling 60 sec 48 500 Sacramento 1 Control 0 000 Sacramento 2 Control 3 171 Sacramento 3 Control 3 171 Sacramento 4 Control 099 Sacramento 1 Percussion 1 min 57 495 Sacramento 2 Percussion 1 min 61 488 Sacramento 3 Percussion 1 min 62 485 Sacramento 4 Percussion 1 min 55 497 Sacramento 1 Percussion 2 min 84 367 Sacramento 2 Percussion 2 min 70 458 Sacramento 3 Percussion 2 min 81 392 Sacramento 4 Percussion 2 min 69 462 Sacramento 1 Percussion 4 min 91 286 Sacramento 2 Percussion 4 min 84 367 Sacramento 3 Percussion 4 min 84 367 Sacramento 4 Percussion 4 min 94 237 Sacramento 1 Percussion 8 min 82 384 Sacramento 2 Percussion 8 min 81 392 Sacramento 3 Percussion 8 min 82 384 Sacramento 4 Percussion 8 min 81 392 Sandia 1 Immersion 76 427 Sandia 2 Immersion 70 458 Sandia 3 Immersion 70 458 Sandia 4 Immersion 81 392 Sandia 1 Boiling 30 sec 78 414 Sandia 2 Boiling 30 sec 71 454 Sandia 3 Boiling 30 sec 63 483 Sandia 4 Boiling 30 sec 78 414 Sandia 1 Boiling 60 sec 67 470 Sandia 2 Boiling 60 sec 65 477 Sandia 3 Boiling 60 sec 74 439 Sandia 4 Boiling 60 sec 66 474 Sandia 1 Control 16 367 Sandia 2 Control 7 255
70
Table B 1 Continued
Source Block Treatment Gennination Percentage Standard Error
Sandia 3 Control 8 271 Sandia 4 Control 16 367 Sandia 1 Percussion 1 min 67 470 Sandia 2 Percussion 1 min 61 488 Sandia 3 Percussion 1 min 66 474 Sandia 4 Percussion 1 min 59 492 Sandia I Percussion 2 min 82 384 Sandia 2 Percussion 2 min 83 376 Sandia 3 Percussion 2 min 81 392 Sandia 4 Percussion 2 min 84 367 Sandia 1 Percussion 4 min 89 313 Sandia 2 Percussion 4 min 86 347 Sandia 3 Percussion 4 min 89 313 Sandia 4 Percussion 4 min 85 357 Sandia 1 Percussion 8 min 80 400 Sandia 2 Percussion 8 min 77 421 Sandia 3 Percussion 8 min 69 462 Sandia 4 Percussion 8 min 66 474
LITERATURE CITED
Ashby WC Vogel WG Rogers NF 1985 Black locust in the reclamation equation General Technical Report Northeastern Forest Experiment Station USDA Forest Service NE-I05 12 p
Ballard LAT 1973 Physical barriers to gennination Seed Sci Technoll 285-303
___ 1976 Strophiolar water conduction in the seeds ofTrifolieae induced by action on the testa at nonstrophiolar sites Austr J Plant Physiol3 465-469
Barton LV 1947 Special studies on seed coat impenneability Contrib Boyce Thompson Inst 14 355-362
Baskin CC Baskin JM 1998 Seeds Ecology biogeography and evolution of donnancy and gennination Academic Press London UK 666 p
Bewley JD 1997 Seed germination and donnancy Plant Ce119 1055-1066
Bewley JD Black M 1994 Seeds physiology of development and germination 2nd ed Plenum Press New York NY 445 p
Briscoe CB 1996 Growing Nitrogen Fixing Trees in Acid Soils In MH Powell (ed) Nitrogen Fixing Trees for Acid Soils- A Field ManuaL Winrock International Morrilton AR 22 p
Copeland LO McDonald MB 1995 Principles of Seed Science and Technology 3rded Chapman amp Hall New York NY 409 p
Cox RE Klett JE 1984 Seed gennination requirements of native Colorado plants for use in the landscape Plant Propag 30(2) 6-10
Dell B 1980 Structure and function ofthe strophiolar plug in seeds ofAlbizia Lophantha Am J Bot 67 556-563
Dick-Peddie WA 1993 New Mexico Vegetation Past Present and Future University ofNew Mexico Press Albuquerque NM 244 p
Dreesen DR 2001 Propagation protocol for production ofcontainer Robinia neomexicana plants Los Lunas Plant Materials Center Los Lunas New Mexico In Native Plant Network URL httpwwwnativeplantnetworkorg (accessed 9 January 2002) Moscow (ID) University ofIdaho CoIl Natural Resources For Res Nursery
72
Dreesen DR Harrington JT 1997 Propagation of native plants for restoration projects in the Southwestern US- Preliminary Investigations In Landis TD Thompson JR tech coords National Proceedings Forest and Conservation Nursery Assocs Gen Tech Rep PNW-GTR-419 Portland OR USDA For Ser PNW Res Sta 77-88
Gottfried GJ 1980 Control ofNew Mexican locust and the effect on planted ponderosa pine in central Arizona US For Ser Res Note RM-386 Rocky Mountain For Range ExpStn Fort Collins CO
Gosling PG Samuel YK Jones SK 1995 A systematic examination of germination temperature chipping and water temperaturesoak duration pretreatments on the seeds ofLeucaena leucocephala Seed Sci amp Technol 23521-532
Hamly DH 1932 Softening of the seeds of Melilotus alba Bot Gazette 93 345shy375
Hanna PJ 1984 Anatomical features of the seed coat ofAcacia kempeana (Mueller) which relate to increased germination rate induced by heat treatment New Phytol 96 23-29
Hanks JP Dick-Peddie WA 1974 Vegetation patterns of the White Mountains New Mexico Southwest Naturalist 18(4) 371-382
Hine S Harrington JT Dreesen DR 1997 Seed scarification requirements for Robinia neomexicana In Harrington JTtech coord Proc Third SouthwestemContainer Growers Conf Feb 12-13 1997 Ft Collins CO
Hopkinson JM 1993 The strophiole of leguminous seeds Newsletter- IntI Herbage Seed Production Res Grp 18 7-8
---- 1997 Differences in hardseededness between Cunningham and Tarramba leucaena In Leucnet News The Newsletter of the International LeucaenaRes and Devlt Netwk No4
ISTA (International Seed Testing Association) 1999 International rules for seed testing rules Seed Sci amp Technol27 Supplement
Kelly KM van Staden J 1987 The lens as the site of permeability in the papilionoid seed Aspalathus linearis J Plant Physiol 128(45) 395-404
73
Keresztesi B 1988 Black locust the tree of agriculture Outlook on Agr 17(2) 77~85
Kigel 1 and Galili G 1995 Seed development and germination Marcel Dekker New York 853 p
Klemmedson 10 1994 New Mexico locust and parent material Influence on macronutrients of forest floor and soil Soil Sci Soc Am 1 58 974~980
Kuo WH Tam AW 1988 The pathway of water absorption of mung bean seeds Seed Sci amp Technol16(1) 139~144
Leadem CL 1997 Dormancy--unlocking seed secrets In Landis TD Thompson lR tech coords National Proceedings Forestand Conservation Nursery Assocs Gen Tech Rep PNW-G TR-419 Portland OR USDA For Ser PNW Res Sta 43-52
Lin Y Wagner MR Seweyestewa A Honahni MD DeGomez M McMillin JD 1996 Influence of cultural treatments on seed germination and seedling growth of Robinia neomexicana Forest Farm and Community Tree Res Reports Volume 1
Marunda CT 1990 Effects of seed pretreatment on the development ofAcacia auriculforinis and A holosericea seedlings ACIAR Proceedings Series No 28
Meginnis HG 1937 Sulphuric acid treatment to increase germination of black locust seed USDA Circ 453 34 p
Miklas PN Towensend CE Ladd SL 1987 Seedcoat anatomy and the scarification ofcicer milkvetch seed Crop Sci 27 766-772
Monsen SB 1984 Use of shrubs on mine spoils In The Challenge ofProducing Native Plants for the Intermountain Area Proceedings Intermountain Nurserymans Association 1983 conference Las Vegas NV compo PM Murphy 26-31 Ogden UT USDA For Ser Gen Tech Report INT-l68
Olson DF 1974 Robinia L Locust In Seeds of Woody Plants in the US tech coord CS Schopmeyer 728-731 USDA For Ser Agr Handbk No450
Rehman S Loescher RNl Harris P1C 1998 Dormancy breaking and germination ofAcacia salicina Lindl seeds Seed Sci amp Techno127 553-557
Rolston MP 1978 Water impermeable seed dormancy Bot Rev 44 365-396
74
SAS Institute Inc 1989 SASSTAT Users Guide Version 6 4th Edition Volume 1 Cary NC SAS Institute Inc 943 p
SerratoValenti G Conara L Ferrando M Modenesi P 1993 Structural and histochemical features ofStylosanthes scabra (Leguminosae Papilionoidae) seed coat as related to water entry Can J Bot 71 834-840
Simpson BJ 1988 A field guide to Texas trees Austin TX Texas Monthly Press 372p
Tran VN Cavanagh AK 1984 Structural aspects ofdormancy In Seed Physiology Vol 2 Murray DR (ed) p1-44 Academic Press Australia
USDA Forest Service 1948 Woody-plant seed manual USDA Misc Pub1654 416p
Vines RA 1960 Trees Shrubs and Woody Vines of the Southwest Austin Univ ofTexas Press 1104 p
Wagner MR Larson RE Craig TP 1992 Ecology and silvics ofNew Mexico locust (Robinia neomexicana) with an emphasis on the arboreal form p53-57 In JW Hanover K Miller and S Plesko (eds) Proc Intemat Confon Black Locust Biology Culture and Utilization Mich State Univ USA
Wilson JK 1944 Immersing seeds of species of Robinia in boiling water hastens germination J For 42 453-454
Zimmerman RW Carpenter SB 1980 First-year coppice production from a 5shyyear old black locust stand on surface mine spoil p309-314 Centr Hardwd For ConfProc University ofMissouri Columbia
damage (which also may be contributing to high imbibition) For the three sources
for which 8 minutes of percussion resulted in maximum germination extended
percussion of seeds necessary to loosen the strophiole may not have excessively
damaged the seed anatomy The Manzano source did not significantly change in
germination from 4 to 8 minutes of percussion suggesting that an increase in
permeable seeds at the 8-minute treatment duration was balanced by the number of
seeds that failed to germinate due to seed damage To summarize the 4-minute
percussion treatment improved germination over 8 minutes ofpercussion for a
majority of sources (five ofnine) the 8-minute percussion treatment improved
germination over 4-minutes ofpercussion for three sources and there was no
difference with the Manzano source Notably for the Raton source 8 minutes of
percussion increased germination by 235 percentage points over 4-minutes of
percussion (7875 vs 5525 respectively) the next best treatment Raton was the
only source to result in germination less than 75 for 4 minutes ofpercussion so a
grower may wish to apply longer percussion durations to lots that do not respond well
to this treatment level
Finally there was an interesting trend in the Jemez and Raton sources seed
that significantly contributed to the source by treatment interaction (Figure 12) A
~u test showed that the Jemez source had a significantly lighter seed weight than
other sources while the Raton source had an average seed weight For the Jemez
the longest duration of boiling increased germination Conversely only short
U4UVll) of percussion were needed to achieve high germination and the longest
41
80 _ Jemez a Raton
iD
~ C iD e iD
Q c 0 ~
c ~ iD 0
20
0------shy
Immersion Boiling 30 sec Boiling 60 sec
Hot Water Scarification
10degTr========~--------~------------------------------
80
60
40
20
_ Jemez Raton
0------shy
1 min 2 min 4 min 8 min
Percussion Scarification
Figure 12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources
42
b
--~----~~~~========~---- duration ofpercussion reduced germination For the Raton source longer durations
ofboiling consistently decreased germination whereas longer percussion treatments
consistently increased germination
A possible explanation is that a thick seed coat is associated with a loose
strophiole structure and that a thin seed coat is associated with a constrained
strophiole This contrast merits further study and may suggest an ecological
adaptation Further investigation ofthis phenomenon may contribute to successful
pre-screening ofunknown seed sources to determine optimal treatment leveL
43
CONCLUSION
Based on results obtained in pilot study (see Appendix) and thesis
experiments 4 minutes ofpercussion is recommended as a standard protocol for
treating an unknown source ofNew Mexico locust seed In addition to laboratory
results the 4-minute percussion treatment has resulted in germination greater than
80 and healthy seedling grow-outs for production purposes (unpublished data) In
laboratory experiments only one hundred seeds per 118 ml (40z) soil tin were
percussed at a time whereas for production we have treated up to five hundred seeds
per tin For large-scale production ofNew Mexico locust seedlings growers may
wish to experiment with larger quantities of seed and larger-size soil tins
Seed storage trials following percussion treatments were not conducted
However germination greater than 80 and healthy seedlings were obtained when
seeds were percussion-treated and subsequently stored at room-temperature (22degC)
for one week (unpublished data) Seed storage trials merit further study as the
standard hot water treatment leaves seeds in a softened condition that is not
conducive to storage
Hydroseeding is an effective method for direct seeding in reclamation
situations Percussion treatment may be conducive to hydro seeding as seeds remain
a dry relatively undamaged yet permeable condition following treatment Sulfuric
seeds also remain dry following treatment but extensive damage to seed coat
poor seedling vigor have been reported with this treatment (Kelly and van Staden
A grow-out study using a hydro seeder on a reclamation site testing percussion
44
against other treatments such as sulfuric acid and mechanical scarification also
merits further study
Papilionoids are the largest subfamily oflegumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust these legumes often play an integral role in the revegetation of
disturbed lands The pilot study (Appendix) indicates that percussion scarification
successfully treats the widely used black locust as well Developing superior
scarification methods to the standard hot water mechanical scarification and acid
treatments may facilitate the use ofpapilionoid legumes in reclamation projects
45
APPENDICES
APPENDIX A
PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR
NEW MEXICO LOCUST AND BLACK LOCUST SEEDS
Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4
Hot water and sulfuric acid soaks are traditional treatments for seeds of many
temperate woody legwnes including locusts However these scarification techniques
often produce inconsistent germination Percussion scarification where seeds are
repeatedly propelled against a hard surface was compared with hot water
scarification to evaluate treatment efficacy for New Mexico locust (Robinia
neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment
seeds were placed in a 98degC water bath which was immediately removed from the
heat source For percussion scarification seeds were placed in a soil sample tin and
agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the
control were followed by 24-hour water soaks Hot water baths resulted in 56 and
41 germination for New Mexico locust and black locust respectively For both
species nearly all durations ofpercussion increased germination over the hot water
treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3
4 and 5 minutes for black locust resulted in at least 90 germination Traditional
scarification treatments randomly degrade the entire seed coat which can lead to
tissue damage during water uptake Percussion scarification specifically weakens the
strophiole the natural source ofwater entry to the seed in papilionoid legwnes
INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom
2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031
48
Following percussion imbibition is controlled through the strophiole and underlying
tissue is protected
Keywords New Mexico locust black locust scarification percussion germination
Introduction
Locust trees (Robinia spp) are aggressive pioneer species that quickly
colonize disturbed land fix atmospheric nitrogen in the soil and stabilize slopes that
are prone to erosion (Klemmedson 1994 Ashby et al 1985) (Figure A1ab)
Recognition of these attributes has contributed to increased demand for locusts in
reclamation projects Locust seeds however exhibit physical dormancy where a
thick seed coat prevents the movement of water and gasses to the embryo (Leadem
1997)
Sulfuric acid or hot water soaks have traditionally been used to break seed
dormancy in macro-propagation of locusts Many growers are moving away from the
use of su1furic acid in treating locust seeds (Dreesen and Harrington 1997) In
addition to being dangerous acid soak durations must be specifically correlated to
seed lot (Olson 1974) Collections of native seed can vary tremendously in seed size
seed weight and hard seededness etc This variability limits the utility of sulfuric
acid soaks as even within a given lot some seeds may be damaged whereas others
are under-treated and fail to imbibe (take up water) A survey of Southwestern
container growers indicates that hot water baths are currently the most common
scarification method for locust seeds (Hine et al 1997) However hot water baths
also produce inconsistent germination (Lin et al 1996)
49
b
Figure AI New Mexico locust a primary invader following disturbance Ala Crown-sprouting at edge ofburn near Los Alamos NM--one year after Cerro Grande fire Alb Colonizing road cut Gila National Forest NM
50
Percussion scarification where seeds are repeatedly propelled against a hard
surface is an alternative dormancy-breaking method that has proven successful in
legumes related to locusts (Hamly 1932 Barton 1947) We compared this alternative
treatment to hot water scarification to evaluate treatment efficacy for New Mexico
locust and black locust seeds
Materials and Methods
Commercial New Mexico locust seeds (Western Native Seed Coaldale CO)
were collected fall 2000 in Huerfano County CO Black locust seeds were collected
September 2000 from Taos County NM by harvesting ripe pods from several trees at
a distance of up to 3 m from ground level Black locust seeds were allowed to air-dry
for 3 weeks and were threshed and separated from large chaffusing a greenhouse
fan A South Dakota Seed Blower (Chicago Illinltois) was used to remove fine chaff
from both seed lots Seeds were storedmiddotat 2-4C until the germination study was
conducted in March 2001
This study compared hot water scarification with percussion scarification
using treatment and species as experimental factors Along with a control
scarification treatments included immersion of seed in boiling water as well as
percussion durations of 1 2 3 45 or 10 minutes Four 100-seed samples were used
to test each species by scarification treatment combination
For the hot water treatment test tubes were filled with 30 mL water and
placed in a water-filled beaker (Figure A2a) The bath was raised to boiling (-98C at
51
b
c
Figure A2 Implementation ofhot water and percussion scarification A2a Hot water bath A2b Paint-shaker set-up A2c Placing soil tin containing seeds at end ofpaint shaker ann
52
Las Cruces NM elevation of 1300 meters) Each 100-seed replicate was placed in a
test tube and each test tube was immediately removed to cool at room temperature
Percussion scarification was implemented using a pneumatic paint shaker
(Central Pneumatic model 00422 Camarillo CA) (Figure A2b) This allowed
standardization of treatment intensity Compressed air pressure was maintained at
530kPa plusmn 30kPa (80psi plusmn 5psi) resulting in approximately 350 oscillations per
minute Each lOO-seed sample was placed in a 118 ml (4 oz) soil tin for shaking To
maximize lateral movement of paint shaker a paint can was used as a spacer to place
soil tins at the end of the shaking arm (Figure A2c) This allowed shaking to occur at
the greatest distance from the pivot point (fulcrum) Following all treatments
including the control seeds were soaked for twenty-four hours in distilled water prior
to plating out
Gennination was evaluated on lab benches at room temperature Each
replicate of seeds was placed in a 100 cm petri dish on moistened filter paper with
humidity maintained by enclosing the petri dishes in ziplock bags Gennination was
monitored daily for 14 days in accordance with International Seed Testing
Association standards for Robinia species (ISTA 1999) Gennination was defined as
protrusion of the radicle from the embryo by at least 1mm as viewed by the naked
eye
Categorical Analysis of Variance using Prpc Catmod (SAS Institute 1989)
was used to detennine treatment differences in germination percentages for each
source This procedure is an extension of a Chi-square test of homogeneity using the
53
natural log of the ratio of germinated to non-germinated seeds for each treatment Pshy
values less than 005 were considered significant A limited set of pairwiseshy
comparisons was conducted to compare treatment mean percents using a
conservative alpha value of 005 divided by the number of comparisons
Results
Percussion maximized germination for both New Mexico locust and black
locust There were three germination response levels for New Mexico locust (Figure
A3) First there was a low response in the control indicating a dormant seed lot
The hot water treatment and I-minute duration of percussion share a second
intermediate response Germination was highest at percussion levels from 2 to 10
minutes
A selected percussion treatment intermediate in time (5 minutes) produced the
steepest germination curve indicating the fastest germination rate (Figure A4) The
hot water treatment produced an intermediate curve or relatively slower germination
rate The control curve was very flat indicating the slowest germination rate for New
Mexico locust
Control seeds germinated poorly for black locust as well (Figure A5) Hot
water scarification improved germination over the control but all levels of percussion
improved germination over hot water The optimal durations of percussion treatment
were bracketed with a significant increase from the 1 to 2 minute percussion level a
high germination response from 2 to 5 minutes of percussion and finally a significant
54
~ d 25 CL)
p t1 0 ~ t1
g Vl ~ Vl
100~---------------------------------------------------------------
80
60
40
20
o Control Hot Water Percussion
I min Percussion
2 min Percussion
3 min
Scarification
Percussion 4 min
Figure A3 Effect of scarification on germination response for New Mexico locust
Percussion 5 min
=significant difference between adjacent treatment bars ns = no significant difference
Percussion 10 min
~ ~ () u agt
Il-t d o ~
~ ~ c3
100middotlr~~~------------------------------------e- Percussion 5min middot 0middot Hot Water -- Control
80-J=middot======
6b-middot-lmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middot--middotmiddot-middot~-middotmiddot -middotmiddot middot middotmiddot-middot 1
40
20
-----~-----------
0 0 0 middotmiddotmiddotmiddotmiddot0middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot0
0 --~-----y-----~------_---~--- T-----~
o shy -TDay 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A4 Effect of selected scarification treatments on germination speed for New Mexico locust First seven days of fourteen-day experiment are shown
Day 7
80
~ cd
5 60 ()
[) Cl d 0 1t1 d
40sect dUl
-J
20
o
ns
Scarification
Figure A5 Effect of scarification on germination response for black locust = significant difference between adjacent treatment bars ns = no significant difference
decrease at the 10-minute level This drop-off suggests that 10 minutes of percussion
over-treats seeds from this lot of-black locust
A selected percussion treatment intermediate in time (5 minutes) also had the
fastest germination rate for black locust (Figure A6)_ The hot water curve is
somewhat flatter indicating a slower germination rate The control curve is very flat
indicating a slow germination rate
A qualitative difference between hot water and percussion-treated seeds was
also observed Along with healthy germinants the hot water treatment produced
over-treated seeds (Figure A 7a) Examples of damaged germinants include seeds
where the radicle did not elongate (Figure A 7b) seeds where the radicle elongated
but was delayed in freeing itself from the seed coat (Figure A7c) and seeds where the
radicle broke off completely from the embryo (Figure A7d) In addition to these
examples of over-treatment there were numerous small dark seeds that failed to
imbibe water For percussion treatment nearly all seeds took up water (Figure A8a)
Germination was rapid and uniform with little damage evident (Figure A8b)
To summarize both New Mexico locust and black locust seed lots were
dormant The standard hot water scarification treatment improved germination but
only to 56 and 41 for New Mexico locust and black locust respectively Nearly
all percussion durations improved total germination as well as germination rate over
the hot water treatment Germination was greater than 90 for percussion durations
of 4 5 and 10 minutes for New Mexico locust and 3 4 and 5 minutes for black locust
58
CI)
sgt t CI) 0
igt 0
sect ttl t E
Vl 0
c3
100r==~==~~=r------------------------------------~ ___ Percussion 5 min
middotmiddotmiddot0middotmiddotmiddot Hot Water -lIf- Control
80r=========~--~~----------------------------~
60
40
20 -~-~---~---------- ---~------------I
omiddot
O
_~_----------lIf-----~bull -----_ shy---shy I
Day 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A6 Effect of selected scarification treatments on germination speed for black locust First seven days of fourteen-day experiment are shown
Day 7
a
b c d
Figure A7 Damage to New Mexico locust seeds observed during germination counts following hot water scarification A7a Hot water replicate including damaged seedlings and numerous unimbibed seeds A7b Close-up ofseed where radicle failed to elongate A7c Close-up ofseed where radicle elongated but was delayed in freeing itself from the seed coat A7d Close-up ofseed where radicle completely broke offfrom embryo
60
a
b
Figure A8 New Mexico locust seeds following percussion treatment at time of germination counts A8a Percussion replicate with uniformly healthy seedlings and near complete imbibition of seeds A8b Close-up ofpercussion-treated seedling
61
Discussion
Papilionoid seeds have a specific anatomical feature known as the strophiole
or lens-the natural site of water entry to the seed (Hamly 1932) The strophiole is
located on the cotyledonary lobe of a locust seed (Figure A9) When a papilionoid
legume seed is percussed for the appropriate amount of time repeated hits on the
integrated seed coat loosen the constrained cells of the strophiolar region without
excessively damaging the rest of the seed coat (Ballard 1976)
When a percussed seed is soaked water enters exclusively through the
strophiole in a controlled manner (Kelly and Staden 1987) This regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues This
contrasts with seeds that have been hot water or acid scarified treatments that can
randomly degrade the seed coat Unlocalized cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rehman et al 1998 Marunda 1990)
Papilionoids are the largest subfamily of legumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust and black locust these legumes often play an integral role in the
revegetation ofdisturbed lands Developing superior scarification methods to the
standard hot water and acid treatments should fadlitate the use ofthese legumes in
restoration projects
62
Figure A9 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (bc)
63
APPENDIXB
GERMINATION PERCENTS AND STANDARD ERRORS FOR SOURCE
BLOCK AND TREATMENT FOR NEW MEXICO LOCUST
Table B 1 Gennination percents and standard errors for source block and treatment for New Mexico locust
Source Block Treatment Germination Percentage Standard Error
Capitan 1 Immersion 74 437 Capitan 2 Immersion 69 462 Capitan 3 Immersion 71 454 Capitan 4 Immersion 67 470 Capitan Boiling 30 sec 79 407 Capitan 2 Boiling 30 sec 76 427 Capitan 3 Boiling 30 sec 72 449 Capitan 4 Boiling 30 sec 76 427 Capitan Boiling 60 sec 71 454 Capitan 2 Boiling 60 sec 54 498 Capitan 3 Boiling 60 sec 67 470 Capitan 4 Boiling 60 sec 76 427 Capitan I Control 5 218 Capitan 2 Control 7 255 Capitan 3 Control 4 196 Capitan 4 Control 5 218 Capitan 1 Percussion I min 47 499 Capitan 2 Percussion I min
470
Capitan Percussion I min 39 488
Capitan 4 Percussion I min 38 485 Capitan I Percussion 2 min 55 497 Capitan Capitan
2
Percussion 2 min Percussion 2 min
71 65
454 477
Capitan 4 Percussion 2 min 56 496 Capitan I Percussion 4 min 86 347 Capitan 2 Percussion 4 min 79 407 Capitan 3 Percussion 4 min 81 392 Capitan 4 Percussion 4 min 82 384 Capitan I Percussion 8 min 90 300 Capitan 2 Percussion 8 min 88 325 Capitan 3 Percussion 8 min 90 300 Capitan 4 Percussion 8 min 90 300 Gila I Immersion 71 454 Gila 2 Immersion 77 421 Gila
Immersion 75 433
Gila 4 Immersion 72 449
Gila I Boiling 30 sec 68 466
Gila 2 Boiling 30 sec 81 392
Gila 3 Boiling 30 sec 54 498
Gila 4 Boiling 30 sec 63 483
Gila I Boiling 60 sec 55 497
Gila 2 Boiling 60 sec 68 466
Gila 3 Boiling 60 sec 57 495
Gila 4 Boiling 60 sec 70 458
65
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Gila 2 Control 13 336 Gila 3 Control 13 336 Gila 4 Control 20 400 Gila 1 Percussion 1 min 66 474 Gila 2 Percussion 1 min 57 495 Gila 3 Percussion 1 min 73 444 Gila 4 Percussion 1 min 82 384 Gila 1 Percussion 2 min 81 392 Gila 2 Percussion 2 min 86 347 Gila 3 Percussion 2 min 85 357 Gila 4 Percussion 2 min 54 498 Gila 1 Percussion 4 min 79 407 Gila 2 Percussion 4 min 83 376 Gila 3 Percussion 4 min 84 367 Gila 4 Percussion 4 min 79 407 Gila 1 Percussion 8 min 51 500 Gila 2 Percussion 8 min 76 427 Gila 3 Percussion 8 min 52 500 Gila 4 Percussion 8 min 74 439 Holman 1 Immersion 83 376 Holman 2 Immersion 86 347 Holman 3 Immersion 81 392 Holman 4 Immersion 90 300 Holman 1 Boiling 30 sec 90 300 Holman 2 Boiling 30 sec 85 357 Holman 3 Boiling 30 sec 87 336 Holman 4 Boiling 30 sec 83 376 Holman 1 Boiling 60 sec 79 407 Holman 2 Boiling 60 sec 80 400 Holman 3 Boiling 60 sec 86 347 Holman 4 Boiling 60 sec 93 255 Holman Control 25 433 Holman 2 Control 19 392 Holman Control 21 407
Holman 4 Control 24 427
Holman Percussion 1 min 77 421
Holman 2 Percussion 1 min 85 357
Holman 3 Percussion 1 min 80 400
Holman 4 Percussion 1 min 89 313
Holman 1 Percussion 2 min 88 325
Holman 2 Percussion 2 min 96 196
Holman 3 Percussion 2 min 93 255
Holman 4 Percussion 2 min 96 196
Holman 1 Percussion 4 min 92 271
Holman 2 Percussion 4 min 97 171
66
I
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Holman 3 Percussion 4 min 91 286 Holman 4 Percussion 4 min 96 196 Holman 1 Percussion 8 min 88 325 Holman 2 Percussion 8 min 85 357 Holman 3 Percussion 8 min 83 376 Holman 4 Percussion 8 min 82 384 Jemez 1 Immersion 53 499 Jemez 2 Immersion 44 496 Jemez 3 Immersion 52 500 Jemez 4 Immersion 70 458 Jemez 1 Boiling 30 sec 58 494 Jemez 2 Boiling 30 sec 55 497 Jemez 3 Boiling 30 sec 48 500 Jemez 4 Boiling 30 sec 65 477 Jemez 1 Boiling 60 sec 68 466 Jemez 2 Boiling 60 sec 63 483 Jemez 3 Boiling 60 sec 73 444 Jemez 4 Boiling 60 sec 89 313 Jemez 1 Control 2 140 Jemez 2 Control 9 286 Jemez 3 Control 6 237 Jemez 4 Control 8 271 Jemez 1 Percussion 1 min 58 494 Jemez 2 Percussion 1 min 63 483 Jemez 3 Percussion 1 min 77 421 Jemez 4 Percussion 1 min 89 313 Jemez 1 Percussion 2 min 68 466 Jemez 2 Percussion 2 min 64 480 Jemez 3 Percussion 2 min 80 400 Jemez 4 Percussion 2 min 83 376 Jemez Percussion 4 min 68 466 Jemez 2 Percussion 4 min 79 407 Jemez 3 Percussion 4 min 70 458 Jemez 4 Percussion 4 min 83 376 Jemez 1 Percussion 8 min 52 500 Jemez 2 Percussion 8 min 59 492 Jemez 3 Percussion 8 min 61 488 Jemez 4 Percussion 8 min 70 458 Magdalena 1 Immersion 69 462 Magdalena 2 Immersion 69 462 Magdalena J Immersion 70 458 Magdalena 4 Immersion 85 357 Magdalena 1 Boiling 30 sec 65 477 Magdalena 2 Boiling 30 sec 71 454 Magdalena 3 Boiling 30 sec 68 466
67
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Magdalena 4 Boiling 30 sec 83 376 Magdalena 1 Boiling 60 sec 63 483 Magdalena 2 Boiling 60 sec 68 466 Magdalena 3 Boiling 60 sec 63 483 Magdalena 4 Boiling 60 sec 82 384 Magdalena 1 Control 11 313 Magdalena 2 Control 6 237 Magdalena 3 Control 5 218 Magdalena 4 Control 4 196 Magdalena Percussion 1 min 32 466 Magdalena 2 Percussion 1 min 45 497 Magdalena 3 Percussion 1 min 46 498 Magdalena 4 Percussion I min 36 480 Magdalena 1 Percussion 2 min 67 470 Magdalena 2 Percussion 2 min 68 466 Magdalena 3 Percussion 2 min 65 477 Magdalena 4 Percussion 2 min 81 392 Magdalena 1 Percussion 4 min 87 336 Magdalena 2 Percussion 4 min 97 171 Magdalena 3 Percussion 4 min 83 376 Magdalena 4 Percussion 4 min 97 171 Magdalena 1 Percussion 8 min 98 lAO Magdalena 2 Percussion 8 min 100 000 Magdalena 3 Percussion 8 min 96 196 Magdalena 4 Percussion 8 min 98 lAO Manzano 1 Immersion 67 470 Manzano 2 Immersion 57 495 Manzano 3 Immersion 72 4049 Manzano 4 Immersion 73 4044 Manzano 1 Boiling 30 sec 70 458 Manzano 2 Boiling 30 sec 64 480 Manzano 3 Boiling 30 sec 71 454 Manzano 4 Boiling 30 sec 80 400 Manzano I Boiling 60 sec 50 500 Manzano 2 Boiling 60 sec 53 499 Manzano 3 Boiling 60 sec 59 492 Manzano 4 Boiling 60 sec 74 439 Manzano I Control 4 196 Manzano 2 Control 8 271 Manzano 3 Control 1 099 Manzano 4 Control 6 237 Manzano 1 Percussion 1 min 52 500 Manzano 2 Percussion 1 min 39 488 Manzano 3 Percussion 1 min 36 480 Manzano 4 Percussion 1 min 51 500
68
Table B 1 Continued
Source Block Treatment Gerrni~ation Percentage Standard Error
Manzano Percussion 2 min 53 499 Manzano 2 Percussion 2 min 58 494 Manzano 3 Percussion 2 min 61 488 Manzano 4 Percussion 2 min 62 485 Manzano 1 Percussion 4 min 76 427 Manzano 2 Percussion 4 min 85 357 Manzano 3 Percussion 4 min 79 407 Manzano 4 Percussion 4 min 83 376 Manzano 1 Percussion 8 min 87 336 Manzano 2 Percussion 8 min 83 376 Manzano 3 Percussion 8 min 83 376 Manzano 4 Percussion 8 min 75 433 Raton 1 Immersion 54 498 Raton 2 Immersion 37 483 Raton 3 Immersion 39 488 Raton 4 Immersion 69 462 Raton 1 Boiling 30 sec 36 480 Raton 2 Boiling 30 sec 20 400 Raton 3 Boiling 30 sec 28 449 Raton 4 Boiling 30 sec 60 490 Raton 1 Boiling 60 sec 15 357 Raton 2 Boiling 60 sec 171 Raton 3 Boiling 60 sec 4 196 Raton 4 Boiling 60 sec 35 477 Raton 1 Control 4 196 Raton 2 Control 12 325 Raton 3 Control 2 140 Raton 4 Control 6 237 Raton 1 Percussion 1 min 52 500 Raton 2 Percussion I min 29 454 Raton 3 Percussion I min 35 477 Raton 4 Percussion 1 min 31 462 Raton 1 Percussion 2 min 55 497 Raton 2 Percussion 2 min 35 477 Raton 3 Percussion 2 min 33 470 Raton 4 Percussion 2 min 42 494 Raton 1 Percussion 4 min 65 477 Raton 2 Percussion 4 min 52 500 Raton Percussion 4 min 51 500 Raton 4 Percussion 4 min 53 499 Raton 1 Percussion 8 min 88 325 Raton 2 Percussion 8 min 80 400 Raton 3 Percussion 8 min 73 444 Raton 4 Percussion 8 min 74 439 Sacramento 1 Immersion 43 495
69
Table B 1 Continued
-Source Block Treatment Germination Percentage Standard Error
Sacramento 2 Immersion 30 458 Sacramento 3 Immersion 38 485 Sacramento 4 Immersion 56 496 Sacramento 1 Boiling 30 sec 47 499 Sacramento 2 Boiling 30 sec 48 500 Sacramento 3 Boiling 30 sec 48 500 Sacramento 4 Boiling 30 sec 56 496 Sacramento 1 Boiling 60 sec 52 500 Sacramento 2 Boiling 60 sec 67 470 Sacramento 3 Boiling 60 sec 43 495 Sacramento 4 Boiling 60 sec 48 500 Sacramento 1 Control 0 000 Sacramento 2 Control 3 171 Sacramento 3 Control 3 171 Sacramento 4 Control 099 Sacramento 1 Percussion 1 min 57 495 Sacramento 2 Percussion 1 min 61 488 Sacramento 3 Percussion 1 min 62 485 Sacramento 4 Percussion 1 min 55 497 Sacramento 1 Percussion 2 min 84 367 Sacramento 2 Percussion 2 min 70 458 Sacramento 3 Percussion 2 min 81 392 Sacramento 4 Percussion 2 min 69 462 Sacramento 1 Percussion 4 min 91 286 Sacramento 2 Percussion 4 min 84 367 Sacramento 3 Percussion 4 min 84 367 Sacramento 4 Percussion 4 min 94 237 Sacramento 1 Percussion 8 min 82 384 Sacramento 2 Percussion 8 min 81 392 Sacramento 3 Percussion 8 min 82 384 Sacramento 4 Percussion 8 min 81 392 Sandia 1 Immersion 76 427 Sandia 2 Immersion 70 458 Sandia 3 Immersion 70 458 Sandia 4 Immersion 81 392 Sandia 1 Boiling 30 sec 78 414 Sandia 2 Boiling 30 sec 71 454 Sandia 3 Boiling 30 sec 63 483 Sandia 4 Boiling 30 sec 78 414 Sandia 1 Boiling 60 sec 67 470 Sandia 2 Boiling 60 sec 65 477 Sandia 3 Boiling 60 sec 74 439 Sandia 4 Boiling 60 sec 66 474 Sandia 1 Control 16 367 Sandia 2 Control 7 255
70
Table B 1 Continued
Source Block Treatment Gennination Percentage Standard Error
Sandia 3 Control 8 271 Sandia 4 Control 16 367 Sandia 1 Percussion 1 min 67 470 Sandia 2 Percussion 1 min 61 488 Sandia 3 Percussion 1 min 66 474 Sandia 4 Percussion 1 min 59 492 Sandia I Percussion 2 min 82 384 Sandia 2 Percussion 2 min 83 376 Sandia 3 Percussion 2 min 81 392 Sandia 4 Percussion 2 min 84 367 Sandia 1 Percussion 4 min 89 313 Sandia 2 Percussion 4 min 86 347 Sandia 3 Percussion 4 min 89 313 Sandia 4 Percussion 4 min 85 357 Sandia 1 Percussion 8 min 80 400 Sandia 2 Percussion 8 min 77 421 Sandia 3 Percussion 8 min 69 462 Sandia 4 Percussion 8 min 66 474
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74
SAS Institute Inc 1989 SASSTAT Users Guide Version 6 4th Edition Volume 1 Cary NC SAS Institute Inc 943 p
SerratoValenti G Conara L Ferrando M Modenesi P 1993 Structural and histochemical features ofStylosanthes scabra (Leguminosae Papilionoidae) seed coat as related to water entry Can J Bot 71 834-840
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USDA Forest Service 1948 Woody-plant seed manual USDA Misc Pub1654 416p
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Wagner MR Larson RE Craig TP 1992 Ecology and silvics ofNew Mexico locust (Robinia neomexicana) with an emphasis on the arboreal form p53-57 In JW Hanover K Miller and S Plesko (eds) Proc Intemat Confon Black Locust Biology Culture and Utilization Mich State Univ USA
Wilson JK 1944 Immersing seeds of species of Robinia in boiling water hastens germination J For 42 453-454
Zimmerman RW Carpenter SB 1980 First-year coppice production from a 5shyyear old black locust stand on surface mine spoil p309-314 Centr Hardwd For ConfProc University ofMissouri Columbia
80 _ Jemez a Raton
iD
~ C iD e iD
Q c 0 ~
c ~ iD 0
20
0------shy
Immersion Boiling 30 sec Boiling 60 sec
Hot Water Scarification
10degTr========~--------~------------------------------
80
60
40
20
_ Jemez Raton
0------shy
1 min 2 min 4 min 8 min
Percussion Scarification
Figure 12 Effect of hot water scarification (a) and percussion scarification (b) on interaction of Jemez and Raton seed sources
42
b
--~----~~~~========~---- duration ofpercussion reduced germination For the Raton source longer durations
ofboiling consistently decreased germination whereas longer percussion treatments
consistently increased germination
A possible explanation is that a thick seed coat is associated with a loose
strophiole structure and that a thin seed coat is associated with a constrained
strophiole This contrast merits further study and may suggest an ecological
adaptation Further investigation ofthis phenomenon may contribute to successful
pre-screening ofunknown seed sources to determine optimal treatment leveL
43
CONCLUSION
Based on results obtained in pilot study (see Appendix) and thesis
experiments 4 minutes ofpercussion is recommended as a standard protocol for
treating an unknown source ofNew Mexico locust seed In addition to laboratory
results the 4-minute percussion treatment has resulted in germination greater than
80 and healthy seedling grow-outs for production purposes (unpublished data) In
laboratory experiments only one hundred seeds per 118 ml (40z) soil tin were
percussed at a time whereas for production we have treated up to five hundred seeds
per tin For large-scale production ofNew Mexico locust seedlings growers may
wish to experiment with larger quantities of seed and larger-size soil tins
Seed storage trials following percussion treatments were not conducted
However germination greater than 80 and healthy seedlings were obtained when
seeds were percussion-treated and subsequently stored at room-temperature (22degC)
for one week (unpublished data) Seed storage trials merit further study as the
standard hot water treatment leaves seeds in a softened condition that is not
conducive to storage
Hydroseeding is an effective method for direct seeding in reclamation
situations Percussion treatment may be conducive to hydro seeding as seeds remain
a dry relatively undamaged yet permeable condition following treatment Sulfuric
seeds also remain dry following treatment but extensive damage to seed coat
poor seedling vigor have been reported with this treatment (Kelly and van Staden
A grow-out study using a hydro seeder on a reclamation site testing percussion
44
against other treatments such as sulfuric acid and mechanical scarification also
merits further study
Papilionoids are the largest subfamily oflegumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust these legumes often play an integral role in the revegetation of
disturbed lands The pilot study (Appendix) indicates that percussion scarification
successfully treats the widely used black locust as well Developing superior
scarification methods to the standard hot water mechanical scarification and acid
treatments may facilitate the use ofpapilionoid legumes in reclamation projects
45
APPENDICES
APPENDIX A
PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR
NEW MEXICO LOCUST AND BLACK LOCUST SEEDS
Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4
Hot water and sulfuric acid soaks are traditional treatments for seeds of many
temperate woody legwnes including locusts However these scarification techniques
often produce inconsistent germination Percussion scarification where seeds are
repeatedly propelled against a hard surface was compared with hot water
scarification to evaluate treatment efficacy for New Mexico locust (Robinia
neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment
seeds were placed in a 98degC water bath which was immediately removed from the
heat source For percussion scarification seeds were placed in a soil sample tin and
agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the
control were followed by 24-hour water soaks Hot water baths resulted in 56 and
41 germination for New Mexico locust and black locust respectively For both
species nearly all durations ofpercussion increased germination over the hot water
treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3
4 and 5 minutes for black locust resulted in at least 90 germination Traditional
scarification treatments randomly degrade the entire seed coat which can lead to
tissue damage during water uptake Percussion scarification specifically weakens the
strophiole the natural source ofwater entry to the seed in papilionoid legwnes
INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom
2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031
48
Following percussion imbibition is controlled through the strophiole and underlying
tissue is protected
Keywords New Mexico locust black locust scarification percussion germination
Introduction
Locust trees (Robinia spp) are aggressive pioneer species that quickly
colonize disturbed land fix atmospheric nitrogen in the soil and stabilize slopes that
are prone to erosion (Klemmedson 1994 Ashby et al 1985) (Figure A1ab)
Recognition of these attributes has contributed to increased demand for locusts in
reclamation projects Locust seeds however exhibit physical dormancy where a
thick seed coat prevents the movement of water and gasses to the embryo (Leadem
1997)
Sulfuric acid or hot water soaks have traditionally been used to break seed
dormancy in macro-propagation of locusts Many growers are moving away from the
use of su1furic acid in treating locust seeds (Dreesen and Harrington 1997) In
addition to being dangerous acid soak durations must be specifically correlated to
seed lot (Olson 1974) Collections of native seed can vary tremendously in seed size
seed weight and hard seededness etc This variability limits the utility of sulfuric
acid soaks as even within a given lot some seeds may be damaged whereas others
are under-treated and fail to imbibe (take up water) A survey of Southwestern
container growers indicates that hot water baths are currently the most common
scarification method for locust seeds (Hine et al 1997) However hot water baths
also produce inconsistent germination (Lin et al 1996)
49
b
Figure AI New Mexico locust a primary invader following disturbance Ala Crown-sprouting at edge ofburn near Los Alamos NM--one year after Cerro Grande fire Alb Colonizing road cut Gila National Forest NM
50
Percussion scarification where seeds are repeatedly propelled against a hard
surface is an alternative dormancy-breaking method that has proven successful in
legumes related to locusts (Hamly 1932 Barton 1947) We compared this alternative
treatment to hot water scarification to evaluate treatment efficacy for New Mexico
locust and black locust seeds
Materials and Methods
Commercial New Mexico locust seeds (Western Native Seed Coaldale CO)
were collected fall 2000 in Huerfano County CO Black locust seeds were collected
September 2000 from Taos County NM by harvesting ripe pods from several trees at
a distance of up to 3 m from ground level Black locust seeds were allowed to air-dry
for 3 weeks and were threshed and separated from large chaffusing a greenhouse
fan A South Dakota Seed Blower (Chicago Illinltois) was used to remove fine chaff
from both seed lots Seeds were storedmiddotat 2-4C until the germination study was
conducted in March 2001
This study compared hot water scarification with percussion scarification
using treatment and species as experimental factors Along with a control
scarification treatments included immersion of seed in boiling water as well as
percussion durations of 1 2 3 45 or 10 minutes Four 100-seed samples were used
to test each species by scarification treatment combination
For the hot water treatment test tubes were filled with 30 mL water and
placed in a water-filled beaker (Figure A2a) The bath was raised to boiling (-98C at
51
b
c
Figure A2 Implementation ofhot water and percussion scarification A2a Hot water bath A2b Paint-shaker set-up A2c Placing soil tin containing seeds at end ofpaint shaker ann
52
Las Cruces NM elevation of 1300 meters) Each 100-seed replicate was placed in a
test tube and each test tube was immediately removed to cool at room temperature
Percussion scarification was implemented using a pneumatic paint shaker
(Central Pneumatic model 00422 Camarillo CA) (Figure A2b) This allowed
standardization of treatment intensity Compressed air pressure was maintained at
530kPa plusmn 30kPa (80psi plusmn 5psi) resulting in approximately 350 oscillations per
minute Each lOO-seed sample was placed in a 118 ml (4 oz) soil tin for shaking To
maximize lateral movement of paint shaker a paint can was used as a spacer to place
soil tins at the end of the shaking arm (Figure A2c) This allowed shaking to occur at
the greatest distance from the pivot point (fulcrum) Following all treatments
including the control seeds were soaked for twenty-four hours in distilled water prior
to plating out
Gennination was evaluated on lab benches at room temperature Each
replicate of seeds was placed in a 100 cm petri dish on moistened filter paper with
humidity maintained by enclosing the petri dishes in ziplock bags Gennination was
monitored daily for 14 days in accordance with International Seed Testing
Association standards for Robinia species (ISTA 1999) Gennination was defined as
protrusion of the radicle from the embryo by at least 1mm as viewed by the naked
eye
Categorical Analysis of Variance using Prpc Catmod (SAS Institute 1989)
was used to detennine treatment differences in germination percentages for each
source This procedure is an extension of a Chi-square test of homogeneity using the
53
natural log of the ratio of germinated to non-germinated seeds for each treatment Pshy
values less than 005 were considered significant A limited set of pairwiseshy
comparisons was conducted to compare treatment mean percents using a
conservative alpha value of 005 divided by the number of comparisons
Results
Percussion maximized germination for both New Mexico locust and black
locust There were three germination response levels for New Mexico locust (Figure
A3) First there was a low response in the control indicating a dormant seed lot
The hot water treatment and I-minute duration of percussion share a second
intermediate response Germination was highest at percussion levels from 2 to 10
minutes
A selected percussion treatment intermediate in time (5 minutes) produced the
steepest germination curve indicating the fastest germination rate (Figure A4) The
hot water treatment produced an intermediate curve or relatively slower germination
rate The control curve was very flat indicating the slowest germination rate for New
Mexico locust
Control seeds germinated poorly for black locust as well (Figure A5) Hot
water scarification improved germination over the control but all levels of percussion
improved germination over hot water The optimal durations of percussion treatment
were bracketed with a significant increase from the 1 to 2 minute percussion level a
high germination response from 2 to 5 minutes of percussion and finally a significant
54
~ d 25 CL)
p t1 0 ~ t1
g Vl ~ Vl
100~---------------------------------------------------------------
80
60
40
20
o Control Hot Water Percussion
I min Percussion
2 min Percussion
3 min
Scarification
Percussion 4 min
Figure A3 Effect of scarification on germination response for New Mexico locust
Percussion 5 min
=significant difference between adjacent treatment bars ns = no significant difference
Percussion 10 min
~ ~ () u agt
Il-t d o ~
~ ~ c3
100middotlr~~~------------------------------------e- Percussion 5min middot 0middot Hot Water -- Control
80-J=middot======
6b-middot-lmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middot--middotmiddot-middot~-middotmiddot -middotmiddot middot middotmiddot-middot 1
40
20
-----~-----------
0 0 0 middotmiddotmiddotmiddotmiddot0middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot0
0 --~-----y-----~------_---~--- T-----~
o shy -TDay 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A4 Effect of selected scarification treatments on germination speed for New Mexico locust First seven days of fourteen-day experiment are shown
Day 7
80
~ cd
5 60 ()
[) Cl d 0 1t1 d
40sect dUl
-J
20
o
ns
Scarification
Figure A5 Effect of scarification on germination response for black locust = significant difference between adjacent treatment bars ns = no significant difference
decrease at the 10-minute level This drop-off suggests that 10 minutes of percussion
over-treats seeds from this lot of-black locust
A selected percussion treatment intermediate in time (5 minutes) also had the
fastest germination rate for black locust (Figure A6)_ The hot water curve is
somewhat flatter indicating a slower germination rate The control curve is very flat
indicating a slow germination rate
A qualitative difference between hot water and percussion-treated seeds was
also observed Along with healthy germinants the hot water treatment produced
over-treated seeds (Figure A 7a) Examples of damaged germinants include seeds
where the radicle did not elongate (Figure A 7b) seeds where the radicle elongated
but was delayed in freeing itself from the seed coat (Figure A7c) and seeds where the
radicle broke off completely from the embryo (Figure A7d) In addition to these
examples of over-treatment there were numerous small dark seeds that failed to
imbibe water For percussion treatment nearly all seeds took up water (Figure A8a)
Germination was rapid and uniform with little damage evident (Figure A8b)
To summarize both New Mexico locust and black locust seed lots were
dormant The standard hot water scarification treatment improved germination but
only to 56 and 41 for New Mexico locust and black locust respectively Nearly
all percussion durations improved total germination as well as germination rate over
the hot water treatment Germination was greater than 90 for percussion durations
of 4 5 and 10 minutes for New Mexico locust and 3 4 and 5 minutes for black locust
58
CI)
sgt t CI) 0
igt 0
sect ttl t E
Vl 0
c3
100r==~==~~=r------------------------------------~ ___ Percussion 5 min
middotmiddotmiddot0middotmiddotmiddot Hot Water -lIf- Control
80r=========~--~~----------------------------~
60
40
20 -~-~---~---------- ---~------------I
omiddot
O
_~_----------lIf-----~bull -----_ shy---shy I
Day 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A6 Effect of selected scarification treatments on germination speed for black locust First seven days of fourteen-day experiment are shown
Day 7
a
b c d
Figure A7 Damage to New Mexico locust seeds observed during germination counts following hot water scarification A7a Hot water replicate including damaged seedlings and numerous unimbibed seeds A7b Close-up ofseed where radicle failed to elongate A7c Close-up ofseed where radicle elongated but was delayed in freeing itself from the seed coat A7d Close-up ofseed where radicle completely broke offfrom embryo
60
a
b
Figure A8 New Mexico locust seeds following percussion treatment at time of germination counts A8a Percussion replicate with uniformly healthy seedlings and near complete imbibition of seeds A8b Close-up ofpercussion-treated seedling
61
Discussion
Papilionoid seeds have a specific anatomical feature known as the strophiole
or lens-the natural site of water entry to the seed (Hamly 1932) The strophiole is
located on the cotyledonary lobe of a locust seed (Figure A9) When a papilionoid
legume seed is percussed for the appropriate amount of time repeated hits on the
integrated seed coat loosen the constrained cells of the strophiolar region without
excessively damaging the rest of the seed coat (Ballard 1976)
When a percussed seed is soaked water enters exclusively through the
strophiole in a controlled manner (Kelly and Staden 1987) This regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues This
contrasts with seeds that have been hot water or acid scarified treatments that can
randomly degrade the seed coat Unlocalized cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rehman et al 1998 Marunda 1990)
Papilionoids are the largest subfamily of legumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust and black locust these legumes often play an integral role in the
revegetation ofdisturbed lands Developing superior scarification methods to the
standard hot water and acid treatments should fadlitate the use ofthese legumes in
restoration projects
62
Figure A9 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (bc)
63
APPENDIXB
GERMINATION PERCENTS AND STANDARD ERRORS FOR SOURCE
BLOCK AND TREATMENT FOR NEW MEXICO LOCUST
Table B 1 Gennination percents and standard errors for source block and treatment for New Mexico locust
Source Block Treatment Germination Percentage Standard Error
Capitan 1 Immersion 74 437 Capitan 2 Immersion 69 462 Capitan 3 Immersion 71 454 Capitan 4 Immersion 67 470 Capitan Boiling 30 sec 79 407 Capitan 2 Boiling 30 sec 76 427 Capitan 3 Boiling 30 sec 72 449 Capitan 4 Boiling 30 sec 76 427 Capitan Boiling 60 sec 71 454 Capitan 2 Boiling 60 sec 54 498 Capitan 3 Boiling 60 sec 67 470 Capitan 4 Boiling 60 sec 76 427 Capitan I Control 5 218 Capitan 2 Control 7 255 Capitan 3 Control 4 196 Capitan 4 Control 5 218 Capitan 1 Percussion I min 47 499 Capitan 2 Percussion I min
470
Capitan Percussion I min 39 488
Capitan 4 Percussion I min 38 485 Capitan I Percussion 2 min 55 497 Capitan Capitan
2
Percussion 2 min Percussion 2 min
71 65
454 477
Capitan 4 Percussion 2 min 56 496 Capitan I Percussion 4 min 86 347 Capitan 2 Percussion 4 min 79 407 Capitan 3 Percussion 4 min 81 392 Capitan 4 Percussion 4 min 82 384 Capitan I Percussion 8 min 90 300 Capitan 2 Percussion 8 min 88 325 Capitan 3 Percussion 8 min 90 300 Capitan 4 Percussion 8 min 90 300 Gila I Immersion 71 454 Gila 2 Immersion 77 421 Gila
Immersion 75 433
Gila 4 Immersion 72 449
Gila I Boiling 30 sec 68 466
Gila 2 Boiling 30 sec 81 392
Gila 3 Boiling 30 sec 54 498
Gila 4 Boiling 30 sec 63 483
Gila I Boiling 60 sec 55 497
Gila 2 Boiling 60 sec 68 466
Gila 3 Boiling 60 sec 57 495
Gila 4 Boiling 60 sec 70 458
65
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Gila 2 Control 13 336 Gila 3 Control 13 336 Gila 4 Control 20 400 Gila 1 Percussion 1 min 66 474 Gila 2 Percussion 1 min 57 495 Gila 3 Percussion 1 min 73 444 Gila 4 Percussion 1 min 82 384 Gila 1 Percussion 2 min 81 392 Gila 2 Percussion 2 min 86 347 Gila 3 Percussion 2 min 85 357 Gila 4 Percussion 2 min 54 498 Gila 1 Percussion 4 min 79 407 Gila 2 Percussion 4 min 83 376 Gila 3 Percussion 4 min 84 367 Gila 4 Percussion 4 min 79 407 Gila 1 Percussion 8 min 51 500 Gila 2 Percussion 8 min 76 427 Gila 3 Percussion 8 min 52 500 Gila 4 Percussion 8 min 74 439 Holman 1 Immersion 83 376 Holman 2 Immersion 86 347 Holman 3 Immersion 81 392 Holman 4 Immersion 90 300 Holman 1 Boiling 30 sec 90 300 Holman 2 Boiling 30 sec 85 357 Holman 3 Boiling 30 sec 87 336 Holman 4 Boiling 30 sec 83 376 Holman 1 Boiling 60 sec 79 407 Holman 2 Boiling 60 sec 80 400 Holman 3 Boiling 60 sec 86 347 Holman 4 Boiling 60 sec 93 255 Holman Control 25 433 Holman 2 Control 19 392 Holman Control 21 407
Holman 4 Control 24 427
Holman Percussion 1 min 77 421
Holman 2 Percussion 1 min 85 357
Holman 3 Percussion 1 min 80 400
Holman 4 Percussion 1 min 89 313
Holman 1 Percussion 2 min 88 325
Holman 2 Percussion 2 min 96 196
Holman 3 Percussion 2 min 93 255
Holman 4 Percussion 2 min 96 196
Holman 1 Percussion 4 min 92 271
Holman 2 Percussion 4 min 97 171
66
I
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Holman 3 Percussion 4 min 91 286 Holman 4 Percussion 4 min 96 196 Holman 1 Percussion 8 min 88 325 Holman 2 Percussion 8 min 85 357 Holman 3 Percussion 8 min 83 376 Holman 4 Percussion 8 min 82 384 Jemez 1 Immersion 53 499 Jemez 2 Immersion 44 496 Jemez 3 Immersion 52 500 Jemez 4 Immersion 70 458 Jemez 1 Boiling 30 sec 58 494 Jemez 2 Boiling 30 sec 55 497 Jemez 3 Boiling 30 sec 48 500 Jemez 4 Boiling 30 sec 65 477 Jemez 1 Boiling 60 sec 68 466 Jemez 2 Boiling 60 sec 63 483 Jemez 3 Boiling 60 sec 73 444 Jemez 4 Boiling 60 sec 89 313 Jemez 1 Control 2 140 Jemez 2 Control 9 286 Jemez 3 Control 6 237 Jemez 4 Control 8 271 Jemez 1 Percussion 1 min 58 494 Jemez 2 Percussion 1 min 63 483 Jemez 3 Percussion 1 min 77 421 Jemez 4 Percussion 1 min 89 313 Jemez 1 Percussion 2 min 68 466 Jemez 2 Percussion 2 min 64 480 Jemez 3 Percussion 2 min 80 400 Jemez 4 Percussion 2 min 83 376 Jemez Percussion 4 min 68 466 Jemez 2 Percussion 4 min 79 407 Jemez 3 Percussion 4 min 70 458 Jemez 4 Percussion 4 min 83 376 Jemez 1 Percussion 8 min 52 500 Jemez 2 Percussion 8 min 59 492 Jemez 3 Percussion 8 min 61 488 Jemez 4 Percussion 8 min 70 458 Magdalena 1 Immersion 69 462 Magdalena 2 Immersion 69 462 Magdalena J Immersion 70 458 Magdalena 4 Immersion 85 357 Magdalena 1 Boiling 30 sec 65 477 Magdalena 2 Boiling 30 sec 71 454 Magdalena 3 Boiling 30 sec 68 466
67
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Magdalena 4 Boiling 30 sec 83 376 Magdalena 1 Boiling 60 sec 63 483 Magdalena 2 Boiling 60 sec 68 466 Magdalena 3 Boiling 60 sec 63 483 Magdalena 4 Boiling 60 sec 82 384 Magdalena 1 Control 11 313 Magdalena 2 Control 6 237 Magdalena 3 Control 5 218 Magdalena 4 Control 4 196 Magdalena Percussion 1 min 32 466 Magdalena 2 Percussion 1 min 45 497 Magdalena 3 Percussion 1 min 46 498 Magdalena 4 Percussion I min 36 480 Magdalena 1 Percussion 2 min 67 470 Magdalena 2 Percussion 2 min 68 466 Magdalena 3 Percussion 2 min 65 477 Magdalena 4 Percussion 2 min 81 392 Magdalena 1 Percussion 4 min 87 336 Magdalena 2 Percussion 4 min 97 171 Magdalena 3 Percussion 4 min 83 376 Magdalena 4 Percussion 4 min 97 171 Magdalena 1 Percussion 8 min 98 lAO Magdalena 2 Percussion 8 min 100 000 Magdalena 3 Percussion 8 min 96 196 Magdalena 4 Percussion 8 min 98 lAO Manzano 1 Immersion 67 470 Manzano 2 Immersion 57 495 Manzano 3 Immersion 72 4049 Manzano 4 Immersion 73 4044 Manzano 1 Boiling 30 sec 70 458 Manzano 2 Boiling 30 sec 64 480 Manzano 3 Boiling 30 sec 71 454 Manzano 4 Boiling 30 sec 80 400 Manzano I Boiling 60 sec 50 500 Manzano 2 Boiling 60 sec 53 499 Manzano 3 Boiling 60 sec 59 492 Manzano 4 Boiling 60 sec 74 439 Manzano I Control 4 196 Manzano 2 Control 8 271 Manzano 3 Control 1 099 Manzano 4 Control 6 237 Manzano 1 Percussion 1 min 52 500 Manzano 2 Percussion 1 min 39 488 Manzano 3 Percussion 1 min 36 480 Manzano 4 Percussion 1 min 51 500
68
Table B 1 Continued
Source Block Treatment Gerrni~ation Percentage Standard Error
Manzano Percussion 2 min 53 499 Manzano 2 Percussion 2 min 58 494 Manzano 3 Percussion 2 min 61 488 Manzano 4 Percussion 2 min 62 485 Manzano 1 Percussion 4 min 76 427 Manzano 2 Percussion 4 min 85 357 Manzano 3 Percussion 4 min 79 407 Manzano 4 Percussion 4 min 83 376 Manzano 1 Percussion 8 min 87 336 Manzano 2 Percussion 8 min 83 376 Manzano 3 Percussion 8 min 83 376 Manzano 4 Percussion 8 min 75 433 Raton 1 Immersion 54 498 Raton 2 Immersion 37 483 Raton 3 Immersion 39 488 Raton 4 Immersion 69 462 Raton 1 Boiling 30 sec 36 480 Raton 2 Boiling 30 sec 20 400 Raton 3 Boiling 30 sec 28 449 Raton 4 Boiling 30 sec 60 490 Raton 1 Boiling 60 sec 15 357 Raton 2 Boiling 60 sec 171 Raton 3 Boiling 60 sec 4 196 Raton 4 Boiling 60 sec 35 477 Raton 1 Control 4 196 Raton 2 Control 12 325 Raton 3 Control 2 140 Raton 4 Control 6 237 Raton 1 Percussion 1 min 52 500 Raton 2 Percussion I min 29 454 Raton 3 Percussion I min 35 477 Raton 4 Percussion 1 min 31 462 Raton 1 Percussion 2 min 55 497 Raton 2 Percussion 2 min 35 477 Raton 3 Percussion 2 min 33 470 Raton 4 Percussion 2 min 42 494 Raton 1 Percussion 4 min 65 477 Raton 2 Percussion 4 min 52 500 Raton Percussion 4 min 51 500 Raton 4 Percussion 4 min 53 499 Raton 1 Percussion 8 min 88 325 Raton 2 Percussion 8 min 80 400 Raton 3 Percussion 8 min 73 444 Raton 4 Percussion 8 min 74 439 Sacramento 1 Immersion 43 495
69
Table B 1 Continued
-Source Block Treatment Germination Percentage Standard Error
Sacramento 2 Immersion 30 458 Sacramento 3 Immersion 38 485 Sacramento 4 Immersion 56 496 Sacramento 1 Boiling 30 sec 47 499 Sacramento 2 Boiling 30 sec 48 500 Sacramento 3 Boiling 30 sec 48 500 Sacramento 4 Boiling 30 sec 56 496 Sacramento 1 Boiling 60 sec 52 500 Sacramento 2 Boiling 60 sec 67 470 Sacramento 3 Boiling 60 sec 43 495 Sacramento 4 Boiling 60 sec 48 500 Sacramento 1 Control 0 000 Sacramento 2 Control 3 171 Sacramento 3 Control 3 171 Sacramento 4 Control 099 Sacramento 1 Percussion 1 min 57 495 Sacramento 2 Percussion 1 min 61 488 Sacramento 3 Percussion 1 min 62 485 Sacramento 4 Percussion 1 min 55 497 Sacramento 1 Percussion 2 min 84 367 Sacramento 2 Percussion 2 min 70 458 Sacramento 3 Percussion 2 min 81 392 Sacramento 4 Percussion 2 min 69 462 Sacramento 1 Percussion 4 min 91 286 Sacramento 2 Percussion 4 min 84 367 Sacramento 3 Percussion 4 min 84 367 Sacramento 4 Percussion 4 min 94 237 Sacramento 1 Percussion 8 min 82 384 Sacramento 2 Percussion 8 min 81 392 Sacramento 3 Percussion 8 min 82 384 Sacramento 4 Percussion 8 min 81 392 Sandia 1 Immersion 76 427 Sandia 2 Immersion 70 458 Sandia 3 Immersion 70 458 Sandia 4 Immersion 81 392 Sandia 1 Boiling 30 sec 78 414 Sandia 2 Boiling 30 sec 71 454 Sandia 3 Boiling 30 sec 63 483 Sandia 4 Boiling 30 sec 78 414 Sandia 1 Boiling 60 sec 67 470 Sandia 2 Boiling 60 sec 65 477 Sandia 3 Boiling 60 sec 74 439 Sandia 4 Boiling 60 sec 66 474 Sandia 1 Control 16 367 Sandia 2 Control 7 255
70
Table B 1 Continued
Source Block Treatment Gennination Percentage Standard Error
Sandia 3 Control 8 271 Sandia 4 Control 16 367 Sandia 1 Percussion 1 min 67 470 Sandia 2 Percussion 1 min 61 488 Sandia 3 Percussion 1 min 66 474 Sandia 4 Percussion 1 min 59 492 Sandia I Percussion 2 min 82 384 Sandia 2 Percussion 2 min 83 376 Sandia 3 Percussion 2 min 81 392 Sandia 4 Percussion 2 min 84 367 Sandia 1 Percussion 4 min 89 313 Sandia 2 Percussion 4 min 86 347 Sandia 3 Percussion 4 min 89 313 Sandia 4 Percussion 4 min 85 357 Sandia 1 Percussion 8 min 80 400 Sandia 2 Percussion 8 min 77 421 Sandia 3 Percussion 8 min 69 462 Sandia 4 Percussion 8 min 66 474
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Kuo WH Tam AW 1988 The pathway of water absorption of mung bean seeds Seed Sci amp Technol16(1) 139~144
Leadem CL 1997 Dormancy--unlocking seed secrets In Landis TD Thompson lR tech coords National Proceedings Forestand Conservation Nursery Assocs Gen Tech Rep PNW-G TR-419 Portland OR USDA For Ser PNW Res Sta 43-52
Lin Y Wagner MR Seweyestewa A Honahni MD DeGomez M McMillin JD 1996 Influence of cultural treatments on seed germination and seedling growth of Robinia neomexicana Forest Farm and Community Tree Res Reports Volume 1
Marunda CT 1990 Effects of seed pretreatment on the development ofAcacia auriculforinis and A holosericea seedlings ACIAR Proceedings Series No 28
Meginnis HG 1937 Sulphuric acid treatment to increase germination of black locust seed USDA Circ 453 34 p
Miklas PN Towensend CE Ladd SL 1987 Seedcoat anatomy and the scarification ofcicer milkvetch seed Crop Sci 27 766-772
Monsen SB 1984 Use of shrubs on mine spoils In The Challenge ofProducing Native Plants for the Intermountain Area Proceedings Intermountain Nurserymans Association 1983 conference Las Vegas NV compo PM Murphy 26-31 Ogden UT USDA For Ser Gen Tech Report INT-l68
Olson DF 1974 Robinia L Locust In Seeds of Woody Plants in the US tech coord CS Schopmeyer 728-731 USDA For Ser Agr Handbk No450
Rehman S Loescher RNl Harris P1C 1998 Dormancy breaking and germination ofAcacia salicina Lindl seeds Seed Sci amp Techno127 553-557
Rolston MP 1978 Water impermeable seed dormancy Bot Rev 44 365-396
74
SAS Institute Inc 1989 SASSTAT Users Guide Version 6 4th Edition Volume 1 Cary NC SAS Institute Inc 943 p
SerratoValenti G Conara L Ferrando M Modenesi P 1993 Structural and histochemical features ofStylosanthes scabra (Leguminosae Papilionoidae) seed coat as related to water entry Can J Bot 71 834-840
Simpson BJ 1988 A field guide to Texas trees Austin TX Texas Monthly Press 372p
Tran VN Cavanagh AK 1984 Structural aspects ofdormancy In Seed Physiology Vol 2 Murray DR (ed) p1-44 Academic Press Australia
USDA Forest Service 1948 Woody-plant seed manual USDA Misc Pub1654 416p
Vines RA 1960 Trees Shrubs and Woody Vines of the Southwest Austin Univ ofTexas Press 1104 p
Wagner MR Larson RE Craig TP 1992 Ecology and silvics ofNew Mexico locust (Robinia neomexicana) with an emphasis on the arboreal form p53-57 In JW Hanover K Miller and S Plesko (eds) Proc Intemat Confon Black Locust Biology Culture and Utilization Mich State Univ USA
Wilson JK 1944 Immersing seeds of species of Robinia in boiling water hastens germination J For 42 453-454
Zimmerman RW Carpenter SB 1980 First-year coppice production from a 5shyyear old black locust stand on surface mine spoil p309-314 Centr Hardwd For ConfProc University ofMissouri Columbia
--~----~~~~========~---- duration ofpercussion reduced germination For the Raton source longer durations
ofboiling consistently decreased germination whereas longer percussion treatments
consistently increased germination
A possible explanation is that a thick seed coat is associated with a loose
strophiole structure and that a thin seed coat is associated with a constrained
strophiole This contrast merits further study and may suggest an ecological
adaptation Further investigation ofthis phenomenon may contribute to successful
pre-screening ofunknown seed sources to determine optimal treatment leveL
43
CONCLUSION
Based on results obtained in pilot study (see Appendix) and thesis
experiments 4 minutes ofpercussion is recommended as a standard protocol for
treating an unknown source ofNew Mexico locust seed In addition to laboratory
results the 4-minute percussion treatment has resulted in germination greater than
80 and healthy seedling grow-outs for production purposes (unpublished data) In
laboratory experiments only one hundred seeds per 118 ml (40z) soil tin were
percussed at a time whereas for production we have treated up to five hundred seeds
per tin For large-scale production ofNew Mexico locust seedlings growers may
wish to experiment with larger quantities of seed and larger-size soil tins
Seed storage trials following percussion treatments were not conducted
However germination greater than 80 and healthy seedlings were obtained when
seeds were percussion-treated and subsequently stored at room-temperature (22degC)
for one week (unpublished data) Seed storage trials merit further study as the
standard hot water treatment leaves seeds in a softened condition that is not
conducive to storage
Hydroseeding is an effective method for direct seeding in reclamation
situations Percussion treatment may be conducive to hydro seeding as seeds remain
a dry relatively undamaged yet permeable condition following treatment Sulfuric
seeds also remain dry following treatment but extensive damage to seed coat
poor seedling vigor have been reported with this treatment (Kelly and van Staden
A grow-out study using a hydro seeder on a reclamation site testing percussion
44
against other treatments such as sulfuric acid and mechanical scarification also
merits further study
Papilionoids are the largest subfamily oflegumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust these legumes often play an integral role in the revegetation of
disturbed lands The pilot study (Appendix) indicates that percussion scarification
successfully treats the widely used black locust as well Developing superior
scarification methods to the standard hot water mechanical scarification and acid
treatments may facilitate the use ofpapilionoid legumes in reclamation projects
45
APPENDICES
APPENDIX A
PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR
NEW MEXICO LOCUST AND BLACK LOCUST SEEDS
Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4
Hot water and sulfuric acid soaks are traditional treatments for seeds of many
temperate woody legwnes including locusts However these scarification techniques
often produce inconsistent germination Percussion scarification where seeds are
repeatedly propelled against a hard surface was compared with hot water
scarification to evaluate treatment efficacy for New Mexico locust (Robinia
neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment
seeds were placed in a 98degC water bath which was immediately removed from the
heat source For percussion scarification seeds were placed in a soil sample tin and
agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the
control were followed by 24-hour water soaks Hot water baths resulted in 56 and
41 germination for New Mexico locust and black locust respectively For both
species nearly all durations ofpercussion increased germination over the hot water
treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3
4 and 5 minutes for black locust resulted in at least 90 germination Traditional
scarification treatments randomly degrade the entire seed coat which can lead to
tissue damage during water uptake Percussion scarification specifically weakens the
strophiole the natural source ofwater entry to the seed in papilionoid legwnes
INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom
2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031
48
Following percussion imbibition is controlled through the strophiole and underlying
tissue is protected
Keywords New Mexico locust black locust scarification percussion germination
Introduction
Locust trees (Robinia spp) are aggressive pioneer species that quickly
colonize disturbed land fix atmospheric nitrogen in the soil and stabilize slopes that
are prone to erosion (Klemmedson 1994 Ashby et al 1985) (Figure A1ab)
Recognition of these attributes has contributed to increased demand for locusts in
reclamation projects Locust seeds however exhibit physical dormancy where a
thick seed coat prevents the movement of water and gasses to the embryo (Leadem
1997)
Sulfuric acid or hot water soaks have traditionally been used to break seed
dormancy in macro-propagation of locusts Many growers are moving away from the
use of su1furic acid in treating locust seeds (Dreesen and Harrington 1997) In
addition to being dangerous acid soak durations must be specifically correlated to
seed lot (Olson 1974) Collections of native seed can vary tremendously in seed size
seed weight and hard seededness etc This variability limits the utility of sulfuric
acid soaks as even within a given lot some seeds may be damaged whereas others
are under-treated and fail to imbibe (take up water) A survey of Southwestern
container growers indicates that hot water baths are currently the most common
scarification method for locust seeds (Hine et al 1997) However hot water baths
also produce inconsistent germination (Lin et al 1996)
49
b
Figure AI New Mexico locust a primary invader following disturbance Ala Crown-sprouting at edge ofburn near Los Alamos NM--one year after Cerro Grande fire Alb Colonizing road cut Gila National Forest NM
50
Percussion scarification where seeds are repeatedly propelled against a hard
surface is an alternative dormancy-breaking method that has proven successful in
legumes related to locusts (Hamly 1932 Barton 1947) We compared this alternative
treatment to hot water scarification to evaluate treatment efficacy for New Mexico
locust and black locust seeds
Materials and Methods
Commercial New Mexico locust seeds (Western Native Seed Coaldale CO)
were collected fall 2000 in Huerfano County CO Black locust seeds were collected
September 2000 from Taos County NM by harvesting ripe pods from several trees at
a distance of up to 3 m from ground level Black locust seeds were allowed to air-dry
for 3 weeks and were threshed and separated from large chaffusing a greenhouse
fan A South Dakota Seed Blower (Chicago Illinltois) was used to remove fine chaff
from both seed lots Seeds were storedmiddotat 2-4C until the germination study was
conducted in March 2001
This study compared hot water scarification with percussion scarification
using treatment and species as experimental factors Along with a control
scarification treatments included immersion of seed in boiling water as well as
percussion durations of 1 2 3 45 or 10 minutes Four 100-seed samples were used
to test each species by scarification treatment combination
For the hot water treatment test tubes were filled with 30 mL water and
placed in a water-filled beaker (Figure A2a) The bath was raised to boiling (-98C at
51
b
c
Figure A2 Implementation ofhot water and percussion scarification A2a Hot water bath A2b Paint-shaker set-up A2c Placing soil tin containing seeds at end ofpaint shaker ann
52
Las Cruces NM elevation of 1300 meters) Each 100-seed replicate was placed in a
test tube and each test tube was immediately removed to cool at room temperature
Percussion scarification was implemented using a pneumatic paint shaker
(Central Pneumatic model 00422 Camarillo CA) (Figure A2b) This allowed
standardization of treatment intensity Compressed air pressure was maintained at
530kPa plusmn 30kPa (80psi plusmn 5psi) resulting in approximately 350 oscillations per
minute Each lOO-seed sample was placed in a 118 ml (4 oz) soil tin for shaking To
maximize lateral movement of paint shaker a paint can was used as a spacer to place
soil tins at the end of the shaking arm (Figure A2c) This allowed shaking to occur at
the greatest distance from the pivot point (fulcrum) Following all treatments
including the control seeds were soaked for twenty-four hours in distilled water prior
to plating out
Gennination was evaluated on lab benches at room temperature Each
replicate of seeds was placed in a 100 cm petri dish on moistened filter paper with
humidity maintained by enclosing the petri dishes in ziplock bags Gennination was
monitored daily for 14 days in accordance with International Seed Testing
Association standards for Robinia species (ISTA 1999) Gennination was defined as
protrusion of the radicle from the embryo by at least 1mm as viewed by the naked
eye
Categorical Analysis of Variance using Prpc Catmod (SAS Institute 1989)
was used to detennine treatment differences in germination percentages for each
source This procedure is an extension of a Chi-square test of homogeneity using the
53
natural log of the ratio of germinated to non-germinated seeds for each treatment Pshy
values less than 005 were considered significant A limited set of pairwiseshy
comparisons was conducted to compare treatment mean percents using a
conservative alpha value of 005 divided by the number of comparisons
Results
Percussion maximized germination for both New Mexico locust and black
locust There were three germination response levels for New Mexico locust (Figure
A3) First there was a low response in the control indicating a dormant seed lot
The hot water treatment and I-minute duration of percussion share a second
intermediate response Germination was highest at percussion levels from 2 to 10
minutes
A selected percussion treatment intermediate in time (5 minutes) produced the
steepest germination curve indicating the fastest germination rate (Figure A4) The
hot water treatment produced an intermediate curve or relatively slower germination
rate The control curve was very flat indicating the slowest germination rate for New
Mexico locust
Control seeds germinated poorly for black locust as well (Figure A5) Hot
water scarification improved germination over the control but all levels of percussion
improved germination over hot water The optimal durations of percussion treatment
were bracketed with a significant increase from the 1 to 2 minute percussion level a
high germination response from 2 to 5 minutes of percussion and finally a significant
54
~ d 25 CL)
p t1 0 ~ t1
g Vl ~ Vl
100~---------------------------------------------------------------
80
60
40
20
o Control Hot Water Percussion
I min Percussion
2 min Percussion
3 min
Scarification
Percussion 4 min
Figure A3 Effect of scarification on germination response for New Mexico locust
Percussion 5 min
=significant difference between adjacent treatment bars ns = no significant difference
Percussion 10 min
~ ~ () u agt
Il-t d o ~
~ ~ c3
100middotlr~~~------------------------------------e- Percussion 5min middot 0middot Hot Water -- Control
80-J=middot======
6b-middot-lmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middot--middotmiddot-middot~-middotmiddot -middotmiddot middot middotmiddot-middot 1
40
20
-----~-----------
0 0 0 middotmiddotmiddotmiddotmiddot0middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot0
0 --~-----y-----~------_---~--- T-----~
o shy -TDay 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A4 Effect of selected scarification treatments on germination speed for New Mexico locust First seven days of fourteen-day experiment are shown
Day 7
80
~ cd
5 60 ()
[) Cl d 0 1t1 d
40sect dUl
-J
20
o
ns
Scarification
Figure A5 Effect of scarification on germination response for black locust = significant difference between adjacent treatment bars ns = no significant difference
decrease at the 10-minute level This drop-off suggests that 10 minutes of percussion
over-treats seeds from this lot of-black locust
A selected percussion treatment intermediate in time (5 minutes) also had the
fastest germination rate for black locust (Figure A6)_ The hot water curve is
somewhat flatter indicating a slower germination rate The control curve is very flat
indicating a slow germination rate
A qualitative difference between hot water and percussion-treated seeds was
also observed Along with healthy germinants the hot water treatment produced
over-treated seeds (Figure A 7a) Examples of damaged germinants include seeds
where the radicle did not elongate (Figure A 7b) seeds where the radicle elongated
but was delayed in freeing itself from the seed coat (Figure A7c) and seeds where the
radicle broke off completely from the embryo (Figure A7d) In addition to these
examples of over-treatment there were numerous small dark seeds that failed to
imbibe water For percussion treatment nearly all seeds took up water (Figure A8a)
Germination was rapid and uniform with little damage evident (Figure A8b)
To summarize both New Mexico locust and black locust seed lots were
dormant The standard hot water scarification treatment improved germination but
only to 56 and 41 for New Mexico locust and black locust respectively Nearly
all percussion durations improved total germination as well as germination rate over
the hot water treatment Germination was greater than 90 for percussion durations
of 4 5 and 10 minutes for New Mexico locust and 3 4 and 5 minutes for black locust
58
CI)
sgt t CI) 0
igt 0
sect ttl t E
Vl 0
c3
100r==~==~~=r------------------------------------~ ___ Percussion 5 min
middotmiddotmiddot0middotmiddotmiddot Hot Water -lIf- Control
80r=========~--~~----------------------------~
60
40
20 -~-~---~---------- ---~------------I
omiddot
O
_~_----------lIf-----~bull -----_ shy---shy I
Day 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A6 Effect of selected scarification treatments on germination speed for black locust First seven days of fourteen-day experiment are shown
Day 7
a
b c d
Figure A7 Damage to New Mexico locust seeds observed during germination counts following hot water scarification A7a Hot water replicate including damaged seedlings and numerous unimbibed seeds A7b Close-up ofseed where radicle failed to elongate A7c Close-up ofseed where radicle elongated but was delayed in freeing itself from the seed coat A7d Close-up ofseed where radicle completely broke offfrom embryo
60
a
b
Figure A8 New Mexico locust seeds following percussion treatment at time of germination counts A8a Percussion replicate with uniformly healthy seedlings and near complete imbibition of seeds A8b Close-up ofpercussion-treated seedling
61
Discussion
Papilionoid seeds have a specific anatomical feature known as the strophiole
or lens-the natural site of water entry to the seed (Hamly 1932) The strophiole is
located on the cotyledonary lobe of a locust seed (Figure A9) When a papilionoid
legume seed is percussed for the appropriate amount of time repeated hits on the
integrated seed coat loosen the constrained cells of the strophiolar region without
excessively damaging the rest of the seed coat (Ballard 1976)
When a percussed seed is soaked water enters exclusively through the
strophiole in a controlled manner (Kelly and Staden 1987) This regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues This
contrasts with seeds that have been hot water or acid scarified treatments that can
randomly degrade the seed coat Unlocalized cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rehman et al 1998 Marunda 1990)
Papilionoids are the largest subfamily of legumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust and black locust these legumes often play an integral role in the
revegetation ofdisturbed lands Developing superior scarification methods to the
standard hot water and acid treatments should fadlitate the use ofthese legumes in
restoration projects
62
Figure A9 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (bc)
63
APPENDIXB
GERMINATION PERCENTS AND STANDARD ERRORS FOR SOURCE
BLOCK AND TREATMENT FOR NEW MEXICO LOCUST
Table B 1 Gennination percents and standard errors for source block and treatment for New Mexico locust
Source Block Treatment Germination Percentage Standard Error
Capitan 1 Immersion 74 437 Capitan 2 Immersion 69 462 Capitan 3 Immersion 71 454 Capitan 4 Immersion 67 470 Capitan Boiling 30 sec 79 407 Capitan 2 Boiling 30 sec 76 427 Capitan 3 Boiling 30 sec 72 449 Capitan 4 Boiling 30 sec 76 427 Capitan Boiling 60 sec 71 454 Capitan 2 Boiling 60 sec 54 498 Capitan 3 Boiling 60 sec 67 470 Capitan 4 Boiling 60 sec 76 427 Capitan I Control 5 218 Capitan 2 Control 7 255 Capitan 3 Control 4 196 Capitan 4 Control 5 218 Capitan 1 Percussion I min 47 499 Capitan 2 Percussion I min
470
Capitan Percussion I min 39 488
Capitan 4 Percussion I min 38 485 Capitan I Percussion 2 min 55 497 Capitan Capitan
2
Percussion 2 min Percussion 2 min
71 65
454 477
Capitan 4 Percussion 2 min 56 496 Capitan I Percussion 4 min 86 347 Capitan 2 Percussion 4 min 79 407 Capitan 3 Percussion 4 min 81 392 Capitan 4 Percussion 4 min 82 384 Capitan I Percussion 8 min 90 300 Capitan 2 Percussion 8 min 88 325 Capitan 3 Percussion 8 min 90 300 Capitan 4 Percussion 8 min 90 300 Gila I Immersion 71 454 Gila 2 Immersion 77 421 Gila
Immersion 75 433
Gila 4 Immersion 72 449
Gila I Boiling 30 sec 68 466
Gila 2 Boiling 30 sec 81 392
Gila 3 Boiling 30 sec 54 498
Gila 4 Boiling 30 sec 63 483
Gila I Boiling 60 sec 55 497
Gila 2 Boiling 60 sec 68 466
Gila 3 Boiling 60 sec 57 495
Gila 4 Boiling 60 sec 70 458
65
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Gila 2 Control 13 336 Gila 3 Control 13 336 Gila 4 Control 20 400 Gila 1 Percussion 1 min 66 474 Gila 2 Percussion 1 min 57 495 Gila 3 Percussion 1 min 73 444 Gila 4 Percussion 1 min 82 384 Gila 1 Percussion 2 min 81 392 Gila 2 Percussion 2 min 86 347 Gila 3 Percussion 2 min 85 357 Gila 4 Percussion 2 min 54 498 Gila 1 Percussion 4 min 79 407 Gila 2 Percussion 4 min 83 376 Gila 3 Percussion 4 min 84 367 Gila 4 Percussion 4 min 79 407 Gila 1 Percussion 8 min 51 500 Gila 2 Percussion 8 min 76 427 Gila 3 Percussion 8 min 52 500 Gila 4 Percussion 8 min 74 439 Holman 1 Immersion 83 376 Holman 2 Immersion 86 347 Holman 3 Immersion 81 392 Holman 4 Immersion 90 300 Holman 1 Boiling 30 sec 90 300 Holman 2 Boiling 30 sec 85 357 Holman 3 Boiling 30 sec 87 336 Holman 4 Boiling 30 sec 83 376 Holman 1 Boiling 60 sec 79 407 Holman 2 Boiling 60 sec 80 400 Holman 3 Boiling 60 sec 86 347 Holman 4 Boiling 60 sec 93 255 Holman Control 25 433 Holman 2 Control 19 392 Holman Control 21 407
Holman 4 Control 24 427
Holman Percussion 1 min 77 421
Holman 2 Percussion 1 min 85 357
Holman 3 Percussion 1 min 80 400
Holman 4 Percussion 1 min 89 313
Holman 1 Percussion 2 min 88 325
Holman 2 Percussion 2 min 96 196
Holman 3 Percussion 2 min 93 255
Holman 4 Percussion 2 min 96 196
Holman 1 Percussion 4 min 92 271
Holman 2 Percussion 4 min 97 171
66
I
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Holman 3 Percussion 4 min 91 286 Holman 4 Percussion 4 min 96 196 Holman 1 Percussion 8 min 88 325 Holman 2 Percussion 8 min 85 357 Holman 3 Percussion 8 min 83 376 Holman 4 Percussion 8 min 82 384 Jemez 1 Immersion 53 499 Jemez 2 Immersion 44 496 Jemez 3 Immersion 52 500 Jemez 4 Immersion 70 458 Jemez 1 Boiling 30 sec 58 494 Jemez 2 Boiling 30 sec 55 497 Jemez 3 Boiling 30 sec 48 500 Jemez 4 Boiling 30 sec 65 477 Jemez 1 Boiling 60 sec 68 466 Jemez 2 Boiling 60 sec 63 483 Jemez 3 Boiling 60 sec 73 444 Jemez 4 Boiling 60 sec 89 313 Jemez 1 Control 2 140 Jemez 2 Control 9 286 Jemez 3 Control 6 237 Jemez 4 Control 8 271 Jemez 1 Percussion 1 min 58 494 Jemez 2 Percussion 1 min 63 483 Jemez 3 Percussion 1 min 77 421 Jemez 4 Percussion 1 min 89 313 Jemez 1 Percussion 2 min 68 466 Jemez 2 Percussion 2 min 64 480 Jemez 3 Percussion 2 min 80 400 Jemez 4 Percussion 2 min 83 376 Jemez Percussion 4 min 68 466 Jemez 2 Percussion 4 min 79 407 Jemez 3 Percussion 4 min 70 458 Jemez 4 Percussion 4 min 83 376 Jemez 1 Percussion 8 min 52 500 Jemez 2 Percussion 8 min 59 492 Jemez 3 Percussion 8 min 61 488 Jemez 4 Percussion 8 min 70 458 Magdalena 1 Immersion 69 462 Magdalena 2 Immersion 69 462 Magdalena J Immersion 70 458 Magdalena 4 Immersion 85 357 Magdalena 1 Boiling 30 sec 65 477 Magdalena 2 Boiling 30 sec 71 454 Magdalena 3 Boiling 30 sec 68 466
67
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Magdalena 4 Boiling 30 sec 83 376 Magdalena 1 Boiling 60 sec 63 483 Magdalena 2 Boiling 60 sec 68 466 Magdalena 3 Boiling 60 sec 63 483 Magdalena 4 Boiling 60 sec 82 384 Magdalena 1 Control 11 313 Magdalena 2 Control 6 237 Magdalena 3 Control 5 218 Magdalena 4 Control 4 196 Magdalena Percussion 1 min 32 466 Magdalena 2 Percussion 1 min 45 497 Magdalena 3 Percussion 1 min 46 498 Magdalena 4 Percussion I min 36 480 Magdalena 1 Percussion 2 min 67 470 Magdalena 2 Percussion 2 min 68 466 Magdalena 3 Percussion 2 min 65 477 Magdalena 4 Percussion 2 min 81 392 Magdalena 1 Percussion 4 min 87 336 Magdalena 2 Percussion 4 min 97 171 Magdalena 3 Percussion 4 min 83 376 Magdalena 4 Percussion 4 min 97 171 Magdalena 1 Percussion 8 min 98 lAO Magdalena 2 Percussion 8 min 100 000 Magdalena 3 Percussion 8 min 96 196 Magdalena 4 Percussion 8 min 98 lAO Manzano 1 Immersion 67 470 Manzano 2 Immersion 57 495 Manzano 3 Immersion 72 4049 Manzano 4 Immersion 73 4044 Manzano 1 Boiling 30 sec 70 458 Manzano 2 Boiling 30 sec 64 480 Manzano 3 Boiling 30 sec 71 454 Manzano 4 Boiling 30 sec 80 400 Manzano I Boiling 60 sec 50 500 Manzano 2 Boiling 60 sec 53 499 Manzano 3 Boiling 60 sec 59 492 Manzano 4 Boiling 60 sec 74 439 Manzano I Control 4 196 Manzano 2 Control 8 271 Manzano 3 Control 1 099 Manzano 4 Control 6 237 Manzano 1 Percussion 1 min 52 500 Manzano 2 Percussion 1 min 39 488 Manzano 3 Percussion 1 min 36 480 Manzano 4 Percussion 1 min 51 500
68
Table B 1 Continued
Source Block Treatment Gerrni~ation Percentage Standard Error
Manzano Percussion 2 min 53 499 Manzano 2 Percussion 2 min 58 494 Manzano 3 Percussion 2 min 61 488 Manzano 4 Percussion 2 min 62 485 Manzano 1 Percussion 4 min 76 427 Manzano 2 Percussion 4 min 85 357 Manzano 3 Percussion 4 min 79 407 Manzano 4 Percussion 4 min 83 376 Manzano 1 Percussion 8 min 87 336 Manzano 2 Percussion 8 min 83 376 Manzano 3 Percussion 8 min 83 376 Manzano 4 Percussion 8 min 75 433 Raton 1 Immersion 54 498 Raton 2 Immersion 37 483 Raton 3 Immersion 39 488 Raton 4 Immersion 69 462 Raton 1 Boiling 30 sec 36 480 Raton 2 Boiling 30 sec 20 400 Raton 3 Boiling 30 sec 28 449 Raton 4 Boiling 30 sec 60 490 Raton 1 Boiling 60 sec 15 357 Raton 2 Boiling 60 sec 171 Raton 3 Boiling 60 sec 4 196 Raton 4 Boiling 60 sec 35 477 Raton 1 Control 4 196 Raton 2 Control 12 325 Raton 3 Control 2 140 Raton 4 Control 6 237 Raton 1 Percussion 1 min 52 500 Raton 2 Percussion I min 29 454 Raton 3 Percussion I min 35 477 Raton 4 Percussion 1 min 31 462 Raton 1 Percussion 2 min 55 497 Raton 2 Percussion 2 min 35 477 Raton 3 Percussion 2 min 33 470 Raton 4 Percussion 2 min 42 494 Raton 1 Percussion 4 min 65 477 Raton 2 Percussion 4 min 52 500 Raton Percussion 4 min 51 500 Raton 4 Percussion 4 min 53 499 Raton 1 Percussion 8 min 88 325 Raton 2 Percussion 8 min 80 400 Raton 3 Percussion 8 min 73 444 Raton 4 Percussion 8 min 74 439 Sacramento 1 Immersion 43 495
69
Table B 1 Continued
-Source Block Treatment Germination Percentage Standard Error
Sacramento 2 Immersion 30 458 Sacramento 3 Immersion 38 485 Sacramento 4 Immersion 56 496 Sacramento 1 Boiling 30 sec 47 499 Sacramento 2 Boiling 30 sec 48 500 Sacramento 3 Boiling 30 sec 48 500 Sacramento 4 Boiling 30 sec 56 496 Sacramento 1 Boiling 60 sec 52 500 Sacramento 2 Boiling 60 sec 67 470 Sacramento 3 Boiling 60 sec 43 495 Sacramento 4 Boiling 60 sec 48 500 Sacramento 1 Control 0 000 Sacramento 2 Control 3 171 Sacramento 3 Control 3 171 Sacramento 4 Control 099 Sacramento 1 Percussion 1 min 57 495 Sacramento 2 Percussion 1 min 61 488 Sacramento 3 Percussion 1 min 62 485 Sacramento 4 Percussion 1 min 55 497 Sacramento 1 Percussion 2 min 84 367 Sacramento 2 Percussion 2 min 70 458 Sacramento 3 Percussion 2 min 81 392 Sacramento 4 Percussion 2 min 69 462 Sacramento 1 Percussion 4 min 91 286 Sacramento 2 Percussion 4 min 84 367 Sacramento 3 Percussion 4 min 84 367 Sacramento 4 Percussion 4 min 94 237 Sacramento 1 Percussion 8 min 82 384 Sacramento 2 Percussion 8 min 81 392 Sacramento 3 Percussion 8 min 82 384 Sacramento 4 Percussion 8 min 81 392 Sandia 1 Immersion 76 427 Sandia 2 Immersion 70 458 Sandia 3 Immersion 70 458 Sandia 4 Immersion 81 392 Sandia 1 Boiling 30 sec 78 414 Sandia 2 Boiling 30 sec 71 454 Sandia 3 Boiling 30 sec 63 483 Sandia 4 Boiling 30 sec 78 414 Sandia 1 Boiling 60 sec 67 470 Sandia 2 Boiling 60 sec 65 477 Sandia 3 Boiling 60 sec 74 439 Sandia 4 Boiling 60 sec 66 474 Sandia 1 Control 16 367 Sandia 2 Control 7 255
70
Table B 1 Continued
Source Block Treatment Gennination Percentage Standard Error
Sandia 3 Control 8 271 Sandia 4 Control 16 367 Sandia 1 Percussion 1 min 67 470 Sandia 2 Percussion 1 min 61 488 Sandia 3 Percussion 1 min 66 474 Sandia 4 Percussion 1 min 59 492 Sandia I Percussion 2 min 82 384 Sandia 2 Percussion 2 min 83 376 Sandia 3 Percussion 2 min 81 392 Sandia 4 Percussion 2 min 84 367 Sandia 1 Percussion 4 min 89 313 Sandia 2 Percussion 4 min 86 347 Sandia 3 Percussion 4 min 89 313 Sandia 4 Percussion 4 min 85 357 Sandia 1 Percussion 8 min 80 400 Sandia 2 Percussion 8 min 77 421 Sandia 3 Percussion 8 min 69 462 Sandia 4 Percussion 8 min 66 474
LITERATURE CITED
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Ballard LAT 1973 Physical barriers to gennination Seed Sci Technoll 285-303
___ 1976 Strophiolar water conduction in the seeds ofTrifolieae induced by action on the testa at nonstrophiolar sites Austr J Plant Physiol3 465-469
Barton LV 1947 Special studies on seed coat impenneability Contrib Boyce Thompson Inst 14 355-362
Baskin CC Baskin JM 1998 Seeds Ecology biogeography and evolution of donnancy and gennination Academic Press London UK 666 p
Bewley JD 1997 Seed germination and donnancy Plant Ce119 1055-1066
Bewley JD Black M 1994 Seeds physiology of development and germination 2nd ed Plenum Press New York NY 445 p
Briscoe CB 1996 Growing Nitrogen Fixing Trees in Acid Soils In MH Powell (ed) Nitrogen Fixing Trees for Acid Soils- A Field ManuaL Winrock International Morrilton AR 22 p
Copeland LO McDonald MB 1995 Principles of Seed Science and Technology 3rded Chapman amp Hall New York NY 409 p
Cox RE Klett JE 1984 Seed gennination requirements of native Colorado plants for use in the landscape Plant Propag 30(2) 6-10
Dell B 1980 Structure and function ofthe strophiolar plug in seeds ofAlbizia Lophantha Am J Bot 67 556-563
Dick-Peddie WA 1993 New Mexico Vegetation Past Present and Future University ofNew Mexico Press Albuquerque NM 244 p
Dreesen DR 2001 Propagation protocol for production ofcontainer Robinia neomexicana plants Los Lunas Plant Materials Center Los Lunas New Mexico In Native Plant Network URL httpwwwnativeplantnetworkorg (accessed 9 January 2002) Moscow (ID) University ofIdaho CoIl Natural Resources For Res Nursery
72
Dreesen DR Harrington JT 1997 Propagation of native plants for restoration projects in the Southwestern US- Preliminary Investigations In Landis TD Thompson JR tech coords National Proceedings Forest and Conservation Nursery Assocs Gen Tech Rep PNW-GTR-419 Portland OR USDA For Ser PNW Res Sta 77-88
Gottfried GJ 1980 Control ofNew Mexican locust and the effect on planted ponderosa pine in central Arizona US For Ser Res Note RM-386 Rocky Mountain For Range ExpStn Fort Collins CO
Gosling PG Samuel YK Jones SK 1995 A systematic examination of germination temperature chipping and water temperaturesoak duration pretreatments on the seeds ofLeucaena leucocephala Seed Sci amp Technol 23521-532
Hamly DH 1932 Softening of the seeds of Melilotus alba Bot Gazette 93 345shy375
Hanna PJ 1984 Anatomical features of the seed coat ofAcacia kempeana (Mueller) which relate to increased germination rate induced by heat treatment New Phytol 96 23-29
Hanks JP Dick-Peddie WA 1974 Vegetation patterns of the White Mountains New Mexico Southwest Naturalist 18(4) 371-382
Hine S Harrington JT Dreesen DR 1997 Seed scarification requirements for Robinia neomexicana In Harrington JTtech coord Proc Third SouthwestemContainer Growers Conf Feb 12-13 1997 Ft Collins CO
Hopkinson JM 1993 The strophiole of leguminous seeds Newsletter- IntI Herbage Seed Production Res Grp 18 7-8
---- 1997 Differences in hardseededness between Cunningham and Tarramba leucaena In Leucnet News The Newsletter of the International LeucaenaRes and Devlt Netwk No4
ISTA (International Seed Testing Association) 1999 International rules for seed testing rules Seed Sci amp Technol27 Supplement
Kelly KM van Staden J 1987 The lens as the site of permeability in the papilionoid seed Aspalathus linearis J Plant Physiol 128(45) 395-404
73
Keresztesi B 1988 Black locust the tree of agriculture Outlook on Agr 17(2) 77~85
Kigel 1 and Galili G 1995 Seed development and germination Marcel Dekker New York 853 p
Klemmedson 10 1994 New Mexico locust and parent material Influence on macronutrients of forest floor and soil Soil Sci Soc Am 1 58 974~980
Kuo WH Tam AW 1988 The pathway of water absorption of mung bean seeds Seed Sci amp Technol16(1) 139~144
Leadem CL 1997 Dormancy--unlocking seed secrets In Landis TD Thompson lR tech coords National Proceedings Forestand Conservation Nursery Assocs Gen Tech Rep PNW-G TR-419 Portland OR USDA For Ser PNW Res Sta 43-52
Lin Y Wagner MR Seweyestewa A Honahni MD DeGomez M McMillin JD 1996 Influence of cultural treatments on seed germination and seedling growth of Robinia neomexicana Forest Farm and Community Tree Res Reports Volume 1
Marunda CT 1990 Effects of seed pretreatment on the development ofAcacia auriculforinis and A holosericea seedlings ACIAR Proceedings Series No 28
Meginnis HG 1937 Sulphuric acid treatment to increase germination of black locust seed USDA Circ 453 34 p
Miklas PN Towensend CE Ladd SL 1987 Seedcoat anatomy and the scarification ofcicer milkvetch seed Crop Sci 27 766-772
Monsen SB 1984 Use of shrubs on mine spoils In The Challenge ofProducing Native Plants for the Intermountain Area Proceedings Intermountain Nurserymans Association 1983 conference Las Vegas NV compo PM Murphy 26-31 Ogden UT USDA For Ser Gen Tech Report INT-l68
Olson DF 1974 Robinia L Locust In Seeds of Woody Plants in the US tech coord CS Schopmeyer 728-731 USDA For Ser Agr Handbk No450
Rehman S Loescher RNl Harris P1C 1998 Dormancy breaking and germination ofAcacia salicina Lindl seeds Seed Sci amp Techno127 553-557
Rolston MP 1978 Water impermeable seed dormancy Bot Rev 44 365-396
74
SAS Institute Inc 1989 SASSTAT Users Guide Version 6 4th Edition Volume 1 Cary NC SAS Institute Inc 943 p
SerratoValenti G Conara L Ferrando M Modenesi P 1993 Structural and histochemical features ofStylosanthes scabra (Leguminosae Papilionoidae) seed coat as related to water entry Can J Bot 71 834-840
Simpson BJ 1988 A field guide to Texas trees Austin TX Texas Monthly Press 372p
Tran VN Cavanagh AK 1984 Structural aspects ofdormancy In Seed Physiology Vol 2 Murray DR (ed) p1-44 Academic Press Australia
USDA Forest Service 1948 Woody-plant seed manual USDA Misc Pub1654 416p
Vines RA 1960 Trees Shrubs and Woody Vines of the Southwest Austin Univ ofTexas Press 1104 p
Wagner MR Larson RE Craig TP 1992 Ecology and silvics ofNew Mexico locust (Robinia neomexicana) with an emphasis on the arboreal form p53-57 In JW Hanover K Miller and S Plesko (eds) Proc Intemat Confon Black Locust Biology Culture and Utilization Mich State Univ USA
Wilson JK 1944 Immersing seeds of species of Robinia in boiling water hastens germination J For 42 453-454
Zimmerman RW Carpenter SB 1980 First-year coppice production from a 5shyyear old black locust stand on surface mine spoil p309-314 Centr Hardwd For ConfProc University ofMissouri Columbia
CONCLUSION
Based on results obtained in pilot study (see Appendix) and thesis
experiments 4 minutes ofpercussion is recommended as a standard protocol for
treating an unknown source ofNew Mexico locust seed In addition to laboratory
results the 4-minute percussion treatment has resulted in germination greater than
80 and healthy seedling grow-outs for production purposes (unpublished data) In
laboratory experiments only one hundred seeds per 118 ml (40z) soil tin were
percussed at a time whereas for production we have treated up to five hundred seeds
per tin For large-scale production ofNew Mexico locust seedlings growers may
wish to experiment with larger quantities of seed and larger-size soil tins
Seed storage trials following percussion treatments were not conducted
However germination greater than 80 and healthy seedlings were obtained when
seeds were percussion-treated and subsequently stored at room-temperature (22degC)
for one week (unpublished data) Seed storage trials merit further study as the
standard hot water treatment leaves seeds in a softened condition that is not
conducive to storage
Hydroseeding is an effective method for direct seeding in reclamation
situations Percussion treatment may be conducive to hydro seeding as seeds remain
a dry relatively undamaged yet permeable condition following treatment Sulfuric
seeds also remain dry following treatment but extensive damage to seed coat
poor seedling vigor have been reported with this treatment (Kelly and van Staden
A grow-out study using a hydro seeder on a reclamation site testing percussion
44
against other treatments such as sulfuric acid and mechanical scarification also
merits further study
Papilionoids are the largest subfamily oflegumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust these legumes often play an integral role in the revegetation of
disturbed lands The pilot study (Appendix) indicates that percussion scarification
successfully treats the widely used black locust as well Developing superior
scarification methods to the standard hot water mechanical scarification and acid
treatments may facilitate the use ofpapilionoid legumes in reclamation projects
45
APPENDICES
APPENDIX A
PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR
NEW MEXICO LOCUST AND BLACK LOCUST SEEDS
Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4
Hot water and sulfuric acid soaks are traditional treatments for seeds of many
temperate woody legwnes including locusts However these scarification techniques
often produce inconsistent germination Percussion scarification where seeds are
repeatedly propelled against a hard surface was compared with hot water
scarification to evaluate treatment efficacy for New Mexico locust (Robinia
neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment
seeds were placed in a 98degC water bath which was immediately removed from the
heat source For percussion scarification seeds were placed in a soil sample tin and
agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the
control were followed by 24-hour water soaks Hot water baths resulted in 56 and
41 germination for New Mexico locust and black locust respectively For both
species nearly all durations ofpercussion increased germination over the hot water
treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3
4 and 5 minutes for black locust resulted in at least 90 germination Traditional
scarification treatments randomly degrade the entire seed coat which can lead to
tissue damage during water uptake Percussion scarification specifically weakens the
strophiole the natural source ofwater entry to the seed in papilionoid legwnes
INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom
2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031
48
Following percussion imbibition is controlled through the strophiole and underlying
tissue is protected
Keywords New Mexico locust black locust scarification percussion germination
Introduction
Locust trees (Robinia spp) are aggressive pioneer species that quickly
colonize disturbed land fix atmospheric nitrogen in the soil and stabilize slopes that
are prone to erosion (Klemmedson 1994 Ashby et al 1985) (Figure A1ab)
Recognition of these attributes has contributed to increased demand for locusts in
reclamation projects Locust seeds however exhibit physical dormancy where a
thick seed coat prevents the movement of water and gasses to the embryo (Leadem
1997)
Sulfuric acid or hot water soaks have traditionally been used to break seed
dormancy in macro-propagation of locusts Many growers are moving away from the
use of su1furic acid in treating locust seeds (Dreesen and Harrington 1997) In
addition to being dangerous acid soak durations must be specifically correlated to
seed lot (Olson 1974) Collections of native seed can vary tremendously in seed size
seed weight and hard seededness etc This variability limits the utility of sulfuric
acid soaks as even within a given lot some seeds may be damaged whereas others
are under-treated and fail to imbibe (take up water) A survey of Southwestern
container growers indicates that hot water baths are currently the most common
scarification method for locust seeds (Hine et al 1997) However hot water baths
also produce inconsistent germination (Lin et al 1996)
49
b
Figure AI New Mexico locust a primary invader following disturbance Ala Crown-sprouting at edge ofburn near Los Alamos NM--one year after Cerro Grande fire Alb Colonizing road cut Gila National Forest NM
50
Percussion scarification where seeds are repeatedly propelled against a hard
surface is an alternative dormancy-breaking method that has proven successful in
legumes related to locusts (Hamly 1932 Barton 1947) We compared this alternative
treatment to hot water scarification to evaluate treatment efficacy for New Mexico
locust and black locust seeds
Materials and Methods
Commercial New Mexico locust seeds (Western Native Seed Coaldale CO)
were collected fall 2000 in Huerfano County CO Black locust seeds were collected
September 2000 from Taos County NM by harvesting ripe pods from several trees at
a distance of up to 3 m from ground level Black locust seeds were allowed to air-dry
for 3 weeks and were threshed and separated from large chaffusing a greenhouse
fan A South Dakota Seed Blower (Chicago Illinltois) was used to remove fine chaff
from both seed lots Seeds were storedmiddotat 2-4C until the germination study was
conducted in March 2001
This study compared hot water scarification with percussion scarification
using treatment and species as experimental factors Along with a control
scarification treatments included immersion of seed in boiling water as well as
percussion durations of 1 2 3 45 or 10 minutes Four 100-seed samples were used
to test each species by scarification treatment combination
For the hot water treatment test tubes were filled with 30 mL water and
placed in a water-filled beaker (Figure A2a) The bath was raised to boiling (-98C at
51
b
c
Figure A2 Implementation ofhot water and percussion scarification A2a Hot water bath A2b Paint-shaker set-up A2c Placing soil tin containing seeds at end ofpaint shaker ann
52
Las Cruces NM elevation of 1300 meters) Each 100-seed replicate was placed in a
test tube and each test tube was immediately removed to cool at room temperature
Percussion scarification was implemented using a pneumatic paint shaker
(Central Pneumatic model 00422 Camarillo CA) (Figure A2b) This allowed
standardization of treatment intensity Compressed air pressure was maintained at
530kPa plusmn 30kPa (80psi plusmn 5psi) resulting in approximately 350 oscillations per
minute Each lOO-seed sample was placed in a 118 ml (4 oz) soil tin for shaking To
maximize lateral movement of paint shaker a paint can was used as a spacer to place
soil tins at the end of the shaking arm (Figure A2c) This allowed shaking to occur at
the greatest distance from the pivot point (fulcrum) Following all treatments
including the control seeds were soaked for twenty-four hours in distilled water prior
to plating out
Gennination was evaluated on lab benches at room temperature Each
replicate of seeds was placed in a 100 cm petri dish on moistened filter paper with
humidity maintained by enclosing the petri dishes in ziplock bags Gennination was
monitored daily for 14 days in accordance with International Seed Testing
Association standards for Robinia species (ISTA 1999) Gennination was defined as
protrusion of the radicle from the embryo by at least 1mm as viewed by the naked
eye
Categorical Analysis of Variance using Prpc Catmod (SAS Institute 1989)
was used to detennine treatment differences in germination percentages for each
source This procedure is an extension of a Chi-square test of homogeneity using the
53
natural log of the ratio of germinated to non-germinated seeds for each treatment Pshy
values less than 005 were considered significant A limited set of pairwiseshy
comparisons was conducted to compare treatment mean percents using a
conservative alpha value of 005 divided by the number of comparisons
Results
Percussion maximized germination for both New Mexico locust and black
locust There were three germination response levels for New Mexico locust (Figure
A3) First there was a low response in the control indicating a dormant seed lot
The hot water treatment and I-minute duration of percussion share a second
intermediate response Germination was highest at percussion levels from 2 to 10
minutes
A selected percussion treatment intermediate in time (5 minutes) produced the
steepest germination curve indicating the fastest germination rate (Figure A4) The
hot water treatment produced an intermediate curve or relatively slower germination
rate The control curve was very flat indicating the slowest germination rate for New
Mexico locust
Control seeds germinated poorly for black locust as well (Figure A5) Hot
water scarification improved germination over the control but all levels of percussion
improved germination over hot water The optimal durations of percussion treatment
were bracketed with a significant increase from the 1 to 2 minute percussion level a
high germination response from 2 to 5 minutes of percussion and finally a significant
54
~ d 25 CL)
p t1 0 ~ t1
g Vl ~ Vl
100~---------------------------------------------------------------
80
60
40
20
o Control Hot Water Percussion
I min Percussion
2 min Percussion
3 min
Scarification
Percussion 4 min
Figure A3 Effect of scarification on germination response for New Mexico locust
Percussion 5 min
=significant difference between adjacent treatment bars ns = no significant difference
Percussion 10 min
~ ~ () u agt
Il-t d o ~
~ ~ c3
100middotlr~~~------------------------------------e- Percussion 5min middot 0middot Hot Water -- Control
80-J=middot======
6b-middot-lmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middot--middotmiddot-middot~-middotmiddot -middotmiddot middot middotmiddot-middot 1
40
20
-----~-----------
0 0 0 middotmiddotmiddotmiddotmiddot0middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot0
0 --~-----y-----~------_---~--- T-----~
o shy -TDay 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A4 Effect of selected scarification treatments on germination speed for New Mexico locust First seven days of fourteen-day experiment are shown
Day 7
80
~ cd
5 60 ()
[) Cl d 0 1t1 d
40sect dUl
-J
20
o
ns
Scarification
Figure A5 Effect of scarification on germination response for black locust = significant difference between adjacent treatment bars ns = no significant difference
decrease at the 10-minute level This drop-off suggests that 10 minutes of percussion
over-treats seeds from this lot of-black locust
A selected percussion treatment intermediate in time (5 minutes) also had the
fastest germination rate for black locust (Figure A6)_ The hot water curve is
somewhat flatter indicating a slower germination rate The control curve is very flat
indicating a slow germination rate
A qualitative difference between hot water and percussion-treated seeds was
also observed Along with healthy germinants the hot water treatment produced
over-treated seeds (Figure A 7a) Examples of damaged germinants include seeds
where the radicle did not elongate (Figure A 7b) seeds where the radicle elongated
but was delayed in freeing itself from the seed coat (Figure A7c) and seeds where the
radicle broke off completely from the embryo (Figure A7d) In addition to these
examples of over-treatment there were numerous small dark seeds that failed to
imbibe water For percussion treatment nearly all seeds took up water (Figure A8a)
Germination was rapid and uniform with little damage evident (Figure A8b)
To summarize both New Mexico locust and black locust seed lots were
dormant The standard hot water scarification treatment improved germination but
only to 56 and 41 for New Mexico locust and black locust respectively Nearly
all percussion durations improved total germination as well as germination rate over
the hot water treatment Germination was greater than 90 for percussion durations
of 4 5 and 10 minutes for New Mexico locust and 3 4 and 5 minutes for black locust
58
CI)
sgt t CI) 0
igt 0
sect ttl t E
Vl 0
c3
100r==~==~~=r------------------------------------~ ___ Percussion 5 min
middotmiddotmiddot0middotmiddotmiddot Hot Water -lIf- Control
80r=========~--~~----------------------------~
60
40
20 -~-~---~---------- ---~------------I
omiddot
O
_~_----------lIf-----~bull -----_ shy---shy I
Day 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A6 Effect of selected scarification treatments on germination speed for black locust First seven days of fourteen-day experiment are shown
Day 7
a
b c d
Figure A7 Damage to New Mexico locust seeds observed during germination counts following hot water scarification A7a Hot water replicate including damaged seedlings and numerous unimbibed seeds A7b Close-up ofseed where radicle failed to elongate A7c Close-up ofseed where radicle elongated but was delayed in freeing itself from the seed coat A7d Close-up ofseed where radicle completely broke offfrom embryo
60
a
b
Figure A8 New Mexico locust seeds following percussion treatment at time of germination counts A8a Percussion replicate with uniformly healthy seedlings and near complete imbibition of seeds A8b Close-up ofpercussion-treated seedling
61
Discussion
Papilionoid seeds have a specific anatomical feature known as the strophiole
or lens-the natural site of water entry to the seed (Hamly 1932) The strophiole is
located on the cotyledonary lobe of a locust seed (Figure A9) When a papilionoid
legume seed is percussed for the appropriate amount of time repeated hits on the
integrated seed coat loosen the constrained cells of the strophiolar region without
excessively damaging the rest of the seed coat (Ballard 1976)
When a percussed seed is soaked water enters exclusively through the
strophiole in a controlled manner (Kelly and Staden 1987) This regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues This
contrasts with seeds that have been hot water or acid scarified treatments that can
randomly degrade the seed coat Unlocalized cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rehman et al 1998 Marunda 1990)
Papilionoids are the largest subfamily of legumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust and black locust these legumes often play an integral role in the
revegetation ofdisturbed lands Developing superior scarification methods to the
standard hot water and acid treatments should fadlitate the use ofthese legumes in
restoration projects
62
Figure A9 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (bc)
63
APPENDIXB
GERMINATION PERCENTS AND STANDARD ERRORS FOR SOURCE
BLOCK AND TREATMENT FOR NEW MEXICO LOCUST
Table B 1 Gennination percents and standard errors for source block and treatment for New Mexico locust
Source Block Treatment Germination Percentage Standard Error
Capitan 1 Immersion 74 437 Capitan 2 Immersion 69 462 Capitan 3 Immersion 71 454 Capitan 4 Immersion 67 470 Capitan Boiling 30 sec 79 407 Capitan 2 Boiling 30 sec 76 427 Capitan 3 Boiling 30 sec 72 449 Capitan 4 Boiling 30 sec 76 427 Capitan Boiling 60 sec 71 454 Capitan 2 Boiling 60 sec 54 498 Capitan 3 Boiling 60 sec 67 470 Capitan 4 Boiling 60 sec 76 427 Capitan I Control 5 218 Capitan 2 Control 7 255 Capitan 3 Control 4 196 Capitan 4 Control 5 218 Capitan 1 Percussion I min 47 499 Capitan 2 Percussion I min
470
Capitan Percussion I min 39 488
Capitan 4 Percussion I min 38 485 Capitan I Percussion 2 min 55 497 Capitan Capitan
2
Percussion 2 min Percussion 2 min
71 65
454 477
Capitan 4 Percussion 2 min 56 496 Capitan I Percussion 4 min 86 347 Capitan 2 Percussion 4 min 79 407 Capitan 3 Percussion 4 min 81 392 Capitan 4 Percussion 4 min 82 384 Capitan I Percussion 8 min 90 300 Capitan 2 Percussion 8 min 88 325 Capitan 3 Percussion 8 min 90 300 Capitan 4 Percussion 8 min 90 300 Gila I Immersion 71 454 Gila 2 Immersion 77 421 Gila
Immersion 75 433
Gila 4 Immersion 72 449
Gila I Boiling 30 sec 68 466
Gila 2 Boiling 30 sec 81 392
Gila 3 Boiling 30 sec 54 498
Gila 4 Boiling 30 sec 63 483
Gila I Boiling 60 sec 55 497
Gila 2 Boiling 60 sec 68 466
Gila 3 Boiling 60 sec 57 495
Gila 4 Boiling 60 sec 70 458
65
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Gila 2 Control 13 336 Gila 3 Control 13 336 Gila 4 Control 20 400 Gila 1 Percussion 1 min 66 474 Gila 2 Percussion 1 min 57 495 Gila 3 Percussion 1 min 73 444 Gila 4 Percussion 1 min 82 384 Gila 1 Percussion 2 min 81 392 Gila 2 Percussion 2 min 86 347 Gila 3 Percussion 2 min 85 357 Gila 4 Percussion 2 min 54 498 Gila 1 Percussion 4 min 79 407 Gila 2 Percussion 4 min 83 376 Gila 3 Percussion 4 min 84 367 Gila 4 Percussion 4 min 79 407 Gila 1 Percussion 8 min 51 500 Gila 2 Percussion 8 min 76 427 Gila 3 Percussion 8 min 52 500 Gila 4 Percussion 8 min 74 439 Holman 1 Immersion 83 376 Holman 2 Immersion 86 347 Holman 3 Immersion 81 392 Holman 4 Immersion 90 300 Holman 1 Boiling 30 sec 90 300 Holman 2 Boiling 30 sec 85 357 Holman 3 Boiling 30 sec 87 336 Holman 4 Boiling 30 sec 83 376 Holman 1 Boiling 60 sec 79 407 Holman 2 Boiling 60 sec 80 400 Holman 3 Boiling 60 sec 86 347 Holman 4 Boiling 60 sec 93 255 Holman Control 25 433 Holman 2 Control 19 392 Holman Control 21 407
Holman 4 Control 24 427
Holman Percussion 1 min 77 421
Holman 2 Percussion 1 min 85 357
Holman 3 Percussion 1 min 80 400
Holman 4 Percussion 1 min 89 313
Holman 1 Percussion 2 min 88 325
Holman 2 Percussion 2 min 96 196
Holman 3 Percussion 2 min 93 255
Holman 4 Percussion 2 min 96 196
Holman 1 Percussion 4 min 92 271
Holman 2 Percussion 4 min 97 171
66
I
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Holman 3 Percussion 4 min 91 286 Holman 4 Percussion 4 min 96 196 Holman 1 Percussion 8 min 88 325 Holman 2 Percussion 8 min 85 357 Holman 3 Percussion 8 min 83 376 Holman 4 Percussion 8 min 82 384 Jemez 1 Immersion 53 499 Jemez 2 Immersion 44 496 Jemez 3 Immersion 52 500 Jemez 4 Immersion 70 458 Jemez 1 Boiling 30 sec 58 494 Jemez 2 Boiling 30 sec 55 497 Jemez 3 Boiling 30 sec 48 500 Jemez 4 Boiling 30 sec 65 477 Jemez 1 Boiling 60 sec 68 466 Jemez 2 Boiling 60 sec 63 483 Jemez 3 Boiling 60 sec 73 444 Jemez 4 Boiling 60 sec 89 313 Jemez 1 Control 2 140 Jemez 2 Control 9 286 Jemez 3 Control 6 237 Jemez 4 Control 8 271 Jemez 1 Percussion 1 min 58 494 Jemez 2 Percussion 1 min 63 483 Jemez 3 Percussion 1 min 77 421 Jemez 4 Percussion 1 min 89 313 Jemez 1 Percussion 2 min 68 466 Jemez 2 Percussion 2 min 64 480 Jemez 3 Percussion 2 min 80 400 Jemez 4 Percussion 2 min 83 376 Jemez Percussion 4 min 68 466 Jemez 2 Percussion 4 min 79 407 Jemez 3 Percussion 4 min 70 458 Jemez 4 Percussion 4 min 83 376 Jemez 1 Percussion 8 min 52 500 Jemez 2 Percussion 8 min 59 492 Jemez 3 Percussion 8 min 61 488 Jemez 4 Percussion 8 min 70 458 Magdalena 1 Immersion 69 462 Magdalena 2 Immersion 69 462 Magdalena J Immersion 70 458 Magdalena 4 Immersion 85 357 Magdalena 1 Boiling 30 sec 65 477 Magdalena 2 Boiling 30 sec 71 454 Magdalena 3 Boiling 30 sec 68 466
67
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Magdalena 4 Boiling 30 sec 83 376 Magdalena 1 Boiling 60 sec 63 483 Magdalena 2 Boiling 60 sec 68 466 Magdalena 3 Boiling 60 sec 63 483 Magdalena 4 Boiling 60 sec 82 384 Magdalena 1 Control 11 313 Magdalena 2 Control 6 237 Magdalena 3 Control 5 218 Magdalena 4 Control 4 196 Magdalena Percussion 1 min 32 466 Magdalena 2 Percussion 1 min 45 497 Magdalena 3 Percussion 1 min 46 498 Magdalena 4 Percussion I min 36 480 Magdalena 1 Percussion 2 min 67 470 Magdalena 2 Percussion 2 min 68 466 Magdalena 3 Percussion 2 min 65 477 Magdalena 4 Percussion 2 min 81 392 Magdalena 1 Percussion 4 min 87 336 Magdalena 2 Percussion 4 min 97 171 Magdalena 3 Percussion 4 min 83 376 Magdalena 4 Percussion 4 min 97 171 Magdalena 1 Percussion 8 min 98 lAO Magdalena 2 Percussion 8 min 100 000 Magdalena 3 Percussion 8 min 96 196 Magdalena 4 Percussion 8 min 98 lAO Manzano 1 Immersion 67 470 Manzano 2 Immersion 57 495 Manzano 3 Immersion 72 4049 Manzano 4 Immersion 73 4044 Manzano 1 Boiling 30 sec 70 458 Manzano 2 Boiling 30 sec 64 480 Manzano 3 Boiling 30 sec 71 454 Manzano 4 Boiling 30 sec 80 400 Manzano I Boiling 60 sec 50 500 Manzano 2 Boiling 60 sec 53 499 Manzano 3 Boiling 60 sec 59 492 Manzano 4 Boiling 60 sec 74 439 Manzano I Control 4 196 Manzano 2 Control 8 271 Manzano 3 Control 1 099 Manzano 4 Control 6 237 Manzano 1 Percussion 1 min 52 500 Manzano 2 Percussion 1 min 39 488 Manzano 3 Percussion 1 min 36 480 Manzano 4 Percussion 1 min 51 500
68
Table B 1 Continued
Source Block Treatment Gerrni~ation Percentage Standard Error
Manzano Percussion 2 min 53 499 Manzano 2 Percussion 2 min 58 494 Manzano 3 Percussion 2 min 61 488 Manzano 4 Percussion 2 min 62 485 Manzano 1 Percussion 4 min 76 427 Manzano 2 Percussion 4 min 85 357 Manzano 3 Percussion 4 min 79 407 Manzano 4 Percussion 4 min 83 376 Manzano 1 Percussion 8 min 87 336 Manzano 2 Percussion 8 min 83 376 Manzano 3 Percussion 8 min 83 376 Manzano 4 Percussion 8 min 75 433 Raton 1 Immersion 54 498 Raton 2 Immersion 37 483 Raton 3 Immersion 39 488 Raton 4 Immersion 69 462 Raton 1 Boiling 30 sec 36 480 Raton 2 Boiling 30 sec 20 400 Raton 3 Boiling 30 sec 28 449 Raton 4 Boiling 30 sec 60 490 Raton 1 Boiling 60 sec 15 357 Raton 2 Boiling 60 sec 171 Raton 3 Boiling 60 sec 4 196 Raton 4 Boiling 60 sec 35 477 Raton 1 Control 4 196 Raton 2 Control 12 325 Raton 3 Control 2 140 Raton 4 Control 6 237 Raton 1 Percussion 1 min 52 500 Raton 2 Percussion I min 29 454 Raton 3 Percussion I min 35 477 Raton 4 Percussion 1 min 31 462 Raton 1 Percussion 2 min 55 497 Raton 2 Percussion 2 min 35 477 Raton 3 Percussion 2 min 33 470 Raton 4 Percussion 2 min 42 494 Raton 1 Percussion 4 min 65 477 Raton 2 Percussion 4 min 52 500 Raton Percussion 4 min 51 500 Raton 4 Percussion 4 min 53 499 Raton 1 Percussion 8 min 88 325 Raton 2 Percussion 8 min 80 400 Raton 3 Percussion 8 min 73 444 Raton 4 Percussion 8 min 74 439 Sacramento 1 Immersion 43 495
69
Table B 1 Continued
-Source Block Treatment Germination Percentage Standard Error
Sacramento 2 Immersion 30 458 Sacramento 3 Immersion 38 485 Sacramento 4 Immersion 56 496 Sacramento 1 Boiling 30 sec 47 499 Sacramento 2 Boiling 30 sec 48 500 Sacramento 3 Boiling 30 sec 48 500 Sacramento 4 Boiling 30 sec 56 496 Sacramento 1 Boiling 60 sec 52 500 Sacramento 2 Boiling 60 sec 67 470 Sacramento 3 Boiling 60 sec 43 495 Sacramento 4 Boiling 60 sec 48 500 Sacramento 1 Control 0 000 Sacramento 2 Control 3 171 Sacramento 3 Control 3 171 Sacramento 4 Control 099 Sacramento 1 Percussion 1 min 57 495 Sacramento 2 Percussion 1 min 61 488 Sacramento 3 Percussion 1 min 62 485 Sacramento 4 Percussion 1 min 55 497 Sacramento 1 Percussion 2 min 84 367 Sacramento 2 Percussion 2 min 70 458 Sacramento 3 Percussion 2 min 81 392 Sacramento 4 Percussion 2 min 69 462 Sacramento 1 Percussion 4 min 91 286 Sacramento 2 Percussion 4 min 84 367 Sacramento 3 Percussion 4 min 84 367 Sacramento 4 Percussion 4 min 94 237 Sacramento 1 Percussion 8 min 82 384 Sacramento 2 Percussion 8 min 81 392 Sacramento 3 Percussion 8 min 82 384 Sacramento 4 Percussion 8 min 81 392 Sandia 1 Immersion 76 427 Sandia 2 Immersion 70 458 Sandia 3 Immersion 70 458 Sandia 4 Immersion 81 392 Sandia 1 Boiling 30 sec 78 414 Sandia 2 Boiling 30 sec 71 454 Sandia 3 Boiling 30 sec 63 483 Sandia 4 Boiling 30 sec 78 414 Sandia 1 Boiling 60 sec 67 470 Sandia 2 Boiling 60 sec 65 477 Sandia 3 Boiling 60 sec 74 439 Sandia 4 Boiling 60 sec 66 474 Sandia 1 Control 16 367 Sandia 2 Control 7 255
70
Table B 1 Continued
Source Block Treatment Gennination Percentage Standard Error
Sandia 3 Control 8 271 Sandia 4 Control 16 367 Sandia 1 Percussion 1 min 67 470 Sandia 2 Percussion 1 min 61 488 Sandia 3 Percussion 1 min 66 474 Sandia 4 Percussion 1 min 59 492 Sandia I Percussion 2 min 82 384 Sandia 2 Percussion 2 min 83 376 Sandia 3 Percussion 2 min 81 392 Sandia 4 Percussion 2 min 84 367 Sandia 1 Percussion 4 min 89 313 Sandia 2 Percussion 4 min 86 347 Sandia 3 Percussion 4 min 89 313 Sandia 4 Percussion 4 min 85 357 Sandia 1 Percussion 8 min 80 400 Sandia 2 Percussion 8 min 77 421 Sandia 3 Percussion 8 min 69 462 Sandia 4 Percussion 8 min 66 474
LITERATURE CITED
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Ballard LAT 1973 Physical barriers to gennination Seed Sci Technoll 285-303
___ 1976 Strophiolar water conduction in the seeds ofTrifolieae induced by action on the testa at nonstrophiolar sites Austr J Plant Physiol3 465-469
Barton LV 1947 Special studies on seed coat impenneability Contrib Boyce Thompson Inst 14 355-362
Baskin CC Baskin JM 1998 Seeds Ecology biogeography and evolution of donnancy and gennination Academic Press London UK 666 p
Bewley JD 1997 Seed germination and donnancy Plant Ce119 1055-1066
Bewley JD Black M 1994 Seeds physiology of development and germination 2nd ed Plenum Press New York NY 445 p
Briscoe CB 1996 Growing Nitrogen Fixing Trees in Acid Soils In MH Powell (ed) Nitrogen Fixing Trees for Acid Soils- A Field ManuaL Winrock International Morrilton AR 22 p
Copeland LO McDonald MB 1995 Principles of Seed Science and Technology 3rded Chapman amp Hall New York NY 409 p
Cox RE Klett JE 1984 Seed gennination requirements of native Colorado plants for use in the landscape Plant Propag 30(2) 6-10
Dell B 1980 Structure and function ofthe strophiolar plug in seeds ofAlbizia Lophantha Am J Bot 67 556-563
Dick-Peddie WA 1993 New Mexico Vegetation Past Present and Future University ofNew Mexico Press Albuquerque NM 244 p
Dreesen DR 2001 Propagation protocol for production ofcontainer Robinia neomexicana plants Los Lunas Plant Materials Center Los Lunas New Mexico In Native Plant Network URL httpwwwnativeplantnetworkorg (accessed 9 January 2002) Moscow (ID) University ofIdaho CoIl Natural Resources For Res Nursery
72
Dreesen DR Harrington JT 1997 Propagation of native plants for restoration projects in the Southwestern US- Preliminary Investigations In Landis TD Thompson JR tech coords National Proceedings Forest and Conservation Nursery Assocs Gen Tech Rep PNW-GTR-419 Portland OR USDA For Ser PNW Res Sta 77-88
Gottfried GJ 1980 Control ofNew Mexican locust and the effect on planted ponderosa pine in central Arizona US For Ser Res Note RM-386 Rocky Mountain For Range ExpStn Fort Collins CO
Gosling PG Samuel YK Jones SK 1995 A systematic examination of germination temperature chipping and water temperaturesoak duration pretreatments on the seeds ofLeucaena leucocephala Seed Sci amp Technol 23521-532
Hamly DH 1932 Softening of the seeds of Melilotus alba Bot Gazette 93 345shy375
Hanna PJ 1984 Anatomical features of the seed coat ofAcacia kempeana (Mueller) which relate to increased germination rate induced by heat treatment New Phytol 96 23-29
Hanks JP Dick-Peddie WA 1974 Vegetation patterns of the White Mountains New Mexico Southwest Naturalist 18(4) 371-382
Hine S Harrington JT Dreesen DR 1997 Seed scarification requirements for Robinia neomexicana In Harrington JTtech coord Proc Third SouthwestemContainer Growers Conf Feb 12-13 1997 Ft Collins CO
Hopkinson JM 1993 The strophiole of leguminous seeds Newsletter- IntI Herbage Seed Production Res Grp 18 7-8
---- 1997 Differences in hardseededness between Cunningham and Tarramba leucaena In Leucnet News The Newsletter of the International LeucaenaRes and Devlt Netwk No4
ISTA (International Seed Testing Association) 1999 International rules for seed testing rules Seed Sci amp Technol27 Supplement
Kelly KM van Staden J 1987 The lens as the site of permeability in the papilionoid seed Aspalathus linearis J Plant Physiol 128(45) 395-404
73
Keresztesi B 1988 Black locust the tree of agriculture Outlook on Agr 17(2) 77~85
Kigel 1 and Galili G 1995 Seed development and germination Marcel Dekker New York 853 p
Klemmedson 10 1994 New Mexico locust and parent material Influence on macronutrients of forest floor and soil Soil Sci Soc Am 1 58 974~980
Kuo WH Tam AW 1988 The pathway of water absorption of mung bean seeds Seed Sci amp Technol16(1) 139~144
Leadem CL 1997 Dormancy--unlocking seed secrets In Landis TD Thompson lR tech coords National Proceedings Forestand Conservation Nursery Assocs Gen Tech Rep PNW-G TR-419 Portland OR USDA For Ser PNW Res Sta 43-52
Lin Y Wagner MR Seweyestewa A Honahni MD DeGomez M McMillin JD 1996 Influence of cultural treatments on seed germination and seedling growth of Robinia neomexicana Forest Farm and Community Tree Res Reports Volume 1
Marunda CT 1990 Effects of seed pretreatment on the development ofAcacia auriculforinis and A holosericea seedlings ACIAR Proceedings Series No 28
Meginnis HG 1937 Sulphuric acid treatment to increase germination of black locust seed USDA Circ 453 34 p
Miklas PN Towensend CE Ladd SL 1987 Seedcoat anatomy and the scarification ofcicer milkvetch seed Crop Sci 27 766-772
Monsen SB 1984 Use of shrubs on mine spoils In The Challenge ofProducing Native Plants for the Intermountain Area Proceedings Intermountain Nurserymans Association 1983 conference Las Vegas NV compo PM Murphy 26-31 Ogden UT USDA For Ser Gen Tech Report INT-l68
Olson DF 1974 Robinia L Locust In Seeds of Woody Plants in the US tech coord CS Schopmeyer 728-731 USDA For Ser Agr Handbk No450
Rehman S Loescher RNl Harris P1C 1998 Dormancy breaking and germination ofAcacia salicina Lindl seeds Seed Sci amp Techno127 553-557
Rolston MP 1978 Water impermeable seed dormancy Bot Rev 44 365-396
74
SAS Institute Inc 1989 SASSTAT Users Guide Version 6 4th Edition Volume 1 Cary NC SAS Institute Inc 943 p
SerratoValenti G Conara L Ferrando M Modenesi P 1993 Structural and histochemical features ofStylosanthes scabra (Leguminosae Papilionoidae) seed coat as related to water entry Can J Bot 71 834-840
Simpson BJ 1988 A field guide to Texas trees Austin TX Texas Monthly Press 372p
Tran VN Cavanagh AK 1984 Structural aspects ofdormancy In Seed Physiology Vol 2 Murray DR (ed) p1-44 Academic Press Australia
USDA Forest Service 1948 Woody-plant seed manual USDA Misc Pub1654 416p
Vines RA 1960 Trees Shrubs and Woody Vines of the Southwest Austin Univ ofTexas Press 1104 p
Wagner MR Larson RE Craig TP 1992 Ecology and silvics ofNew Mexico locust (Robinia neomexicana) with an emphasis on the arboreal form p53-57 In JW Hanover K Miller and S Plesko (eds) Proc Intemat Confon Black Locust Biology Culture and Utilization Mich State Univ USA
Wilson JK 1944 Immersing seeds of species of Robinia in boiling water hastens germination J For 42 453-454
Zimmerman RW Carpenter SB 1980 First-year coppice production from a 5shyyear old black locust stand on surface mine spoil p309-314 Centr Hardwd For ConfProc University ofMissouri Columbia
against other treatments such as sulfuric acid and mechanical scarification also
merits further study
Papilionoids are the largest subfamily oflegumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust these legumes often play an integral role in the revegetation of
disturbed lands The pilot study (Appendix) indicates that percussion scarification
successfully treats the widely used black locust as well Developing superior
scarification methods to the standard hot water mechanical scarification and acid
treatments may facilitate the use ofpapilionoid legumes in reclamation projects
45
APPENDICES
APPENDIX A
PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR
NEW MEXICO LOCUST AND BLACK LOCUST SEEDS
Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4
Hot water and sulfuric acid soaks are traditional treatments for seeds of many
temperate woody legwnes including locusts However these scarification techniques
often produce inconsistent germination Percussion scarification where seeds are
repeatedly propelled against a hard surface was compared with hot water
scarification to evaluate treatment efficacy for New Mexico locust (Robinia
neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment
seeds were placed in a 98degC water bath which was immediately removed from the
heat source For percussion scarification seeds were placed in a soil sample tin and
agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the
control were followed by 24-hour water soaks Hot water baths resulted in 56 and
41 germination for New Mexico locust and black locust respectively For both
species nearly all durations ofpercussion increased germination over the hot water
treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3
4 and 5 minutes for black locust resulted in at least 90 germination Traditional
scarification treatments randomly degrade the entire seed coat which can lead to
tissue damage during water uptake Percussion scarification specifically weakens the
strophiole the natural source ofwater entry to the seed in papilionoid legwnes
INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom
2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031
48
Following percussion imbibition is controlled through the strophiole and underlying
tissue is protected
Keywords New Mexico locust black locust scarification percussion germination
Introduction
Locust trees (Robinia spp) are aggressive pioneer species that quickly
colonize disturbed land fix atmospheric nitrogen in the soil and stabilize slopes that
are prone to erosion (Klemmedson 1994 Ashby et al 1985) (Figure A1ab)
Recognition of these attributes has contributed to increased demand for locusts in
reclamation projects Locust seeds however exhibit physical dormancy where a
thick seed coat prevents the movement of water and gasses to the embryo (Leadem
1997)
Sulfuric acid or hot water soaks have traditionally been used to break seed
dormancy in macro-propagation of locusts Many growers are moving away from the
use of su1furic acid in treating locust seeds (Dreesen and Harrington 1997) In
addition to being dangerous acid soak durations must be specifically correlated to
seed lot (Olson 1974) Collections of native seed can vary tremendously in seed size
seed weight and hard seededness etc This variability limits the utility of sulfuric
acid soaks as even within a given lot some seeds may be damaged whereas others
are under-treated and fail to imbibe (take up water) A survey of Southwestern
container growers indicates that hot water baths are currently the most common
scarification method for locust seeds (Hine et al 1997) However hot water baths
also produce inconsistent germination (Lin et al 1996)
49
b
Figure AI New Mexico locust a primary invader following disturbance Ala Crown-sprouting at edge ofburn near Los Alamos NM--one year after Cerro Grande fire Alb Colonizing road cut Gila National Forest NM
50
Percussion scarification where seeds are repeatedly propelled against a hard
surface is an alternative dormancy-breaking method that has proven successful in
legumes related to locusts (Hamly 1932 Barton 1947) We compared this alternative
treatment to hot water scarification to evaluate treatment efficacy for New Mexico
locust and black locust seeds
Materials and Methods
Commercial New Mexico locust seeds (Western Native Seed Coaldale CO)
were collected fall 2000 in Huerfano County CO Black locust seeds were collected
September 2000 from Taos County NM by harvesting ripe pods from several trees at
a distance of up to 3 m from ground level Black locust seeds were allowed to air-dry
for 3 weeks and were threshed and separated from large chaffusing a greenhouse
fan A South Dakota Seed Blower (Chicago Illinltois) was used to remove fine chaff
from both seed lots Seeds were storedmiddotat 2-4C until the germination study was
conducted in March 2001
This study compared hot water scarification with percussion scarification
using treatment and species as experimental factors Along with a control
scarification treatments included immersion of seed in boiling water as well as
percussion durations of 1 2 3 45 or 10 minutes Four 100-seed samples were used
to test each species by scarification treatment combination
For the hot water treatment test tubes were filled with 30 mL water and
placed in a water-filled beaker (Figure A2a) The bath was raised to boiling (-98C at
51
b
c
Figure A2 Implementation ofhot water and percussion scarification A2a Hot water bath A2b Paint-shaker set-up A2c Placing soil tin containing seeds at end ofpaint shaker ann
52
Las Cruces NM elevation of 1300 meters) Each 100-seed replicate was placed in a
test tube and each test tube was immediately removed to cool at room temperature
Percussion scarification was implemented using a pneumatic paint shaker
(Central Pneumatic model 00422 Camarillo CA) (Figure A2b) This allowed
standardization of treatment intensity Compressed air pressure was maintained at
530kPa plusmn 30kPa (80psi plusmn 5psi) resulting in approximately 350 oscillations per
minute Each lOO-seed sample was placed in a 118 ml (4 oz) soil tin for shaking To
maximize lateral movement of paint shaker a paint can was used as a spacer to place
soil tins at the end of the shaking arm (Figure A2c) This allowed shaking to occur at
the greatest distance from the pivot point (fulcrum) Following all treatments
including the control seeds were soaked for twenty-four hours in distilled water prior
to plating out
Gennination was evaluated on lab benches at room temperature Each
replicate of seeds was placed in a 100 cm petri dish on moistened filter paper with
humidity maintained by enclosing the petri dishes in ziplock bags Gennination was
monitored daily for 14 days in accordance with International Seed Testing
Association standards for Robinia species (ISTA 1999) Gennination was defined as
protrusion of the radicle from the embryo by at least 1mm as viewed by the naked
eye
Categorical Analysis of Variance using Prpc Catmod (SAS Institute 1989)
was used to detennine treatment differences in germination percentages for each
source This procedure is an extension of a Chi-square test of homogeneity using the
53
natural log of the ratio of germinated to non-germinated seeds for each treatment Pshy
values less than 005 were considered significant A limited set of pairwiseshy
comparisons was conducted to compare treatment mean percents using a
conservative alpha value of 005 divided by the number of comparisons
Results
Percussion maximized germination for both New Mexico locust and black
locust There were three germination response levels for New Mexico locust (Figure
A3) First there was a low response in the control indicating a dormant seed lot
The hot water treatment and I-minute duration of percussion share a second
intermediate response Germination was highest at percussion levels from 2 to 10
minutes
A selected percussion treatment intermediate in time (5 minutes) produced the
steepest germination curve indicating the fastest germination rate (Figure A4) The
hot water treatment produced an intermediate curve or relatively slower germination
rate The control curve was very flat indicating the slowest germination rate for New
Mexico locust
Control seeds germinated poorly for black locust as well (Figure A5) Hot
water scarification improved germination over the control but all levels of percussion
improved germination over hot water The optimal durations of percussion treatment
were bracketed with a significant increase from the 1 to 2 minute percussion level a
high germination response from 2 to 5 minutes of percussion and finally a significant
54
~ d 25 CL)
p t1 0 ~ t1
g Vl ~ Vl
100~---------------------------------------------------------------
80
60
40
20
o Control Hot Water Percussion
I min Percussion
2 min Percussion
3 min
Scarification
Percussion 4 min
Figure A3 Effect of scarification on germination response for New Mexico locust
Percussion 5 min
=significant difference between adjacent treatment bars ns = no significant difference
Percussion 10 min
~ ~ () u agt
Il-t d o ~
~ ~ c3
100middotlr~~~------------------------------------e- Percussion 5min middot 0middot Hot Water -- Control
80-J=middot======
6b-middot-lmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middot--middotmiddot-middot~-middotmiddot -middotmiddot middot middotmiddot-middot 1
40
20
-----~-----------
0 0 0 middotmiddotmiddotmiddotmiddot0middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot0
0 --~-----y-----~------_---~--- T-----~
o shy -TDay 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A4 Effect of selected scarification treatments on germination speed for New Mexico locust First seven days of fourteen-day experiment are shown
Day 7
80
~ cd
5 60 ()
[) Cl d 0 1t1 d
40sect dUl
-J
20
o
ns
Scarification
Figure A5 Effect of scarification on germination response for black locust = significant difference between adjacent treatment bars ns = no significant difference
decrease at the 10-minute level This drop-off suggests that 10 minutes of percussion
over-treats seeds from this lot of-black locust
A selected percussion treatment intermediate in time (5 minutes) also had the
fastest germination rate for black locust (Figure A6)_ The hot water curve is
somewhat flatter indicating a slower germination rate The control curve is very flat
indicating a slow germination rate
A qualitative difference between hot water and percussion-treated seeds was
also observed Along with healthy germinants the hot water treatment produced
over-treated seeds (Figure A 7a) Examples of damaged germinants include seeds
where the radicle did not elongate (Figure A 7b) seeds where the radicle elongated
but was delayed in freeing itself from the seed coat (Figure A7c) and seeds where the
radicle broke off completely from the embryo (Figure A7d) In addition to these
examples of over-treatment there were numerous small dark seeds that failed to
imbibe water For percussion treatment nearly all seeds took up water (Figure A8a)
Germination was rapid and uniform with little damage evident (Figure A8b)
To summarize both New Mexico locust and black locust seed lots were
dormant The standard hot water scarification treatment improved germination but
only to 56 and 41 for New Mexico locust and black locust respectively Nearly
all percussion durations improved total germination as well as germination rate over
the hot water treatment Germination was greater than 90 for percussion durations
of 4 5 and 10 minutes for New Mexico locust and 3 4 and 5 minutes for black locust
58
CI)
sgt t CI) 0
igt 0
sect ttl t E
Vl 0
c3
100r==~==~~=r------------------------------------~ ___ Percussion 5 min
middotmiddotmiddot0middotmiddotmiddot Hot Water -lIf- Control
80r=========~--~~----------------------------~
60
40
20 -~-~---~---------- ---~------------I
omiddot
O
_~_----------lIf-----~bull -----_ shy---shy I
Day 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A6 Effect of selected scarification treatments on germination speed for black locust First seven days of fourteen-day experiment are shown
Day 7
a
b c d
Figure A7 Damage to New Mexico locust seeds observed during germination counts following hot water scarification A7a Hot water replicate including damaged seedlings and numerous unimbibed seeds A7b Close-up ofseed where radicle failed to elongate A7c Close-up ofseed where radicle elongated but was delayed in freeing itself from the seed coat A7d Close-up ofseed where radicle completely broke offfrom embryo
60
a
b
Figure A8 New Mexico locust seeds following percussion treatment at time of germination counts A8a Percussion replicate with uniformly healthy seedlings and near complete imbibition of seeds A8b Close-up ofpercussion-treated seedling
61
Discussion
Papilionoid seeds have a specific anatomical feature known as the strophiole
or lens-the natural site of water entry to the seed (Hamly 1932) The strophiole is
located on the cotyledonary lobe of a locust seed (Figure A9) When a papilionoid
legume seed is percussed for the appropriate amount of time repeated hits on the
integrated seed coat loosen the constrained cells of the strophiolar region without
excessively damaging the rest of the seed coat (Ballard 1976)
When a percussed seed is soaked water enters exclusively through the
strophiole in a controlled manner (Kelly and Staden 1987) This regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues This
contrasts with seeds that have been hot water or acid scarified treatments that can
randomly degrade the seed coat Unlocalized cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rehman et al 1998 Marunda 1990)
Papilionoids are the largest subfamily of legumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust and black locust these legumes often play an integral role in the
revegetation ofdisturbed lands Developing superior scarification methods to the
standard hot water and acid treatments should fadlitate the use ofthese legumes in
restoration projects
62
Figure A9 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (bc)
63
APPENDIXB
GERMINATION PERCENTS AND STANDARD ERRORS FOR SOURCE
BLOCK AND TREATMENT FOR NEW MEXICO LOCUST
Table B 1 Gennination percents and standard errors for source block and treatment for New Mexico locust
Source Block Treatment Germination Percentage Standard Error
Capitan 1 Immersion 74 437 Capitan 2 Immersion 69 462 Capitan 3 Immersion 71 454 Capitan 4 Immersion 67 470 Capitan Boiling 30 sec 79 407 Capitan 2 Boiling 30 sec 76 427 Capitan 3 Boiling 30 sec 72 449 Capitan 4 Boiling 30 sec 76 427 Capitan Boiling 60 sec 71 454 Capitan 2 Boiling 60 sec 54 498 Capitan 3 Boiling 60 sec 67 470 Capitan 4 Boiling 60 sec 76 427 Capitan I Control 5 218 Capitan 2 Control 7 255 Capitan 3 Control 4 196 Capitan 4 Control 5 218 Capitan 1 Percussion I min 47 499 Capitan 2 Percussion I min
470
Capitan Percussion I min 39 488
Capitan 4 Percussion I min 38 485 Capitan I Percussion 2 min 55 497 Capitan Capitan
2
Percussion 2 min Percussion 2 min
71 65
454 477
Capitan 4 Percussion 2 min 56 496 Capitan I Percussion 4 min 86 347 Capitan 2 Percussion 4 min 79 407 Capitan 3 Percussion 4 min 81 392 Capitan 4 Percussion 4 min 82 384 Capitan I Percussion 8 min 90 300 Capitan 2 Percussion 8 min 88 325 Capitan 3 Percussion 8 min 90 300 Capitan 4 Percussion 8 min 90 300 Gila I Immersion 71 454 Gila 2 Immersion 77 421 Gila
Immersion 75 433
Gila 4 Immersion 72 449
Gila I Boiling 30 sec 68 466
Gila 2 Boiling 30 sec 81 392
Gila 3 Boiling 30 sec 54 498
Gila 4 Boiling 30 sec 63 483
Gila I Boiling 60 sec 55 497
Gila 2 Boiling 60 sec 68 466
Gila 3 Boiling 60 sec 57 495
Gila 4 Boiling 60 sec 70 458
65
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Gila 2 Control 13 336 Gila 3 Control 13 336 Gila 4 Control 20 400 Gila 1 Percussion 1 min 66 474 Gila 2 Percussion 1 min 57 495 Gila 3 Percussion 1 min 73 444 Gila 4 Percussion 1 min 82 384 Gila 1 Percussion 2 min 81 392 Gila 2 Percussion 2 min 86 347 Gila 3 Percussion 2 min 85 357 Gila 4 Percussion 2 min 54 498 Gila 1 Percussion 4 min 79 407 Gila 2 Percussion 4 min 83 376 Gila 3 Percussion 4 min 84 367 Gila 4 Percussion 4 min 79 407 Gila 1 Percussion 8 min 51 500 Gila 2 Percussion 8 min 76 427 Gila 3 Percussion 8 min 52 500 Gila 4 Percussion 8 min 74 439 Holman 1 Immersion 83 376 Holman 2 Immersion 86 347 Holman 3 Immersion 81 392 Holman 4 Immersion 90 300 Holman 1 Boiling 30 sec 90 300 Holman 2 Boiling 30 sec 85 357 Holman 3 Boiling 30 sec 87 336 Holman 4 Boiling 30 sec 83 376 Holman 1 Boiling 60 sec 79 407 Holman 2 Boiling 60 sec 80 400 Holman 3 Boiling 60 sec 86 347 Holman 4 Boiling 60 sec 93 255 Holman Control 25 433 Holman 2 Control 19 392 Holman Control 21 407
Holman 4 Control 24 427
Holman Percussion 1 min 77 421
Holman 2 Percussion 1 min 85 357
Holman 3 Percussion 1 min 80 400
Holman 4 Percussion 1 min 89 313
Holman 1 Percussion 2 min 88 325
Holman 2 Percussion 2 min 96 196
Holman 3 Percussion 2 min 93 255
Holman 4 Percussion 2 min 96 196
Holman 1 Percussion 4 min 92 271
Holman 2 Percussion 4 min 97 171
66
I
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Holman 3 Percussion 4 min 91 286 Holman 4 Percussion 4 min 96 196 Holman 1 Percussion 8 min 88 325 Holman 2 Percussion 8 min 85 357 Holman 3 Percussion 8 min 83 376 Holman 4 Percussion 8 min 82 384 Jemez 1 Immersion 53 499 Jemez 2 Immersion 44 496 Jemez 3 Immersion 52 500 Jemez 4 Immersion 70 458 Jemez 1 Boiling 30 sec 58 494 Jemez 2 Boiling 30 sec 55 497 Jemez 3 Boiling 30 sec 48 500 Jemez 4 Boiling 30 sec 65 477 Jemez 1 Boiling 60 sec 68 466 Jemez 2 Boiling 60 sec 63 483 Jemez 3 Boiling 60 sec 73 444 Jemez 4 Boiling 60 sec 89 313 Jemez 1 Control 2 140 Jemez 2 Control 9 286 Jemez 3 Control 6 237 Jemez 4 Control 8 271 Jemez 1 Percussion 1 min 58 494 Jemez 2 Percussion 1 min 63 483 Jemez 3 Percussion 1 min 77 421 Jemez 4 Percussion 1 min 89 313 Jemez 1 Percussion 2 min 68 466 Jemez 2 Percussion 2 min 64 480 Jemez 3 Percussion 2 min 80 400 Jemez 4 Percussion 2 min 83 376 Jemez Percussion 4 min 68 466 Jemez 2 Percussion 4 min 79 407 Jemez 3 Percussion 4 min 70 458 Jemez 4 Percussion 4 min 83 376 Jemez 1 Percussion 8 min 52 500 Jemez 2 Percussion 8 min 59 492 Jemez 3 Percussion 8 min 61 488 Jemez 4 Percussion 8 min 70 458 Magdalena 1 Immersion 69 462 Magdalena 2 Immersion 69 462 Magdalena J Immersion 70 458 Magdalena 4 Immersion 85 357 Magdalena 1 Boiling 30 sec 65 477 Magdalena 2 Boiling 30 sec 71 454 Magdalena 3 Boiling 30 sec 68 466
67
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Magdalena 4 Boiling 30 sec 83 376 Magdalena 1 Boiling 60 sec 63 483 Magdalena 2 Boiling 60 sec 68 466 Magdalena 3 Boiling 60 sec 63 483 Magdalena 4 Boiling 60 sec 82 384 Magdalena 1 Control 11 313 Magdalena 2 Control 6 237 Magdalena 3 Control 5 218 Magdalena 4 Control 4 196 Magdalena Percussion 1 min 32 466 Magdalena 2 Percussion 1 min 45 497 Magdalena 3 Percussion 1 min 46 498 Magdalena 4 Percussion I min 36 480 Magdalena 1 Percussion 2 min 67 470 Magdalena 2 Percussion 2 min 68 466 Magdalena 3 Percussion 2 min 65 477 Magdalena 4 Percussion 2 min 81 392 Magdalena 1 Percussion 4 min 87 336 Magdalena 2 Percussion 4 min 97 171 Magdalena 3 Percussion 4 min 83 376 Magdalena 4 Percussion 4 min 97 171 Magdalena 1 Percussion 8 min 98 lAO Magdalena 2 Percussion 8 min 100 000 Magdalena 3 Percussion 8 min 96 196 Magdalena 4 Percussion 8 min 98 lAO Manzano 1 Immersion 67 470 Manzano 2 Immersion 57 495 Manzano 3 Immersion 72 4049 Manzano 4 Immersion 73 4044 Manzano 1 Boiling 30 sec 70 458 Manzano 2 Boiling 30 sec 64 480 Manzano 3 Boiling 30 sec 71 454 Manzano 4 Boiling 30 sec 80 400 Manzano I Boiling 60 sec 50 500 Manzano 2 Boiling 60 sec 53 499 Manzano 3 Boiling 60 sec 59 492 Manzano 4 Boiling 60 sec 74 439 Manzano I Control 4 196 Manzano 2 Control 8 271 Manzano 3 Control 1 099 Manzano 4 Control 6 237 Manzano 1 Percussion 1 min 52 500 Manzano 2 Percussion 1 min 39 488 Manzano 3 Percussion 1 min 36 480 Manzano 4 Percussion 1 min 51 500
68
Table B 1 Continued
Source Block Treatment Gerrni~ation Percentage Standard Error
Manzano Percussion 2 min 53 499 Manzano 2 Percussion 2 min 58 494 Manzano 3 Percussion 2 min 61 488 Manzano 4 Percussion 2 min 62 485 Manzano 1 Percussion 4 min 76 427 Manzano 2 Percussion 4 min 85 357 Manzano 3 Percussion 4 min 79 407 Manzano 4 Percussion 4 min 83 376 Manzano 1 Percussion 8 min 87 336 Manzano 2 Percussion 8 min 83 376 Manzano 3 Percussion 8 min 83 376 Manzano 4 Percussion 8 min 75 433 Raton 1 Immersion 54 498 Raton 2 Immersion 37 483 Raton 3 Immersion 39 488 Raton 4 Immersion 69 462 Raton 1 Boiling 30 sec 36 480 Raton 2 Boiling 30 sec 20 400 Raton 3 Boiling 30 sec 28 449 Raton 4 Boiling 30 sec 60 490 Raton 1 Boiling 60 sec 15 357 Raton 2 Boiling 60 sec 171 Raton 3 Boiling 60 sec 4 196 Raton 4 Boiling 60 sec 35 477 Raton 1 Control 4 196 Raton 2 Control 12 325 Raton 3 Control 2 140 Raton 4 Control 6 237 Raton 1 Percussion 1 min 52 500 Raton 2 Percussion I min 29 454 Raton 3 Percussion I min 35 477 Raton 4 Percussion 1 min 31 462 Raton 1 Percussion 2 min 55 497 Raton 2 Percussion 2 min 35 477 Raton 3 Percussion 2 min 33 470 Raton 4 Percussion 2 min 42 494 Raton 1 Percussion 4 min 65 477 Raton 2 Percussion 4 min 52 500 Raton Percussion 4 min 51 500 Raton 4 Percussion 4 min 53 499 Raton 1 Percussion 8 min 88 325 Raton 2 Percussion 8 min 80 400 Raton 3 Percussion 8 min 73 444 Raton 4 Percussion 8 min 74 439 Sacramento 1 Immersion 43 495
69
Table B 1 Continued
-Source Block Treatment Germination Percentage Standard Error
Sacramento 2 Immersion 30 458 Sacramento 3 Immersion 38 485 Sacramento 4 Immersion 56 496 Sacramento 1 Boiling 30 sec 47 499 Sacramento 2 Boiling 30 sec 48 500 Sacramento 3 Boiling 30 sec 48 500 Sacramento 4 Boiling 30 sec 56 496 Sacramento 1 Boiling 60 sec 52 500 Sacramento 2 Boiling 60 sec 67 470 Sacramento 3 Boiling 60 sec 43 495 Sacramento 4 Boiling 60 sec 48 500 Sacramento 1 Control 0 000 Sacramento 2 Control 3 171 Sacramento 3 Control 3 171 Sacramento 4 Control 099 Sacramento 1 Percussion 1 min 57 495 Sacramento 2 Percussion 1 min 61 488 Sacramento 3 Percussion 1 min 62 485 Sacramento 4 Percussion 1 min 55 497 Sacramento 1 Percussion 2 min 84 367 Sacramento 2 Percussion 2 min 70 458 Sacramento 3 Percussion 2 min 81 392 Sacramento 4 Percussion 2 min 69 462 Sacramento 1 Percussion 4 min 91 286 Sacramento 2 Percussion 4 min 84 367 Sacramento 3 Percussion 4 min 84 367 Sacramento 4 Percussion 4 min 94 237 Sacramento 1 Percussion 8 min 82 384 Sacramento 2 Percussion 8 min 81 392 Sacramento 3 Percussion 8 min 82 384 Sacramento 4 Percussion 8 min 81 392 Sandia 1 Immersion 76 427 Sandia 2 Immersion 70 458 Sandia 3 Immersion 70 458 Sandia 4 Immersion 81 392 Sandia 1 Boiling 30 sec 78 414 Sandia 2 Boiling 30 sec 71 454 Sandia 3 Boiling 30 sec 63 483 Sandia 4 Boiling 30 sec 78 414 Sandia 1 Boiling 60 sec 67 470 Sandia 2 Boiling 60 sec 65 477 Sandia 3 Boiling 60 sec 74 439 Sandia 4 Boiling 60 sec 66 474 Sandia 1 Control 16 367 Sandia 2 Control 7 255
70
Table B 1 Continued
Source Block Treatment Gennination Percentage Standard Error
Sandia 3 Control 8 271 Sandia 4 Control 16 367 Sandia 1 Percussion 1 min 67 470 Sandia 2 Percussion 1 min 61 488 Sandia 3 Percussion 1 min 66 474 Sandia 4 Percussion 1 min 59 492 Sandia I Percussion 2 min 82 384 Sandia 2 Percussion 2 min 83 376 Sandia 3 Percussion 2 min 81 392 Sandia 4 Percussion 2 min 84 367 Sandia 1 Percussion 4 min 89 313 Sandia 2 Percussion 4 min 86 347 Sandia 3 Percussion 4 min 89 313 Sandia 4 Percussion 4 min 85 357 Sandia 1 Percussion 8 min 80 400 Sandia 2 Percussion 8 min 77 421 Sandia 3 Percussion 8 min 69 462 Sandia 4 Percussion 8 min 66 474
LITERATURE CITED
Ashby WC Vogel WG Rogers NF 1985 Black locust in the reclamation equation General Technical Report Northeastern Forest Experiment Station USDA Forest Service NE-I05 12 p
Ballard LAT 1973 Physical barriers to gennination Seed Sci Technoll 285-303
___ 1976 Strophiolar water conduction in the seeds ofTrifolieae induced by action on the testa at nonstrophiolar sites Austr J Plant Physiol3 465-469
Barton LV 1947 Special studies on seed coat impenneability Contrib Boyce Thompson Inst 14 355-362
Baskin CC Baskin JM 1998 Seeds Ecology biogeography and evolution of donnancy and gennination Academic Press London UK 666 p
Bewley JD 1997 Seed germination and donnancy Plant Ce119 1055-1066
Bewley JD Black M 1994 Seeds physiology of development and germination 2nd ed Plenum Press New York NY 445 p
Briscoe CB 1996 Growing Nitrogen Fixing Trees in Acid Soils In MH Powell (ed) Nitrogen Fixing Trees for Acid Soils- A Field ManuaL Winrock International Morrilton AR 22 p
Copeland LO McDonald MB 1995 Principles of Seed Science and Technology 3rded Chapman amp Hall New York NY 409 p
Cox RE Klett JE 1984 Seed gennination requirements of native Colorado plants for use in the landscape Plant Propag 30(2) 6-10
Dell B 1980 Structure and function ofthe strophiolar plug in seeds ofAlbizia Lophantha Am J Bot 67 556-563
Dick-Peddie WA 1993 New Mexico Vegetation Past Present and Future University ofNew Mexico Press Albuquerque NM 244 p
Dreesen DR 2001 Propagation protocol for production ofcontainer Robinia neomexicana plants Los Lunas Plant Materials Center Los Lunas New Mexico In Native Plant Network URL httpwwwnativeplantnetworkorg (accessed 9 January 2002) Moscow (ID) University ofIdaho CoIl Natural Resources For Res Nursery
72
Dreesen DR Harrington JT 1997 Propagation of native plants for restoration projects in the Southwestern US- Preliminary Investigations In Landis TD Thompson JR tech coords National Proceedings Forest and Conservation Nursery Assocs Gen Tech Rep PNW-GTR-419 Portland OR USDA For Ser PNW Res Sta 77-88
Gottfried GJ 1980 Control ofNew Mexican locust and the effect on planted ponderosa pine in central Arizona US For Ser Res Note RM-386 Rocky Mountain For Range ExpStn Fort Collins CO
Gosling PG Samuel YK Jones SK 1995 A systematic examination of germination temperature chipping and water temperaturesoak duration pretreatments on the seeds ofLeucaena leucocephala Seed Sci amp Technol 23521-532
Hamly DH 1932 Softening of the seeds of Melilotus alba Bot Gazette 93 345shy375
Hanna PJ 1984 Anatomical features of the seed coat ofAcacia kempeana (Mueller) which relate to increased germination rate induced by heat treatment New Phytol 96 23-29
Hanks JP Dick-Peddie WA 1974 Vegetation patterns of the White Mountains New Mexico Southwest Naturalist 18(4) 371-382
Hine S Harrington JT Dreesen DR 1997 Seed scarification requirements for Robinia neomexicana In Harrington JTtech coord Proc Third SouthwestemContainer Growers Conf Feb 12-13 1997 Ft Collins CO
Hopkinson JM 1993 The strophiole of leguminous seeds Newsletter- IntI Herbage Seed Production Res Grp 18 7-8
---- 1997 Differences in hardseededness between Cunningham and Tarramba leucaena In Leucnet News The Newsletter of the International LeucaenaRes and Devlt Netwk No4
ISTA (International Seed Testing Association) 1999 International rules for seed testing rules Seed Sci amp Technol27 Supplement
Kelly KM van Staden J 1987 The lens as the site of permeability in the papilionoid seed Aspalathus linearis J Plant Physiol 128(45) 395-404
73
Keresztesi B 1988 Black locust the tree of agriculture Outlook on Agr 17(2) 77~85
Kigel 1 and Galili G 1995 Seed development and germination Marcel Dekker New York 853 p
Klemmedson 10 1994 New Mexico locust and parent material Influence on macronutrients of forest floor and soil Soil Sci Soc Am 1 58 974~980
Kuo WH Tam AW 1988 The pathway of water absorption of mung bean seeds Seed Sci amp Technol16(1) 139~144
Leadem CL 1997 Dormancy--unlocking seed secrets In Landis TD Thompson lR tech coords National Proceedings Forestand Conservation Nursery Assocs Gen Tech Rep PNW-G TR-419 Portland OR USDA For Ser PNW Res Sta 43-52
Lin Y Wagner MR Seweyestewa A Honahni MD DeGomez M McMillin JD 1996 Influence of cultural treatments on seed germination and seedling growth of Robinia neomexicana Forest Farm and Community Tree Res Reports Volume 1
Marunda CT 1990 Effects of seed pretreatment on the development ofAcacia auriculforinis and A holosericea seedlings ACIAR Proceedings Series No 28
Meginnis HG 1937 Sulphuric acid treatment to increase germination of black locust seed USDA Circ 453 34 p
Miklas PN Towensend CE Ladd SL 1987 Seedcoat anatomy and the scarification ofcicer milkvetch seed Crop Sci 27 766-772
Monsen SB 1984 Use of shrubs on mine spoils In The Challenge ofProducing Native Plants for the Intermountain Area Proceedings Intermountain Nurserymans Association 1983 conference Las Vegas NV compo PM Murphy 26-31 Ogden UT USDA For Ser Gen Tech Report INT-l68
Olson DF 1974 Robinia L Locust In Seeds of Woody Plants in the US tech coord CS Schopmeyer 728-731 USDA For Ser Agr Handbk No450
Rehman S Loescher RNl Harris P1C 1998 Dormancy breaking and germination ofAcacia salicina Lindl seeds Seed Sci amp Techno127 553-557
Rolston MP 1978 Water impermeable seed dormancy Bot Rev 44 365-396
74
SAS Institute Inc 1989 SASSTAT Users Guide Version 6 4th Edition Volume 1 Cary NC SAS Institute Inc 943 p
SerratoValenti G Conara L Ferrando M Modenesi P 1993 Structural and histochemical features ofStylosanthes scabra (Leguminosae Papilionoidae) seed coat as related to water entry Can J Bot 71 834-840
Simpson BJ 1988 A field guide to Texas trees Austin TX Texas Monthly Press 372p
Tran VN Cavanagh AK 1984 Structural aspects ofdormancy In Seed Physiology Vol 2 Murray DR (ed) p1-44 Academic Press Australia
USDA Forest Service 1948 Woody-plant seed manual USDA Misc Pub1654 416p
Vines RA 1960 Trees Shrubs and Woody Vines of the Southwest Austin Univ ofTexas Press 1104 p
Wagner MR Larson RE Craig TP 1992 Ecology and silvics ofNew Mexico locust (Robinia neomexicana) with an emphasis on the arboreal form p53-57 In JW Hanover K Miller and S Plesko (eds) Proc Intemat Confon Black Locust Biology Culture and Utilization Mich State Univ USA
Wilson JK 1944 Immersing seeds of species of Robinia in boiling water hastens germination J For 42 453-454
Zimmerman RW Carpenter SB 1980 First-year coppice production from a 5shyyear old black locust stand on surface mine spoil p309-314 Centr Hardwd For ConfProc University ofMissouri Columbia
APPENDICES
APPENDIX A
PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR
NEW MEXICO LOCUST AND BLACK LOCUST SEEDS
Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4
Hot water and sulfuric acid soaks are traditional treatments for seeds of many
temperate woody legwnes including locusts However these scarification techniques
often produce inconsistent germination Percussion scarification where seeds are
repeatedly propelled against a hard surface was compared with hot water
scarification to evaluate treatment efficacy for New Mexico locust (Robinia
neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment
seeds were placed in a 98degC water bath which was immediately removed from the
heat source For percussion scarification seeds were placed in a soil sample tin and
agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the
control were followed by 24-hour water soaks Hot water baths resulted in 56 and
41 germination for New Mexico locust and black locust respectively For both
species nearly all durations ofpercussion increased germination over the hot water
treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3
4 and 5 minutes for black locust resulted in at least 90 germination Traditional
scarification treatments randomly degrade the entire seed coat which can lead to
tissue damage during water uptake Percussion scarification specifically weakens the
strophiole the natural source ofwater entry to the seed in papilionoid legwnes
INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom
2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031
48
Following percussion imbibition is controlled through the strophiole and underlying
tissue is protected
Keywords New Mexico locust black locust scarification percussion germination
Introduction
Locust trees (Robinia spp) are aggressive pioneer species that quickly
colonize disturbed land fix atmospheric nitrogen in the soil and stabilize slopes that
are prone to erosion (Klemmedson 1994 Ashby et al 1985) (Figure A1ab)
Recognition of these attributes has contributed to increased demand for locusts in
reclamation projects Locust seeds however exhibit physical dormancy where a
thick seed coat prevents the movement of water and gasses to the embryo (Leadem
1997)
Sulfuric acid or hot water soaks have traditionally been used to break seed
dormancy in macro-propagation of locusts Many growers are moving away from the
use of su1furic acid in treating locust seeds (Dreesen and Harrington 1997) In
addition to being dangerous acid soak durations must be specifically correlated to
seed lot (Olson 1974) Collections of native seed can vary tremendously in seed size
seed weight and hard seededness etc This variability limits the utility of sulfuric
acid soaks as even within a given lot some seeds may be damaged whereas others
are under-treated and fail to imbibe (take up water) A survey of Southwestern
container growers indicates that hot water baths are currently the most common
scarification method for locust seeds (Hine et al 1997) However hot water baths
also produce inconsistent germination (Lin et al 1996)
49
b
Figure AI New Mexico locust a primary invader following disturbance Ala Crown-sprouting at edge ofburn near Los Alamos NM--one year after Cerro Grande fire Alb Colonizing road cut Gila National Forest NM
50
Percussion scarification where seeds are repeatedly propelled against a hard
surface is an alternative dormancy-breaking method that has proven successful in
legumes related to locusts (Hamly 1932 Barton 1947) We compared this alternative
treatment to hot water scarification to evaluate treatment efficacy for New Mexico
locust and black locust seeds
Materials and Methods
Commercial New Mexico locust seeds (Western Native Seed Coaldale CO)
were collected fall 2000 in Huerfano County CO Black locust seeds were collected
September 2000 from Taos County NM by harvesting ripe pods from several trees at
a distance of up to 3 m from ground level Black locust seeds were allowed to air-dry
for 3 weeks and were threshed and separated from large chaffusing a greenhouse
fan A South Dakota Seed Blower (Chicago Illinltois) was used to remove fine chaff
from both seed lots Seeds were storedmiddotat 2-4C until the germination study was
conducted in March 2001
This study compared hot water scarification with percussion scarification
using treatment and species as experimental factors Along with a control
scarification treatments included immersion of seed in boiling water as well as
percussion durations of 1 2 3 45 or 10 minutes Four 100-seed samples were used
to test each species by scarification treatment combination
For the hot water treatment test tubes were filled with 30 mL water and
placed in a water-filled beaker (Figure A2a) The bath was raised to boiling (-98C at
51
b
c
Figure A2 Implementation ofhot water and percussion scarification A2a Hot water bath A2b Paint-shaker set-up A2c Placing soil tin containing seeds at end ofpaint shaker ann
52
Las Cruces NM elevation of 1300 meters) Each 100-seed replicate was placed in a
test tube and each test tube was immediately removed to cool at room temperature
Percussion scarification was implemented using a pneumatic paint shaker
(Central Pneumatic model 00422 Camarillo CA) (Figure A2b) This allowed
standardization of treatment intensity Compressed air pressure was maintained at
530kPa plusmn 30kPa (80psi plusmn 5psi) resulting in approximately 350 oscillations per
minute Each lOO-seed sample was placed in a 118 ml (4 oz) soil tin for shaking To
maximize lateral movement of paint shaker a paint can was used as a spacer to place
soil tins at the end of the shaking arm (Figure A2c) This allowed shaking to occur at
the greatest distance from the pivot point (fulcrum) Following all treatments
including the control seeds were soaked for twenty-four hours in distilled water prior
to plating out
Gennination was evaluated on lab benches at room temperature Each
replicate of seeds was placed in a 100 cm petri dish on moistened filter paper with
humidity maintained by enclosing the petri dishes in ziplock bags Gennination was
monitored daily for 14 days in accordance with International Seed Testing
Association standards for Robinia species (ISTA 1999) Gennination was defined as
protrusion of the radicle from the embryo by at least 1mm as viewed by the naked
eye
Categorical Analysis of Variance using Prpc Catmod (SAS Institute 1989)
was used to detennine treatment differences in germination percentages for each
source This procedure is an extension of a Chi-square test of homogeneity using the
53
natural log of the ratio of germinated to non-germinated seeds for each treatment Pshy
values less than 005 were considered significant A limited set of pairwiseshy
comparisons was conducted to compare treatment mean percents using a
conservative alpha value of 005 divided by the number of comparisons
Results
Percussion maximized germination for both New Mexico locust and black
locust There were three germination response levels for New Mexico locust (Figure
A3) First there was a low response in the control indicating a dormant seed lot
The hot water treatment and I-minute duration of percussion share a second
intermediate response Germination was highest at percussion levels from 2 to 10
minutes
A selected percussion treatment intermediate in time (5 minutes) produced the
steepest germination curve indicating the fastest germination rate (Figure A4) The
hot water treatment produced an intermediate curve or relatively slower germination
rate The control curve was very flat indicating the slowest germination rate for New
Mexico locust
Control seeds germinated poorly for black locust as well (Figure A5) Hot
water scarification improved germination over the control but all levels of percussion
improved germination over hot water The optimal durations of percussion treatment
were bracketed with a significant increase from the 1 to 2 minute percussion level a
high germination response from 2 to 5 minutes of percussion and finally a significant
54
~ d 25 CL)
p t1 0 ~ t1
g Vl ~ Vl
100~---------------------------------------------------------------
80
60
40
20
o Control Hot Water Percussion
I min Percussion
2 min Percussion
3 min
Scarification
Percussion 4 min
Figure A3 Effect of scarification on germination response for New Mexico locust
Percussion 5 min
=significant difference between adjacent treatment bars ns = no significant difference
Percussion 10 min
~ ~ () u agt
Il-t d o ~
~ ~ c3
100middotlr~~~------------------------------------e- Percussion 5min middot 0middot Hot Water -- Control
80-J=middot======
6b-middot-lmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middot--middotmiddot-middot~-middotmiddot -middotmiddot middot middotmiddot-middot 1
40
20
-----~-----------
0 0 0 middotmiddotmiddotmiddotmiddot0middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot0
0 --~-----y-----~------_---~--- T-----~
o shy -TDay 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A4 Effect of selected scarification treatments on germination speed for New Mexico locust First seven days of fourteen-day experiment are shown
Day 7
80
~ cd
5 60 ()
[) Cl d 0 1t1 d
40sect dUl
-J
20
o
ns
Scarification
Figure A5 Effect of scarification on germination response for black locust = significant difference between adjacent treatment bars ns = no significant difference
decrease at the 10-minute level This drop-off suggests that 10 minutes of percussion
over-treats seeds from this lot of-black locust
A selected percussion treatment intermediate in time (5 minutes) also had the
fastest germination rate for black locust (Figure A6)_ The hot water curve is
somewhat flatter indicating a slower germination rate The control curve is very flat
indicating a slow germination rate
A qualitative difference between hot water and percussion-treated seeds was
also observed Along with healthy germinants the hot water treatment produced
over-treated seeds (Figure A 7a) Examples of damaged germinants include seeds
where the radicle did not elongate (Figure A 7b) seeds where the radicle elongated
but was delayed in freeing itself from the seed coat (Figure A7c) and seeds where the
radicle broke off completely from the embryo (Figure A7d) In addition to these
examples of over-treatment there were numerous small dark seeds that failed to
imbibe water For percussion treatment nearly all seeds took up water (Figure A8a)
Germination was rapid and uniform with little damage evident (Figure A8b)
To summarize both New Mexico locust and black locust seed lots were
dormant The standard hot water scarification treatment improved germination but
only to 56 and 41 for New Mexico locust and black locust respectively Nearly
all percussion durations improved total germination as well as germination rate over
the hot water treatment Germination was greater than 90 for percussion durations
of 4 5 and 10 minutes for New Mexico locust and 3 4 and 5 minutes for black locust
58
CI)
sgt t CI) 0
igt 0
sect ttl t E
Vl 0
c3
100r==~==~~=r------------------------------------~ ___ Percussion 5 min
middotmiddotmiddot0middotmiddotmiddot Hot Water -lIf- Control
80r=========~--~~----------------------------~
60
40
20 -~-~---~---------- ---~------------I
omiddot
O
_~_----------lIf-----~bull -----_ shy---shy I
Day 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A6 Effect of selected scarification treatments on germination speed for black locust First seven days of fourteen-day experiment are shown
Day 7
a
b c d
Figure A7 Damage to New Mexico locust seeds observed during germination counts following hot water scarification A7a Hot water replicate including damaged seedlings and numerous unimbibed seeds A7b Close-up ofseed where radicle failed to elongate A7c Close-up ofseed where radicle elongated but was delayed in freeing itself from the seed coat A7d Close-up ofseed where radicle completely broke offfrom embryo
60
a
b
Figure A8 New Mexico locust seeds following percussion treatment at time of germination counts A8a Percussion replicate with uniformly healthy seedlings and near complete imbibition of seeds A8b Close-up ofpercussion-treated seedling
61
Discussion
Papilionoid seeds have a specific anatomical feature known as the strophiole
or lens-the natural site of water entry to the seed (Hamly 1932) The strophiole is
located on the cotyledonary lobe of a locust seed (Figure A9) When a papilionoid
legume seed is percussed for the appropriate amount of time repeated hits on the
integrated seed coat loosen the constrained cells of the strophiolar region without
excessively damaging the rest of the seed coat (Ballard 1976)
When a percussed seed is soaked water enters exclusively through the
strophiole in a controlled manner (Kelly and Staden 1987) This regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues This
contrasts with seeds that have been hot water or acid scarified treatments that can
randomly degrade the seed coat Unlocalized cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rehman et al 1998 Marunda 1990)
Papilionoids are the largest subfamily of legumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust and black locust these legumes often play an integral role in the
revegetation ofdisturbed lands Developing superior scarification methods to the
standard hot water and acid treatments should fadlitate the use ofthese legumes in
restoration projects
62
Figure A9 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (bc)
63
APPENDIXB
GERMINATION PERCENTS AND STANDARD ERRORS FOR SOURCE
BLOCK AND TREATMENT FOR NEW MEXICO LOCUST
Table B 1 Gennination percents and standard errors for source block and treatment for New Mexico locust
Source Block Treatment Germination Percentage Standard Error
Capitan 1 Immersion 74 437 Capitan 2 Immersion 69 462 Capitan 3 Immersion 71 454 Capitan 4 Immersion 67 470 Capitan Boiling 30 sec 79 407 Capitan 2 Boiling 30 sec 76 427 Capitan 3 Boiling 30 sec 72 449 Capitan 4 Boiling 30 sec 76 427 Capitan Boiling 60 sec 71 454 Capitan 2 Boiling 60 sec 54 498 Capitan 3 Boiling 60 sec 67 470 Capitan 4 Boiling 60 sec 76 427 Capitan I Control 5 218 Capitan 2 Control 7 255 Capitan 3 Control 4 196 Capitan 4 Control 5 218 Capitan 1 Percussion I min 47 499 Capitan 2 Percussion I min
470
Capitan Percussion I min 39 488
Capitan 4 Percussion I min 38 485 Capitan I Percussion 2 min 55 497 Capitan Capitan
2
Percussion 2 min Percussion 2 min
71 65
454 477
Capitan 4 Percussion 2 min 56 496 Capitan I Percussion 4 min 86 347 Capitan 2 Percussion 4 min 79 407 Capitan 3 Percussion 4 min 81 392 Capitan 4 Percussion 4 min 82 384 Capitan I Percussion 8 min 90 300 Capitan 2 Percussion 8 min 88 325 Capitan 3 Percussion 8 min 90 300 Capitan 4 Percussion 8 min 90 300 Gila I Immersion 71 454 Gila 2 Immersion 77 421 Gila
Immersion 75 433
Gila 4 Immersion 72 449
Gila I Boiling 30 sec 68 466
Gila 2 Boiling 30 sec 81 392
Gila 3 Boiling 30 sec 54 498
Gila 4 Boiling 30 sec 63 483
Gila I Boiling 60 sec 55 497
Gila 2 Boiling 60 sec 68 466
Gila 3 Boiling 60 sec 57 495
Gila 4 Boiling 60 sec 70 458
65
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Gila 2 Control 13 336 Gila 3 Control 13 336 Gila 4 Control 20 400 Gila 1 Percussion 1 min 66 474 Gila 2 Percussion 1 min 57 495 Gila 3 Percussion 1 min 73 444 Gila 4 Percussion 1 min 82 384 Gila 1 Percussion 2 min 81 392 Gila 2 Percussion 2 min 86 347 Gila 3 Percussion 2 min 85 357 Gila 4 Percussion 2 min 54 498 Gila 1 Percussion 4 min 79 407 Gila 2 Percussion 4 min 83 376 Gila 3 Percussion 4 min 84 367 Gila 4 Percussion 4 min 79 407 Gila 1 Percussion 8 min 51 500 Gila 2 Percussion 8 min 76 427 Gila 3 Percussion 8 min 52 500 Gila 4 Percussion 8 min 74 439 Holman 1 Immersion 83 376 Holman 2 Immersion 86 347 Holman 3 Immersion 81 392 Holman 4 Immersion 90 300 Holman 1 Boiling 30 sec 90 300 Holman 2 Boiling 30 sec 85 357 Holman 3 Boiling 30 sec 87 336 Holman 4 Boiling 30 sec 83 376 Holman 1 Boiling 60 sec 79 407 Holman 2 Boiling 60 sec 80 400 Holman 3 Boiling 60 sec 86 347 Holman 4 Boiling 60 sec 93 255 Holman Control 25 433 Holman 2 Control 19 392 Holman Control 21 407
Holman 4 Control 24 427
Holman Percussion 1 min 77 421
Holman 2 Percussion 1 min 85 357
Holman 3 Percussion 1 min 80 400
Holman 4 Percussion 1 min 89 313
Holman 1 Percussion 2 min 88 325
Holman 2 Percussion 2 min 96 196
Holman 3 Percussion 2 min 93 255
Holman 4 Percussion 2 min 96 196
Holman 1 Percussion 4 min 92 271
Holman 2 Percussion 4 min 97 171
66
I
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Holman 3 Percussion 4 min 91 286 Holman 4 Percussion 4 min 96 196 Holman 1 Percussion 8 min 88 325 Holman 2 Percussion 8 min 85 357 Holman 3 Percussion 8 min 83 376 Holman 4 Percussion 8 min 82 384 Jemez 1 Immersion 53 499 Jemez 2 Immersion 44 496 Jemez 3 Immersion 52 500 Jemez 4 Immersion 70 458 Jemez 1 Boiling 30 sec 58 494 Jemez 2 Boiling 30 sec 55 497 Jemez 3 Boiling 30 sec 48 500 Jemez 4 Boiling 30 sec 65 477 Jemez 1 Boiling 60 sec 68 466 Jemez 2 Boiling 60 sec 63 483 Jemez 3 Boiling 60 sec 73 444 Jemez 4 Boiling 60 sec 89 313 Jemez 1 Control 2 140 Jemez 2 Control 9 286 Jemez 3 Control 6 237 Jemez 4 Control 8 271 Jemez 1 Percussion 1 min 58 494 Jemez 2 Percussion 1 min 63 483 Jemez 3 Percussion 1 min 77 421 Jemez 4 Percussion 1 min 89 313 Jemez 1 Percussion 2 min 68 466 Jemez 2 Percussion 2 min 64 480 Jemez 3 Percussion 2 min 80 400 Jemez 4 Percussion 2 min 83 376 Jemez Percussion 4 min 68 466 Jemez 2 Percussion 4 min 79 407 Jemez 3 Percussion 4 min 70 458 Jemez 4 Percussion 4 min 83 376 Jemez 1 Percussion 8 min 52 500 Jemez 2 Percussion 8 min 59 492 Jemez 3 Percussion 8 min 61 488 Jemez 4 Percussion 8 min 70 458 Magdalena 1 Immersion 69 462 Magdalena 2 Immersion 69 462 Magdalena J Immersion 70 458 Magdalena 4 Immersion 85 357 Magdalena 1 Boiling 30 sec 65 477 Magdalena 2 Boiling 30 sec 71 454 Magdalena 3 Boiling 30 sec 68 466
67
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Magdalena 4 Boiling 30 sec 83 376 Magdalena 1 Boiling 60 sec 63 483 Magdalena 2 Boiling 60 sec 68 466 Magdalena 3 Boiling 60 sec 63 483 Magdalena 4 Boiling 60 sec 82 384 Magdalena 1 Control 11 313 Magdalena 2 Control 6 237 Magdalena 3 Control 5 218 Magdalena 4 Control 4 196 Magdalena Percussion 1 min 32 466 Magdalena 2 Percussion 1 min 45 497 Magdalena 3 Percussion 1 min 46 498 Magdalena 4 Percussion I min 36 480 Magdalena 1 Percussion 2 min 67 470 Magdalena 2 Percussion 2 min 68 466 Magdalena 3 Percussion 2 min 65 477 Magdalena 4 Percussion 2 min 81 392 Magdalena 1 Percussion 4 min 87 336 Magdalena 2 Percussion 4 min 97 171 Magdalena 3 Percussion 4 min 83 376 Magdalena 4 Percussion 4 min 97 171 Magdalena 1 Percussion 8 min 98 lAO Magdalena 2 Percussion 8 min 100 000 Magdalena 3 Percussion 8 min 96 196 Magdalena 4 Percussion 8 min 98 lAO Manzano 1 Immersion 67 470 Manzano 2 Immersion 57 495 Manzano 3 Immersion 72 4049 Manzano 4 Immersion 73 4044 Manzano 1 Boiling 30 sec 70 458 Manzano 2 Boiling 30 sec 64 480 Manzano 3 Boiling 30 sec 71 454 Manzano 4 Boiling 30 sec 80 400 Manzano I Boiling 60 sec 50 500 Manzano 2 Boiling 60 sec 53 499 Manzano 3 Boiling 60 sec 59 492 Manzano 4 Boiling 60 sec 74 439 Manzano I Control 4 196 Manzano 2 Control 8 271 Manzano 3 Control 1 099 Manzano 4 Control 6 237 Manzano 1 Percussion 1 min 52 500 Manzano 2 Percussion 1 min 39 488 Manzano 3 Percussion 1 min 36 480 Manzano 4 Percussion 1 min 51 500
68
Table B 1 Continued
Source Block Treatment Gerrni~ation Percentage Standard Error
Manzano Percussion 2 min 53 499 Manzano 2 Percussion 2 min 58 494 Manzano 3 Percussion 2 min 61 488 Manzano 4 Percussion 2 min 62 485 Manzano 1 Percussion 4 min 76 427 Manzano 2 Percussion 4 min 85 357 Manzano 3 Percussion 4 min 79 407 Manzano 4 Percussion 4 min 83 376 Manzano 1 Percussion 8 min 87 336 Manzano 2 Percussion 8 min 83 376 Manzano 3 Percussion 8 min 83 376 Manzano 4 Percussion 8 min 75 433 Raton 1 Immersion 54 498 Raton 2 Immersion 37 483 Raton 3 Immersion 39 488 Raton 4 Immersion 69 462 Raton 1 Boiling 30 sec 36 480 Raton 2 Boiling 30 sec 20 400 Raton 3 Boiling 30 sec 28 449 Raton 4 Boiling 30 sec 60 490 Raton 1 Boiling 60 sec 15 357 Raton 2 Boiling 60 sec 171 Raton 3 Boiling 60 sec 4 196 Raton 4 Boiling 60 sec 35 477 Raton 1 Control 4 196 Raton 2 Control 12 325 Raton 3 Control 2 140 Raton 4 Control 6 237 Raton 1 Percussion 1 min 52 500 Raton 2 Percussion I min 29 454 Raton 3 Percussion I min 35 477 Raton 4 Percussion 1 min 31 462 Raton 1 Percussion 2 min 55 497 Raton 2 Percussion 2 min 35 477 Raton 3 Percussion 2 min 33 470 Raton 4 Percussion 2 min 42 494 Raton 1 Percussion 4 min 65 477 Raton 2 Percussion 4 min 52 500 Raton Percussion 4 min 51 500 Raton 4 Percussion 4 min 53 499 Raton 1 Percussion 8 min 88 325 Raton 2 Percussion 8 min 80 400 Raton 3 Percussion 8 min 73 444 Raton 4 Percussion 8 min 74 439 Sacramento 1 Immersion 43 495
69
Table B 1 Continued
-Source Block Treatment Germination Percentage Standard Error
Sacramento 2 Immersion 30 458 Sacramento 3 Immersion 38 485 Sacramento 4 Immersion 56 496 Sacramento 1 Boiling 30 sec 47 499 Sacramento 2 Boiling 30 sec 48 500 Sacramento 3 Boiling 30 sec 48 500 Sacramento 4 Boiling 30 sec 56 496 Sacramento 1 Boiling 60 sec 52 500 Sacramento 2 Boiling 60 sec 67 470 Sacramento 3 Boiling 60 sec 43 495 Sacramento 4 Boiling 60 sec 48 500 Sacramento 1 Control 0 000 Sacramento 2 Control 3 171 Sacramento 3 Control 3 171 Sacramento 4 Control 099 Sacramento 1 Percussion 1 min 57 495 Sacramento 2 Percussion 1 min 61 488 Sacramento 3 Percussion 1 min 62 485 Sacramento 4 Percussion 1 min 55 497 Sacramento 1 Percussion 2 min 84 367 Sacramento 2 Percussion 2 min 70 458 Sacramento 3 Percussion 2 min 81 392 Sacramento 4 Percussion 2 min 69 462 Sacramento 1 Percussion 4 min 91 286 Sacramento 2 Percussion 4 min 84 367 Sacramento 3 Percussion 4 min 84 367 Sacramento 4 Percussion 4 min 94 237 Sacramento 1 Percussion 8 min 82 384 Sacramento 2 Percussion 8 min 81 392 Sacramento 3 Percussion 8 min 82 384 Sacramento 4 Percussion 8 min 81 392 Sandia 1 Immersion 76 427 Sandia 2 Immersion 70 458 Sandia 3 Immersion 70 458 Sandia 4 Immersion 81 392 Sandia 1 Boiling 30 sec 78 414 Sandia 2 Boiling 30 sec 71 454 Sandia 3 Boiling 30 sec 63 483 Sandia 4 Boiling 30 sec 78 414 Sandia 1 Boiling 60 sec 67 470 Sandia 2 Boiling 60 sec 65 477 Sandia 3 Boiling 60 sec 74 439 Sandia 4 Boiling 60 sec 66 474 Sandia 1 Control 16 367 Sandia 2 Control 7 255
70
Table B 1 Continued
Source Block Treatment Gennination Percentage Standard Error
Sandia 3 Control 8 271 Sandia 4 Control 16 367 Sandia 1 Percussion 1 min 67 470 Sandia 2 Percussion 1 min 61 488 Sandia 3 Percussion 1 min 66 474 Sandia 4 Percussion 1 min 59 492 Sandia I Percussion 2 min 82 384 Sandia 2 Percussion 2 min 83 376 Sandia 3 Percussion 2 min 81 392 Sandia 4 Percussion 2 min 84 367 Sandia 1 Percussion 4 min 89 313 Sandia 2 Percussion 4 min 86 347 Sandia 3 Percussion 4 min 89 313 Sandia 4 Percussion 4 min 85 357 Sandia 1 Percussion 8 min 80 400 Sandia 2 Percussion 8 min 77 421 Sandia 3 Percussion 8 min 69 462 Sandia 4 Percussion 8 min 66 474
LITERATURE CITED
Ashby WC Vogel WG Rogers NF 1985 Black locust in the reclamation equation General Technical Report Northeastern Forest Experiment Station USDA Forest Service NE-I05 12 p
Ballard LAT 1973 Physical barriers to gennination Seed Sci Technoll 285-303
___ 1976 Strophiolar water conduction in the seeds ofTrifolieae induced by action on the testa at nonstrophiolar sites Austr J Plant Physiol3 465-469
Barton LV 1947 Special studies on seed coat impenneability Contrib Boyce Thompson Inst 14 355-362
Baskin CC Baskin JM 1998 Seeds Ecology biogeography and evolution of donnancy and gennination Academic Press London UK 666 p
Bewley JD 1997 Seed germination and donnancy Plant Ce119 1055-1066
Bewley JD Black M 1994 Seeds physiology of development and germination 2nd ed Plenum Press New York NY 445 p
Briscoe CB 1996 Growing Nitrogen Fixing Trees in Acid Soils In MH Powell (ed) Nitrogen Fixing Trees for Acid Soils- A Field ManuaL Winrock International Morrilton AR 22 p
Copeland LO McDonald MB 1995 Principles of Seed Science and Technology 3rded Chapman amp Hall New York NY 409 p
Cox RE Klett JE 1984 Seed gennination requirements of native Colorado plants for use in the landscape Plant Propag 30(2) 6-10
Dell B 1980 Structure and function ofthe strophiolar plug in seeds ofAlbizia Lophantha Am J Bot 67 556-563
Dick-Peddie WA 1993 New Mexico Vegetation Past Present and Future University ofNew Mexico Press Albuquerque NM 244 p
Dreesen DR 2001 Propagation protocol for production ofcontainer Robinia neomexicana plants Los Lunas Plant Materials Center Los Lunas New Mexico In Native Plant Network URL httpwwwnativeplantnetworkorg (accessed 9 January 2002) Moscow (ID) University ofIdaho CoIl Natural Resources For Res Nursery
72
Dreesen DR Harrington JT 1997 Propagation of native plants for restoration projects in the Southwestern US- Preliminary Investigations In Landis TD Thompson JR tech coords National Proceedings Forest and Conservation Nursery Assocs Gen Tech Rep PNW-GTR-419 Portland OR USDA For Ser PNW Res Sta 77-88
Gottfried GJ 1980 Control ofNew Mexican locust and the effect on planted ponderosa pine in central Arizona US For Ser Res Note RM-386 Rocky Mountain For Range ExpStn Fort Collins CO
Gosling PG Samuel YK Jones SK 1995 A systematic examination of germination temperature chipping and water temperaturesoak duration pretreatments on the seeds ofLeucaena leucocephala Seed Sci amp Technol 23521-532
Hamly DH 1932 Softening of the seeds of Melilotus alba Bot Gazette 93 345shy375
Hanna PJ 1984 Anatomical features of the seed coat ofAcacia kempeana (Mueller) which relate to increased germination rate induced by heat treatment New Phytol 96 23-29
Hanks JP Dick-Peddie WA 1974 Vegetation patterns of the White Mountains New Mexico Southwest Naturalist 18(4) 371-382
Hine S Harrington JT Dreesen DR 1997 Seed scarification requirements for Robinia neomexicana In Harrington JTtech coord Proc Third SouthwestemContainer Growers Conf Feb 12-13 1997 Ft Collins CO
Hopkinson JM 1993 The strophiole of leguminous seeds Newsletter- IntI Herbage Seed Production Res Grp 18 7-8
---- 1997 Differences in hardseededness between Cunningham and Tarramba leucaena In Leucnet News The Newsletter of the International LeucaenaRes and Devlt Netwk No4
ISTA (International Seed Testing Association) 1999 International rules for seed testing rules Seed Sci amp Technol27 Supplement
Kelly KM van Staden J 1987 The lens as the site of permeability in the papilionoid seed Aspalathus linearis J Plant Physiol 128(45) 395-404
73
Keresztesi B 1988 Black locust the tree of agriculture Outlook on Agr 17(2) 77~85
Kigel 1 and Galili G 1995 Seed development and germination Marcel Dekker New York 853 p
Klemmedson 10 1994 New Mexico locust and parent material Influence on macronutrients of forest floor and soil Soil Sci Soc Am 1 58 974~980
Kuo WH Tam AW 1988 The pathway of water absorption of mung bean seeds Seed Sci amp Technol16(1) 139~144
Leadem CL 1997 Dormancy--unlocking seed secrets In Landis TD Thompson lR tech coords National Proceedings Forestand Conservation Nursery Assocs Gen Tech Rep PNW-G TR-419 Portland OR USDA For Ser PNW Res Sta 43-52
Lin Y Wagner MR Seweyestewa A Honahni MD DeGomez M McMillin JD 1996 Influence of cultural treatments on seed germination and seedling growth of Robinia neomexicana Forest Farm and Community Tree Res Reports Volume 1
Marunda CT 1990 Effects of seed pretreatment on the development ofAcacia auriculforinis and A holosericea seedlings ACIAR Proceedings Series No 28
Meginnis HG 1937 Sulphuric acid treatment to increase germination of black locust seed USDA Circ 453 34 p
Miklas PN Towensend CE Ladd SL 1987 Seedcoat anatomy and the scarification ofcicer milkvetch seed Crop Sci 27 766-772
Monsen SB 1984 Use of shrubs on mine spoils In The Challenge ofProducing Native Plants for the Intermountain Area Proceedings Intermountain Nurserymans Association 1983 conference Las Vegas NV compo PM Murphy 26-31 Ogden UT USDA For Ser Gen Tech Report INT-l68
Olson DF 1974 Robinia L Locust In Seeds of Woody Plants in the US tech coord CS Schopmeyer 728-731 USDA For Ser Agr Handbk No450
Rehman S Loescher RNl Harris P1C 1998 Dormancy breaking and germination ofAcacia salicina Lindl seeds Seed Sci amp Techno127 553-557
Rolston MP 1978 Water impermeable seed dormancy Bot Rev 44 365-396
74
SAS Institute Inc 1989 SASSTAT Users Guide Version 6 4th Edition Volume 1 Cary NC SAS Institute Inc 943 p
SerratoValenti G Conara L Ferrando M Modenesi P 1993 Structural and histochemical features ofStylosanthes scabra (Leguminosae Papilionoidae) seed coat as related to water entry Can J Bot 71 834-840
Simpson BJ 1988 A field guide to Texas trees Austin TX Texas Monthly Press 372p
Tran VN Cavanagh AK 1984 Structural aspects ofdormancy In Seed Physiology Vol 2 Murray DR (ed) p1-44 Academic Press Australia
USDA Forest Service 1948 Woody-plant seed manual USDA Misc Pub1654 416p
Vines RA 1960 Trees Shrubs and Woody Vines of the Southwest Austin Univ ofTexas Press 1104 p
Wagner MR Larson RE Craig TP 1992 Ecology and silvics ofNew Mexico locust (Robinia neomexicana) with an emphasis on the arboreal form p53-57 In JW Hanover K Miller and S Plesko (eds) Proc Intemat Confon Black Locust Biology Culture and Utilization Mich State Univ USA
Wilson JK 1944 Immersing seeds of species of Robinia in boiling water hastens germination J For 42 453-454
Zimmerman RW Carpenter SB 1980 First-year coppice production from a 5shyyear old black locust stand on surface mine spoil p309-314 Centr Hardwd For ConfProc University ofMissouri Columbia
APPENDIX A
PILOT STUDY PERCUSSION AS AN ALTERNATIVE SCARIFICATION FOR
NEW MEXICO LOCUST AND BLACK LOCUST SEEDS
Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4
Hot water and sulfuric acid soaks are traditional treatments for seeds of many
temperate woody legwnes including locusts However these scarification techniques
often produce inconsistent germination Percussion scarification where seeds are
repeatedly propelled against a hard surface was compared with hot water
scarification to evaluate treatment efficacy for New Mexico locust (Robinia
neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment
seeds were placed in a 98degC water bath which was immediately removed from the
heat source For percussion scarification seeds were placed in a soil sample tin and
agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the
control were followed by 24-hour water soaks Hot water baths resulted in 56 and
41 germination for New Mexico locust and black locust respectively For both
species nearly all durations ofpercussion increased germination over the hot water
treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3
4 and 5 minutes for black locust resulted in at least 90 germination Traditional
scarification treatments randomly degrade the entire seed coat which can lead to
tissue damage during water uptake Percussion scarification specifically weakens the
strophiole the natural source ofwater entry to the seed in papilionoid legwnes
INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom
2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031
48
Following percussion imbibition is controlled through the strophiole and underlying
tissue is protected
Keywords New Mexico locust black locust scarification percussion germination
Introduction
Locust trees (Robinia spp) are aggressive pioneer species that quickly
colonize disturbed land fix atmospheric nitrogen in the soil and stabilize slopes that
are prone to erosion (Klemmedson 1994 Ashby et al 1985) (Figure A1ab)
Recognition of these attributes has contributed to increased demand for locusts in
reclamation projects Locust seeds however exhibit physical dormancy where a
thick seed coat prevents the movement of water and gasses to the embryo (Leadem
1997)
Sulfuric acid or hot water soaks have traditionally been used to break seed
dormancy in macro-propagation of locusts Many growers are moving away from the
use of su1furic acid in treating locust seeds (Dreesen and Harrington 1997) In
addition to being dangerous acid soak durations must be specifically correlated to
seed lot (Olson 1974) Collections of native seed can vary tremendously in seed size
seed weight and hard seededness etc This variability limits the utility of sulfuric
acid soaks as even within a given lot some seeds may be damaged whereas others
are under-treated and fail to imbibe (take up water) A survey of Southwestern
container growers indicates that hot water baths are currently the most common
scarification method for locust seeds (Hine et al 1997) However hot water baths
also produce inconsistent germination (Lin et al 1996)
49
b
Figure AI New Mexico locust a primary invader following disturbance Ala Crown-sprouting at edge ofburn near Los Alamos NM--one year after Cerro Grande fire Alb Colonizing road cut Gila National Forest NM
50
Percussion scarification where seeds are repeatedly propelled against a hard
surface is an alternative dormancy-breaking method that has proven successful in
legumes related to locusts (Hamly 1932 Barton 1947) We compared this alternative
treatment to hot water scarification to evaluate treatment efficacy for New Mexico
locust and black locust seeds
Materials and Methods
Commercial New Mexico locust seeds (Western Native Seed Coaldale CO)
were collected fall 2000 in Huerfano County CO Black locust seeds were collected
September 2000 from Taos County NM by harvesting ripe pods from several trees at
a distance of up to 3 m from ground level Black locust seeds were allowed to air-dry
for 3 weeks and were threshed and separated from large chaffusing a greenhouse
fan A South Dakota Seed Blower (Chicago Illinltois) was used to remove fine chaff
from both seed lots Seeds were storedmiddotat 2-4C until the germination study was
conducted in March 2001
This study compared hot water scarification with percussion scarification
using treatment and species as experimental factors Along with a control
scarification treatments included immersion of seed in boiling water as well as
percussion durations of 1 2 3 45 or 10 minutes Four 100-seed samples were used
to test each species by scarification treatment combination
For the hot water treatment test tubes were filled with 30 mL water and
placed in a water-filled beaker (Figure A2a) The bath was raised to boiling (-98C at
51
b
c
Figure A2 Implementation ofhot water and percussion scarification A2a Hot water bath A2b Paint-shaker set-up A2c Placing soil tin containing seeds at end ofpaint shaker ann
52
Las Cruces NM elevation of 1300 meters) Each 100-seed replicate was placed in a
test tube and each test tube was immediately removed to cool at room temperature
Percussion scarification was implemented using a pneumatic paint shaker
(Central Pneumatic model 00422 Camarillo CA) (Figure A2b) This allowed
standardization of treatment intensity Compressed air pressure was maintained at
530kPa plusmn 30kPa (80psi plusmn 5psi) resulting in approximately 350 oscillations per
minute Each lOO-seed sample was placed in a 118 ml (4 oz) soil tin for shaking To
maximize lateral movement of paint shaker a paint can was used as a spacer to place
soil tins at the end of the shaking arm (Figure A2c) This allowed shaking to occur at
the greatest distance from the pivot point (fulcrum) Following all treatments
including the control seeds were soaked for twenty-four hours in distilled water prior
to plating out
Gennination was evaluated on lab benches at room temperature Each
replicate of seeds was placed in a 100 cm petri dish on moistened filter paper with
humidity maintained by enclosing the petri dishes in ziplock bags Gennination was
monitored daily for 14 days in accordance with International Seed Testing
Association standards for Robinia species (ISTA 1999) Gennination was defined as
protrusion of the radicle from the embryo by at least 1mm as viewed by the naked
eye
Categorical Analysis of Variance using Prpc Catmod (SAS Institute 1989)
was used to detennine treatment differences in germination percentages for each
source This procedure is an extension of a Chi-square test of homogeneity using the
53
natural log of the ratio of germinated to non-germinated seeds for each treatment Pshy
values less than 005 were considered significant A limited set of pairwiseshy
comparisons was conducted to compare treatment mean percents using a
conservative alpha value of 005 divided by the number of comparisons
Results
Percussion maximized germination for both New Mexico locust and black
locust There were three germination response levels for New Mexico locust (Figure
A3) First there was a low response in the control indicating a dormant seed lot
The hot water treatment and I-minute duration of percussion share a second
intermediate response Germination was highest at percussion levels from 2 to 10
minutes
A selected percussion treatment intermediate in time (5 minutes) produced the
steepest germination curve indicating the fastest germination rate (Figure A4) The
hot water treatment produced an intermediate curve or relatively slower germination
rate The control curve was very flat indicating the slowest germination rate for New
Mexico locust
Control seeds germinated poorly for black locust as well (Figure A5) Hot
water scarification improved germination over the control but all levels of percussion
improved germination over hot water The optimal durations of percussion treatment
were bracketed with a significant increase from the 1 to 2 minute percussion level a
high germination response from 2 to 5 minutes of percussion and finally a significant
54
~ d 25 CL)
p t1 0 ~ t1
g Vl ~ Vl
100~---------------------------------------------------------------
80
60
40
20
o Control Hot Water Percussion
I min Percussion
2 min Percussion
3 min
Scarification
Percussion 4 min
Figure A3 Effect of scarification on germination response for New Mexico locust
Percussion 5 min
=significant difference between adjacent treatment bars ns = no significant difference
Percussion 10 min
~ ~ () u agt
Il-t d o ~
~ ~ c3
100middotlr~~~------------------------------------e- Percussion 5min middot 0middot Hot Water -- Control
80-J=middot======
6b-middot-lmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middot--middotmiddot-middot~-middotmiddot -middotmiddot middot middotmiddot-middot 1
40
20
-----~-----------
0 0 0 middotmiddotmiddotmiddotmiddot0middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot0
0 --~-----y-----~------_---~--- T-----~
o shy -TDay 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A4 Effect of selected scarification treatments on germination speed for New Mexico locust First seven days of fourteen-day experiment are shown
Day 7
80
~ cd
5 60 ()
[) Cl d 0 1t1 d
40sect dUl
-J
20
o
ns
Scarification
Figure A5 Effect of scarification on germination response for black locust = significant difference between adjacent treatment bars ns = no significant difference
decrease at the 10-minute level This drop-off suggests that 10 minutes of percussion
over-treats seeds from this lot of-black locust
A selected percussion treatment intermediate in time (5 minutes) also had the
fastest germination rate for black locust (Figure A6)_ The hot water curve is
somewhat flatter indicating a slower germination rate The control curve is very flat
indicating a slow germination rate
A qualitative difference between hot water and percussion-treated seeds was
also observed Along with healthy germinants the hot water treatment produced
over-treated seeds (Figure A 7a) Examples of damaged germinants include seeds
where the radicle did not elongate (Figure A 7b) seeds where the radicle elongated
but was delayed in freeing itself from the seed coat (Figure A7c) and seeds where the
radicle broke off completely from the embryo (Figure A7d) In addition to these
examples of over-treatment there were numerous small dark seeds that failed to
imbibe water For percussion treatment nearly all seeds took up water (Figure A8a)
Germination was rapid and uniform with little damage evident (Figure A8b)
To summarize both New Mexico locust and black locust seed lots were
dormant The standard hot water scarification treatment improved germination but
only to 56 and 41 for New Mexico locust and black locust respectively Nearly
all percussion durations improved total germination as well as germination rate over
the hot water treatment Germination was greater than 90 for percussion durations
of 4 5 and 10 minutes for New Mexico locust and 3 4 and 5 minutes for black locust
58
CI)
sgt t CI) 0
igt 0
sect ttl t E
Vl 0
c3
100r==~==~~=r------------------------------------~ ___ Percussion 5 min
middotmiddotmiddot0middotmiddotmiddot Hot Water -lIf- Control
80r=========~--~~----------------------------~
60
40
20 -~-~---~---------- ---~------------I
omiddot
O
_~_----------lIf-----~bull -----_ shy---shy I
Day 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A6 Effect of selected scarification treatments on germination speed for black locust First seven days of fourteen-day experiment are shown
Day 7
a
b c d
Figure A7 Damage to New Mexico locust seeds observed during germination counts following hot water scarification A7a Hot water replicate including damaged seedlings and numerous unimbibed seeds A7b Close-up ofseed where radicle failed to elongate A7c Close-up ofseed where radicle elongated but was delayed in freeing itself from the seed coat A7d Close-up ofseed where radicle completely broke offfrom embryo
60
a
b
Figure A8 New Mexico locust seeds following percussion treatment at time of germination counts A8a Percussion replicate with uniformly healthy seedlings and near complete imbibition of seeds A8b Close-up ofpercussion-treated seedling
61
Discussion
Papilionoid seeds have a specific anatomical feature known as the strophiole
or lens-the natural site of water entry to the seed (Hamly 1932) The strophiole is
located on the cotyledonary lobe of a locust seed (Figure A9) When a papilionoid
legume seed is percussed for the appropriate amount of time repeated hits on the
integrated seed coat loosen the constrained cells of the strophiolar region without
excessively damaging the rest of the seed coat (Ballard 1976)
When a percussed seed is soaked water enters exclusively through the
strophiole in a controlled manner (Kelly and Staden 1987) This regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues This
contrasts with seeds that have been hot water or acid scarified treatments that can
randomly degrade the seed coat Unlocalized cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rehman et al 1998 Marunda 1990)
Papilionoids are the largest subfamily of legumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust and black locust these legumes often play an integral role in the
revegetation ofdisturbed lands Developing superior scarification methods to the
standard hot water and acid treatments should fadlitate the use ofthese legumes in
restoration projects
62
Figure A9 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (bc)
63
APPENDIXB
GERMINATION PERCENTS AND STANDARD ERRORS FOR SOURCE
BLOCK AND TREATMENT FOR NEW MEXICO LOCUST
Table B 1 Gennination percents and standard errors for source block and treatment for New Mexico locust
Source Block Treatment Germination Percentage Standard Error
Capitan 1 Immersion 74 437 Capitan 2 Immersion 69 462 Capitan 3 Immersion 71 454 Capitan 4 Immersion 67 470 Capitan Boiling 30 sec 79 407 Capitan 2 Boiling 30 sec 76 427 Capitan 3 Boiling 30 sec 72 449 Capitan 4 Boiling 30 sec 76 427 Capitan Boiling 60 sec 71 454 Capitan 2 Boiling 60 sec 54 498 Capitan 3 Boiling 60 sec 67 470 Capitan 4 Boiling 60 sec 76 427 Capitan I Control 5 218 Capitan 2 Control 7 255 Capitan 3 Control 4 196 Capitan 4 Control 5 218 Capitan 1 Percussion I min 47 499 Capitan 2 Percussion I min
470
Capitan Percussion I min 39 488
Capitan 4 Percussion I min 38 485 Capitan I Percussion 2 min 55 497 Capitan Capitan
2
Percussion 2 min Percussion 2 min
71 65
454 477
Capitan 4 Percussion 2 min 56 496 Capitan I Percussion 4 min 86 347 Capitan 2 Percussion 4 min 79 407 Capitan 3 Percussion 4 min 81 392 Capitan 4 Percussion 4 min 82 384 Capitan I Percussion 8 min 90 300 Capitan 2 Percussion 8 min 88 325 Capitan 3 Percussion 8 min 90 300 Capitan 4 Percussion 8 min 90 300 Gila I Immersion 71 454 Gila 2 Immersion 77 421 Gila
Immersion 75 433
Gila 4 Immersion 72 449
Gila I Boiling 30 sec 68 466
Gila 2 Boiling 30 sec 81 392
Gila 3 Boiling 30 sec 54 498
Gila 4 Boiling 30 sec 63 483
Gila I Boiling 60 sec 55 497
Gila 2 Boiling 60 sec 68 466
Gila 3 Boiling 60 sec 57 495
Gila 4 Boiling 60 sec 70 458
65
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Gila 2 Control 13 336 Gila 3 Control 13 336 Gila 4 Control 20 400 Gila 1 Percussion 1 min 66 474 Gila 2 Percussion 1 min 57 495 Gila 3 Percussion 1 min 73 444 Gila 4 Percussion 1 min 82 384 Gila 1 Percussion 2 min 81 392 Gila 2 Percussion 2 min 86 347 Gila 3 Percussion 2 min 85 357 Gila 4 Percussion 2 min 54 498 Gila 1 Percussion 4 min 79 407 Gila 2 Percussion 4 min 83 376 Gila 3 Percussion 4 min 84 367 Gila 4 Percussion 4 min 79 407 Gila 1 Percussion 8 min 51 500 Gila 2 Percussion 8 min 76 427 Gila 3 Percussion 8 min 52 500 Gila 4 Percussion 8 min 74 439 Holman 1 Immersion 83 376 Holman 2 Immersion 86 347 Holman 3 Immersion 81 392 Holman 4 Immersion 90 300 Holman 1 Boiling 30 sec 90 300 Holman 2 Boiling 30 sec 85 357 Holman 3 Boiling 30 sec 87 336 Holman 4 Boiling 30 sec 83 376 Holman 1 Boiling 60 sec 79 407 Holman 2 Boiling 60 sec 80 400 Holman 3 Boiling 60 sec 86 347 Holman 4 Boiling 60 sec 93 255 Holman Control 25 433 Holman 2 Control 19 392 Holman Control 21 407
Holman 4 Control 24 427
Holman Percussion 1 min 77 421
Holman 2 Percussion 1 min 85 357
Holman 3 Percussion 1 min 80 400
Holman 4 Percussion 1 min 89 313
Holman 1 Percussion 2 min 88 325
Holman 2 Percussion 2 min 96 196
Holman 3 Percussion 2 min 93 255
Holman 4 Percussion 2 min 96 196
Holman 1 Percussion 4 min 92 271
Holman 2 Percussion 4 min 97 171
66
I
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Holman 3 Percussion 4 min 91 286 Holman 4 Percussion 4 min 96 196 Holman 1 Percussion 8 min 88 325 Holman 2 Percussion 8 min 85 357 Holman 3 Percussion 8 min 83 376 Holman 4 Percussion 8 min 82 384 Jemez 1 Immersion 53 499 Jemez 2 Immersion 44 496 Jemez 3 Immersion 52 500 Jemez 4 Immersion 70 458 Jemez 1 Boiling 30 sec 58 494 Jemez 2 Boiling 30 sec 55 497 Jemez 3 Boiling 30 sec 48 500 Jemez 4 Boiling 30 sec 65 477 Jemez 1 Boiling 60 sec 68 466 Jemez 2 Boiling 60 sec 63 483 Jemez 3 Boiling 60 sec 73 444 Jemez 4 Boiling 60 sec 89 313 Jemez 1 Control 2 140 Jemez 2 Control 9 286 Jemez 3 Control 6 237 Jemez 4 Control 8 271 Jemez 1 Percussion 1 min 58 494 Jemez 2 Percussion 1 min 63 483 Jemez 3 Percussion 1 min 77 421 Jemez 4 Percussion 1 min 89 313 Jemez 1 Percussion 2 min 68 466 Jemez 2 Percussion 2 min 64 480 Jemez 3 Percussion 2 min 80 400 Jemez 4 Percussion 2 min 83 376 Jemez Percussion 4 min 68 466 Jemez 2 Percussion 4 min 79 407 Jemez 3 Percussion 4 min 70 458 Jemez 4 Percussion 4 min 83 376 Jemez 1 Percussion 8 min 52 500 Jemez 2 Percussion 8 min 59 492 Jemez 3 Percussion 8 min 61 488 Jemez 4 Percussion 8 min 70 458 Magdalena 1 Immersion 69 462 Magdalena 2 Immersion 69 462 Magdalena J Immersion 70 458 Magdalena 4 Immersion 85 357 Magdalena 1 Boiling 30 sec 65 477 Magdalena 2 Boiling 30 sec 71 454 Magdalena 3 Boiling 30 sec 68 466
67
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Magdalena 4 Boiling 30 sec 83 376 Magdalena 1 Boiling 60 sec 63 483 Magdalena 2 Boiling 60 sec 68 466 Magdalena 3 Boiling 60 sec 63 483 Magdalena 4 Boiling 60 sec 82 384 Magdalena 1 Control 11 313 Magdalena 2 Control 6 237 Magdalena 3 Control 5 218 Magdalena 4 Control 4 196 Magdalena Percussion 1 min 32 466 Magdalena 2 Percussion 1 min 45 497 Magdalena 3 Percussion 1 min 46 498 Magdalena 4 Percussion I min 36 480 Magdalena 1 Percussion 2 min 67 470 Magdalena 2 Percussion 2 min 68 466 Magdalena 3 Percussion 2 min 65 477 Magdalena 4 Percussion 2 min 81 392 Magdalena 1 Percussion 4 min 87 336 Magdalena 2 Percussion 4 min 97 171 Magdalena 3 Percussion 4 min 83 376 Magdalena 4 Percussion 4 min 97 171 Magdalena 1 Percussion 8 min 98 lAO Magdalena 2 Percussion 8 min 100 000 Magdalena 3 Percussion 8 min 96 196 Magdalena 4 Percussion 8 min 98 lAO Manzano 1 Immersion 67 470 Manzano 2 Immersion 57 495 Manzano 3 Immersion 72 4049 Manzano 4 Immersion 73 4044 Manzano 1 Boiling 30 sec 70 458 Manzano 2 Boiling 30 sec 64 480 Manzano 3 Boiling 30 sec 71 454 Manzano 4 Boiling 30 sec 80 400 Manzano I Boiling 60 sec 50 500 Manzano 2 Boiling 60 sec 53 499 Manzano 3 Boiling 60 sec 59 492 Manzano 4 Boiling 60 sec 74 439 Manzano I Control 4 196 Manzano 2 Control 8 271 Manzano 3 Control 1 099 Manzano 4 Control 6 237 Manzano 1 Percussion 1 min 52 500 Manzano 2 Percussion 1 min 39 488 Manzano 3 Percussion 1 min 36 480 Manzano 4 Percussion 1 min 51 500
68
Table B 1 Continued
Source Block Treatment Gerrni~ation Percentage Standard Error
Manzano Percussion 2 min 53 499 Manzano 2 Percussion 2 min 58 494 Manzano 3 Percussion 2 min 61 488 Manzano 4 Percussion 2 min 62 485 Manzano 1 Percussion 4 min 76 427 Manzano 2 Percussion 4 min 85 357 Manzano 3 Percussion 4 min 79 407 Manzano 4 Percussion 4 min 83 376 Manzano 1 Percussion 8 min 87 336 Manzano 2 Percussion 8 min 83 376 Manzano 3 Percussion 8 min 83 376 Manzano 4 Percussion 8 min 75 433 Raton 1 Immersion 54 498 Raton 2 Immersion 37 483 Raton 3 Immersion 39 488 Raton 4 Immersion 69 462 Raton 1 Boiling 30 sec 36 480 Raton 2 Boiling 30 sec 20 400 Raton 3 Boiling 30 sec 28 449 Raton 4 Boiling 30 sec 60 490 Raton 1 Boiling 60 sec 15 357 Raton 2 Boiling 60 sec 171 Raton 3 Boiling 60 sec 4 196 Raton 4 Boiling 60 sec 35 477 Raton 1 Control 4 196 Raton 2 Control 12 325 Raton 3 Control 2 140 Raton 4 Control 6 237 Raton 1 Percussion 1 min 52 500 Raton 2 Percussion I min 29 454 Raton 3 Percussion I min 35 477 Raton 4 Percussion 1 min 31 462 Raton 1 Percussion 2 min 55 497 Raton 2 Percussion 2 min 35 477 Raton 3 Percussion 2 min 33 470 Raton 4 Percussion 2 min 42 494 Raton 1 Percussion 4 min 65 477 Raton 2 Percussion 4 min 52 500 Raton Percussion 4 min 51 500 Raton 4 Percussion 4 min 53 499 Raton 1 Percussion 8 min 88 325 Raton 2 Percussion 8 min 80 400 Raton 3 Percussion 8 min 73 444 Raton 4 Percussion 8 min 74 439 Sacramento 1 Immersion 43 495
69
Table B 1 Continued
-Source Block Treatment Germination Percentage Standard Error
Sacramento 2 Immersion 30 458 Sacramento 3 Immersion 38 485 Sacramento 4 Immersion 56 496 Sacramento 1 Boiling 30 sec 47 499 Sacramento 2 Boiling 30 sec 48 500 Sacramento 3 Boiling 30 sec 48 500 Sacramento 4 Boiling 30 sec 56 496 Sacramento 1 Boiling 60 sec 52 500 Sacramento 2 Boiling 60 sec 67 470 Sacramento 3 Boiling 60 sec 43 495 Sacramento 4 Boiling 60 sec 48 500 Sacramento 1 Control 0 000 Sacramento 2 Control 3 171 Sacramento 3 Control 3 171 Sacramento 4 Control 099 Sacramento 1 Percussion 1 min 57 495 Sacramento 2 Percussion 1 min 61 488 Sacramento 3 Percussion 1 min 62 485 Sacramento 4 Percussion 1 min 55 497 Sacramento 1 Percussion 2 min 84 367 Sacramento 2 Percussion 2 min 70 458 Sacramento 3 Percussion 2 min 81 392 Sacramento 4 Percussion 2 min 69 462 Sacramento 1 Percussion 4 min 91 286 Sacramento 2 Percussion 4 min 84 367 Sacramento 3 Percussion 4 min 84 367 Sacramento 4 Percussion 4 min 94 237 Sacramento 1 Percussion 8 min 82 384 Sacramento 2 Percussion 8 min 81 392 Sacramento 3 Percussion 8 min 82 384 Sacramento 4 Percussion 8 min 81 392 Sandia 1 Immersion 76 427 Sandia 2 Immersion 70 458 Sandia 3 Immersion 70 458 Sandia 4 Immersion 81 392 Sandia 1 Boiling 30 sec 78 414 Sandia 2 Boiling 30 sec 71 454 Sandia 3 Boiling 30 sec 63 483 Sandia 4 Boiling 30 sec 78 414 Sandia 1 Boiling 60 sec 67 470 Sandia 2 Boiling 60 sec 65 477 Sandia 3 Boiling 60 sec 74 439 Sandia 4 Boiling 60 sec 66 474 Sandia 1 Control 16 367 Sandia 2 Control 7 255
70
Table B 1 Continued
Source Block Treatment Gennination Percentage Standard Error
Sandia 3 Control 8 271 Sandia 4 Control 16 367 Sandia 1 Percussion 1 min 67 470 Sandia 2 Percussion 1 min 61 488 Sandia 3 Percussion 1 min 66 474 Sandia 4 Percussion 1 min 59 492 Sandia I Percussion 2 min 82 384 Sandia 2 Percussion 2 min 83 376 Sandia 3 Percussion 2 min 81 392 Sandia 4 Percussion 2 min 84 367 Sandia 1 Percussion 4 min 89 313 Sandia 2 Percussion 4 min 86 347 Sandia 3 Percussion 4 min 89 313 Sandia 4 Percussion 4 min 85 357 Sandia 1 Percussion 8 min 80 400 Sandia 2 Percussion 8 min 77 421 Sandia 3 Percussion 8 min 69 462 Sandia 4 Percussion 8 min 66 474
LITERATURE CITED
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Ballard LAT 1973 Physical barriers to gennination Seed Sci Technoll 285-303
___ 1976 Strophiolar water conduction in the seeds ofTrifolieae induced by action on the testa at nonstrophiolar sites Austr J Plant Physiol3 465-469
Barton LV 1947 Special studies on seed coat impenneability Contrib Boyce Thompson Inst 14 355-362
Baskin CC Baskin JM 1998 Seeds Ecology biogeography and evolution of donnancy and gennination Academic Press London UK 666 p
Bewley JD 1997 Seed germination and donnancy Plant Ce119 1055-1066
Bewley JD Black M 1994 Seeds physiology of development and germination 2nd ed Plenum Press New York NY 445 p
Briscoe CB 1996 Growing Nitrogen Fixing Trees in Acid Soils In MH Powell (ed) Nitrogen Fixing Trees for Acid Soils- A Field ManuaL Winrock International Morrilton AR 22 p
Copeland LO McDonald MB 1995 Principles of Seed Science and Technology 3rded Chapman amp Hall New York NY 409 p
Cox RE Klett JE 1984 Seed gennination requirements of native Colorado plants for use in the landscape Plant Propag 30(2) 6-10
Dell B 1980 Structure and function ofthe strophiolar plug in seeds ofAlbizia Lophantha Am J Bot 67 556-563
Dick-Peddie WA 1993 New Mexico Vegetation Past Present and Future University ofNew Mexico Press Albuquerque NM 244 p
Dreesen DR 2001 Propagation protocol for production ofcontainer Robinia neomexicana plants Los Lunas Plant Materials Center Los Lunas New Mexico In Native Plant Network URL httpwwwnativeplantnetworkorg (accessed 9 January 2002) Moscow (ID) University ofIdaho CoIl Natural Resources For Res Nursery
72
Dreesen DR Harrington JT 1997 Propagation of native plants for restoration projects in the Southwestern US- Preliminary Investigations In Landis TD Thompson JR tech coords National Proceedings Forest and Conservation Nursery Assocs Gen Tech Rep PNW-GTR-419 Portland OR USDA For Ser PNW Res Sta 77-88
Gottfried GJ 1980 Control ofNew Mexican locust and the effect on planted ponderosa pine in central Arizona US For Ser Res Note RM-386 Rocky Mountain For Range ExpStn Fort Collins CO
Gosling PG Samuel YK Jones SK 1995 A systematic examination of germination temperature chipping and water temperaturesoak duration pretreatments on the seeds ofLeucaena leucocephala Seed Sci amp Technol 23521-532
Hamly DH 1932 Softening of the seeds of Melilotus alba Bot Gazette 93 345shy375
Hanna PJ 1984 Anatomical features of the seed coat ofAcacia kempeana (Mueller) which relate to increased germination rate induced by heat treatment New Phytol 96 23-29
Hanks JP Dick-Peddie WA 1974 Vegetation patterns of the White Mountains New Mexico Southwest Naturalist 18(4) 371-382
Hine S Harrington JT Dreesen DR 1997 Seed scarification requirements for Robinia neomexicana In Harrington JTtech coord Proc Third SouthwestemContainer Growers Conf Feb 12-13 1997 Ft Collins CO
Hopkinson JM 1993 The strophiole of leguminous seeds Newsletter- IntI Herbage Seed Production Res Grp 18 7-8
---- 1997 Differences in hardseededness between Cunningham and Tarramba leucaena In Leucnet News The Newsletter of the International LeucaenaRes and Devlt Netwk No4
ISTA (International Seed Testing Association) 1999 International rules for seed testing rules Seed Sci amp Technol27 Supplement
Kelly KM van Staden J 1987 The lens as the site of permeability in the papilionoid seed Aspalathus linearis J Plant Physiol 128(45) 395-404
73
Keresztesi B 1988 Black locust the tree of agriculture Outlook on Agr 17(2) 77~85
Kigel 1 and Galili G 1995 Seed development and germination Marcel Dekker New York 853 p
Klemmedson 10 1994 New Mexico locust and parent material Influence on macronutrients of forest floor and soil Soil Sci Soc Am 1 58 974~980
Kuo WH Tam AW 1988 The pathway of water absorption of mung bean seeds Seed Sci amp Technol16(1) 139~144
Leadem CL 1997 Dormancy--unlocking seed secrets In Landis TD Thompson lR tech coords National Proceedings Forestand Conservation Nursery Assocs Gen Tech Rep PNW-G TR-419 Portland OR USDA For Ser PNW Res Sta 43-52
Lin Y Wagner MR Seweyestewa A Honahni MD DeGomez M McMillin JD 1996 Influence of cultural treatments on seed germination and seedling growth of Robinia neomexicana Forest Farm and Community Tree Res Reports Volume 1
Marunda CT 1990 Effects of seed pretreatment on the development ofAcacia auriculforinis and A holosericea seedlings ACIAR Proceedings Series No 28
Meginnis HG 1937 Sulphuric acid treatment to increase germination of black locust seed USDA Circ 453 34 p
Miklas PN Towensend CE Ladd SL 1987 Seedcoat anatomy and the scarification ofcicer milkvetch seed Crop Sci 27 766-772
Monsen SB 1984 Use of shrubs on mine spoils In The Challenge ofProducing Native Plants for the Intermountain Area Proceedings Intermountain Nurserymans Association 1983 conference Las Vegas NV compo PM Murphy 26-31 Ogden UT USDA For Ser Gen Tech Report INT-l68
Olson DF 1974 Robinia L Locust In Seeds of Woody Plants in the US tech coord CS Schopmeyer 728-731 USDA For Ser Agr Handbk No450
Rehman S Loescher RNl Harris P1C 1998 Dormancy breaking and germination ofAcacia salicina Lindl seeds Seed Sci amp Techno127 553-557
Rolston MP 1978 Water impermeable seed dormancy Bot Rev 44 365-396
74
SAS Institute Inc 1989 SASSTAT Users Guide Version 6 4th Edition Volume 1 Cary NC SAS Institute Inc 943 p
SerratoValenti G Conara L Ferrando M Modenesi P 1993 Structural and histochemical features ofStylosanthes scabra (Leguminosae Papilionoidae) seed coat as related to water entry Can J Bot 71 834-840
Simpson BJ 1988 A field guide to Texas trees Austin TX Texas Monthly Press 372p
Tran VN Cavanagh AK 1984 Structural aspects ofdormancy In Seed Physiology Vol 2 Murray DR (ed) p1-44 Academic Press Australia
USDA Forest Service 1948 Woody-plant seed manual USDA Misc Pub1654 416p
Vines RA 1960 Trees Shrubs and Woody Vines of the Southwest Austin Univ ofTexas Press 1104 p
Wagner MR Larson RE Craig TP 1992 Ecology and silvics ofNew Mexico locust (Robinia neomexicana) with an emphasis on the arboreal form p53-57 In JW Hanover K Miller and S Plesko (eds) Proc Intemat Confon Black Locust Biology Culture and Utilization Mich State Univ USA
Wilson JK 1944 Immersing seeds of species of Robinia in boiling water hastens germination J For 42 453-454
Zimmerman RW Carpenter SB 1980 First-year coppice production from a 5shyyear old black locust stand on surface mine spoil p309-314 Centr Hardwd For ConfProc University ofMissouri Columbia
Nabil Y Khadduri John T Harrington2 Lee S Rosnet and David R Dreesen4
Hot water and sulfuric acid soaks are traditional treatments for seeds of many
temperate woody legwnes including locusts However these scarification techniques
often produce inconsistent germination Percussion scarification where seeds are
repeatedly propelled against a hard surface was compared with hot water
scarification to evaluate treatment efficacy for New Mexico locust (Robinia
neomexicana) and black locust CR pseudoacacia) seeds In the hot water treatment
seeds were placed in a 98degC water bath which was immediately removed from the
heat source For percussion scarification seeds were placed in a soil sample tin and
agitated in a paint shaker for 1 2 34 5 or 10 minutes All treatments including the
control were followed by 24-hour water soaks Hot water baths resulted in 56 and
41 germination for New Mexico locust and black locust respectively For both
species nearly all durations ofpercussion increased germination over the hot water
treatment Percussion durations of 4 5 and 10 minutes for New Mexico locust and 3
4 and 5 minutes for black locust resulted in at least 90 germination Traditional
scarification treatments randomly degrade the entire seed coat which can lead to
tissue damage during water uptake Percussion scarification specifically weakens the
strophiole the natural source ofwater entry to the seed in papilionoid legwnes
INabil Y Khadduri Graduate Research Assistant New Mexico State University Las Cruces NM 88003 nkhaddurihotmaiLcom
2John T Harrington Associate Professor New Mexico State University Mora Research Center Mora NM 87332 joharrinnmslledu 3Lee S Rosner Science Specialist New Mexico State University Mora NM 87332 Irosnernmsuedu 4David R Dreesen Agronomist USDA Natural Resources Conservation Service Los Lunas Plant Materials Center Los Lunas NM 87031
48
Following percussion imbibition is controlled through the strophiole and underlying
tissue is protected
Keywords New Mexico locust black locust scarification percussion germination
Introduction
Locust trees (Robinia spp) are aggressive pioneer species that quickly
colonize disturbed land fix atmospheric nitrogen in the soil and stabilize slopes that
are prone to erosion (Klemmedson 1994 Ashby et al 1985) (Figure A1ab)
Recognition of these attributes has contributed to increased demand for locusts in
reclamation projects Locust seeds however exhibit physical dormancy where a
thick seed coat prevents the movement of water and gasses to the embryo (Leadem
1997)
Sulfuric acid or hot water soaks have traditionally been used to break seed
dormancy in macro-propagation of locusts Many growers are moving away from the
use of su1furic acid in treating locust seeds (Dreesen and Harrington 1997) In
addition to being dangerous acid soak durations must be specifically correlated to
seed lot (Olson 1974) Collections of native seed can vary tremendously in seed size
seed weight and hard seededness etc This variability limits the utility of sulfuric
acid soaks as even within a given lot some seeds may be damaged whereas others
are under-treated and fail to imbibe (take up water) A survey of Southwestern
container growers indicates that hot water baths are currently the most common
scarification method for locust seeds (Hine et al 1997) However hot water baths
also produce inconsistent germination (Lin et al 1996)
49
b
Figure AI New Mexico locust a primary invader following disturbance Ala Crown-sprouting at edge ofburn near Los Alamos NM--one year after Cerro Grande fire Alb Colonizing road cut Gila National Forest NM
50
Percussion scarification where seeds are repeatedly propelled against a hard
surface is an alternative dormancy-breaking method that has proven successful in
legumes related to locusts (Hamly 1932 Barton 1947) We compared this alternative
treatment to hot water scarification to evaluate treatment efficacy for New Mexico
locust and black locust seeds
Materials and Methods
Commercial New Mexico locust seeds (Western Native Seed Coaldale CO)
were collected fall 2000 in Huerfano County CO Black locust seeds were collected
September 2000 from Taos County NM by harvesting ripe pods from several trees at
a distance of up to 3 m from ground level Black locust seeds were allowed to air-dry
for 3 weeks and were threshed and separated from large chaffusing a greenhouse
fan A South Dakota Seed Blower (Chicago Illinltois) was used to remove fine chaff
from both seed lots Seeds were storedmiddotat 2-4C until the germination study was
conducted in March 2001
This study compared hot water scarification with percussion scarification
using treatment and species as experimental factors Along with a control
scarification treatments included immersion of seed in boiling water as well as
percussion durations of 1 2 3 45 or 10 minutes Four 100-seed samples were used
to test each species by scarification treatment combination
For the hot water treatment test tubes were filled with 30 mL water and
placed in a water-filled beaker (Figure A2a) The bath was raised to boiling (-98C at
51
b
c
Figure A2 Implementation ofhot water and percussion scarification A2a Hot water bath A2b Paint-shaker set-up A2c Placing soil tin containing seeds at end ofpaint shaker ann
52
Las Cruces NM elevation of 1300 meters) Each 100-seed replicate was placed in a
test tube and each test tube was immediately removed to cool at room temperature
Percussion scarification was implemented using a pneumatic paint shaker
(Central Pneumatic model 00422 Camarillo CA) (Figure A2b) This allowed
standardization of treatment intensity Compressed air pressure was maintained at
530kPa plusmn 30kPa (80psi plusmn 5psi) resulting in approximately 350 oscillations per
minute Each lOO-seed sample was placed in a 118 ml (4 oz) soil tin for shaking To
maximize lateral movement of paint shaker a paint can was used as a spacer to place
soil tins at the end of the shaking arm (Figure A2c) This allowed shaking to occur at
the greatest distance from the pivot point (fulcrum) Following all treatments
including the control seeds were soaked for twenty-four hours in distilled water prior
to plating out
Gennination was evaluated on lab benches at room temperature Each
replicate of seeds was placed in a 100 cm petri dish on moistened filter paper with
humidity maintained by enclosing the petri dishes in ziplock bags Gennination was
monitored daily for 14 days in accordance with International Seed Testing
Association standards for Robinia species (ISTA 1999) Gennination was defined as
protrusion of the radicle from the embryo by at least 1mm as viewed by the naked
eye
Categorical Analysis of Variance using Prpc Catmod (SAS Institute 1989)
was used to detennine treatment differences in germination percentages for each
source This procedure is an extension of a Chi-square test of homogeneity using the
53
natural log of the ratio of germinated to non-germinated seeds for each treatment Pshy
values less than 005 were considered significant A limited set of pairwiseshy
comparisons was conducted to compare treatment mean percents using a
conservative alpha value of 005 divided by the number of comparisons
Results
Percussion maximized germination for both New Mexico locust and black
locust There were three germination response levels for New Mexico locust (Figure
A3) First there was a low response in the control indicating a dormant seed lot
The hot water treatment and I-minute duration of percussion share a second
intermediate response Germination was highest at percussion levels from 2 to 10
minutes
A selected percussion treatment intermediate in time (5 minutes) produced the
steepest germination curve indicating the fastest germination rate (Figure A4) The
hot water treatment produced an intermediate curve or relatively slower germination
rate The control curve was very flat indicating the slowest germination rate for New
Mexico locust
Control seeds germinated poorly for black locust as well (Figure A5) Hot
water scarification improved germination over the control but all levels of percussion
improved germination over hot water The optimal durations of percussion treatment
were bracketed with a significant increase from the 1 to 2 minute percussion level a
high germination response from 2 to 5 minutes of percussion and finally a significant
54
~ d 25 CL)
p t1 0 ~ t1
g Vl ~ Vl
100~---------------------------------------------------------------
80
60
40
20
o Control Hot Water Percussion
I min Percussion
2 min Percussion
3 min
Scarification
Percussion 4 min
Figure A3 Effect of scarification on germination response for New Mexico locust
Percussion 5 min
=significant difference between adjacent treatment bars ns = no significant difference
Percussion 10 min
~ ~ () u agt
Il-t d o ~
~ ~ c3
100middotlr~~~------------------------------------e- Percussion 5min middot 0middot Hot Water -- Control
80-J=middot======
6b-middot-lmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middot--middotmiddot-middot~-middotmiddot -middotmiddot middot middotmiddot-middot 1
40
20
-----~-----------
0 0 0 middotmiddotmiddotmiddotmiddot0middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot0
0 --~-----y-----~------_---~--- T-----~
o shy -TDay 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A4 Effect of selected scarification treatments on germination speed for New Mexico locust First seven days of fourteen-day experiment are shown
Day 7
80
~ cd
5 60 ()
[) Cl d 0 1t1 d
40sect dUl
-J
20
o
ns
Scarification
Figure A5 Effect of scarification on germination response for black locust = significant difference between adjacent treatment bars ns = no significant difference
decrease at the 10-minute level This drop-off suggests that 10 minutes of percussion
over-treats seeds from this lot of-black locust
A selected percussion treatment intermediate in time (5 minutes) also had the
fastest germination rate for black locust (Figure A6)_ The hot water curve is
somewhat flatter indicating a slower germination rate The control curve is very flat
indicating a slow germination rate
A qualitative difference between hot water and percussion-treated seeds was
also observed Along with healthy germinants the hot water treatment produced
over-treated seeds (Figure A 7a) Examples of damaged germinants include seeds
where the radicle did not elongate (Figure A 7b) seeds where the radicle elongated
but was delayed in freeing itself from the seed coat (Figure A7c) and seeds where the
radicle broke off completely from the embryo (Figure A7d) In addition to these
examples of over-treatment there were numerous small dark seeds that failed to
imbibe water For percussion treatment nearly all seeds took up water (Figure A8a)
Germination was rapid and uniform with little damage evident (Figure A8b)
To summarize both New Mexico locust and black locust seed lots were
dormant The standard hot water scarification treatment improved germination but
only to 56 and 41 for New Mexico locust and black locust respectively Nearly
all percussion durations improved total germination as well as germination rate over
the hot water treatment Germination was greater than 90 for percussion durations
of 4 5 and 10 minutes for New Mexico locust and 3 4 and 5 minutes for black locust
58
CI)
sgt t CI) 0
igt 0
sect ttl t E
Vl 0
c3
100r==~==~~=r------------------------------------~ ___ Percussion 5 min
middotmiddotmiddot0middotmiddotmiddot Hot Water -lIf- Control
80r=========~--~~----------------------------~
60
40
20 -~-~---~---------- ---~------------I
omiddot
O
_~_----------lIf-----~bull -----_ shy---shy I
Day 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A6 Effect of selected scarification treatments on germination speed for black locust First seven days of fourteen-day experiment are shown
Day 7
a
b c d
Figure A7 Damage to New Mexico locust seeds observed during germination counts following hot water scarification A7a Hot water replicate including damaged seedlings and numerous unimbibed seeds A7b Close-up ofseed where radicle failed to elongate A7c Close-up ofseed where radicle elongated but was delayed in freeing itself from the seed coat A7d Close-up ofseed where radicle completely broke offfrom embryo
60
a
b
Figure A8 New Mexico locust seeds following percussion treatment at time of germination counts A8a Percussion replicate with uniformly healthy seedlings and near complete imbibition of seeds A8b Close-up ofpercussion-treated seedling
61
Discussion
Papilionoid seeds have a specific anatomical feature known as the strophiole
or lens-the natural site of water entry to the seed (Hamly 1932) The strophiole is
located on the cotyledonary lobe of a locust seed (Figure A9) When a papilionoid
legume seed is percussed for the appropriate amount of time repeated hits on the
integrated seed coat loosen the constrained cells of the strophiolar region without
excessively damaging the rest of the seed coat (Ballard 1976)
When a percussed seed is soaked water enters exclusively through the
strophiole in a controlled manner (Kelly and Staden 1987) This regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues This
contrasts with seeds that have been hot water or acid scarified treatments that can
randomly degrade the seed coat Unlocalized cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rehman et al 1998 Marunda 1990)
Papilionoids are the largest subfamily of legumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust and black locust these legumes often play an integral role in the
revegetation ofdisturbed lands Developing superior scarification methods to the
standard hot water and acid treatments should fadlitate the use ofthese legumes in
restoration projects
62
Figure A9 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (bc)
63
APPENDIXB
GERMINATION PERCENTS AND STANDARD ERRORS FOR SOURCE
BLOCK AND TREATMENT FOR NEW MEXICO LOCUST
Table B 1 Gennination percents and standard errors for source block and treatment for New Mexico locust
Source Block Treatment Germination Percentage Standard Error
Capitan 1 Immersion 74 437 Capitan 2 Immersion 69 462 Capitan 3 Immersion 71 454 Capitan 4 Immersion 67 470 Capitan Boiling 30 sec 79 407 Capitan 2 Boiling 30 sec 76 427 Capitan 3 Boiling 30 sec 72 449 Capitan 4 Boiling 30 sec 76 427 Capitan Boiling 60 sec 71 454 Capitan 2 Boiling 60 sec 54 498 Capitan 3 Boiling 60 sec 67 470 Capitan 4 Boiling 60 sec 76 427 Capitan I Control 5 218 Capitan 2 Control 7 255 Capitan 3 Control 4 196 Capitan 4 Control 5 218 Capitan 1 Percussion I min 47 499 Capitan 2 Percussion I min
470
Capitan Percussion I min 39 488
Capitan 4 Percussion I min 38 485 Capitan I Percussion 2 min 55 497 Capitan Capitan
2
Percussion 2 min Percussion 2 min
71 65
454 477
Capitan 4 Percussion 2 min 56 496 Capitan I Percussion 4 min 86 347 Capitan 2 Percussion 4 min 79 407 Capitan 3 Percussion 4 min 81 392 Capitan 4 Percussion 4 min 82 384 Capitan I Percussion 8 min 90 300 Capitan 2 Percussion 8 min 88 325 Capitan 3 Percussion 8 min 90 300 Capitan 4 Percussion 8 min 90 300 Gila I Immersion 71 454 Gila 2 Immersion 77 421 Gila
Immersion 75 433
Gila 4 Immersion 72 449
Gila I Boiling 30 sec 68 466
Gila 2 Boiling 30 sec 81 392
Gila 3 Boiling 30 sec 54 498
Gila 4 Boiling 30 sec 63 483
Gila I Boiling 60 sec 55 497
Gila 2 Boiling 60 sec 68 466
Gila 3 Boiling 60 sec 57 495
Gila 4 Boiling 60 sec 70 458
65
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Gila 2 Control 13 336 Gila 3 Control 13 336 Gila 4 Control 20 400 Gila 1 Percussion 1 min 66 474 Gila 2 Percussion 1 min 57 495 Gila 3 Percussion 1 min 73 444 Gila 4 Percussion 1 min 82 384 Gila 1 Percussion 2 min 81 392 Gila 2 Percussion 2 min 86 347 Gila 3 Percussion 2 min 85 357 Gila 4 Percussion 2 min 54 498 Gila 1 Percussion 4 min 79 407 Gila 2 Percussion 4 min 83 376 Gila 3 Percussion 4 min 84 367 Gila 4 Percussion 4 min 79 407 Gila 1 Percussion 8 min 51 500 Gila 2 Percussion 8 min 76 427 Gila 3 Percussion 8 min 52 500 Gila 4 Percussion 8 min 74 439 Holman 1 Immersion 83 376 Holman 2 Immersion 86 347 Holman 3 Immersion 81 392 Holman 4 Immersion 90 300 Holman 1 Boiling 30 sec 90 300 Holman 2 Boiling 30 sec 85 357 Holman 3 Boiling 30 sec 87 336 Holman 4 Boiling 30 sec 83 376 Holman 1 Boiling 60 sec 79 407 Holman 2 Boiling 60 sec 80 400 Holman 3 Boiling 60 sec 86 347 Holman 4 Boiling 60 sec 93 255 Holman Control 25 433 Holman 2 Control 19 392 Holman Control 21 407
Holman 4 Control 24 427
Holman Percussion 1 min 77 421
Holman 2 Percussion 1 min 85 357
Holman 3 Percussion 1 min 80 400
Holman 4 Percussion 1 min 89 313
Holman 1 Percussion 2 min 88 325
Holman 2 Percussion 2 min 96 196
Holman 3 Percussion 2 min 93 255
Holman 4 Percussion 2 min 96 196
Holman 1 Percussion 4 min 92 271
Holman 2 Percussion 4 min 97 171
66
I
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Holman 3 Percussion 4 min 91 286 Holman 4 Percussion 4 min 96 196 Holman 1 Percussion 8 min 88 325 Holman 2 Percussion 8 min 85 357 Holman 3 Percussion 8 min 83 376 Holman 4 Percussion 8 min 82 384 Jemez 1 Immersion 53 499 Jemez 2 Immersion 44 496 Jemez 3 Immersion 52 500 Jemez 4 Immersion 70 458 Jemez 1 Boiling 30 sec 58 494 Jemez 2 Boiling 30 sec 55 497 Jemez 3 Boiling 30 sec 48 500 Jemez 4 Boiling 30 sec 65 477 Jemez 1 Boiling 60 sec 68 466 Jemez 2 Boiling 60 sec 63 483 Jemez 3 Boiling 60 sec 73 444 Jemez 4 Boiling 60 sec 89 313 Jemez 1 Control 2 140 Jemez 2 Control 9 286 Jemez 3 Control 6 237 Jemez 4 Control 8 271 Jemez 1 Percussion 1 min 58 494 Jemez 2 Percussion 1 min 63 483 Jemez 3 Percussion 1 min 77 421 Jemez 4 Percussion 1 min 89 313 Jemez 1 Percussion 2 min 68 466 Jemez 2 Percussion 2 min 64 480 Jemez 3 Percussion 2 min 80 400 Jemez 4 Percussion 2 min 83 376 Jemez Percussion 4 min 68 466 Jemez 2 Percussion 4 min 79 407 Jemez 3 Percussion 4 min 70 458 Jemez 4 Percussion 4 min 83 376 Jemez 1 Percussion 8 min 52 500 Jemez 2 Percussion 8 min 59 492 Jemez 3 Percussion 8 min 61 488 Jemez 4 Percussion 8 min 70 458 Magdalena 1 Immersion 69 462 Magdalena 2 Immersion 69 462 Magdalena J Immersion 70 458 Magdalena 4 Immersion 85 357 Magdalena 1 Boiling 30 sec 65 477 Magdalena 2 Boiling 30 sec 71 454 Magdalena 3 Boiling 30 sec 68 466
67
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Magdalena 4 Boiling 30 sec 83 376 Magdalena 1 Boiling 60 sec 63 483 Magdalena 2 Boiling 60 sec 68 466 Magdalena 3 Boiling 60 sec 63 483 Magdalena 4 Boiling 60 sec 82 384 Magdalena 1 Control 11 313 Magdalena 2 Control 6 237 Magdalena 3 Control 5 218 Magdalena 4 Control 4 196 Magdalena Percussion 1 min 32 466 Magdalena 2 Percussion 1 min 45 497 Magdalena 3 Percussion 1 min 46 498 Magdalena 4 Percussion I min 36 480 Magdalena 1 Percussion 2 min 67 470 Magdalena 2 Percussion 2 min 68 466 Magdalena 3 Percussion 2 min 65 477 Magdalena 4 Percussion 2 min 81 392 Magdalena 1 Percussion 4 min 87 336 Magdalena 2 Percussion 4 min 97 171 Magdalena 3 Percussion 4 min 83 376 Magdalena 4 Percussion 4 min 97 171 Magdalena 1 Percussion 8 min 98 lAO Magdalena 2 Percussion 8 min 100 000 Magdalena 3 Percussion 8 min 96 196 Magdalena 4 Percussion 8 min 98 lAO Manzano 1 Immersion 67 470 Manzano 2 Immersion 57 495 Manzano 3 Immersion 72 4049 Manzano 4 Immersion 73 4044 Manzano 1 Boiling 30 sec 70 458 Manzano 2 Boiling 30 sec 64 480 Manzano 3 Boiling 30 sec 71 454 Manzano 4 Boiling 30 sec 80 400 Manzano I Boiling 60 sec 50 500 Manzano 2 Boiling 60 sec 53 499 Manzano 3 Boiling 60 sec 59 492 Manzano 4 Boiling 60 sec 74 439 Manzano I Control 4 196 Manzano 2 Control 8 271 Manzano 3 Control 1 099 Manzano 4 Control 6 237 Manzano 1 Percussion 1 min 52 500 Manzano 2 Percussion 1 min 39 488 Manzano 3 Percussion 1 min 36 480 Manzano 4 Percussion 1 min 51 500
68
Table B 1 Continued
Source Block Treatment Gerrni~ation Percentage Standard Error
Manzano Percussion 2 min 53 499 Manzano 2 Percussion 2 min 58 494 Manzano 3 Percussion 2 min 61 488 Manzano 4 Percussion 2 min 62 485 Manzano 1 Percussion 4 min 76 427 Manzano 2 Percussion 4 min 85 357 Manzano 3 Percussion 4 min 79 407 Manzano 4 Percussion 4 min 83 376 Manzano 1 Percussion 8 min 87 336 Manzano 2 Percussion 8 min 83 376 Manzano 3 Percussion 8 min 83 376 Manzano 4 Percussion 8 min 75 433 Raton 1 Immersion 54 498 Raton 2 Immersion 37 483 Raton 3 Immersion 39 488 Raton 4 Immersion 69 462 Raton 1 Boiling 30 sec 36 480 Raton 2 Boiling 30 sec 20 400 Raton 3 Boiling 30 sec 28 449 Raton 4 Boiling 30 sec 60 490 Raton 1 Boiling 60 sec 15 357 Raton 2 Boiling 60 sec 171 Raton 3 Boiling 60 sec 4 196 Raton 4 Boiling 60 sec 35 477 Raton 1 Control 4 196 Raton 2 Control 12 325 Raton 3 Control 2 140 Raton 4 Control 6 237 Raton 1 Percussion 1 min 52 500 Raton 2 Percussion I min 29 454 Raton 3 Percussion I min 35 477 Raton 4 Percussion 1 min 31 462 Raton 1 Percussion 2 min 55 497 Raton 2 Percussion 2 min 35 477 Raton 3 Percussion 2 min 33 470 Raton 4 Percussion 2 min 42 494 Raton 1 Percussion 4 min 65 477 Raton 2 Percussion 4 min 52 500 Raton Percussion 4 min 51 500 Raton 4 Percussion 4 min 53 499 Raton 1 Percussion 8 min 88 325 Raton 2 Percussion 8 min 80 400 Raton 3 Percussion 8 min 73 444 Raton 4 Percussion 8 min 74 439 Sacramento 1 Immersion 43 495
69
Table B 1 Continued
-Source Block Treatment Germination Percentage Standard Error
Sacramento 2 Immersion 30 458 Sacramento 3 Immersion 38 485 Sacramento 4 Immersion 56 496 Sacramento 1 Boiling 30 sec 47 499 Sacramento 2 Boiling 30 sec 48 500 Sacramento 3 Boiling 30 sec 48 500 Sacramento 4 Boiling 30 sec 56 496 Sacramento 1 Boiling 60 sec 52 500 Sacramento 2 Boiling 60 sec 67 470 Sacramento 3 Boiling 60 sec 43 495 Sacramento 4 Boiling 60 sec 48 500 Sacramento 1 Control 0 000 Sacramento 2 Control 3 171 Sacramento 3 Control 3 171 Sacramento 4 Control 099 Sacramento 1 Percussion 1 min 57 495 Sacramento 2 Percussion 1 min 61 488 Sacramento 3 Percussion 1 min 62 485 Sacramento 4 Percussion 1 min 55 497 Sacramento 1 Percussion 2 min 84 367 Sacramento 2 Percussion 2 min 70 458 Sacramento 3 Percussion 2 min 81 392 Sacramento 4 Percussion 2 min 69 462 Sacramento 1 Percussion 4 min 91 286 Sacramento 2 Percussion 4 min 84 367 Sacramento 3 Percussion 4 min 84 367 Sacramento 4 Percussion 4 min 94 237 Sacramento 1 Percussion 8 min 82 384 Sacramento 2 Percussion 8 min 81 392 Sacramento 3 Percussion 8 min 82 384 Sacramento 4 Percussion 8 min 81 392 Sandia 1 Immersion 76 427 Sandia 2 Immersion 70 458 Sandia 3 Immersion 70 458 Sandia 4 Immersion 81 392 Sandia 1 Boiling 30 sec 78 414 Sandia 2 Boiling 30 sec 71 454 Sandia 3 Boiling 30 sec 63 483 Sandia 4 Boiling 30 sec 78 414 Sandia 1 Boiling 60 sec 67 470 Sandia 2 Boiling 60 sec 65 477 Sandia 3 Boiling 60 sec 74 439 Sandia 4 Boiling 60 sec 66 474 Sandia 1 Control 16 367 Sandia 2 Control 7 255
70
Table B 1 Continued
Source Block Treatment Gennination Percentage Standard Error
Sandia 3 Control 8 271 Sandia 4 Control 16 367 Sandia 1 Percussion 1 min 67 470 Sandia 2 Percussion 1 min 61 488 Sandia 3 Percussion 1 min 66 474 Sandia 4 Percussion 1 min 59 492 Sandia I Percussion 2 min 82 384 Sandia 2 Percussion 2 min 83 376 Sandia 3 Percussion 2 min 81 392 Sandia 4 Percussion 2 min 84 367 Sandia 1 Percussion 4 min 89 313 Sandia 2 Percussion 4 min 86 347 Sandia 3 Percussion 4 min 89 313 Sandia 4 Percussion 4 min 85 357 Sandia 1 Percussion 8 min 80 400 Sandia 2 Percussion 8 min 77 421 Sandia 3 Percussion 8 min 69 462 Sandia 4 Percussion 8 min 66 474
LITERATURE CITED
Ashby WC Vogel WG Rogers NF 1985 Black locust in the reclamation equation General Technical Report Northeastern Forest Experiment Station USDA Forest Service NE-I05 12 p
Ballard LAT 1973 Physical barriers to gennination Seed Sci Technoll 285-303
___ 1976 Strophiolar water conduction in the seeds ofTrifolieae induced by action on the testa at nonstrophiolar sites Austr J Plant Physiol3 465-469
Barton LV 1947 Special studies on seed coat impenneability Contrib Boyce Thompson Inst 14 355-362
Baskin CC Baskin JM 1998 Seeds Ecology biogeography and evolution of donnancy and gennination Academic Press London UK 666 p
Bewley JD 1997 Seed germination and donnancy Plant Ce119 1055-1066
Bewley JD Black M 1994 Seeds physiology of development and germination 2nd ed Plenum Press New York NY 445 p
Briscoe CB 1996 Growing Nitrogen Fixing Trees in Acid Soils In MH Powell (ed) Nitrogen Fixing Trees for Acid Soils- A Field ManuaL Winrock International Morrilton AR 22 p
Copeland LO McDonald MB 1995 Principles of Seed Science and Technology 3rded Chapman amp Hall New York NY 409 p
Cox RE Klett JE 1984 Seed gennination requirements of native Colorado plants for use in the landscape Plant Propag 30(2) 6-10
Dell B 1980 Structure and function ofthe strophiolar plug in seeds ofAlbizia Lophantha Am J Bot 67 556-563
Dick-Peddie WA 1993 New Mexico Vegetation Past Present and Future University ofNew Mexico Press Albuquerque NM 244 p
Dreesen DR 2001 Propagation protocol for production ofcontainer Robinia neomexicana plants Los Lunas Plant Materials Center Los Lunas New Mexico In Native Plant Network URL httpwwwnativeplantnetworkorg (accessed 9 January 2002) Moscow (ID) University ofIdaho CoIl Natural Resources For Res Nursery
72
Dreesen DR Harrington JT 1997 Propagation of native plants for restoration projects in the Southwestern US- Preliminary Investigations In Landis TD Thompson JR tech coords National Proceedings Forest and Conservation Nursery Assocs Gen Tech Rep PNW-GTR-419 Portland OR USDA For Ser PNW Res Sta 77-88
Gottfried GJ 1980 Control ofNew Mexican locust and the effect on planted ponderosa pine in central Arizona US For Ser Res Note RM-386 Rocky Mountain For Range ExpStn Fort Collins CO
Gosling PG Samuel YK Jones SK 1995 A systematic examination of germination temperature chipping and water temperaturesoak duration pretreatments on the seeds ofLeucaena leucocephala Seed Sci amp Technol 23521-532
Hamly DH 1932 Softening of the seeds of Melilotus alba Bot Gazette 93 345shy375
Hanna PJ 1984 Anatomical features of the seed coat ofAcacia kempeana (Mueller) which relate to increased germination rate induced by heat treatment New Phytol 96 23-29
Hanks JP Dick-Peddie WA 1974 Vegetation patterns of the White Mountains New Mexico Southwest Naturalist 18(4) 371-382
Hine S Harrington JT Dreesen DR 1997 Seed scarification requirements for Robinia neomexicana In Harrington JTtech coord Proc Third SouthwestemContainer Growers Conf Feb 12-13 1997 Ft Collins CO
Hopkinson JM 1993 The strophiole of leguminous seeds Newsletter- IntI Herbage Seed Production Res Grp 18 7-8
---- 1997 Differences in hardseededness between Cunningham and Tarramba leucaena In Leucnet News The Newsletter of the International LeucaenaRes and Devlt Netwk No4
ISTA (International Seed Testing Association) 1999 International rules for seed testing rules Seed Sci amp Technol27 Supplement
Kelly KM van Staden J 1987 The lens as the site of permeability in the papilionoid seed Aspalathus linearis J Plant Physiol 128(45) 395-404
73
Keresztesi B 1988 Black locust the tree of agriculture Outlook on Agr 17(2) 77~85
Kigel 1 and Galili G 1995 Seed development and germination Marcel Dekker New York 853 p
Klemmedson 10 1994 New Mexico locust and parent material Influence on macronutrients of forest floor and soil Soil Sci Soc Am 1 58 974~980
Kuo WH Tam AW 1988 The pathway of water absorption of mung bean seeds Seed Sci amp Technol16(1) 139~144
Leadem CL 1997 Dormancy--unlocking seed secrets In Landis TD Thompson lR tech coords National Proceedings Forestand Conservation Nursery Assocs Gen Tech Rep PNW-G TR-419 Portland OR USDA For Ser PNW Res Sta 43-52
Lin Y Wagner MR Seweyestewa A Honahni MD DeGomez M McMillin JD 1996 Influence of cultural treatments on seed germination and seedling growth of Robinia neomexicana Forest Farm and Community Tree Res Reports Volume 1
Marunda CT 1990 Effects of seed pretreatment on the development ofAcacia auriculforinis and A holosericea seedlings ACIAR Proceedings Series No 28
Meginnis HG 1937 Sulphuric acid treatment to increase germination of black locust seed USDA Circ 453 34 p
Miklas PN Towensend CE Ladd SL 1987 Seedcoat anatomy and the scarification ofcicer milkvetch seed Crop Sci 27 766-772
Monsen SB 1984 Use of shrubs on mine spoils In The Challenge ofProducing Native Plants for the Intermountain Area Proceedings Intermountain Nurserymans Association 1983 conference Las Vegas NV compo PM Murphy 26-31 Ogden UT USDA For Ser Gen Tech Report INT-l68
Olson DF 1974 Robinia L Locust In Seeds of Woody Plants in the US tech coord CS Schopmeyer 728-731 USDA For Ser Agr Handbk No450
Rehman S Loescher RNl Harris P1C 1998 Dormancy breaking and germination ofAcacia salicina Lindl seeds Seed Sci amp Techno127 553-557
Rolston MP 1978 Water impermeable seed dormancy Bot Rev 44 365-396
74
SAS Institute Inc 1989 SASSTAT Users Guide Version 6 4th Edition Volume 1 Cary NC SAS Institute Inc 943 p
SerratoValenti G Conara L Ferrando M Modenesi P 1993 Structural and histochemical features ofStylosanthes scabra (Leguminosae Papilionoidae) seed coat as related to water entry Can J Bot 71 834-840
Simpson BJ 1988 A field guide to Texas trees Austin TX Texas Monthly Press 372p
Tran VN Cavanagh AK 1984 Structural aspects ofdormancy In Seed Physiology Vol 2 Murray DR (ed) p1-44 Academic Press Australia
USDA Forest Service 1948 Woody-plant seed manual USDA Misc Pub1654 416p
Vines RA 1960 Trees Shrubs and Woody Vines of the Southwest Austin Univ ofTexas Press 1104 p
Wagner MR Larson RE Craig TP 1992 Ecology and silvics ofNew Mexico locust (Robinia neomexicana) with an emphasis on the arboreal form p53-57 In JW Hanover K Miller and S Plesko (eds) Proc Intemat Confon Black Locust Biology Culture and Utilization Mich State Univ USA
Wilson JK 1944 Immersing seeds of species of Robinia in boiling water hastens germination J For 42 453-454
Zimmerman RW Carpenter SB 1980 First-year coppice production from a 5shyyear old black locust stand on surface mine spoil p309-314 Centr Hardwd For ConfProc University ofMissouri Columbia
Following percussion imbibition is controlled through the strophiole and underlying
tissue is protected
Keywords New Mexico locust black locust scarification percussion germination
Introduction
Locust trees (Robinia spp) are aggressive pioneer species that quickly
colonize disturbed land fix atmospheric nitrogen in the soil and stabilize slopes that
are prone to erosion (Klemmedson 1994 Ashby et al 1985) (Figure A1ab)
Recognition of these attributes has contributed to increased demand for locusts in
reclamation projects Locust seeds however exhibit physical dormancy where a
thick seed coat prevents the movement of water and gasses to the embryo (Leadem
1997)
Sulfuric acid or hot water soaks have traditionally been used to break seed
dormancy in macro-propagation of locusts Many growers are moving away from the
use of su1furic acid in treating locust seeds (Dreesen and Harrington 1997) In
addition to being dangerous acid soak durations must be specifically correlated to
seed lot (Olson 1974) Collections of native seed can vary tremendously in seed size
seed weight and hard seededness etc This variability limits the utility of sulfuric
acid soaks as even within a given lot some seeds may be damaged whereas others
are under-treated and fail to imbibe (take up water) A survey of Southwestern
container growers indicates that hot water baths are currently the most common
scarification method for locust seeds (Hine et al 1997) However hot water baths
also produce inconsistent germination (Lin et al 1996)
49
b
Figure AI New Mexico locust a primary invader following disturbance Ala Crown-sprouting at edge ofburn near Los Alamos NM--one year after Cerro Grande fire Alb Colonizing road cut Gila National Forest NM
50
Percussion scarification where seeds are repeatedly propelled against a hard
surface is an alternative dormancy-breaking method that has proven successful in
legumes related to locusts (Hamly 1932 Barton 1947) We compared this alternative
treatment to hot water scarification to evaluate treatment efficacy for New Mexico
locust and black locust seeds
Materials and Methods
Commercial New Mexico locust seeds (Western Native Seed Coaldale CO)
were collected fall 2000 in Huerfano County CO Black locust seeds were collected
September 2000 from Taos County NM by harvesting ripe pods from several trees at
a distance of up to 3 m from ground level Black locust seeds were allowed to air-dry
for 3 weeks and were threshed and separated from large chaffusing a greenhouse
fan A South Dakota Seed Blower (Chicago Illinltois) was used to remove fine chaff
from both seed lots Seeds were storedmiddotat 2-4C until the germination study was
conducted in March 2001
This study compared hot water scarification with percussion scarification
using treatment and species as experimental factors Along with a control
scarification treatments included immersion of seed in boiling water as well as
percussion durations of 1 2 3 45 or 10 minutes Four 100-seed samples were used
to test each species by scarification treatment combination
For the hot water treatment test tubes were filled with 30 mL water and
placed in a water-filled beaker (Figure A2a) The bath was raised to boiling (-98C at
51
b
c
Figure A2 Implementation ofhot water and percussion scarification A2a Hot water bath A2b Paint-shaker set-up A2c Placing soil tin containing seeds at end ofpaint shaker ann
52
Las Cruces NM elevation of 1300 meters) Each 100-seed replicate was placed in a
test tube and each test tube was immediately removed to cool at room temperature
Percussion scarification was implemented using a pneumatic paint shaker
(Central Pneumatic model 00422 Camarillo CA) (Figure A2b) This allowed
standardization of treatment intensity Compressed air pressure was maintained at
530kPa plusmn 30kPa (80psi plusmn 5psi) resulting in approximately 350 oscillations per
minute Each lOO-seed sample was placed in a 118 ml (4 oz) soil tin for shaking To
maximize lateral movement of paint shaker a paint can was used as a spacer to place
soil tins at the end of the shaking arm (Figure A2c) This allowed shaking to occur at
the greatest distance from the pivot point (fulcrum) Following all treatments
including the control seeds were soaked for twenty-four hours in distilled water prior
to plating out
Gennination was evaluated on lab benches at room temperature Each
replicate of seeds was placed in a 100 cm petri dish on moistened filter paper with
humidity maintained by enclosing the petri dishes in ziplock bags Gennination was
monitored daily for 14 days in accordance with International Seed Testing
Association standards for Robinia species (ISTA 1999) Gennination was defined as
protrusion of the radicle from the embryo by at least 1mm as viewed by the naked
eye
Categorical Analysis of Variance using Prpc Catmod (SAS Institute 1989)
was used to detennine treatment differences in germination percentages for each
source This procedure is an extension of a Chi-square test of homogeneity using the
53
natural log of the ratio of germinated to non-germinated seeds for each treatment Pshy
values less than 005 were considered significant A limited set of pairwiseshy
comparisons was conducted to compare treatment mean percents using a
conservative alpha value of 005 divided by the number of comparisons
Results
Percussion maximized germination for both New Mexico locust and black
locust There were three germination response levels for New Mexico locust (Figure
A3) First there was a low response in the control indicating a dormant seed lot
The hot water treatment and I-minute duration of percussion share a second
intermediate response Germination was highest at percussion levels from 2 to 10
minutes
A selected percussion treatment intermediate in time (5 minutes) produced the
steepest germination curve indicating the fastest germination rate (Figure A4) The
hot water treatment produced an intermediate curve or relatively slower germination
rate The control curve was very flat indicating the slowest germination rate for New
Mexico locust
Control seeds germinated poorly for black locust as well (Figure A5) Hot
water scarification improved germination over the control but all levels of percussion
improved germination over hot water The optimal durations of percussion treatment
were bracketed with a significant increase from the 1 to 2 minute percussion level a
high germination response from 2 to 5 minutes of percussion and finally a significant
54
~ d 25 CL)
p t1 0 ~ t1
g Vl ~ Vl
100~---------------------------------------------------------------
80
60
40
20
o Control Hot Water Percussion
I min Percussion
2 min Percussion
3 min
Scarification
Percussion 4 min
Figure A3 Effect of scarification on germination response for New Mexico locust
Percussion 5 min
=significant difference between adjacent treatment bars ns = no significant difference
Percussion 10 min
~ ~ () u agt
Il-t d o ~
~ ~ c3
100middotlr~~~------------------------------------e- Percussion 5min middot 0middot Hot Water -- Control
80-J=middot======
6b-middot-lmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middot--middotmiddot-middot~-middotmiddot -middotmiddot middot middotmiddot-middot 1
40
20
-----~-----------
0 0 0 middotmiddotmiddotmiddotmiddot0middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot0
0 --~-----y-----~------_---~--- T-----~
o shy -TDay 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A4 Effect of selected scarification treatments on germination speed for New Mexico locust First seven days of fourteen-day experiment are shown
Day 7
80
~ cd
5 60 ()
[) Cl d 0 1t1 d
40sect dUl
-J
20
o
ns
Scarification
Figure A5 Effect of scarification on germination response for black locust = significant difference between adjacent treatment bars ns = no significant difference
decrease at the 10-minute level This drop-off suggests that 10 minutes of percussion
over-treats seeds from this lot of-black locust
A selected percussion treatment intermediate in time (5 minutes) also had the
fastest germination rate for black locust (Figure A6)_ The hot water curve is
somewhat flatter indicating a slower germination rate The control curve is very flat
indicating a slow germination rate
A qualitative difference between hot water and percussion-treated seeds was
also observed Along with healthy germinants the hot water treatment produced
over-treated seeds (Figure A 7a) Examples of damaged germinants include seeds
where the radicle did not elongate (Figure A 7b) seeds where the radicle elongated
but was delayed in freeing itself from the seed coat (Figure A7c) and seeds where the
radicle broke off completely from the embryo (Figure A7d) In addition to these
examples of over-treatment there were numerous small dark seeds that failed to
imbibe water For percussion treatment nearly all seeds took up water (Figure A8a)
Germination was rapid and uniform with little damage evident (Figure A8b)
To summarize both New Mexico locust and black locust seed lots were
dormant The standard hot water scarification treatment improved germination but
only to 56 and 41 for New Mexico locust and black locust respectively Nearly
all percussion durations improved total germination as well as germination rate over
the hot water treatment Germination was greater than 90 for percussion durations
of 4 5 and 10 minutes for New Mexico locust and 3 4 and 5 minutes for black locust
58
CI)
sgt t CI) 0
igt 0
sect ttl t E
Vl 0
c3
100r==~==~~=r------------------------------------~ ___ Percussion 5 min
middotmiddotmiddot0middotmiddotmiddot Hot Water -lIf- Control
80r=========~--~~----------------------------~
60
40
20 -~-~---~---------- ---~------------I
omiddot
O
_~_----------lIf-----~bull -----_ shy---shy I
Day 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A6 Effect of selected scarification treatments on germination speed for black locust First seven days of fourteen-day experiment are shown
Day 7
a
b c d
Figure A7 Damage to New Mexico locust seeds observed during germination counts following hot water scarification A7a Hot water replicate including damaged seedlings and numerous unimbibed seeds A7b Close-up ofseed where radicle failed to elongate A7c Close-up ofseed where radicle elongated but was delayed in freeing itself from the seed coat A7d Close-up ofseed where radicle completely broke offfrom embryo
60
a
b
Figure A8 New Mexico locust seeds following percussion treatment at time of germination counts A8a Percussion replicate with uniformly healthy seedlings and near complete imbibition of seeds A8b Close-up ofpercussion-treated seedling
61
Discussion
Papilionoid seeds have a specific anatomical feature known as the strophiole
or lens-the natural site of water entry to the seed (Hamly 1932) The strophiole is
located on the cotyledonary lobe of a locust seed (Figure A9) When a papilionoid
legume seed is percussed for the appropriate amount of time repeated hits on the
integrated seed coat loosen the constrained cells of the strophiolar region without
excessively damaging the rest of the seed coat (Ballard 1976)
When a percussed seed is soaked water enters exclusively through the
strophiole in a controlled manner (Kelly and Staden 1987) This regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues This
contrasts with seeds that have been hot water or acid scarified treatments that can
randomly degrade the seed coat Unlocalized cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rehman et al 1998 Marunda 1990)
Papilionoids are the largest subfamily of legumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust and black locust these legumes often play an integral role in the
revegetation ofdisturbed lands Developing superior scarification methods to the
standard hot water and acid treatments should fadlitate the use ofthese legumes in
restoration projects
62
Figure A9 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (bc)
63
APPENDIXB
GERMINATION PERCENTS AND STANDARD ERRORS FOR SOURCE
BLOCK AND TREATMENT FOR NEW MEXICO LOCUST
Table B 1 Gennination percents and standard errors for source block and treatment for New Mexico locust
Source Block Treatment Germination Percentage Standard Error
Capitan 1 Immersion 74 437 Capitan 2 Immersion 69 462 Capitan 3 Immersion 71 454 Capitan 4 Immersion 67 470 Capitan Boiling 30 sec 79 407 Capitan 2 Boiling 30 sec 76 427 Capitan 3 Boiling 30 sec 72 449 Capitan 4 Boiling 30 sec 76 427 Capitan Boiling 60 sec 71 454 Capitan 2 Boiling 60 sec 54 498 Capitan 3 Boiling 60 sec 67 470 Capitan 4 Boiling 60 sec 76 427 Capitan I Control 5 218 Capitan 2 Control 7 255 Capitan 3 Control 4 196 Capitan 4 Control 5 218 Capitan 1 Percussion I min 47 499 Capitan 2 Percussion I min
470
Capitan Percussion I min 39 488
Capitan 4 Percussion I min 38 485 Capitan I Percussion 2 min 55 497 Capitan Capitan
2
Percussion 2 min Percussion 2 min
71 65
454 477
Capitan 4 Percussion 2 min 56 496 Capitan I Percussion 4 min 86 347 Capitan 2 Percussion 4 min 79 407 Capitan 3 Percussion 4 min 81 392 Capitan 4 Percussion 4 min 82 384 Capitan I Percussion 8 min 90 300 Capitan 2 Percussion 8 min 88 325 Capitan 3 Percussion 8 min 90 300 Capitan 4 Percussion 8 min 90 300 Gila I Immersion 71 454 Gila 2 Immersion 77 421 Gila
Immersion 75 433
Gila 4 Immersion 72 449
Gila I Boiling 30 sec 68 466
Gila 2 Boiling 30 sec 81 392
Gila 3 Boiling 30 sec 54 498
Gila 4 Boiling 30 sec 63 483
Gila I Boiling 60 sec 55 497
Gila 2 Boiling 60 sec 68 466
Gila 3 Boiling 60 sec 57 495
Gila 4 Boiling 60 sec 70 458
65
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Gila 2 Control 13 336 Gila 3 Control 13 336 Gila 4 Control 20 400 Gila 1 Percussion 1 min 66 474 Gila 2 Percussion 1 min 57 495 Gila 3 Percussion 1 min 73 444 Gila 4 Percussion 1 min 82 384 Gila 1 Percussion 2 min 81 392 Gila 2 Percussion 2 min 86 347 Gila 3 Percussion 2 min 85 357 Gila 4 Percussion 2 min 54 498 Gila 1 Percussion 4 min 79 407 Gila 2 Percussion 4 min 83 376 Gila 3 Percussion 4 min 84 367 Gila 4 Percussion 4 min 79 407 Gila 1 Percussion 8 min 51 500 Gila 2 Percussion 8 min 76 427 Gila 3 Percussion 8 min 52 500 Gila 4 Percussion 8 min 74 439 Holman 1 Immersion 83 376 Holman 2 Immersion 86 347 Holman 3 Immersion 81 392 Holman 4 Immersion 90 300 Holman 1 Boiling 30 sec 90 300 Holman 2 Boiling 30 sec 85 357 Holman 3 Boiling 30 sec 87 336 Holman 4 Boiling 30 sec 83 376 Holman 1 Boiling 60 sec 79 407 Holman 2 Boiling 60 sec 80 400 Holman 3 Boiling 60 sec 86 347 Holman 4 Boiling 60 sec 93 255 Holman Control 25 433 Holman 2 Control 19 392 Holman Control 21 407
Holman 4 Control 24 427
Holman Percussion 1 min 77 421
Holman 2 Percussion 1 min 85 357
Holman 3 Percussion 1 min 80 400
Holman 4 Percussion 1 min 89 313
Holman 1 Percussion 2 min 88 325
Holman 2 Percussion 2 min 96 196
Holman 3 Percussion 2 min 93 255
Holman 4 Percussion 2 min 96 196
Holman 1 Percussion 4 min 92 271
Holman 2 Percussion 4 min 97 171
66
I
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Holman 3 Percussion 4 min 91 286 Holman 4 Percussion 4 min 96 196 Holman 1 Percussion 8 min 88 325 Holman 2 Percussion 8 min 85 357 Holman 3 Percussion 8 min 83 376 Holman 4 Percussion 8 min 82 384 Jemez 1 Immersion 53 499 Jemez 2 Immersion 44 496 Jemez 3 Immersion 52 500 Jemez 4 Immersion 70 458 Jemez 1 Boiling 30 sec 58 494 Jemez 2 Boiling 30 sec 55 497 Jemez 3 Boiling 30 sec 48 500 Jemez 4 Boiling 30 sec 65 477 Jemez 1 Boiling 60 sec 68 466 Jemez 2 Boiling 60 sec 63 483 Jemez 3 Boiling 60 sec 73 444 Jemez 4 Boiling 60 sec 89 313 Jemez 1 Control 2 140 Jemez 2 Control 9 286 Jemez 3 Control 6 237 Jemez 4 Control 8 271 Jemez 1 Percussion 1 min 58 494 Jemez 2 Percussion 1 min 63 483 Jemez 3 Percussion 1 min 77 421 Jemez 4 Percussion 1 min 89 313 Jemez 1 Percussion 2 min 68 466 Jemez 2 Percussion 2 min 64 480 Jemez 3 Percussion 2 min 80 400 Jemez 4 Percussion 2 min 83 376 Jemez Percussion 4 min 68 466 Jemez 2 Percussion 4 min 79 407 Jemez 3 Percussion 4 min 70 458 Jemez 4 Percussion 4 min 83 376 Jemez 1 Percussion 8 min 52 500 Jemez 2 Percussion 8 min 59 492 Jemez 3 Percussion 8 min 61 488 Jemez 4 Percussion 8 min 70 458 Magdalena 1 Immersion 69 462 Magdalena 2 Immersion 69 462 Magdalena J Immersion 70 458 Magdalena 4 Immersion 85 357 Magdalena 1 Boiling 30 sec 65 477 Magdalena 2 Boiling 30 sec 71 454 Magdalena 3 Boiling 30 sec 68 466
67
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Magdalena 4 Boiling 30 sec 83 376 Magdalena 1 Boiling 60 sec 63 483 Magdalena 2 Boiling 60 sec 68 466 Magdalena 3 Boiling 60 sec 63 483 Magdalena 4 Boiling 60 sec 82 384 Magdalena 1 Control 11 313 Magdalena 2 Control 6 237 Magdalena 3 Control 5 218 Magdalena 4 Control 4 196 Magdalena Percussion 1 min 32 466 Magdalena 2 Percussion 1 min 45 497 Magdalena 3 Percussion 1 min 46 498 Magdalena 4 Percussion I min 36 480 Magdalena 1 Percussion 2 min 67 470 Magdalena 2 Percussion 2 min 68 466 Magdalena 3 Percussion 2 min 65 477 Magdalena 4 Percussion 2 min 81 392 Magdalena 1 Percussion 4 min 87 336 Magdalena 2 Percussion 4 min 97 171 Magdalena 3 Percussion 4 min 83 376 Magdalena 4 Percussion 4 min 97 171 Magdalena 1 Percussion 8 min 98 lAO Magdalena 2 Percussion 8 min 100 000 Magdalena 3 Percussion 8 min 96 196 Magdalena 4 Percussion 8 min 98 lAO Manzano 1 Immersion 67 470 Manzano 2 Immersion 57 495 Manzano 3 Immersion 72 4049 Manzano 4 Immersion 73 4044 Manzano 1 Boiling 30 sec 70 458 Manzano 2 Boiling 30 sec 64 480 Manzano 3 Boiling 30 sec 71 454 Manzano 4 Boiling 30 sec 80 400 Manzano I Boiling 60 sec 50 500 Manzano 2 Boiling 60 sec 53 499 Manzano 3 Boiling 60 sec 59 492 Manzano 4 Boiling 60 sec 74 439 Manzano I Control 4 196 Manzano 2 Control 8 271 Manzano 3 Control 1 099 Manzano 4 Control 6 237 Manzano 1 Percussion 1 min 52 500 Manzano 2 Percussion 1 min 39 488 Manzano 3 Percussion 1 min 36 480 Manzano 4 Percussion 1 min 51 500
68
Table B 1 Continued
Source Block Treatment Gerrni~ation Percentage Standard Error
Manzano Percussion 2 min 53 499 Manzano 2 Percussion 2 min 58 494 Manzano 3 Percussion 2 min 61 488 Manzano 4 Percussion 2 min 62 485 Manzano 1 Percussion 4 min 76 427 Manzano 2 Percussion 4 min 85 357 Manzano 3 Percussion 4 min 79 407 Manzano 4 Percussion 4 min 83 376 Manzano 1 Percussion 8 min 87 336 Manzano 2 Percussion 8 min 83 376 Manzano 3 Percussion 8 min 83 376 Manzano 4 Percussion 8 min 75 433 Raton 1 Immersion 54 498 Raton 2 Immersion 37 483 Raton 3 Immersion 39 488 Raton 4 Immersion 69 462 Raton 1 Boiling 30 sec 36 480 Raton 2 Boiling 30 sec 20 400 Raton 3 Boiling 30 sec 28 449 Raton 4 Boiling 30 sec 60 490 Raton 1 Boiling 60 sec 15 357 Raton 2 Boiling 60 sec 171 Raton 3 Boiling 60 sec 4 196 Raton 4 Boiling 60 sec 35 477 Raton 1 Control 4 196 Raton 2 Control 12 325 Raton 3 Control 2 140 Raton 4 Control 6 237 Raton 1 Percussion 1 min 52 500 Raton 2 Percussion I min 29 454 Raton 3 Percussion I min 35 477 Raton 4 Percussion 1 min 31 462 Raton 1 Percussion 2 min 55 497 Raton 2 Percussion 2 min 35 477 Raton 3 Percussion 2 min 33 470 Raton 4 Percussion 2 min 42 494 Raton 1 Percussion 4 min 65 477 Raton 2 Percussion 4 min 52 500 Raton Percussion 4 min 51 500 Raton 4 Percussion 4 min 53 499 Raton 1 Percussion 8 min 88 325 Raton 2 Percussion 8 min 80 400 Raton 3 Percussion 8 min 73 444 Raton 4 Percussion 8 min 74 439 Sacramento 1 Immersion 43 495
69
Table B 1 Continued
-Source Block Treatment Germination Percentage Standard Error
Sacramento 2 Immersion 30 458 Sacramento 3 Immersion 38 485 Sacramento 4 Immersion 56 496 Sacramento 1 Boiling 30 sec 47 499 Sacramento 2 Boiling 30 sec 48 500 Sacramento 3 Boiling 30 sec 48 500 Sacramento 4 Boiling 30 sec 56 496 Sacramento 1 Boiling 60 sec 52 500 Sacramento 2 Boiling 60 sec 67 470 Sacramento 3 Boiling 60 sec 43 495 Sacramento 4 Boiling 60 sec 48 500 Sacramento 1 Control 0 000 Sacramento 2 Control 3 171 Sacramento 3 Control 3 171 Sacramento 4 Control 099 Sacramento 1 Percussion 1 min 57 495 Sacramento 2 Percussion 1 min 61 488 Sacramento 3 Percussion 1 min 62 485 Sacramento 4 Percussion 1 min 55 497 Sacramento 1 Percussion 2 min 84 367 Sacramento 2 Percussion 2 min 70 458 Sacramento 3 Percussion 2 min 81 392 Sacramento 4 Percussion 2 min 69 462 Sacramento 1 Percussion 4 min 91 286 Sacramento 2 Percussion 4 min 84 367 Sacramento 3 Percussion 4 min 84 367 Sacramento 4 Percussion 4 min 94 237 Sacramento 1 Percussion 8 min 82 384 Sacramento 2 Percussion 8 min 81 392 Sacramento 3 Percussion 8 min 82 384 Sacramento 4 Percussion 8 min 81 392 Sandia 1 Immersion 76 427 Sandia 2 Immersion 70 458 Sandia 3 Immersion 70 458 Sandia 4 Immersion 81 392 Sandia 1 Boiling 30 sec 78 414 Sandia 2 Boiling 30 sec 71 454 Sandia 3 Boiling 30 sec 63 483 Sandia 4 Boiling 30 sec 78 414 Sandia 1 Boiling 60 sec 67 470 Sandia 2 Boiling 60 sec 65 477 Sandia 3 Boiling 60 sec 74 439 Sandia 4 Boiling 60 sec 66 474 Sandia 1 Control 16 367 Sandia 2 Control 7 255
70
Table B 1 Continued
Source Block Treatment Gennination Percentage Standard Error
Sandia 3 Control 8 271 Sandia 4 Control 16 367 Sandia 1 Percussion 1 min 67 470 Sandia 2 Percussion 1 min 61 488 Sandia 3 Percussion 1 min 66 474 Sandia 4 Percussion 1 min 59 492 Sandia I Percussion 2 min 82 384 Sandia 2 Percussion 2 min 83 376 Sandia 3 Percussion 2 min 81 392 Sandia 4 Percussion 2 min 84 367 Sandia 1 Percussion 4 min 89 313 Sandia 2 Percussion 4 min 86 347 Sandia 3 Percussion 4 min 89 313 Sandia 4 Percussion 4 min 85 357 Sandia 1 Percussion 8 min 80 400 Sandia 2 Percussion 8 min 77 421 Sandia 3 Percussion 8 min 69 462 Sandia 4 Percussion 8 min 66 474
LITERATURE CITED
Ashby WC Vogel WG Rogers NF 1985 Black locust in the reclamation equation General Technical Report Northeastern Forest Experiment Station USDA Forest Service NE-I05 12 p
Ballard LAT 1973 Physical barriers to gennination Seed Sci Technoll 285-303
___ 1976 Strophiolar water conduction in the seeds ofTrifolieae induced by action on the testa at nonstrophiolar sites Austr J Plant Physiol3 465-469
Barton LV 1947 Special studies on seed coat impenneability Contrib Boyce Thompson Inst 14 355-362
Baskin CC Baskin JM 1998 Seeds Ecology biogeography and evolution of donnancy and gennination Academic Press London UK 666 p
Bewley JD 1997 Seed germination and donnancy Plant Ce119 1055-1066
Bewley JD Black M 1994 Seeds physiology of development and germination 2nd ed Plenum Press New York NY 445 p
Briscoe CB 1996 Growing Nitrogen Fixing Trees in Acid Soils In MH Powell (ed) Nitrogen Fixing Trees for Acid Soils- A Field ManuaL Winrock International Morrilton AR 22 p
Copeland LO McDonald MB 1995 Principles of Seed Science and Technology 3rded Chapman amp Hall New York NY 409 p
Cox RE Klett JE 1984 Seed gennination requirements of native Colorado plants for use in the landscape Plant Propag 30(2) 6-10
Dell B 1980 Structure and function ofthe strophiolar plug in seeds ofAlbizia Lophantha Am J Bot 67 556-563
Dick-Peddie WA 1993 New Mexico Vegetation Past Present and Future University ofNew Mexico Press Albuquerque NM 244 p
Dreesen DR 2001 Propagation protocol for production ofcontainer Robinia neomexicana plants Los Lunas Plant Materials Center Los Lunas New Mexico In Native Plant Network URL httpwwwnativeplantnetworkorg (accessed 9 January 2002) Moscow (ID) University ofIdaho CoIl Natural Resources For Res Nursery
72
Dreesen DR Harrington JT 1997 Propagation of native plants for restoration projects in the Southwestern US- Preliminary Investigations In Landis TD Thompson JR tech coords National Proceedings Forest and Conservation Nursery Assocs Gen Tech Rep PNW-GTR-419 Portland OR USDA For Ser PNW Res Sta 77-88
Gottfried GJ 1980 Control ofNew Mexican locust and the effect on planted ponderosa pine in central Arizona US For Ser Res Note RM-386 Rocky Mountain For Range ExpStn Fort Collins CO
Gosling PG Samuel YK Jones SK 1995 A systematic examination of germination temperature chipping and water temperaturesoak duration pretreatments on the seeds ofLeucaena leucocephala Seed Sci amp Technol 23521-532
Hamly DH 1932 Softening of the seeds of Melilotus alba Bot Gazette 93 345shy375
Hanna PJ 1984 Anatomical features of the seed coat ofAcacia kempeana (Mueller) which relate to increased germination rate induced by heat treatment New Phytol 96 23-29
Hanks JP Dick-Peddie WA 1974 Vegetation patterns of the White Mountains New Mexico Southwest Naturalist 18(4) 371-382
Hine S Harrington JT Dreesen DR 1997 Seed scarification requirements for Robinia neomexicana In Harrington JTtech coord Proc Third SouthwestemContainer Growers Conf Feb 12-13 1997 Ft Collins CO
Hopkinson JM 1993 The strophiole of leguminous seeds Newsletter- IntI Herbage Seed Production Res Grp 18 7-8
---- 1997 Differences in hardseededness between Cunningham and Tarramba leucaena In Leucnet News The Newsletter of the International LeucaenaRes and Devlt Netwk No4
ISTA (International Seed Testing Association) 1999 International rules for seed testing rules Seed Sci amp Technol27 Supplement
Kelly KM van Staden J 1987 The lens as the site of permeability in the papilionoid seed Aspalathus linearis J Plant Physiol 128(45) 395-404
73
Keresztesi B 1988 Black locust the tree of agriculture Outlook on Agr 17(2) 77~85
Kigel 1 and Galili G 1995 Seed development and germination Marcel Dekker New York 853 p
Klemmedson 10 1994 New Mexico locust and parent material Influence on macronutrients of forest floor and soil Soil Sci Soc Am 1 58 974~980
Kuo WH Tam AW 1988 The pathway of water absorption of mung bean seeds Seed Sci amp Technol16(1) 139~144
Leadem CL 1997 Dormancy--unlocking seed secrets In Landis TD Thompson lR tech coords National Proceedings Forestand Conservation Nursery Assocs Gen Tech Rep PNW-G TR-419 Portland OR USDA For Ser PNW Res Sta 43-52
Lin Y Wagner MR Seweyestewa A Honahni MD DeGomez M McMillin JD 1996 Influence of cultural treatments on seed germination and seedling growth of Robinia neomexicana Forest Farm and Community Tree Res Reports Volume 1
Marunda CT 1990 Effects of seed pretreatment on the development ofAcacia auriculforinis and A holosericea seedlings ACIAR Proceedings Series No 28
Meginnis HG 1937 Sulphuric acid treatment to increase germination of black locust seed USDA Circ 453 34 p
Miklas PN Towensend CE Ladd SL 1987 Seedcoat anatomy and the scarification ofcicer milkvetch seed Crop Sci 27 766-772
Monsen SB 1984 Use of shrubs on mine spoils In The Challenge ofProducing Native Plants for the Intermountain Area Proceedings Intermountain Nurserymans Association 1983 conference Las Vegas NV compo PM Murphy 26-31 Ogden UT USDA For Ser Gen Tech Report INT-l68
Olson DF 1974 Robinia L Locust In Seeds of Woody Plants in the US tech coord CS Schopmeyer 728-731 USDA For Ser Agr Handbk No450
Rehman S Loescher RNl Harris P1C 1998 Dormancy breaking and germination ofAcacia salicina Lindl seeds Seed Sci amp Techno127 553-557
Rolston MP 1978 Water impermeable seed dormancy Bot Rev 44 365-396
74
SAS Institute Inc 1989 SASSTAT Users Guide Version 6 4th Edition Volume 1 Cary NC SAS Institute Inc 943 p
SerratoValenti G Conara L Ferrando M Modenesi P 1993 Structural and histochemical features ofStylosanthes scabra (Leguminosae Papilionoidae) seed coat as related to water entry Can J Bot 71 834-840
Simpson BJ 1988 A field guide to Texas trees Austin TX Texas Monthly Press 372p
Tran VN Cavanagh AK 1984 Structural aspects ofdormancy In Seed Physiology Vol 2 Murray DR (ed) p1-44 Academic Press Australia
USDA Forest Service 1948 Woody-plant seed manual USDA Misc Pub1654 416p
Vines RA 1960 Trees Shrubs and Woody Vines of the Southwest Austin Univ ofTexas Press 1104 p
Wagner MR Larson RE Craig TP 1992 Ecology and silvics ofNew Mexico locust (Robinia neomexicana) with an emphasis on the arboreal form p53-57 In JW Hanover K Miller and S Plesko (eds) Proc Intemat Confon Black Locust Biology Culture and Utilization Mich State Univ USA
Wilson JK 1944 Immersing seeds of species of Robinia in boiling water hastens germination J For 42 453-454
Zimmerman RW Carpenter SB 1980 First-year coppice production from a 5shyyear old black locust stand on surface mine spoil p309-314 Centr Hardwd For ConfProc University ofMissouri Columbia
b
Figure AI New Mexico locust a primary invader following disturbance Ala Crown-sprouting at edge ofburn near Los Alamos NM--one year after Cerro Grande fire Alb Colonizing road cut Gila National Forest NM
50
Percussion scarification where seeds are repeatedly propelled against a hard
surface is an alternative dormancy-breaking method that has proven successful in
legumes related to locusts (Hamly 1932 Barton 1947) We compared this alternative
treatment to hot water scarification to evaluate treatment efficacy for New Mexico
locust and black locust seeds
Materials and Methods
Commercial New Mexico locust seeds (Western Native Seed Coaldale CO)
were collected fall 2000 in Huerfano County CO Black locust seeds were collected
September 2000 from Taos County NM by harvesting ripe pods from several trees at
a distance of up to 3 m from ground level Black locust seeds were allowed to air-dry
for 3 weeks and were threshed and separated from large chaffusing a greenhouse
fan A South Dakota Seed Blower (Chicago Illinltois) was used to remove fine chaff
from both seed lots Seeds were storedmiddotat 2-4C until the germination study was
conducted in March 2001
This study compared hot water scarification with percussion scarification
using treatment and species as experimental factors Along with a control
scarification treatments included immersion of seed in boiling water as well as
percussion durations of 1 2 3 45 or 10 minutes Four 100-seed samples were used
to test each species by scarification treatment combination
For the hot water treatment test tubes were filled with 30 mL water and
placed in a water-filled beaker (Figure A2a) The bath was raised to boiling (-98C at
51
b
c
Figure A2 Implementation ofhot water and percussion scarification A2a Hot water bath A2b Paint-shaker set-up A2c Placing soil tin containing seeds at end ofpaint shaker ann
52
Las Cruces NM elevation of 1300 meters) Each 100-seed replicate was placed in a
test tube and each test tube was immediately removed to cool at room temperature
Percussion scarification was implemented using a pneumatic paint shaker
(Central Pneumatic model 00422 Camarillo CA) (Figure A2b) This allowed
standardization of treatment intensity Compressed air pressure was maintained at
530kPa plusmn 30kPa (80psi plusmn 5psi) resulting in approximately 350 oscillations per
minute Each lOO-seed sample was placed in a 118 ml (4 oz) soil tin for shaking To
maximize lateral movement of paint shaker a paint can was used as a spacer to place
soil tins at the end of the shaking arm (Figure A2c) This allowed shaking to occur at
the greatest distance from the pivot point (fulcrum) Following all treatments
including the control seeds were soaked for twenty-four hours in distilled water prior
to plating out
Gennination was evaluated on lab benches at room temperature Each
replicate of seeds was placed in a 100 cm petri dish on moistened filter paper with
humidity maintained by enclosing the petri dishes in ziplock bags Gennination was
monitored daily for 14 days in accordance with International Seed Testing
Association standards for Robinia species (ISTA 1999) Gennination was defined as
protrusion of the radicle from the embryo by at least 1mm as viewed by the naked
eye
Categorical Analysis of Variance using Prpc Catmod (SAS Institute 1989)
was used to detennine treatment differences in germination percentages for each
source This procedure is an extension of a Chi-square test of homogeneity using the
53
natural log of the ratio of germinated to non-germinated seeds for each treatment Pshy
values less than 005 were considered significant A limited set of pairwiseshy
comparisons was conducted to compare treatment mean percents using a
conservative alpha value of 005 divided by the number of comparisons
Results
Percussion maximized germination for both New Mexico locust and black
locust There were three germination response levels for New Mexico locust (Figure
A3) First there was a low response in the control indicating a dormant seed lot
The hot water treatment and I-minute duration of percussion share a second
intermediate response Germination was highest at percussion levels from 2 to 10
minutes
A selected percussion treatment intermediate in time (5 minutes) produced the
steepest germination curve indicating the fastest germination rate (Figure A4) The
hot water treatment produced an intermediate curve or relatively slower germination
rate The control curve was very flat indicating the slowest germination rate for New
Mexico locust
Control seeds germinated poorly for black locust as well (Figure A5) Hot
water scarification improved germination over the control but all levels of percussion
improved germination over hot water The optimal durations of percussion treatment
were bracketed with a significant increase from the 1 to 2 minute percussion level a
high germination response from 2 to 5 minutes of percussion and finally a significant
54
~ d 25 CL)
p t1 0 ~ t1
g Vl ~ Vl
100~---------------------------------------------------------------
80
60
40
20
o Control Hot Water Percussion
I min Percussion
2 min Percussion
3 min
Scarification
Percussion 4 min
Figure A3 Effect of scarification on germination response for New Mexico locust
Percussion 5 min
=significant difference between adjacent treatment bars ns = no significant difference
Percussion 10 min
~ ~ () u agt
Il-t d o ~
~ ~ c3
100middotlr~~~------------------------------------e- Percussion 5min middot 0middot Hot Water -- Control
80-J=middot======
6b-middot-lmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middot--middotmiddot-middot~-middotmiddot -middotmiddot middot middotmiddot-middot 1
40
20
-----~-----------
0 0 0 middotmiddotmiddotmiddotmiddot0middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot0
0 --~-----y-----~------_---~--- T-----~
o shy -TDay 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A4 Effect of selected scarification treatments on germination speed for New Mexico locust First seven days of fourteen-day experiment are shown
Day 7
80
~ cd
5 60 ()
[) Cl d 0 1t1 d
40sect dUl
-J
20
o
ns
Scarification
Figure A5 Effect of scarification on germination response for black locust = significant difference between adjacent treatment bars ns = no significant difference
decrease at the 10-minute level This drop-off suggests that 10 minutes of percussion
over-treats seeds from this lot of-black locust
A selected percussion treatment intermediate in time (5 minutes) also had the
fastest germination rate for black locust (Figure A6)_ The hot water curve is
somewhat flatter indicating a slower germination rate The control curve is very flat
indicating a slow germination rate
A qualitative difference between hot water and percussion-treated seeds was
also observed Along with healthy germinants the hot water treatment produced
over-treated seeds (Figure A 7a) Examples of damaged germinants include seeds
where the radicle did not elongate (Figure A 7b) seeds where the radicle elongated
but was delayed in freeing itself from the seed coat (Figure A7c) and seeds where the
radicle broke off completely from the embryo (Figure A7d) In addition to these
examples of over-treatment there were numerous small dark seeds that failed to
imbibe water For percussion treatment nearly all seeds took up water (Figure A8a)
Germination was rapid and uniform with little damage evident (Figure A8b)
To summarize both New Mexico locust and black locust seed lots were
dormant The standard hot water scarification treatment improved germination but
only to 56 and 41 for New Mexico locust and black locust respectively Nearly
all percussion durations improved total germination as well as germination rate over
the hot water treatment Germination was greater than 90 for percussion durations
of 4 5 and 10 minutes for New Mexico locust and 3 4 and 5 minutes for black locust
58
CI)
sgt t CI) 0
igt 0
sect ttl t E
Vl 0
c3
100r==~==~~=r------------------------------------~ ___ Percussion 5 min
middotmiddotmiddot0middotmiddotmiddot Hot Water -lIf- Control
80r=========~--~~----------------------------~
60
40
20 -~-~---~---------- ---~------------I
omiddot
O
_~_----------lIf-----~bull -----_ shy---shy I
Day 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A6 Effect of selected scarification treatments on germination speed for black locust First seven days of fourteen-day experiment are shown
Day 7
a
b c d
Figure A7 Damage to New Mexico locust seeds observed during germination counts following hot water scarification A7a Hot water replicate including damaged seedlings and numerous unimbibed seeds A7b Close-up ofseed where radicle failed to elongate A7c Close-up ofseed where radicle elongated but was delayed in freeing itself from the seed coat A7d Close-up ofseed where radicle completely broke offfrom embryo
60
a
b
Figure A8 New Mexico locust seeds following percussion treatment at time of germination counts A8a Percussion replicate with uniformly healthy seedlings and near complete imbibition of seeds A8b Close-up ofpercussion-treated seedling
61
Discussion
Papilionoid seeds have a specific anatomical feature known as the strophiole
or lens-the natural site of water entry to the seed (Hamly 1932) The strophiole is
located on the cotyledonary lobe of a locust seed (Figure A9) When a papilionoid
legume seed is percussed for the appropriate amount of time repeated hits on the
integrated seed coat loosen the constrained cells of the strophiolar region without
excessively damaging the rest of the seed coat (Ballard 1976)
When a percussed seed is soaked water enters exclusively through the
strophiole in a controlled manner (Kelly and Staden 1987) This regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues This
contrasts with seeds that have been hot water or acid scarified treatments that can
randomly degrade the seed coat Unlocalized cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rehman et al 1998 Marunda 1990)
Papilionoids are the largest subfamily of legumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust and black locust these legumes often play an integral role in the
revegetation ofdisturbed lands Developing superior scarification methods to the
standard hot water and acid treatments should fadlitate the use ofthese legumes in
restoration projects
62
Figure A9 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (bc)
63
APPENDIXB
GERMINATION PERCENTS AND STANDARD ERRORS FOR SOURCE
BLOCK AND TREATMENT FOR NEW MEXICO LOCUST
Table B 1 Gennination percents and standard errors for source block and treatment for New Mexico locust
Source Block Treatment Germination Percentage Standard Error
Capitan 1 Immersion 74 437 Capitan 2 Immersion 69 462 Capitan 3 Immersion 71 454 Capitan 4 Immersion 67 470 Capitan Boiling 30 sec 79 407 Capitan 2 Boiling 30 sec 76 427 Capitan 3 Boiling 30 sec 72 449 Capitan 4 Boiling 30 sec 76 427 Capitan Boiling 60 sec 71 454 Capitan 2 Boiling 60 sec 54 498 Capitan 3 Boiling 60 sec 67 470 Capitan 4 Boiling 60 sec 76 427 Capitan I Control 5 218 Capitan 2 Control 7 255 Capitan 3 Control 4 196 Capitan 4 Control 5 218 Capitan 1 Percussion I min 47 499 Capitan 2 Percussion I min
470
Capitan Percussion I min 39 488
Capitan 4 Percussion I min 38 485 Capitan I Percussion 2 min 55 497 Capitan Capitan
2
Percussion 2 min Percussion 2 min
71 65
454 477
Capitan 4 Percussion 2 min 56 496 Capitan I Percussion 4 min 86 347 Capitan 2 Percussion 4 min 79 407 Capitan 3 Percussion 4 min 81 392 Capitan 4 Percussion 4 min 82 384 Capitan I Percussion 8 min 90 300 Capitan 2 Percussion 8 min 88 325 Capitan 3 Percussion 8 min 90 300 Capitan 4 Percussion 8 min 90 300 Gila I Immersion 71 454 Gila 2 Immersion 77 421 Gila
Immersion 75 433
Gila 4 Immersion 72 449
Gila I Boiling 30 sec 68 466
Gila 2 Boiling 30 sec 81 392
Gila 3 Boiling 30 sec 54 498
Gila 4 Boiling 30 sec 63 483
Gila I Boiling 60 sec 55 497
Gila 2 Boiling 60 sec 68 466
Gila 3 Boiling 60 sec 57 495
Gila 4 Boiling 60 sec 70 458
65
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Gila 2 Control 13 336 Gila 3 Control 13 336 Gila 4 Control 20 400 Gila 1 Percussion 1 min 66 474 Gila 2 Percussion 1 min 57 495 Gila 3 Percussion 1 min 73 444 Gila 4 Percussion 1 min 82 384 Gila 1 Percussion 2 min 81 392 Gila 2 Percussion 2 min 86 347 Gila 3 Percussion 2 min 85 357 Gila 4 Percussion 2 min 54 498 Gila 1 Percussion 4 min 79 407 Gila 2 Percussion 4 min 83 376 Gila 3 Percussion 4 min 84 367 Gila 4 Percussion 4 min 79 407 Gila 1 Percussion 8 min 51 500 Gila 2 Percussion 8 min 76 427 Gila 3 Percussion 8 min 52 500 Gila 4 Percussion 8 min 74 439 Holman 1 Immersion 83 376 Holman 2 Immersion 86 347 Holman 3 Immersion 81 392 Holman 4 Immersion 90 300 Holman 1 Boiling 30 sec 90 300 Holman 2 Boiling 30 sec 85 357 Holman 3 Boiling 30 sec 87 336 Holman 4 Boiling 30 sec 83 376 Holman 1 Boiling 60 sec 79 407 Holman 2 Boiling 60 sec 80 400 Holman 3 Boiling 60 sec 86 347 Holman 4 Boiling 60 sec 93 255 Holman Control 25 433 Holman 2 Control 19 392 Holman Control 21 407
Holman 4 Control 24 427
Holman Percussion 1 min 77 421
Holman 2 Percussion 1 min 85 357
Holman 3 Percussion 1 min 80 400
Holman 4 Percussion 1 min 89 313
Holman 1 Percussion 2 min 88 325
Holman 2 Percussion 2 min 96 196
Holman 3 Percussion 2 min 93 255
Holman 4 Percussion 2 min 96 196
Holman 1 Percussion 4 min 92 271
Holman 2 Percussion 4 min 97 171
66
I
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Holman 3 Percussion 4 min 91 286 Holman 4 Percussion 4 min 96 196 Holman 1 Percussion 8 min 88 325 Holman 2 Percussion 8 min 85 357 Holman 3 Percussion 8 min 83 376 Holman 4 Percussion 8 min 82 384 Jemez 1 Immersion 53 499 Jemez 2 Immersion 44 496 Jemez 3 Immersion 52 500 Jemez 4 Immersion 70 458 Jemez 1 Boiling 30 sec 58 494 Jemez 2 Boiling 30 sec 55 497 Jemez 3 Boiling 30 sec 48 500 Jemez 4 Boiling 30 sec 65 477 Jemez 1 Boiling 60 sec 68 466 Jemez 2 Boiling 60 sec 63 483 Jemez 3 Boiling 60 sec 73 444 Jemez 4 Boiling 60 sec 89 313 Jemez 1 Control 2 140 Jemez 2 Control 9 286 Jemez 3 Control 6 237 Jemez 4 Control 8 271 Jemez 1 Percussion 1 min 58 494 Jemez 2 Percussion 1 min 63 483 Jemez 3 Percussion 1 min 77 421 Jemez 4 Percussion 1 min 89 313 Jemez 1 Percussion 2 min 68 466 Jemez 2 Percussion 2 min 64 480 Jemez 3 Percussion 2 min 80 400 Jemez 4 Percussion 2 min 83 376 Jemez Percussion 4 min 68 466 Jemez 2 Percussion 4 min 79 407 Jemez 3 Percussion 4 min 70 458 Jemez 4 Percussion 4 min 83 376 Jemez 1 Percussion 8 min 52 500 Jemez 2 Percussion 8 min 59 492 Jemez 3 Percussion 8 min 61 488 Jemez 4 Percussion 8 min 70 458 Magdalena 1 Immersion 69 462 Magdalena 2 Immersion 69 462 Magdalena J Immersion 70 458 Magdalena 4 Immersion 85 357 Magdalena 1 Boiling 30 sec 65 477 Magdalena 2 Boiling 30 sec 71 454 Magdalena 3 Boiling 30 sec 68 466
67
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Magdalena 4 Boiling 30 sec 83 376 Magdalena 1 Boiling 60 sec 63 483 Magdalena 2 Boiling 60 sec 68 466 Magdalena 3 Boiling 60 sec 63 483 Magdalena 4 Boiling 60 sec 82 384 Magdalena 1 Control 11 313 Magdalena 2 Control 6 237 Magdalena 3 Control 5 218 Magdalena 4 Control 4 196 Magdalena Percussion 1 min 32 466 Magdalena 2 Percussion 1 min 45 497 Magdalena 3 Percussion 1 min 46 498 Magdalena 4 Percussion I min 36 480 Magdalena 1 Percussion 2 min 67 470 Magdalena 2 Percussion 2 min 68 466 Magdalena 3 Percussion 2 min 65 477 Magdalena 4 Percussion 2 min 81 392 Magdalena 1 Percussion 4 min 87 336 Magdalena 2 Percussion 4 min 97 171 Magdalena 3 Percussion 4 min 83 376 Magdalena 4 Percussion 4 min 97 171 Magdalena 1 Percussion 8 min 98 lAO Magdalena 2 Percussion 8 min 100 000 Magdalena 3 Percussion 8 min 96 196 Magdalena 4 Percussion 8 min 98 lAO Manzano 1 Immersion 67 470 Manzano 2 Immersion 57 495 Manzano 3 Immersion 72 4049 Manzano 4 Immersion 73 4044 Manzano 1 Boiling 30 sec 70 458 Manzano 2 Boiling 30 sec 64 480 Manzano 3 Boiling 30 sec 71 454 Manzano 4 Boiling 30 sec 80 400 Manzano I Boiling 60 sec 50 500 Manzano 2 Boiling 60 sec 53 499 Manzano 3 Boiling 60 sec 59 492 Manzano 4 Boiling 60 sec 74 439 Manzano I Control 4 196 Manzano 2 Control 8 271 Manzano 3 Control 1 099 Manzano 4 Control 6 237 Manzano 1 Percussion 1 min 52 500 Manzano 2 Percussion 1 min 39 488 Manzano 3 Percussion 1 min 36 480 Manzano 4 Percussion 1 min 51 500
68
Table B 1 Continued
Source Block Treatment Gerrni~ation Percentage Standard Error
Manzano Percussion 2 min 53 499 Manzano 2 Percussion 2 min 58 494 Manzano 3 Percussion 2 min 61 488 Manzano 4 Percussion 2 min 62 485 Manzano 1 Percussion 4 min 76 427 Manzano 2 Percussion 4 min 85 357 Manzano 3 Percussion 4 min 79 407 Manzano 4 Percussion 4 min 83 376 Manzano 1 Percussion 8 min 87 336 Manzano 2 Percussion 8 min 83 376 Manzano 3 Percussion 8 min 83 376 Manzano 4 Percussion 8 min 75 433 Raton 1 Immersion 54 498 Raton 2 Immersion 37 483 Raton 3 Immersion 39 488 Raton 4 Immersion 69 462 Raton 1 Boiling 30 sec 36 480 Raton 2 Boiling 30 sec 20 400 Raton 3 Boiling 30 sec 28 449 Raton 4 Boiling 30 sec 60 490 Raton 1 Boiling 60 sec 15 357 Raton 2 Boiling 60 sec 171 Raton 3 Boiling 60 sec 4 196 Raton 4 Boiling 60 sec 35 477 Raton 1 Control 4 196 Raton 2 Control 12 325 Raton 3 Control 2 140 Raton 4 Control 6 237 Raton 1 Percussion 1 min 52 500 Raton 2 Percussion I min 29 454 Raton 3 Percussion I min 35 477 Raton 4 Percussion 1 min 31 462 Raton 1 Percussion 2 min 55 497 Raton 2 Percussion 2 min 35 477 Raton 3 Percussion 2 min 33 470 Raton 4 Percussion 2 min 42 494 Raton 1 Percussion 4 min 65 477 Raton 2 Percussion 4 min 52 500 Raton Percussion 4 min 51 500 Raton 4 Percussion 4 min 53 499 Raton 1 Percussion 8 min 88 325 Raton 2 Percussion 8 min 80 400 Raton 3 Percussion 8 min 73 444 Raton 4 Percussion 8 min 74 439 Sacramento 1 Immersion 43 495
69
Table B 1 Continued
-Source Block Treatment Germination Percentage Standard Error
Sacramento 2 Immersion 30 458 Sacramento 3 Immersion 38 485 Sacramento 4 Immersion 56 496 Sacramento 1 Boiling 30 sec 47 499 Sacramento 2 Boiling 30 sec 48 500 Sacramento 3 Boiling 30 sec 48 500 Sacramento 4 Boiling 30 sec 56 496 Sacramento 1 Boiling 60 sec 52 500 Sacramento 2 Boiling 60 sec 67 470 Sacramento 3 Boiling 60 sec 43 495 Sacramento 4 Boiling 60 sec 48 500 Sacramento 1 Control 0 000 Sacramento 2 Control 3 171 Sacramento 3 Control 3 171 Sacramento 4 Control 099 Sacramento 1 Percussion 1 min 57 495 Sacramento 2 Percussion 1 min 61 488 Sacramento 3 Percussion 1 min 62 485 Sacramento 4 Percussion 1 min 55 497 Sacramento 1 Percussion 2 min 84 367 Sacramento 2 Percussion 2 min 70 458 Sacramento 3 Percussion 2 min 81 392 Sacramento 4 Percussion 2 min 69 462 Sacramento 1 Percussion 4 min 91 286 Sacramento 2 Percussion 4 min 84 367 Sacramento 3 Percussion 4 min 84 367 Sacramento 4 Percussion 4 min 94 237 Sacramento 1 Percussion 8 min 82 384 Sacramento 2 Percussion 8 min 81 392 Sacramento 3 Percussion 8 min 82 384 Sacramento 4 Percussion 8 min 81 392 Sandia 1 Immersion 76 427 Sandia 2 Immersion 70 458 Sandia 3 Immersion 70 458 Sandia 4 Immersion 81 392 Sandia 1 Boiling 30 sec 78 414 Sandia 2 Boiling 30 sec 71 454 Sandia 3 Boiling 30 sec 63 483 Sandia 4 Boiling 30 sec 78 414 Sandia 1 Boiling 60 sec 67 470 Sandia 2 Boiling 60 sec 65 477 Sandia 3 Boiling 60 sec 74 439 Sandia 4 Boiling 60 sec 66 474 Sandia 1 Control 16 367 Sandia 2 Control 7 255
70
Table B 1 Continued
Source Block Treatment Gennination Percentage Standard Error
Sandia 3 Control 8 271 Sandia 4 Control 16 367 Sandia 1 Percussion 1 min 67 470 Sandia 2 Percussion 1 min 61 488 Sandia 3 Percussion 1 min 66 474 Sandia 4 Percussion 1 min 59 492 Sandia I Percussion 2 min 82 384 Sandia 2 Percussion 2 min 83 376 Sandia 3 Percussion 2 min 81 392 Sandia 4 Percussion 2 min 84 367 Sandia 1 Percussion 4 min 89 313 Sandia 2 Percussion 4 min 86 347 Sandia 3 Percussion 4 min 89 313 Sandia 4 Percussion 4 min 85 357 Sandia 1 Percussion 8 min 80 400 Sandia 2 Percussion 8 min 77 421 Sandia 3 Percussion 8 min 69 462 Sandia 4 Percussion 8 min 66 474
LITERATURE CITED
Ashby WC Vogel WG Rogers NF 1985 Black locust in the reclamation equation General Technical Report Northeastern Forest Experiment Station USDA Forest Service NE-I05 12 p
Ballard LAT 1973 Physical barriers to gennination Seed Sci Technoll 285-303
___ 1976 Strophiolar water conduction in the seeds ofTrifolieae induced by action on the testa at nonstrophiolar sites Austr J Plant Physiol3 465-469
Barton LV 1947 Special studies on seed coat impenneability Contrib Boyce Thompson Inst 14 355-362
Baskin CC Baskin JM 1998 Seeds Ecology biogeography and evolution of donnancy and gennination Academic Press London UK 666 p
Bewley JD 1997 Seed germination and donnancy Plant Ce119 1055-1066
Bewley JD Black M 1994 Seeds physiology of development and germination 2nd ed Plenum Press New York NY 445 p
Briscoe CB 1996 Growing Nitrogen Fixing Trees in Acid Soils In MH Powell (ed) Nitrogen Fixing Trees for Acid Soils- A Field ManuaL Winrock International Morrilton AR 22 p
Copeland LO McDonald MB 1995 Principles of Seed Science and Technology 3rded Chapman amp Hall New York NY 409 p
Cox RE Klett JE 1984 Seed gennination requirements of native Colorado plants for use in the landscape Plant Propag 30(2) 6-10
Dell B 1980 Structure and function ofthe strophiolar plug in seeds ofAlbizia Lophantha Am J Bot 67 556-563
Dick-Peddie WA 1993 New Mexico Vegetation Past Present and Future University ofNew Mexico Press Albuquerque NM 244 p
Dreesen DR 2001 Propagation protocol for production ofcontainer Robinia neomexicana plants Los Lunas Plant Materials Center Los Lunas New Mexico In Native Plant Network URL httpwwwnativeplantnetworkorg (accessed 9 January 2002) Moscow (ID) University ofIdaho CoIl Natural Resources For Res Nursery
72
Dreesen DR Harrington JT 1997 Propagation of native plants for restoration projects in the Southwestern US- Preliminary Investigations In Landis TD Thompson JR tech coords National Proceedings Forest and Conservation Nursery Assocs Gen Tech Rep PNW-GTR-419 Portland OR USDA For Ser PNW Res Sta 77-88
Gottfried GJ 1980 Control ofNew Mexican locust and the effect on planted ponderosa pine in central Arizona US For Ser Res Note RM-386 Rocky Mountain For Range ExpStn Fort Collins CO
Gosling PG Samuel YK Jones SK 1995 A systematic examination of germination temperature chipping and water temperaturesoak duration pretreatments on the seeds ofLeucaena leucocephala Seed Sci amp Technol 23521-532
Hamly DH 1932 Softening of the seeds of Melilotus alba Bot Gazette 93 345shy375
Hanna PJ 1984 Anatomical features of the seed coat ofAcacia kempeana (Mueller) which relate to increased germination rate induced by heat treatment New Phytol 96 23-29
Hanks JP Dick-Peddie WA 1974 Vegetation patterns of the White Mountains New Mexico Southwest Naturalist 18(4) 371-382
Hine S Harrington JT Dreesen DR 1997 Seed scarification requirements for Robinia neomexicana In Harrington JTtech coord Proc Third SouthwestemContainer Growers Conf Feb 12-13 1997 Ft Collins CO
Hopkinson JM 1993 The strophiole of leguminous seeds Newsletter- IntI Herbage Seed Production Res Grp 18 7-8
---- 1997 Differences in hardseededness between Cunningham and Tarramba leucaena In Leucnet News The Newsletter of the International LeucaenaRes and Devlt Netwk No4
ISTA (International Seed Testing Association) 1999 International rules for seed testing rules Seed Sci amp Technol27 Supplement
Kelly KM van Staden J 1987 The lens as the site of permeability in the papilionoid seed Aspalathus linearis J Plant Physiol 128(45) 395-404
73
Keresztesi B 1988 Black locust the tree of agriculture Outlook on Agr 17(2) 77~85
Kigel 1 and Galili G 1995 Seed development and germination Marcel Dekker New York 853 p
Klemmedson 10 1994 New Mexico locust and parent material Influence on macronutrients of forest floor and soil Soil Sci Soc Am 1 58 974~980
Kuo WH Tam AW 1988 The pathway of water absorption of mung bean seeds Seed Sci amp Technol16(1) 139~144
Leadem CL 1997 Dormancy--unlocking seed secrets In Landis TD Thompson lR tech coords National Proceedings Forestand Conservation Nursery Assocs Gen Tech Rep PNW-G TR-419 Portland OR USDA For Ser PNW Res Sta 43-52
Lin Y Wagner MR Seweyestewa A Honahni MD DeGomez M McMillin JD 1996 Influence of cultural treatments on seed germination and seedling growth of Robinia neomexicana Forest Farm and Community Tree Res Reports Volume 1
Marunda CT 1990 Effects of seed pretreatment on the development ofAcacia auriculforinis and A holosericea seedlings ACIAR Proceedings Series No 28
Meginnis HG 1937 Sulphuric acid treatment to increase germination of black locust seed USDA Circ 453 34 p
Miklas PN Towensend CE Ladd SL 1987 Seedcoat anatomy and the scarification ofcicer milkvetch seed Crop Sci 27 766-772
Monsen SB 1984 Use of shrubs on mine spoils In The Challenge ofProducing Native Plants for the Intermountain Area Proceedings Intermountain Nurserymans Association 1983 conference Las Vegas NV compo PM Murphy 26-31 Ogden UT USDA For Ser Gen Tech Report INT-l68
Olson DF 1974 Robinia L Locust In Seeds of Woody Plants in the US tech coord CS Schopmeyer 728-731 USDA For Ser Agr Handbk No450
Rehman S Loescher RNl Harris P1C 1998 Dormancy breaking and germination ofAcacia salicina Lindl seeds Seed Sci amp Techno127 553-557
Rolston MP 1978 Water impermeable seed dormancy Bot Rev 44 365-396
74
SAS Institute Inc 1989 SASSTAT Users Guide Version 6 4th Edition Volume 1 Cary NC SAS Institute Inc 943 p
SerratoValenti G Conara L Ferrando M Modenesi P 1993 Structural and histochemical features ofStylosanthes scabra (Leguminosae Papilionoidae) seed coat as related to water entry Can J Bot 71 834-840
Simpson BJ 1988 A field guide to Texas trees Austin TX Texas Monthly Press 372p
Tran VN Cavanagh AK 1984 Structural aspects ofdormancy In Seed Physiology Vol 2 Murray DR (ed) p1-44 Academic Press Australia
USDA Forest Service 1948 Woody-plant seed manual USDA Misc Pub1654 416p
Vines RA 1960 Trees Shrubs and Woody Vines of the Southwest Austin Univ ofTexas Press 1104 p
Wagner MR Larson RE Craig TP 1992 Ecology and silvics ofNew Mexico locust (Robinia neomexicana) with an emphasis on the arboreal form p53-57 In JW Hanover K Miller and S Plesko (eds) Proc Intemat Confon Black Locust Biology Culture and Utilization Mich State Univ USA
Wilson JK 1944 Immersing seeds of species of Robinia in boiling water hastens germination J For 42 453-454
Zimmerman RW Carpenter SB 1980 First-year coppice production from a 5shyyear old black locust stand on surface mine spoil p309-314 Centr Hardwd For ConfProc University ofMissouri Columbia
Percussion scarification where seeds are repeatedly propelled against a hard
surface is an alternative dormancy-breaking method that has proven successful in
legumes related to locusts (Hamly 1932 Barton 1947) We compared this alternative
treatment to hot water scarification to evaluate treatment efficacy for New Mexico
locust and black locust seeds
Materials and Methods
Commercial New Mexico locust seeds (Western Native Seed Coaldale CO)
were collected fall 2000 in Huerfano County CO Black locust seeds were collected
September 2000 from Taos County NM by harvesting ripe pods from several trees at
a distance of up to 3 m from ground level Black locust seeds were allowed to air-dry
for 3 weeks and were threshed and separated from large chaffusing a greenhouse
fan A South Dakota Seed Blower (Chicago Illinltois) was used to remove fine chaff
from both seed lots Seeds were storedmiddotat 2-4C until the germination study was
conducted in March 2001
This study compared hot water scarification with percussion scarification
using treatment and species as experimental factors Along with a control
scarification treatments included immersion of seed in boiling water as well as
percussion durations of 1 2 3 45 or 10 minutes Four 100-seed samples were used
to test each species by scarification treatment combination
For the hot water treatment test tubes were filled with 30 mL water and
placed in a water-filled beaker (Figure A2a) The bath was raised to boiling (-98C at
51
b
c
Figure A2 Implementation ofhot water and percussion scarification A2a Hot water bath A2b Paint-shaker set-up A2c Placing soil tin containing seeds at end ofpaint shaker ann
52
Las Cruces NM elevation of 1300 meters) Each 100-seed replicate was placed in a
test tube and each test tube was immediately removed to cool at room temperature
Percussion scarification was implemented using a pneumatic paint shaker
(Central Pneumatic model 00422 Camarillo CA) (Figure A2b) This allowed
standardization of treatment intensity Compressed air pressure was maintained at
530kPa plusmn 30kPa (80psi plusmn 5psi) resulting in approximately 350 oscillations per
minute Each lOO-seed sample was placed in a 118 ml (4 oz) soil tin for shaking To
maximize lateral movement of paint shaker a paint can was used as a spacer to place
soil tins at the end of the shaking arm (Figure A2c) This allowed shaking to occur at
the greatest distance from the pivot point (fulcrum) Following all treatments
including the control seeds were soaked for twenty-four hours in distilled water prior
to plating out
Gennination was evaluated on lab benches at room temperature Each
replicate of seeds was placed in a 100 cm petri dish on moistened filter paper with
humidity maintained by enclosing the petri dishes in ziplock bags Gennination was
monitored daily for 14 days in accordance with International Seed Testing
Association standards for Robinia species (ISTA 1999) Gennination was defined as
protrusion of the radicle from the embryo by at least 1mm as viewed by the naked
eye
Categorical Analysis of Variance using Prpc Catmod (SAS Institute 1989)
was used to detennine treatment differences in germination percentages for each
source This procedure is an extension of a Chi-square test of homogeneity using the
53
natural log of the ratio of germinated to non-germinated seeds for each treatment Pshy
values less than 005 were considered significant A limited set of pairwiseshy
comparisons was conducted to compare treatment mean percents using a
conservative alpha value of 005 divided by the number of comparisons
Results
Percussion maximized germination for both New Mexico locust and black
locust There were three germination response levels for New Mexico locust (Figure
A3) First there was a low response in the control indicating a dormant seed lot
The hot water treatment and I-minute duration of percussion share a second
intermediate response Germination was highest at percussion levels from 2 to 10
minutes
A selected percussion treatment intermediate in time (5 minutes) produced the
steepest germination curve indicating the fastest germination rate (Figure A4) The
hot water treatment produced an intermediate curve or relatively slower germination
rate The control curve was very flat indicating the slowest germination rate for New
Mexico locust
Control seeds germinated poorly for black locust as well (Figure A5) Hot
water scarification improved germination over the control but all levels of percussion
improved germination over hot water The optimal durations of percussion treatment
were bracketed with a significant increase from the 1 to 2 minute percussion level a
high germination response from 2 to 5 minutes of percussion and finally a significant
54
~ d 25 CL)
p t1 0 ~ t1
g Vl ~ Vl
100~---------------------------------------------------------------
80
60
40
20
o Control Hot Water Percussion
I min Percussion
2 min Percussion
3 min
Scarification
Percussion 4 min
Figure A3 Effect of scarification on germination response for New Mexico locust
Percussion 5 min
=significant difference between adjacent treatment bars ns = no significant difference
Percussion 10 min
~ ~ () u agt
Il-t d o ~
~ ~ c3
100middotlr~~~------------------------------------e- Percussion 5min middot 0middot Hot Water -- Control
80-J=middot======
6b-middot-lmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middot--middotmiddot-middot~-middotmiddot -middotmiddot middot middotmiddot-middot 1
40
20
-----~-----------
0 0 0 middotmiddotmiddotmiddotmiddot0middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot0
0 --~-----y-----~------_---~--- T-----~
o shy -TDay 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A4 Effect of selected scarification treatments on germination speed for New Mexico locust First seven days of fourteen-day experiment are shown
Day 7
80
~ cd
5 60 ()
[) Cl d 0 1t1 d
40sect dUl
-J
20
o
ns
Scarification
Figure A5 Effect of scarification on germination response for black locust = significant difference between adjacent treatment bars ns = no significant difference
decrease at the 10-minute level This drop-off suggests that 10 minutes of percussion
over-treats seeds from this lot of-black locust
A selected percussion treatment intermediate in time (5 minutes) also had the
fastest germination rate for black locust (Figure A6)_ The hot water curve is
somewhat flatter indicating a slower germination rate The control curve is very flat
indicating a slow germination rate
A qualitative difference between hot water and percussion-treated seeds was
also observed Along with healthy germinants the hot water treatment produced
over-treated seeds (Figure A 7a) Examples of damaged germinants include seeds
where the radicle did not elongate (Figure A 7b) seeds where the radicle elongated
but was delayed in freeing itself from the seed coat (Figure A7c) and seeds where the
radicle broke off completely from the embryo (Figure A7d) In addition to these
examples of over-treatment there were numerous small dark seeds that failed to
imbibe water For percussion treatment nearly all seeds took up water (Figure A8a)
Germination was rapid and uniform with little damage evident (Figure A8b)
To summarize both New Mexico locust and black locust seed lots were
dormant The standard hot water scarification treatment improved germination but
only to 56 and 41 for New Mexico locust and black locust respectively Nearly
all percussion durations improved total germination as well as germination rate over
the hot water treatment Germination was greater than 90 for percussion durations
of 4 5 and 10 minutes for New Mexico locust and 3 4 and 5 minutes for black locust
58
CI)
sgt t CI) 0
igt 0
sect ttl t E
Vl 0
c3
100r==~==~~=r------------------------------------~ ___ Percussion 5 min
middotmiddotmiddot0middotmiddotmiddot Hot Water -lIf- Control
80r=========~--~~----------------------------~
60
40
20 -~-~---~---------- ---~------------I
omiddot
O
_~_----------lIf-----~bull -----_ shy---shy I
Day 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A6 Effect of selected scarification treatments on germination speed for black locust First seven days of fourteen-day experiment are shown
Day 7
a
b c d
Figure A7 Damage to New Mexico locust seeds observed during germination counts following hot water scarification A7a Hot water replicate including damaged seedlings and numerous unimbibed seeds A7b Close-up ofseed where radicle failed to elongate A7c Close-up ofseed where radicle elongated but was delayed in freeing itself from the seed coat A7d Close-up ofseed where radicle completely broke offfrom embryo
60
a
b
Figure A8 New Mexico locust seeds following percussion treatment at time of germination counts A8a Percussion replicate with uniformly healthy seedlings and near complete imbibition of seeds A8b Close-up ofpercussion-treated seedling
61
Discussion
Papilionoid seeds have a specific anatomical feature known as the strophiole
or lens-the natural site of water entry to the seed (Hamly 1932) The strophiole is
located on the cotyledonary lobe of a locust seed (Figure A9) When a papilionoid
legume seed is percussed for the appropriate amount of time repeated hits on the
integrated seed coat loosen the constrained cells of the strophiolar region without
excessively damaging the rest of the seed coat (Ballard 1976)
When a percussed seed is soaked water enters exclusively through the
strophiole in a controlled manner (Kelly and Staden 1987) This regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues This
contrasts with seeds that have been hot water or acid scarified treatments that can
randomly degrade the seed coat Unlocalized cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rehman et al 1998 Marunda 1990)
Papilionoids are the largest subfamily of legumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust and black locust these legumes often play an integral role in the
revegetation ofdisturbed lands Developing superior scarification methods to the
standard hot water and acid treatments should fadlitate the use ofthese legumes in
restoration projects
62
Figure A9 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (bc)
63
APPENDIXB
GERMINATION PERCENTS AND STANDARD ERRORS FOR SOURCE
BLOCK AND TREATMENT FOR NEW MEXICO LOCUST
Table B 1 Gennination percents and standard errors for source block and treatment for New Mexico locust
Source Block Treatment Germination Percentage Standard Error
Capitan 1 Immersion 74 437 Capitan 2 Immersion 69 462 Capitan 3 Immersion 71 454 Capitan 4 Immersion 67 470 Capitan Boiling 30 sec 79 407 Capitan 2 Boiling 30 sec 76 427 Capitan 3 Boiling 30 sec 72 449 Capitan 4 Boiling 30 sec 76 427 Capitan Boiling 60 sec 71 454 Capitan 2 Boiling 60 sec 54 498 Capitan 3 Boiling 60 sec 67 470 Capitan 4 Boiling 60 sec 76 427 Capitan I Control 5 218 Capitan 2 Control 7 255 Capitan 3 Control 4 196 Capitan 4 Control 5 218 Capitan 1 Percussion I min 47 499 Capitan 2 Percussion I min
470
Capitan Percussion I min 39 488
Capitan 4 Percussion I min 38 485 Capitan I Percussion 2 min 55 497 Capitan Capitan
2
Percussion 2 min Percussion 2 min
71 65
454 477
Capitan 4 Percussion 2 min 56 496 Capitan I Percussion 4 min 86 347 Capitan 2 Percussion 4 min 79 407 Capitan 3 Percussion 4 min 81 392 Capitan 4 Percussion 4 min 82 384 Capitan I Percussion 8 min 90 300 Capitan 2 Percussion 8 min 88 325 Capitan 3 Percussion 8 min 90 300 Capitan 4 Percussion 8 min 90 300 Gila I Immersion 71 454 Gila 2 Immersion 77 421 Gila
Immersion 75 433
Gila 4 Immersion 72 449
Gila I Boiling 30 sec 68 466
Gila 2 Boiling 30 sec 81 392
Gila 3 Boiling 30 sec 54 498
Gila 4 Boiling 30 sec 63 483
Gila I Boiling 60 sec 55 497
Gila 2 Boiling 60 sec 68 466
Gila 3 Boiling 60 sec 57 495
Gila 4 Boiling 60 sec 70 458
65
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Gila 2 Control 13 336 Gila 3 Control 13 336 Gila 4 Control 20 400 Gila 1 Percussion 1 min 66 474 Gila 2 Percussion 1 min 57 495 Gila 3 Percussion 1 min 73 444 Gila 4 Percussion 1 min 82 384 Gila 1 Percussion 2 min 81 392 Gila 2 Percussion 2 min 86 347 Gila 3 Percussion 2 min 85 357 Gila 4 Percussion 2 min 54 498 Gila 1 Percussion 4 min 79 407 Gila 2 Percussion 4 min 83 376 Gila 3 Percussion 4 min 84 367 Gila 4 Percussion 4 min 79 407 Gila 1 Percussion 8 min 51 500 Gila 2 Percussion 8 min 76 427 Gila 3 Percussion 8 min 52 500 Gila 4 Percussion 8 min 74 439 Holman 1 Immersion 83 376 Holman 2 Immersion 86 347 Holman 3 Immersion 81 392 Holman 4 Immersion 90 300 Holman 1 Boiling 30 sec 90 300 Holman 2 Boiling 30 sec 85 357 Holman 3 Boiling 30 sec 87 336 Holman 4 Boiling 30 sec 83 376 Holman 1 Boiling 60 sec 79 407 Holman 2 Boiling 60 sec 80 400 Holman 3 Boiling 60 sec 86 347 Holman 4 Boiling 60 sec 93 255 Holman Control 25 433 Holman 2 Control 19 392 Holman Control 21 407
Holman 4 Control 24 427
Holman Percussion 1 min 77 421
Holman 2 Percussion 1 min 85 357
Holman 3 Percussion 1 min 80 400
Holman 4 Percussion 1 min 89 313
Holman 1 Percussion 2 min 88 325
Holman 2 Percussion 2 min 96 196
Holman 3 Percussion 2 min 93 255
Holman 4 Percussion 2 min 96 196
Holman 1 Percussion 4 min 92 271
Holman 2 Percussion 4 min 97 171
66
I
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Holman 3 Percussion 4 min 91 286 Holman 4 Percussion 4 min 96 196 Holman 1 Percussion 8 min 88 325 Holman 2 Percussion 8 min 85 357 Holman 3 Percussion 8 min 83 376 Holman 4 Percussion 8 min 82 384 Jemez 1 Immersion 53 499 Jemez 2 Immersion 44 496 Jemez 3 Immersion 52 500 Jemez 4 Immersion 70 458 Jemez 1 Boiling 30 sec 58 494 Jemez 2 Boiling 30 sec 55 497 Jemez 3 Boiling 30 sec 48 500 Jemez 4 Boiling 30 sec 65 477 Jemez 1 Boiling 60 sec 68 466 Jemez 2 Boiling 60 sec 63 483 Jemez 3 Boiling 60 sec 73 444 Jemez 4 Boiling 60 sec 89 313 Jemez 1 Control 2 140 Jemez 2 Control 9 286 Jemez 3 Control 6 237 Jemez 4 Control 8 271 Jemez 1 Percussion 1 min 58 494 Jemez 2 Percussion 1 min 63 483 Jemez 3 Percussion 1 min 77 421 Jemez 4 Percussion 1 min 89 313 Jemez 1 Percussion 2 min 68 466 Jemez 2 Percussion 2 min 64 480 Jemez 3 Percussion 2 min 80 400 Jemez 4 Percussion 2 min 83 376 Jemez Percussion 4 min 68 466 Jemez 2 Percussion 4 min 79 407 Jemez 3 Percussion 4 min 70 458 Jemez 4 Percussion 4 min 83 376 Jemez 1 Percussion 8 min 52 500 Jemez 2 Percussion 8 min 59 492 Jemez 3 Percussion 8 min 61 488 Jemez 4 Percussion 8 min 70 458 Magdalena 1 Immersion 69 462 Magdalena 2 Immersion 69 462 Magdalena J Immersion 70 458 Magdalena 4 Immersion 85 357 Magdalena 1 Boiling 30 sec 65 477 Magdalena 2 Boiling 30 sec 71 454 Magdalena 3 Boiling 30 sec 68 466
67
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Magdalena 4 Boiling 30 sec 83 376 Magdalena 1 Boiling 60 sec 63 483 Magdalena 2 Boiling 60 sec 68 466 Magdalena 3 Boiling 60 sec 63 483 Magdalena 4 Boiling 60 sec 82 384 Magdalena 1 Control 11 313 Magdalena 2 Control 6 237 Magdalena 3 Control 5 218 Magdalena 4 Control 4 196 Magdalena Percussion 1 min 32 466 Magdalena 2 Percussion 1 min 45 497 Magdalena 3 Percussion 1 min 46 498 Magdalena 4 Percussion I min 36 480 Magdalena 1 Percussion 2 min 67 470 Magdalena 2 Percussion 2 min 68 466 Magdalena 3 Percussion 2 min 65 477 Magdalena 4 Percussion 2 min 81 392 Magdalena 1 Percussion 4 min 87 336 Magdalena 2 Percussion 4 min 97 171 Magdalena 3 Percussion 4 min 83 376 Magdalena 4 Percussion 4 min 97 171 Magdalena 1 Percussion 8 min 98 lAO Magdalena 2 Percussion 8 min 100 000 Magdalena 3 Percussion 8 min 96 196 Magdalena 4 Percussion 8 min 98 lAO Manzano 1 Immersion 67 470 Manzano 2 Immersion 57 495 Manzano 3 Immersion 72 4049 Manzano 4 Immersion 73 4044 Manzano 1 Boiling 30 sec 70 458 Manzano 2 Boiling 30 sec 64 480 Manzano 3 Boiling 30 sec 71 454 Manzano 4 Boiling 30 sec 80 400 Manzano I Boiling 60 sec 50 500 Manzano 2 Boiling 60 sec 53 499 Manzano 3 Boiling 60 sec 59 492 Manzano 4 Boiling 60 sec 74 439 Manzano I Control 4 196 Manzano 2 Control 8 271 Manzano 3 Control 1 099 Manzano 4 Control 6 237 Manzano 1 Percussion 1 min 52 500 Manzano 2 Percussion 1 min 39 488 Manzano 3 Percussion 1 min 36 480 Manzano 4 Percussion 1 min 51 500
68
Table B 1 Continued
Source Block Treatment Gerrni~ation Percentage Standard Error
Manzano Percussion 2 min 53 499 Manzano 2 Percussion 2 min 58 494 Manzano 3 Percussion 2 min 61 488 Manzano 4 Percussion 2 min 62 485 Manzano 1 Percussion 4 min 76 427 Manzano 2 Percussion 4 min 85 357 Manzano 3 Percussion 4 min 79 407 Manzano 4 Percussion 4 min 83 376 Manzano 1 Percussion 8 min 87 336 Manzano 2 Percussion 8 min 83 376 Manzano 3 Percussion 8 min 83 376 Manzano 4 Percussion 8 min 75 433 Raton 1 Immersion 54 498 Raton 2 Immersion 37 483 Raton 3 Immersion 39 488 Raton 4 Immersion 69 462 Raton 1 Boiling 30 sec 36 480 Raton 2 Boiling 30 sec 20 400 Raton 3 Boiling 30 sec 28 449 Raton 4 Boiling 30 sec 60 490 Raton 1 Boiling 60 sec 15 357 Raton 2 Boiling 60 sec 171 Raton 3 Boiling 60 sec 4 196 Raton 4 Boiling 60 sec 35 477 Raton 1 Control 4 196 Raton 2 Control 12 325 Raton 3 Control 2 140 Raton 4 Control 6 237 Raton 1 Percussion 1 min 52 500 Raton 2 Percussion I min 29 454 Raton 3 Percussion I min 35 477 Raton 4 Percussion 1 min 31 462 Raton 1 Percussion 2 min 55 497 Raton 2 Percussion 2 min 35 477 Raton 3 Percussion 2 min 33 470 Raton 4 Percussion 2 min 42 494 Raton 1 Percussion 4 min 65 477 Raton 2 Percussion 4 min 52 500 Raton Percussion 4 min 51 500 Raton 4 Percussion 4 min 53 499 Raton 1 Percussion 8 min 88 325 Raton 2 Percussion 8 min 80 400 Raton 3 Percussion 8 min 73 444 Raton 4 Percussion 8 min 74 439 Sacramento 1 Immersion 43 495
69
Table B 1 Continued
-Source Block Treatment Germination Percentage Standard Error
Sacramento 2 Immersion 30 458 Sacramento 3 Immersion 38 485 Sacramento 4 Immersion 56 496 Sacramento 1 Boiling 30 sec 47 499 Sacramento 2 Boiling 30 sec 48 500 Sacramento 3 Boiling 30 sec 48 500 Sacramento 4 Boiling 30 sec 56 496 Sacramento 1 Boiling 60 sec 52 500 Sacramento 2 Boiling 60 sec 67 470 Sacramento 3 Boiling 60 sec 43 495 Sacramento 4 Boiling 60 sec 48 500 Sacramento 1 Control 0 000 Sacramento 2 Control 3 171 Sacramento 3 Control 3 171 Sacramento 4 Control 099 Sacramento 1 Percussion 1 min 57 495 Sacramento 2 Percussion 1 min 61 488 Sacramento 3 Percussion 1 min 62 485 Sacramento 4 Percussion 1 min 55 497 Sacramento 1 Percussion 2 min 84 367 Sacramento 2 Percussion 2 min 70 458 Sacramento 3 Percussion 2 min 81 392 Sacramento 4 Percussion 2 min 69 462 Sacramento 1 Percussion 4 min 91 286 Sacramento 2 Percussion 4 min 84 367 Sacramento 3 Percussion 4 min 84 367 Sacramento 4 Percussion 4 min 94 237 Sacramento 1 Percussion 8 min 82 384 Sacramento 2 Percussion 8 min 81 392 Sacramento 3 Percussion 8 min 82 384 Sacramento 4 Percussion 8 min 81 392 Sandia 1 Immersion 76 427 Sandia 2 Immersion 70 458 Sandia 3 Immersion 70 458 Sandia 4 Immersion 81 392 Sandia 1 Boiling 30 sec 78 414 Sandia 2 Boiling 30 sec 71 454 Sandia 3 Boiling 30 sec 63 483 Sandia 4 Boiling 30 sec 78 414 Sandia 1 Boiling 60 sec 67 470 Sandia 2 Boiling 60 sec 65 477 Sandia 3 Boiling 60 sec 74 439 Sandia 4 Boiling 60 sec 66 474 Sandia 1 Control 16 367 Sandia 2 Control 7 255
70
Table B 1 Continued
Source Block Treatment Gennination Percentage Standard Error
Sandia 3 Control 8 271 Sandia 4 Control 16 367 Sandia 1 Percussion 1 min 67 470 Sandia 2 Percussion 1 min 61 488 Sandia 3 Percussion 1 min 66 474 Sandia 4 Percussion 1 min 59 492 Sandia I Percussion 2 min 82 384 Sandia 2 Percussion 2 min 83 376 Sandia 3 Percussion 2 min 81 392 Sandia 4 Percussion 2 min 84 367 Sandia 1 Percussion 4 min 89 313 Sandia 2 Percussion 4 min 86 347 Sandia 3 Percussion 4 min 89 313 Sandia 4 Percussion 4 min 85 357 Sandia 1 Percussion 8 min 80 400 Sandia 2 Percussion 8 min 77 421 Sandia 3 Percussion 8 min 69 462 Sandia 4 Percussion 8 min 66 474
LITERATURE CITED
Ashby WC Vogel WG Rogers NF 1985 Black locust in the reclamation equation General Technical Report Northeastern Forest Experiment Station USDA Forest Service NE-I05 12 p
Ballard LAT 1973 Physical barriers to gennination Seed Sci Technoll 285-303
___ 1976 Strophiolar water conduction in the seeds ofTrifolieae induced by action on the testa at nonstrophiolar sites Austr J Plant Physiol3 465-469
Barton LV 1947 Special studies on seed coat impenneability Contrib Boyce Thompson Inst 14 355-362
Baskin CC Baskin JM 1998 Seeds Ecology biogeography and evolution of donnancy and gennination Academic Press London UK 666 p
Bewley JD 1997 Seed germination and donnancy Plant Ce119 1055-1066
Bewley JD Black M 1994 Seeds physiology of development and germination 2nd ed Plenum Press New York NY 445 p
Briscoe CB 1996 Growing Nitrogen Fixing Trees in Acid Soils In MH Powell (ed) Nitrogen Fixing Trees for Acid Soils- A Field ManuaL Winrock International Morrilton AR 22 p
Copeland LO McDonald MB 1995 Principles of Seed Science and Technology 3rded Chapman amp Hall New York NY 409 p
Cox RE Klett JE 1984 Seed gennination requirements of native Colorado plants for use in the landscape Plant Propag 30(2) 6-10
Dell B 1980 Structure and function ofthe strophiolar plug in seeds ofAlbizia Lophantha Am J Bot 67 556-563
Dick-Peddie WA 1993 New Mexico Vegetation Past Present and Future University ofNew Mexico Press Albuquerque NM 244 p
Dreesen DR 2001 Propagation protocol for production ofcontainer Robinia neomexicana plants Los Lunas Plant Materials Center Los Lunas New Mexico In Native Plant Network URL httpwwwnativeplantnetworkorg (accessed 9 January 2002) Moscow (ID) University ofIdaho CoIl Natural Resources For Res Nursery
72
Dreesen DR Harrington JT 1997 Propagation of native plants for restoration projects in the Southwestern US- Preliminary Investigations In Landis TD Thompson JR tech coords National Proceedings Forest and Conservation Nursery Assocs Gen Tech Rep PNW-GTR-419 Portland OR USDA For Ser PNW Res Sta 77-88
Gottfried GJ 1980 Control ofNew Mexican locust and the effect on planted ponderosa pine in central Arizona US For Ser Res Note RM-386 Rocky Mountain For Range ExpStn Fort Collins CO
Gosling PG Samuel YK Jones SK 1995 A systematic examination of germination temperature chipping and water temperaturesoak duration pretreatments on the seeds ofLeucaena leucocephala Seed Sci amp Technol 23521-532
Hamly DH 1932 Softening of the seeds of Melilotus alba Bot Gazette 93 345shy375
Hanna PJ 1984 Anatomical features of the seed coat ofAcacia kempeana (Mueller) which relate to increased germination rate induced by heat treatment New Phytol 96 23-29
Hanks JP Dick-Peddie WA 1974 Vegetation patterns of the White Mountains New Mexico Southwest Naturalist 18(4) 371-382
Hine S Harrington JT Dreesen DR 1997 Seed scarification requirements for Robinia neomexicana In Harrington JTtech coord Proc Third SouthwestemContainer Growers Conf Feb 12-13 1997 Ft Collins CO
Hopkinson JM 1993 The strophiole of leguminous seeds Newsletter- IntI Herbage Seed Production Res Grp 18 7-8
---- 1997 Differences in hardseededness between Cunningham and Tarramba leucaena In Leucnet News The Newsletter of the International LeucaenaRes and Devlt Netwk No4
ISTA (International Seed Testing Association) 1999 International rules for seed testing rules Seed Sci amp Technol27 Supplement
Kelly KM van Staden J 1987 The lens as the site of permeability in the papilionoid seed Aspalathus linearis J Plant Physiol 128(45) 395-404
73
Keresztesi B 1988 Black locust the tree of agriculture Outlook on Agr 17(2) 77~85
Kigel 1 and Galili G 1995 Seed development and germination Marcel Dekker New York 853 p
Klemmedson 10 1994 New Mexico locust and parent material Influence on macronutrients of forest floor and soil Soil Sci Soc Am 1 58 974~980
Kuo WH Tam AW 1988 The pathway of water absorption of mung bean seeds Seed Sci amp Technol16(1) 139~144
Leadem CL 1997 Dormancy--unlocking seed secrets In Landis TD Thompson lR tech coords National Proceedings Forestand Conservation Nursery Assocs Gen Tech Rep PNW-G TR-419 Portland OR USDA For Ser PNW Res Sta 43-52
Lin Y Wagner MR Seweyestewa A Honahni MD DeGomez M McMillin JD 1996 Influence of cultural treatments on seed germination and seedling growth of Robinia neomexicana Forest Farm and Community Tree Res Reports Volume 1
Marunda CT 1990 Effects of seed pretreatment on the development ofAcacia auriculforinis and A holosericea seedlings ACIAR Proceedings Series No 28
Meginnis HG 1937 Sulphuric acid treatment to increase germination of black locust seed USDA Circ 453 34 p
Miklas PN Towensend CE Ladd SL 1987 Seedcoat anatomy and the scarification ofcicer milkvetch seed Crop Sci 27 766-772
Monsen SB 1984 Use of shrubs on mine spoils In The Challenge ofProducing Native Plants for the Intermountain Area Proceedings Intermountain Nurserymans Association 1983 conference Las Vegas NV compo PM Murphy 26-31 Ogden UT USDA For Ser Gen Tech Report INT-l68
Olson DF 1974 Robinia L Locust In Seeds of Woody Plants in the US tech coord CS Schopmeyer 728-731 USDA For Ser Agr Handbk No450
Rehman S Loescher RNl Harris P1C 1998 Dormancy breaking and germination ofAcacia salicina Lindl seeds Seed Sci amp Techno127 553-557
Rolston MP 1978 Water impermeable seed dormancy Bot Rev 44 365-396
74
SAS Institute Inc 1989 SASSTAT Users Guide Version 6 4th Edition Volume 1 Cary NC SAS Institute Inc 943 p
SerratoValenti G Conara L Ferrando M Modenesi P 1993 Structural and histochemical features ofStylosanthes scabra (Leguminosae Papilionoidae) seed coat as related to water entry Can J Bot 71 834-840
Simpson BJ 1988 A field guide to Texas trees Austin TX Texas Monthly Press 372p
Tran VN Cavanagh AK 1984 Structural aspects ofdormancy In Seed Physiology Vol 2 Murray DR (ed) p1-44 Academic Press Australia
USDA Forest Service 1948 Woody-plant seed manual USDA Misc Pub1654 416p
Vines RA 1960 Trees Shrubs and Woody Vines of the Southwest Austin Univ ofTexas Press 1104 p
Wagner MR Larson RE Craig TP 1992 Ecology and silvics ofNew Mexico locust (Robinia neomexicana) with an emphasis on the arboreal form p53-57 In JW Hanover K Miller and S Plesko (eds) Proc Intemat Confon Black Locust Biology Culture and Utilization Mich State Univ USA
Wilson JK 1944 Immersing seeds of species of Robinia in boiling water hastens germination J For 42 453-454
Zimmerman RW Carpenter SB 1980 First-year coppice production from a 5shyyear old black locust stand on surface mine spoil p309-314 Centr Hardwd For ConfProc University ofMissouri Columbia
b
c
Figure A2 Implementation ofhot water and percussion scarification A2a Hot water bath A2b Paint-shaker set-up A2c Placing soil tin containing seeds at end ofpaint shaker ann
52
Las Cruces NM elevation of 1300 meters) Each 100-seed replicate was placed in a
test tube and each test tube was immediately removed to cool at room temperature
Percussion scarification was implemented using a pneumatic paint shaker
(Central Pneumatic model 00422 Camarillo CA) (Figure A2b) This allowed
standardization of treatment intensity Compressed air pressure was maintained at
530kPa plusmn 30kPa (80psi plusmn 5psi) resulting in approximately 350 oscillations per
minute Each lOO-seed sample was placed in a 118 ml (4 oz) soil tin for shaking To
maximize lateral movement of paint shaker a paint can was used as a spacer to place
soil tins at the end of the shaking arm (Figure A2c) This allowed shaking to occur at
the greatest distance from the pivot point (fulcrum) Following all treatments
including the control seeds were soaked for twenty-four hours in distilled water prior
to plating out
Gennination was evaluated on lab benches at room temperature Each
replicate of seeds was placed in a 100 cm petri dish on moistened filter paper with
humidity maintained by enclosing the petri dishes in ziplock bags Gennination was
monitored daily for 14 days in accordance with International Seed Testing
Association standards for Robinia species (ISTA 1999) Gennination was defined as
protrusion of the radicle from the embryo by at least 1mm as viewed by the naked
eye
Categorical Analysis of Variance using Prpc Catmod (SAS Institute 1989)
was used to detennine treatment differences in germination percentages for each
source This procedure is an extension of a Chi-square test of homogeneity using the
53
natural log of the ratio of germinated to non-germinated seeds for each treatment Pshy
values less than 005 were considered significant A limited set of pairwiseshy
comparisons was conducted to compare treatment mean percents using a
conservative alpha value of 005 divided by the number of comparisons
Results
Percussion maximized germination for both New Mexico locust and black
locust There were three germination response levels for New Mexico locust (Figure
A3) First there was a low response in the control indicating a dormant seed lot
The hot water treatment and I-minute duration of percussion share a second
intermediate response Germination was highest at percussion levels from 2 to 10
minutes
A selected percussion treatment intermediate in time (5 minutes) produced the
steepest germination curve indicating the fastest germination rate (Figure A4) The
hot water treatment produced an intermediate curve or relatively slower germination
rate The control curve was very flat indicating the slowest germination rate for New
Mexico locust
Control seeds germinated poorly for black locust as well (Figure A5) Hot
water scarification improved germination over the control but all levels of percussion
improved germination over hot water The optimal durations of percussion treatment
were bracketed with a significant increase from the 1 to 2 minute percussion level a
high germination response from 2 to 5 minutes of percussion and finally a significant
54
~ d 25 CL)
p t1 0 ~ t1
g Vl ~ Vl
100~---------------------------------------------------------------
80
60
40
20
o Control Hot Water Percussion
I min Percussion
2 min Percussion
3 min
Scarification
Percussion 4 min
Figure A3 Effect of scarification on germination response for New Mexico locust
Percussion 5 min
=significant difference between adjacent treatment bars ns = no significant difference
Percussion 10 min
~ ~ () u agt
Il-t d o ~
~ ~ c3
100middotlr~~~------------------------------------e- Percussion 5min middot 0middot Hot Water -- Control
80-J=middot======
6b-middot-lmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middot--middotmiddot-middot~-middotmiddot -middotmiddot middot middotmiddot-middot 1
40
20
-----~-----------
0 0 0 middotmiddotmiddotmiddotmiddot0middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot0
0 --~-----y-----~------_---~--- T-----~
o shy -TDay 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A4 Effect of selected scarification treatments on germination speed for New Mexico locust First seven days of fourteen-day experiment are shown
Day 7
80
~ cd
5 60 ()
[) Cl d 0 1t1 d
40sect dUl
-J
20
o
ns
Scarification
Figure A5 Effect of scarification on germination response for black locust = significant difference between adjacent treatment bars ns = no significant difference
decrease at the 10-minute level This drop-off suggests that 10 minutes of percussion
over-treats seeds from this lot of-black locust
A selected percussion treatment intermediate in time (5 minutes) also had the
fastest germination rate for black locust (Figure A6)_ The hot water curve is
somewhat flatter indicating a slower germination rate The control curve is very flat
indicating a slow germination rate
A qualitative difference between hot water and percussion-treated seeds was
also observed Along with healthy germinants the hot water treatment produced
over-treated seeds (Figure A 7a) Examples of damaged germinants include seeds
where the radicle did not elongate (Figure A 7b) seeds where the radicle elongated
but was delayed in freeing itself from the seed coat (Figure A7c) and seeds where the
radicle broke off completely from the embryo (Figure A7d) In addition to these
examples of over-treatment there were numerous small dark seeds that failed to
imbibe water For percussion treatment nearly all seeds took up water (Figure A8a)
Germination was rapid and uniform with little damage evident (Figure A8b)
To summarize both New Mexico locust and black locust seed lots were
dormant The standard hot water scarification treatment improved germination but
only to 56 and 41 for New Mexico locust and black locust respectively Nearly
all percussion durations improved total germination as well as germination rate over
the hot water treatment Germination was greater than 90 for percussion durations
of 4 5 and 10 minutes for New Mexico locust and 3 4 and 5 minutes for black locust
58
CI)
sgt t CI) 0
igt 0
sect ttl t E
Vl 0
c3
100r==~==~~=r------------------------------------~ ___ Percussion 5 min
middotmiddotmiddot0middotmiddotmiddot Hot Water -lIf- Control
80r=========~--~~----------------------------~
60
40
20 -~-~---~---------- ---~------------I
omiddot
O
_~_----------lIf-----~bull -----_ shy---shy I
Day 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A6 Effect of selected scarification treatments on germination speed for black locust First seven days of fourteen-day experiment are shown
Day 7
a
b c d
Figure A7 Damage to New Mexico locust seeds observed during germination counts following hot water scarification A7a Hot water replicate including damaged seedlings and numerous unimbibed seeds A7b Close-up ofseed where radicle failed to elongate A7c Close-up ofseed where radicle elongated but was delayed in freeing itself from the seed coat A7d Close-up ofseed where radicle completely broke offfrom embryo
60
a
b
Figure A8 New Mexico locust seeds following percussion treatment at time of germination counts A8a Percussion replicate with uniformly healthy seedlings and near complete imbibition of seeds A8b Close-up ofpercussion-treated seedling
61
Discussion
Papilionoid seeds have a specific anatomical feature known as the strophiole
or lens-the natural site of water entry to the seed (Hamly 1932) The strophiole is
located on the cotyledonary lobe of a locust seed (Figure A9) When a papilionoid
legume seed is percussed for the appropriate amount of time repeated hits on the
integrated seed coat loosen the constrained cells of the strophiolar region without
excessively damaging the rest of the seed coat (Ballard 1976)
When a percussed seed is soaked water enters exclusively through the
strophiole in a controlled manner (Kelly and Staden 1987) This regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues This
contrasts with seeds that have been hot water or acid scarified treatments that can
randomly degrade the seed coat Unlocalized cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rehman et al 1998 Marunda 1990)
Papilionoids are the largest subfamily of legumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust and black locust these legumes often play an integral role in the
revegetation ofdisturbed lands Developing superior scarification methods to the
standard hot water and acid treatments should fadlitate the use ofthese legumes in
restoration projects
62
Figure A9 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (bc)
63
APPENDIXB
GERMINATION PERCENTS AND STANDARD ERRORS FOR SOURCE
BLOCK AND TREATMENT FOR NEW MEXICO LOCUST
Table B 1 Gennination percents and standard errors for source block and treatment for New Mexico locust
Source Block Treatment Germination Percentage Standard Error
Capitan 1 Immersion 74 437 Capitan 2 Immersion 69 462 Capitan 3 Immersion 71 454 Capitan 4 Immersion 67 470 Capitan Boiling 30 sec 79 407 Capitan 2 Boiling 30 sec 76 427 Capitan 3 Boiling 30 sec 72 449 Capitan 4 Boiling 30 sec 76 427 Capitan Boiling 60 sec 71 454 Capitan 2 Boiling 60 sec 54 498 Capitan 3 Boiling 60 sec 67 470 Capitan 4 Boiling 60 sec 76 427 Capitan I Control 5 218 Capitan 2 Control 7 255 Capitan 3 Control 4 196 Capitan 4 Control 5 218 Capitan 1 Percussion I min 47 499 Capitan 2 Percussion I min
470
Capitan Percussion I min 39 488
Capitan 4 Percussion I min 38 485 Capitan I Percussion 2 min 55 497 Capitan Capitan
2
Percussion 2 min Percussion 2 min
71 65
454 477
Capitan 4 Percussion 2 min 56 496 Capitan I Percussion 4 min 86 347 Capitan 2 Percussion 4 min 79 407 Capitan 3 Percussion 4 min 81 392 Capitan 4 Percussion 4 min 82 384 Capitan I Percussion 8 min 90 300 Capitan 2 Percussion 8 min 88 325 Capitan 3 Percussion 8 min 90 300 Capitan 4 Percussion 8 min 90 300 Gila I Immersion 71 454 Gila 2 Immersion 77 421 Gila
Immersion 75 433
Gila 4 Immersion 72 449
Gila I Boiling 30 sec 68 466
Gila 2 Boiling 30 sec 81 392
Gila 3 Boiling 30 sec 54 498
Gila 4 Boiling 30 sec 63 483
Gila I Boiling 60 sec 55 497
Gila 2 Boiling 60 sec 68 466
Gila 3 Boiling 60 sec 57 495
Gila 4 Boiling 60 sec 70 458
65
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Gila 2 Control 13 336 Gila 3 Control 13 336 Gila 4 Control 20 400 Gila 1 Percussion 1 min 66 474 Gila 2 Percussion 1 min 57 495 Gila 3 Percussion 1 min 73 444 Gila 4 Percussion 1 min 82 384 Gila 1 Percussion 2 min 81 392 Gila 2 Percussion 2 min 86 347 Gila 3 Percussion 2 min 85 357 Gila 4 Percussion 2 min 54 498 Gila 1 Percussion 4 min 79 407 Gila 2 Percussion 4 min 83 376 Gila 3 Percussion 4 min 84 367 Gila 4 Percussion 4 min 79 407 Gila 1 Percussion 8 min 51 500 Gila 2 Percussion 8 min 76 427 Gila 3 Percussion 8 min 52 500 Gila 4 Percussion 8 min 74 439 Holman 1 Immersion 83 376 Holman 2 Immersion 86 347 Holman 3 Immersion 81 392 Holman 4 Immersion 90 300 Holman 1 Boiling 30 sec 90 300 Holman 2 Boiling 30 sec 85 357 Holman 3 Boiling 30 sec 87 336 Holman 4 Boiling 30 sec 83 376 Holman 1 Boiling 60 sec 79 407 Holman 2 Boiling 60 sec 80 400 Holman 3 Boiling 60 sec 86 347 Holman 4 Boiling 60 sec 93 255 Holman Control 25 433 Holman 2 Control 19 392 Holman Control 21 407
Holman 4 Control 24 427
Holman Percussion 1 min 77 421
Holman 2 Percussion 1 min 85 357
Holman 3 Percussion 1 min 80 400
Holman 4 Percussion 1 min 89 313
Holman 1 Percussion 2 min 88 325
Holman 2 Percussion 2 min 96 196
Holman 3 Percussion 2 min 93 255
Holman 4 Percussion 2 min 96 196
Holman 1 Percussion 4 min 92 271
Holman 2 Percussion 4 min 97 171
66
I
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Holman 3 Percussion 4 min 91 286 Holman 4 Percussion 4 min 96 196 Holman 1 Percussion 8 min 88 325 Holman 2 Percussion 8 min 85 357 Holman 3 Percussion 8 min 83 376 Holman 4 Percussion 8 min 82 384 Jemez 1 Immersion 53 499 Jemez 2 Immersion 44 496 Jemez 3 Immersion 52 500 Jemez 4 Immersion 70 458 Jemez 1 Boiling 30 sec 58 494 Jemez 2 Boiling 30 sec 55 497 Jemez 3 Boiling 30 sec 48 500 Jemez 4 Boiling 30 sec 65 477 Jemez 1 Boiling 60 sec 68 466 Jemez 2 Boiling 60 sec 63 483 Jemez 3 Boiling 60 sec 73 444 Jemez 4 Boiling 60 sec 89 313 Jemez 1 Control 2 140 Jemez 2 Control 9 286 Jemez 3 Control 6 237 Jemez 4 Control 8 271 Jemez 1 Percussion 1 min 58 494 Jemez 2 Percussion 1 min 63 483 Jemez 3 Percussion 1 min 77 421 Jemez 4 Percussion 1 min 89 313 Jemez 1 Percussion 2 min 68 466 Jemez 2 Percussion 2 min 64 480 Jemez 3 Percussion 2 min 80 400 Jemez 4 Percussion 2 min 83 376 Jemez Percussion 4 min 68 466 Jemez 2 Percussion 4 min 79 407 Jemez 3 Percussion 4 min 70 458 Jemez 4 Percussion 4 min 83 376 Jemez 1 Percussion 8 min 52 500 Jemez 2 Percussion 8 min 59 492 Jemez 3 Percussion 8 min 61 488 Jemez 4 Percussion 8 min 70 458 Magdalena 1 Immersion 69 462 Magdalena 2 Immersion 69 462 Magdalena J Immersion 70 458 Magdalena 4 Immersion 85 357 Magdalena 1 Boiling 30 sec 65 477 Magdalena 2 Boiling 30 sec 71 454 Magdalena 3 Boiling 30 sec 68 466
67
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Magdalena 4 Boiling 30 sec 83 376 Magdalena 1 Boiling 60 sec 63 483 Magdalena 2 Boiling 60 sec 68 466 Magdalena 3 Boiling 60 sec 63 483 Magdalena 4 Boiling 60 sec 82 384 Magdalena 1 Control 11 313 Magdalena 2 Control 6 237 Magdalena 3 Control 5 218 Magdalena 4 Control 4 196 Magdalena Percussion 1 min 32 466 Magdalena 2 Percussion 1 min 45 497 Magdalena 3 Percussion 1 min 46 498 Magdalena 4 Percussion I min 36 480 Magdalena 1 Percussion 2 min 67 470 Magdalena 2 Percussion 2 min 68 466 Magdalena 3 Percussion 2 min 65 477 Magdalena 4 Percussion 2 min 81 392 Magdalena 1 Percussion 4 min 87 336 Magdalena 2 Percussion 4 min 97 171 Magdalena 3 Percussion 4 min 83 376 Magdalena 4 Percussion 4 min 97 171 Magdalena 1 Percussion 8 min 98 lAO Magdalena 2 Percussion 8 min 100 000 Magdalena 3 Percussion 8 min 96 196 Magdalena 4 Percussion 8 min 98 lAO Manzano 1 Immersion 67 470 Manzano 2 Immersion 57 495 Manzano 3 Immersion 72 4049 Manzano 4 Immersion 73 4044 Manzano 1 Boiling 30 sec 70 458 Manzano 2 Boiling 30 sec 64 480 Manzano 3 Boiling 30 sec 71 454 Manzano 4 Boiling 30 sec 80 400 Manzano I Boiling 60 sec 50 500 Manzano 2 Boiling 60 sec 53 499 Manzano 3 Boiling 60 sec 59 492 Manzano 4 Boiling 60 sec 74 439 Manzano I Control 4 196 Manzano 2 Control 8 271 Manzano 3 Control 1 099 Manzano 4 Control 6 237 Manzano 1 Percussion 1 min 52 500 Manzano 2 Percussion 1 min 39 488 Manzano 3 Percussion 1 min 36 480 Manzano 4 Percussion 1 min 51 500
68
Table B 1 Continued
Source Block Treatment Gerrni~ation Percentage Standard Error
Manzano Percussion 2 min 53 499 Manzano 2 Percussion 2 min 58 494 Manzano 3 Percussion 2 min 61 488 Manzano 4 Percussion 2 min 62 485 Manzano 1 Percussion 4 min 76 427 Manzano 2 Percussion 4 min 85 357 Manzano 3 Percussion 4 min 79 407 Manzano 4 Percussion 4 min 83 376 Manzano 1 Percussion 8 min 87 336 Manzano 2 Percussion 8 min 83 376 Manzano 3 Percussion 8 min 83 376 Manzano 4 Percussion 8 min 75 433 Raton 1 Immersion 54 498 Raton 2 Immersion 37 483 Raton 3 Immersion 39 488 Raton 4 Immersion 69 462 Raton 1 Boiling 30 sec 36 480 Raton 2 Boiling 30 sec 20 400 Raton 3 Boiling 30 sec 28 449 Raton 4 Boiling 30 sec 60 490 Raton 1 Boiling 60 sec 15 357 Raton 2 Boiling 60 sec 171 Raton 3 Boiling 60 sec 4 196 Raton 4 Boiling 60 sec 35 477 Raton 1 Control 4 196 Raton 2 Control 12 325 Raton 3 Control 2 140 Raton 4 Control 6 237 Raton 1 Percussion 1 min 52 500 Raton 2 Percussion I min 29 454 Raton 3 Percussion I min 35 477 Raton 4 Percussion 1 min 31 462 Raton 1 Percussion 2 min 55 497 Raton 2 Percussion 2 min 35 477 Raton 3 Percussion 2 min 33 470 Raton 4 Percussion 2 min 42 494 Raton 1 Percussion 4 min 65 477 Raton 2 Percussion 4 min 52 500 Raton Percussion 4 min 51 500 Raton 4 Percussion 4 min 53 499 Raton 1 Percussion 8 min 88 325 Raton 2 Percussion 8 min 80 400 Raton 3 Percussion 8 min 73 444 Raton 4 Percussion 8 min 74 439 Sacramento 1 Immersion 43 495
69
Table B 1 Continued
-Source Block Treatment Germination Percentage Standard Error
Sacramento 2 Immersion 30 458 Sacramento 3 Immersion 38 485 Sacramento 4 Immersion 56 496 Sacramento 1 Boiling 30 sec 47 499 Sacramento 2 Boiling 30 sec 48 500 Sacramento 3 Boiling 30 sec 48 500 Sacramento 4 Boiling 30 sec 56 496 Sacramento 1 Boiling 60 sec 52 500 Sacramento 2 Boiling 60 sec 67 470 Sacramento 3 Boiling 60 sec 43 495 Sacramento 4 Boiling 60 sec 48 500 Sacramento 1 Control 0 000 Sacramento 2 Control 3 171 Sacramento 3 Control 3 171 Sacramento 4 Control 099 Sacramento 1 Percussion 1 min 57 495 Sacramento 2 Percussion 1 min 61 488 Sacramento 3 Percussion 1 min 62 485 Sacramento 4 Percussion 1 min 55 497 Sacramento 1 Percussion 2 min 84 367 Sacramento 2 Percussion 2 min 70 458 Sacramento 3 Percussion 2 min 81 392 Sacramento 4 Percussion 2 min 69 462 Sacramento 1 Percussion 4 min 91 286 Sacramento 2 Percussion 4 min 84 367 Sacramento 3 Percussion 4 min 84 367 Sacramento 4 Percussion 4 min 94 237 Sacramento 1 Percussion 8 min 82 384 Sacramento 2 Percussion 8 min 81 392 Sacramento 3 Percussion 8 min 82 384 Sacramento 4 Percussion 8 min 81 392 Sandia 1 Immersion 76 427 Sandia 2 Immersion 70 458 Sandia 3 Immersion 70 458 Sandia 4 Immersion 81 392 Sandia 1 Boiling 30 sec 78 414 Sandia 2 Boiling 30 sec 71 454 Sandia 3 Boiling 30 sec 63 483 Sandia 4 Boiling 30 sec 78 414 Sandia 1 Boiling 60 sec 67 470 Sandia 2 Boiling 60 sec 65 477 Sandia 3 Boiling 60 sec 74 439 Sandia 4 Boiling 60 sec 66 474 Sandia 1 Control 16 367 Sandia 2 Control 7 255
70
Table B 1 Continued
Source Block Treatment Gennination Percentage Standard Error
Sandia 3 Control 8 271 Sandia 4 Control 16 367 Sandia 1 Percussion 1 min 67 470 Sandia 2 Percussion 1 min 61 488 Sandia 3 Percussion 1 min 66 474 Sandia 4 Percussion 1 min 59 492 Sandia I Percussion 2 min 82 384 Sandia 2 Percussion 2 min 83 376 Sandia 3 Percussion 2 min 81 392 Sandia 4 Percussion 2 min 84 367 Sandia 1 Percussion 4 min 89 313 Sandia 2 Percussion 4 min 86 347 Sandia 3 Percussion 4 min 89 313 Sandia 4 Percussion 4 min 85 357 Sandia 1 Percussion 8 min 80 400 Sandia 2 Percussion 8 min 77 421 Sandia 3 Percussion 8 min 69 462 Sandia 4 Percussion 8 min 66 474
LITERATURE CITED
Ashby WC Vogel WG Rogers NF 1985 Black locust in the reclamation equation General Technical Report Northeastern Forest Experiment Station USDA Forest Service NE-I05 12 p
Ballard LAT 1973 Physical barriers to gennination Seed Sci Technoll 285-303
___ 1976 Strophiolar water conduction in the seeds ofTrifolieae induced by action on the testa at nonstrophiolar sites Austr J Plant Physiol3 465-469
Barton LV 1947 Special studies on seed coat impenneability Contrib Boyce Thompson Inst 14 355-362
Baskin CC Baskin JM 1998 Seeds Ecology biogeography and evolution of donnancy and gennination Academic Press London UK 666 p
Bewley JD 1997 Seed germination and donnancy Plant Ce119 1055-1066
Bewley JD Black M 1994 Seeds physiology of development and germination 2nd ed Plenum Press New York NY 445 p
Briscoe CB 1996 Growing Nitrogen Fixing Trees in Acid Soils In MH Powell (ed) Nitrogen Fixing Trees for Acid Soils- A Field ManuaL Winrock International Morrilton AR 22 p
Copeland LO McDonald MB 1995 Principles of Seed Science and Technology 3rded Chapman amp Hall New York NY 409 p
Cox RE Klett JE 1984 Seed gennination requirements of native Colorado plants for use in the landscape Plant Propag 30(2) 6-10
Dell B 1980 Structure and function ofthe strophiolar plug in seeds ofAlbizia Lophantha Am J Bot 67 556-563
Dick-Peddie WA 1993 New Mexico Vegetation Past Present and Future University ofNew Mexico Press Albuquerque NM 244 p
Dreesen DR 2001 Propagation protocol for production ofcontainer Robinia neomexicana plants Los Lunas Plant Materials Center Los Lunas New Mexico In Native Plant Network URL httpwwwnativeplantnetworkorg (accessed 9 January 2002) Moscow (ID) University ofIdaho CoIl Natural Resources For Res Nursery
72
Dreesen DR Harrington JT 1997 Propagation of native plants for restoration projects in the Southwestern US- Preliminary Investigations In Landis TD Thompson JR tech coords National Proceedings Forest and Conservation Nursery Assocs Gen Tech Rep PNW-GTR-419 Portland OR USDA For Ser PNW Res Sta 77-88
Gottfried GJ 1980 Control ofNew Mexican locust and the effect on planted ponderosa pine in central Arizona US For Ser Res Note RM-386 Rocky Mountain For Range ExpStn Fort Collins CO
Gosling PG Samuel YK Jones SK 1995 A systematic examination of germination temperature chipping and water temperaturesoak duration pretreatments on the seeds ofLeucaena leucocephala Seed Sci amp Technol 23521-532
Hamly DH 1932 Softening of the seeds of Melilotus alba Bot Gazette 93 345shy375
Hanna PJ 1984 Anatomical features of the seed coat ofAcacia kempeana (Mueller) which relate to increased germination rate induced by heat treatment New Phytol 96 23-29
Hanks JP Dick-Peddie WA 1974 Vegetation patterns of the White Mountains New Mexico Southwest Naturalist 18(4) 371-382
Hine S Harrington JT Dreesen DR 1997 Seed scarification requirements for Robinia neomexicana In Harrington JTtech coord Proc Third SouthwestemContainer Growers Conf Feb 12-13 1997 Ft Collins CO
Hopkinson JM 1993 The strophiole of leguminous seeds Newsletter- IntI Herbage Seed Production Res Grp 18 7-8
---- 1997 Differences in hardseededness between Cunningham and Tarramba leucaena In Leucnet News The Newsletter of the International LeucaenaRes and Devlt Netwk No4
ISTA (International Seed Testing Association) 1999 International rules for seed testing rules Seed Sci amp Technol27 Supplement
Kelly KM van Staden J 1987 The lens as the site of permeability in the papilionoid seed Aspalathus linearis J Plant Physiol 128(45) 395-404
73
Keresztesi B 1988 Black locust the tree of agriculture Outlook on Agr 17(2) 77~85
Kigel 1 and Galili G 1995 Seed development and germination Marcel Dekker New York 853 p
Klemmedson 10 1994 New Mexico locust and parent material Influence on macronutrients of forest floor and soil Soil Sci Soc Am 1 58 974~980
Kuo WH Tam AW 1988 The pathway of water absorption of mung bean seeds Seed Sci amp Technol16(1) 139~144
Leadem CL 1997 Dormancy--unlocking seed secrets In Landis TD Thompson lR tech coords National Proceedings Forestand Conservation Nursery Assocs Gen Tech Rep PNW-G TR-419 Portland OR USDA For Ser PNW Res Sta 43-52
Lin Y Wagner MR Seweyestewa A Honahni MD DeGomez M McMillin JD 1996 Influence of cultural treatments on seed germination and seedling growth of Robinia neomexicana Forest Farm and Community Tree Res Reports Volume 1
Marunda CT 1990 Effects of seed pretreatment on the development ofAcacia auriculforinis and A holosericea seedlings ACIAR Proceedings Series No 28
Meginnis HG 1937 Sulphuric acid treatment to increase germination of black locust seed USDA Circ 453 34 p
Miklas PN Towensend CE Ladd SL 1987 Seedcoat anatomy and the scarification ofcicer milkvetch seed Crop Sci 27 766-772
Monsen SB 1984 Use of shrubs on mine spoils In The Challenge ofProducing Native Plants for the Intermountain Area Proceedings Intermountain Nurserymans Association 1983 conference Las Vegas NV compo PM Murphy 26-31 Ogden UT USDA For Ser Gen Tech Report INT-l68
Olson DF 1974 Robinia L Locust In Seeds of Woody Plants in the US tech coord CS Schopmeyer 728-731 USDA For Ser Agr Handbk No450
Rehman S Loescher RNl Harris P1C 1998 Dormancy breaking and germination ofAcacia salicina Lindl seeds Seed Sci amp Techno127 553-557
Rolston MP 1978 Water impermeable seed dormancy Bot Rev 44 365-396
74
SAS Institute Inc 1989 SASSTAT Users Guide Version 6 4th Edition Volume 1 Cary NC SAS Institute Inc 943 p
SerratoValenti G Conara L Ferrando M Modenesi P 1993 Structural and histochemical features ofStylosanthes scabra (Leguminosae Papilionoidae) seed coat as related to water entry Can J Bot 71 834-840
Simpson BJ 1988 A field guide to Texas trees Austin TX Texas Monthly Press 372p
Tran VN Cavanagh AK 1984 Structural aspects ofdormancy In Seed Physiology Vol 2 Murray DR (ed) p1-44 Academic Press Australia
USDA Forest Service 1948 Woody-plant seed manual USDA Misc Pub1654 416p
Vines RA 1960 Trees Shrubs and Woody Vines of the Southwest Austin Univ ofTexas Press 1104 p
Wagner MR Larson RE Craig TP 1992 Ecology and silvics ofNew Mexico locust (Robinia neomexicana) with an emphasis on the arboreal form p53-57 In JW Hanover K Miller and S Plesko (eds) Proc Intemat Confon Black Locust Biology Culture and Utilization Mich State Univ USA
Wilson JK 1944 Immersing seeds of species of Robinia in boiling water hastens germination J For 42 453-454
Zimmerman RW Carpenter SB 1980 First-year coppice production from a 5shyyear old black locust stand on surface mine spoil p309-314 Centr Hardwd For ConfProc University ofMissouri Columbia
Las Cruces NM elevation of 1300 meters) Each 100-seed replicate was placed in a
test tube and each test tube was immediately removed to cool at room temperature
Percussion scarification was implemented using a pneumatic paint shaker
(Central Pneumatic model 00422 Camarillo CA) (Figure A2b) This allowed
standardization of treatment intensity Compressed air pressure was maintained at
530kPa plusmn 30kPa (80psi plusmn 5psi) resulting in approximately 350 oscillations per
minute Each lOO-seed sample was placed in a 118 ml (4 oz) soil tin for shaking To
maximize lateral movement of paint shaker a paint can was used as a spacer to place
soil tins at the end of the shaking arm (Figure A2c) This allowed shaking to occur at
the greatest distance from the pivot point (fulcrum) Following all treatments
including the control seeds were soaked for twenty-four hours in distilled water prior
to plating out
Gennination was evaluated on lab benches at room temperature Each
replicate of seeds was placed in a 100 cm petri dish on moistened filter paper with
humidity maintained by enclosing the petri dishes in ziplock bags Gennination was
monitored daily for 14 days in accordance with International Seed Testing
Association standards for Robinia species (ISTA 1999) Gennination was defined as
protrusion of the radicle from the embryo by at least 1mm as viewed by the naked
eye
Categorical Analysis of Variance using Prpc Catmod (SAS Institute 1989)
was used to detennine treatment differences in germination percentages for each
source This procedure is an extension of a Chi-square test of homogeneity using the
53
natural log of the ratio of germinated to non-germinated seeds for each treatment Pshy
values less than 005 were considered significant A limited set of pairwiseshy
comparisons was conducted to compare treatment mean percents using a
conservative alpha value of 005 divided by the number of comparisons
Results
Percussion maximized germination for both New Mexico locust and black
locust There were three germination response levels for New Mexico locust (Figure
A3) First there was a low response in the control indicating a dormant seed lot
The hot water treatment and I-minute duration of percussion share a second
intermediate response Germination was highest at percussion levels from 2 to 10
minutes
A selected percussion treatment intermediate in time (5 minutes) produced the
steepest germination curve indicating the fastest germination rate (Figure A4) The
hot water treatment produced an intermediate curve or relatively slower germination
rate The control curve was very flat indicating the slowest germination rate for New
Mexico locust
Control seeds germinated poorly for black locust as well (Figure A5) Hot
water scarification improved germination over the control but all levels of percussion
improved germination over hot water The optimal durations of percussion treatment
were bracketed with a significant increase from the 1 to 2 minute percussion level a
high germination response from 2 to 5 minutes of percussion and finally a significant
54
~ d 25 CL)
p t1 0 ~ t1
g Vl ~ Vl
100~---------------------------------------------------------------
80
60
40
20
o Control Hot Water Percussion
I min Percussion
2 min Percussion
3 min
Scarification
Percussion 4 min
Figure A3 Effect of scarification on germination response for New Mexico locust
Percussion 5 min
=significant difference between adjacent treatment bars ns = no significant difference
Percussion 10 min
~ ~ () u agt
Il-t d o ~
~ ~ c3
100middotlr~~~------------------------------------e- Percussion 5min middot 0middot Hot Water -- Control
80-J=middot======
6b-middot-lmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middot--middotmiddot-middot~-middotmiddot -middotmiddot middot middotmiddot-middot 1
40
20
-----~-----------
0 0 0 middotmiddotmiddotmiddotmiddot0middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot0
0 --~-----y-----~------_---~--- T-----~
o shy -TDay 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A4 Effect of selected scarification treatments on germination speed for New Mexico locust First seven days of fourteen-day experiment are shown
Day 7
80
~ cd
5 60 ()
[) Cl d 0 1t1 d
40sect dUl
-J
20
o
ns
Scarification
Figure A5 Effect of scarification on germination response for black locust = significant difference between adjacent treatment bars ns = no significant difference
decrease at the 10-minute level This drop-off suggests that 10 minutes of percussion
over-treats seeds from this lot of-black locust
A selected percussion treatment intermediate in time (5 minutes) also had the
fastest germination rate for black locust (Figure A6)_ The hot water curve is
somewhat flatter indicating a slower germination rate The control curve is very flat
indicating a slow germination rate
A qualitative difference between hot water and percussion-treated seeds was
also observed Along with healthy germinants the hot water treatment produced
over-treated seeds (Figure A 7a) Examples of damaged germinants include seeds
where the radicle did not elongate (Figure A 7b) seeds where the radicle elongated
but was delayed in freeing itself from the seed coat (Figure A7c) and seeds where the
radicle broke off completely from the embryo (Figure A7d) In addition to these
examples of over-treatment there were numerous small dark seeds that failed to
imbibe water For percussion treatment nearly all seeds took up water (Figure A8a)
Germination was rapid and uniform with little damage evident (Figure A8b)
To summarize both New Mexico locust and black locust seed lots were
dormant The standard hot water scarification treatment improved germination but
only to 56 and 41 for New Mexico locust and black locust respectively Nearly
all percussion durations improved total germination as well as germination rate over
the hot water treatment Germination was greater than 90 for percussion durations
of 4 5 and 10 minutes for New Mexico locust and 3 4 and 5 minutes for black locust
58
CI)
sgt t CI) 0
igt 0
sect ttl t E
Vl 0
c3
100r==~==~~=r------------------------------------~ ___ Percussion 5 min
middotmiddotmiddot0middotmiddotmiddot Hot Water -lIf- Control
80r=========~--~~----------------------------~
60
40
20 -~-~---~---------- ---~------------I
omiddot
O
_~_----------lIf-----~bull -----_ shy---shy I
Day 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A6 Effect of selected scarification treatments on germination speed for black locust First seven days of fourteen-day experiment are shown
Day 7
a
b c d
Figure A7 Damage to New Mexico locust seeds observed during germination counts following hot water scarification A7a Hot water replicate including damaged seedlings and numerous unimbibed seeds A7b Close-up ofseed where radicle failed to elongate A7c Close-up ofseed where radicle elongated but was delayed in freeing itself from the seed coat A7d Close-up ofseed where radicle completely broke offfrom embryo
60
a
b
Figure A8 New Mexico locust seeds following percussion treatment at time of germination counts A8a Percussion replicate with uniformly healthy seedlings and near complete imbibition of seeds A8b Close-up ofpercussion-treated seedling
61
Discussion
Papilionoid seeds have a specific anatomical feature known as the strophiole
or lens-the natural site of water entry to the seed (Hamly 1932) The strophiole is
located on the cotyledonary lobe of a locust seed (Figure A9) When a papilionoid
legume seed is percussed for the appropriate amount of time repeated hits on the
integrated seed coat loosen the constrained cells of the strophiolar region without
excessively damaging the rest of the seed coat (Ballard 1976)
When a percussed seed is soaked water enters exclusively through the
strophiole in a controlled manner (Kelly and Staden 1987) This regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues This
contrasts with seeds that have been hot water or acid scarified treatments that can
randomly degrade the seed coat Unlocalized cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rehman et al 1998 Marunda 1990)
Papilionoids are the largest subfamily of legumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust and black locust these legumes often play an integral role in the
revegetation ofdisturbed lands Developing superior scarification methods to the
standard hot water and acid treatments should fadlitate the use ofthese legumes in
restoration projects
62
Figure A9 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (bc)
63
APPENDIXB
GERMINATION PERCENTS AND STANDARD ERRORS FOR SOURCE
BLOCK AND TREATMENT FOR NEW MEXICO LOCUST
Table B 1 Gennination percents and standard errors for source block and treatment for New Mexico locust
Source Block Treatment Germination Percentage Standard Error
Capitan 1 Immersion 74 437 Capitan 2 Immersion 69 462 Capitan 3 Immersion 71 454 Capitan 4 Immersion 67 470 Capitan Boiling 30 sec 79 407 Capitan 2 Boiling 30 sec 76 427 Capitan 3 Boiling 30 sec 72 449 Capitan 4 Boiling 30 sec 76 427 Capitan Boiling 60 sec 71 454 Capitan 2 Boiling 60 sec 54 498 Capitan 3 Boiling 60 sec 67 470 Capitan 4 Boiling 60 sec 76 427 Capitan I Control 5 218 Capitan 2 Control 7 255 Capitan 3 Control 4 196 Capitan 4 Control 5 218 Capitan 1 Percussion I min 47 499 Capitan 2 Percussion I min
470
Capitan Percussion I min 39 488
Capitan 4 Percussion I min 38 485 Capitan I Percussion 2 min 55 497 Capitan Capitan
2
Percussion 2 min Percussion 2 min
71 65
454 477
Capitan 4 Percussion 2 min 56 496 Capitan I Percussion 4 min 86 347 Capitan 2 Percussion 4 min 79 407 Capitan 3 Percussion 4 min 81 392 Capitan 4 Percussion 4 min 82 384 Capitan I Percussion 8 min 90 300 Capitan 2 Percussion 8 min 88 325 Capitan 3 Percussion 8 min 90 300 Capitan 4 Percussion 8 min 90 300 Gila I Immersion 71 454 Gila 2 Immersion 77 421 Gila
Immersion 75 433
Gila 4 Immersion 72 449
Gila I Boiling 30 sec 68 466
Gila 2 Boiling 30 sec 81 392
Gila 3 Boiling 30 sec 54 498
Gila 4 Boiling 30 sec 63 483
Gila I Boiling 60 sec 55 497
Gila 2 Boiling 60 sec 68 466
Gila 3 Boiling 60 sec 57 495
Gila 4 Boiling 60 sec 70 458
65
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Gila 2 Control 13 336 Gila 3 Control 13 336 Gila 4 Control 20 400 Gila 1 Percussion 1 min 66 474 Gila 2 Percussion 1 min 57 495 Gila 3 Percussion 1 min 73 444 Gila 4 Percussion 1 min 82 384 Gila 1 Percussion 2 min 81 392 Gila 2 Percussion 2 min 86 347 Gila 3 Percussion 2 min 85 357 Gila 4 Percussion 2 min 54 498 Gila 1 Percussion 4 min 79 407 Gila 2 Percussion 4 min 83 376 Gila 3 Percussion 4 min 84 367 Gila 4 Percussion 4 min 79 407 Gila 1 Percussion 8 min 51 500 Gila 2 Percussion 8 min 76 427 Gila 3 Percussion 8 min 52 500 Gila 4 Percussion 8 min 74 439 Holman 1 Immersion 83 376 Holman 2 Immersion 86 347 Holman 3 Immersion 81 392 Holman 4 Immersion 90 300 Holman 1 Boiling 30 sec 90 300 Holman 2 Boiling 30 sec 85 357 Holman 3 Boiling 30 sec 87 336 Holman 4 Boiling 30 sec 83 376 Holman 1 Boiling 60 sec 79 407 Holman 2 Boiling 60 sec 80 400 Holman 3 Boiling 60 sec 86 347 Holman 4 Boiling 60 sec 93 255 Holman Control 25 433 Holman 2 Control 19 392 Holman Control 21 407
Holman 4 Control 24 427
Holman Percussion 1 min 77 421
Holman 2 Percussion 1 min 85 357
Holman 3 Percussion 1 min 80 400
Holman 4 Percussion 1 min 89 313
Holman 1 Percussion 2 min 88 325
Holman 2 Percussion 2 min 96 196
Holman 3 Percussion 2 min 93 255
Holman 4 Percussion 2 min 96 196
Holman 1 Percussion 4 min 92 271
Holman 2 Percussion 4 min 97 171
66
I
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Holman 3 Percussion 4 min 91 286 Holman 4 Percussion 4 min 96 196 Holman 1 Percussion 8 min 88 325 Holman 2 Percussion 8 min 85 357 Holman 3 Percussion 8 min 83 376 Holman 4 Percussion 8 min 82 384 Jemez 1 Immersion 53 499 Jemez 2 Immersion 44 496 Jemez 3 Immersion 52 500 Jemez 4 Immersion 70 458 Jemez 1 Boiling 30 sec 58 494 Jemez 2 Boiling 30 sec 55 497 Jemez 3 Boiling 30 sec 48 500 Jemez 4 Boiling 30 sec 65 477 Jemez 1 Boiling 60 sec 68 466 Jemez 2 Boiling 60 sec 63 483 Jemez 3 Boiling 60 sec 73 444 Jemez 4 Boiling 60 sec 89 313 Jemez 1 Control 2 140 Jemez 2 Control 9 286 Jemez 3 Control 6 237 Jemez 4 Control 8 271 Jemez 1 Percussion 1 min 58 494 Jemez 2 Percussion 1 min 63 483 Jemez 3 Percussion 1 min 77 421 Jemez 4 Percussion 1 min 89 313 Jemez 1 Percussion 2 min 68 466 Jemez 2 Percussion 2 min 64 480 Jemez 3 Percussion 2 min 80 400 Jemez 4 Percussion 2 min 83 376 Jemez Percussion 4 min 68 466 Jemez 2 Percussion 4 min 79 407 Jemez 3 Percussion 4 min 70 458 Jemez 4 Percussion 4 min 83 376 Jemez 1 Percussion 8 min 52 500 Jemez 2 Percussion 8 min 59 492 Jemez 3 Percussion 8 min 61 488 Jemez 4 Percussion 8 min 70 458 Magdalena 1 Immersion 69 462 Magdalena 2 Immersion 69 462 Magdalena J Immersion 70 458 Magdalena 4 Immersion 85 357 Magdalena 1 Boiling 30 sec 65 477 Magdalena 2 Boiling 30 sec 71 454 Magdalena 3 Boiling 30 sec 68 466
67
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Magdalena 4 Boiling 30 sec 83 376 Magdalena 1 Boiling 60 sec 63 483 Magdalena 2 Boiling 60 sec 68 466 Magdalena 3 Boiling 60 sec 63 483 Magdalena 4 Boiling 60 sec 82 384 Magdalena 1 Control 11 313 Magdalena 2 Control 6 237 Magdalena 3 Control 5 218 Magdalena 4 Control 4 196 Magdalena Percussion 1 min 32 466 Magdalena 2 Percussion 1 min 45 497 Magdalena 3 Percussion 1 min 46 498 Magdalena 4 Percussion I min 36 480 Magdalena 1 Percussion 2 min 67 470 Magdalena 2 Percussion 2 min 68 466 Magdalena 3 Percussion 2 min 65 477 Magdalena 4 Percussion 2 min 81 392 Magdalena 1 Percussion 4 min 87 336 Magdalena 2 Percussion 4 min 97 171 Magdalena 3 Percussion 4 min 83 376 Magdalena 4 Percussion 4 min 97 171 Magdalena 1 Percussion 8 min 98 lAO Magdalena 2 Percussion 8 min 100 000 Magdalena 3 Percussion 8 min 96 196 Magdalena 4 Percussion 8 min 98 lAO Manzano 1 Immersion 67 470 Manzano 2 Immersion 57 495 Manzano 3 Immersion 72 4049 Manzano 4 Immersion 73 4044 Manzano 1 Boiling 30 sec 70 458 Manzano 2 Boiling 30 sec 64 480 Manzano 3 Boiling 30 sec 71 454 Manzano 4 Boiling 30 sec 80 400 Manzano I Boiling 60 sec 50 500 Manzano 2 Boiling 60 sec 53 499 Manzano 3 Boiling 60 sec 59 492 Manzano 4 Boiling 60 sec 74 439 Manzano I Control 4 196 Manzano 2 Control 8 271 Manzano 3 Control 1 099 Manzano 4 Control 6 237 Manzano 1 Percussion 1 min 52 500 Manzano 2 Percussion 1 min 39 488 Manzano 3 Percussion 1 min 36 480 Manzano 4 Percussion 1 min 51 500
68
Table B 1 Continued
Source Block Treatment Gerrni~ation Percentage Standard Error
Manzano Percussion 2 min 53 499 Manzano 2 Percussion 2 min 58 494 Manzano 3 Percussion 2 min 61 488 Manzano 4 Percussion 2 min 62 485 Manzano 1 Percussion 4 min 76 427 Manzano 2 Percussion 4 min 85 357 Manzano 3 Percussion 4 min 79 407 Manzano 4 Percussion 4 min 83 376 Manzano 1 Percussion 8 min 87 336 Manzano 2 Percussion 8 min 83 376 Manzano 3 Percussion 8 min 83 376 Manzano 4 Percussion 8 min 75 433 Raton 1 Immersion 54 498 Raton 2 Immersion 37 483 Raton 3 Immersion 39 488 Raton 4 Immersion 69 462 Raton 1 Boiling 30 sec 36 480 Raton 2 Boiling 30 sec 20 400 Raton 3 Boiling 30 sec 28 449 Raton 4 Boiling 30 sec 60 490 Raton 1 Boiling 60 sec 15 357 Raton 2 Boiling 60 sec 171 Raton 3 Boiling 60 sec 4 196 Raton 4 Boiling 60 sec 35 477 Raton 1 Control 4 196 Raton 2 Control 12 325 Raton 3 Control 2 140 Raton 4 Control 6 237 Raton 1 Percussion 1 min 52 500 Raton 2 Percussion I min 29 454 Raton 3 Percussion I min 35 477 Raton 4 Percussion 1 min 31 462 Raton 1 Percussion 2 min 55 497 Raton 2 Percussion 2 min 35 477 Raton 3 Percussion 2 min 33 470 Raton 4 Percussion 2 min 42 494 Raton 1 Percussion 4 min 65 477 Raton 2 Percussion 4 min 52 500 Raton Percussion 4 min 51 500 Raton 4 Percussion 4 min 53 499 Raton 1 Percussion 8 min 88 325 Raton 2 Percussion 8 min 80 400 Raton 3 Percussion 8 min 73 444 Raton 4 Percussion 8 min 74 439 Sacramento 1 Immersion 43 495
69
Table B 1 Continued
-Source Block Treatment Germination Percentage Standard Error
Sacramento 2 Immersion 30 458 Sacramento 3 Immersion 38 485 Sacramento 4 Immersion 56 496 Sacramento 1 Boiling 30 sec 47 499 Sacramento 2 Boiling 30 sec 48 500 Sacramento 3 Boiling 30 sec 48 500 Sacramento 4 Boiling 30 sec 56 496 Sacramento 1 Boiling 60 sec 52 500 Sacramento 2 Boiling 60 sec 67 470 Sacramento 3 Boiling 60 sec 43 495 Sacramento 4 Boiling 60 sec 48 500 Sacramento 1 Control 0 000 Sacramento 2 Control 3 171 Sacramento 3 Control 3 171 Sacramento 4 Control 099 Sacramento 1 Percussion 1 min 57 495 Sacramento 2 Percussion 1 min 61 488 Sacramento 3 Percussion 1 min 62 485 Sacramento 4 Percussion 1 min 55 497 Sacramento 1 Percussion 2 min 84 367 Sacramento 2 Percussion 2 min 70 458 Sacramento 3 Percussion 2 min 81 392 Sacramento 4 Percussion 2 min 69 462 Sacramento 1 Percussion 4 min 91 286 Sacramento 2 Percussion 4 min 84 367 Sacramento 3 Percussion 4 min 84 367 Sacramento 4 Percussion 4 min 94 237 Sacramento 1 Percussion 8 min 82 384 Sacramento 2 Percussion 8 min 81 392 Sacramento 3 Percussion 8 min 82 384 Sacramento 4 Percussion 8 min 81 392 Sandia 1 Immersion 76 427 Sandia 2 Immersion 70 458 Sandia 3 Immersion 70 458 Sandia 4 Immersion 81 392 Sandia 1 Boiling 30 sec 78 414 Sandia 2 Boiling 30 sec 71 454 Sandia 3 Boiling 30 sec 63 483 Sandia 4 Boiling 30 sec 78 414 Sandia 1 Boiling 60 sec 67 470 Sandia 2 Boiling 60 sec 65 477 Sandia 3 Boiling 60 sec 74 439 Sandia 4 Boiling 60 sec 66 474 Sandia 1 Control 16 367 Sandia 2 Control 7 255
70
Table B 1 Continued
Source Block Treatment Gennination Percentage Standard Error
Sandia 3 Control 8 271 Sandia 4 Control 16 367 Sandia 1 Percussion 1 min 67 470 Sandia 2 Percussion 1 min 61 488 Sandia 3 Percussion 1 min 66 474 Sandia 4 Percussion 1 min 59 492 Sandia I Percussion 2 min 82 384 Sandia 2 Percussion 2 min 83 376 Sandia 3 Percussion 2 min 81 392 Sandia 4 Percussion 2 min 84 367 Sandia 1 Percussion 4 min 89 313 Sandia 2 Percussion 4 min 86 347 Sandia 3 Percussion 4 min 89 313 Sandia 4 Percussion 4 min 85 357 Sandia 1 Percussion 8 min 80 400 Sandia 2 Percussion 8 min 77 421 Sandia 3 Percussion 8 min 69 462 Sandia 4 Percussion 8 min 66 474
LITERATURE CITED
Ashby WC Vogel WG Rogers NF 1985 Black locust in the reclamation equation General Technical Report Northeastern Forest Experiment Station USDA Forest Service NE-I05 12 p
Ballard LAT 1973 Physical barriers to gennination Seed Sci Technoll 285-303
___ 1976 Strophiolar water conduction in the seeds ofTrifolieae induced by action on the testa at nonstrophiolar sites Austr J Plant Physiol3 465-469
Barton LV 1947 Special studies on seed coat impenneability Contrib Boyce Thompson Inst 14 355-362
Baskin CC Baskin JM 1998 Seeds Ecology biogeography and evolution of donnancy and gennination Academic Press London UK 666 p
Bewley JD 1997 Seed germination and donnancy Plant Ce119 1055-1066
Bewley JD Black M 1994 Seeds physiology of development and germination 2nd ed Plenum Press New York NY 445 p
Briscoe CB 1996 Growing Nitrogen Fixing Trees in Acid Soils In MH Powell (ed) Nitrogen Fixing Trees for Acid Soils- A Field ManuaL Winrock International Morrilton AR 22 p
Copeland LO McDonald MB 1995 Principles of Seed Science and Technology 3rded Chapman amp Hall New York NY 409 p
Cox RE Klett JE 1984 Seed gennination requirements of native Colorado plants for use in the landscape Plant Propag 30(2) 6-10
Dell B 1980 Structure and function ofthe strophiolar plug in seeds ofAlbizia Lophantha Am J Bot 67 556-563
Dick-Peddie WA 1993 New Mexico Vegetation Past Present and Future University ofNew Mexico Press Albuquerque NM 244 p
Dreesen DR 2001 Propagation protocol for production ofcontainer Robinia neomexicana plants Los Lunas Plant Materials Center Los Lunas New Mexico In Native Plant Network URL httpwwwnativeplantnetworkorg (accessed 9 January 2002) Moscow (ID) University ofIdaho CoIl Natural Resources For Res Nursery
72
Dreesen DR Harrington JT 1997 Propagation of native plants for restoration projects in the Southwestern US- Preliminary Investigations In Landis TD Thompson JR tech coords National Proceedings Forest and Conservation Nursery Assocs Gen Tech Rep PNW-GTR-419 Portland OR USDA For Ser PNW Res Sta 77-88
Gottfried GJ 1980 Control ofNew Mexican locust and the effect on planted ponderosa pine in central Arizona US For Ser Res Note RM-386 Rocky Mountain For Range ExpStn Fort Collins CO
Gosling PG Samuel YK Jones SK 1995 A systematic examination of germination temperature chipping and water temperaturesoak duration pretreatments on the seeds ofLeucaena leucocephala Seed Sci amp Technol 23521-532
Hamly DH 1932 Softening of the seeds of Melilotus alba Bot Gazette 93 345shy375
Hanna PJ 1984 Anatomical features of the seed coat ofAcacia kempeana (Mueller) which relate to increased germination rate induced by heat treatment New Phytol 96 23-29
Hanks JP Dick-Peddie WA 1974 Vegetation patterns of the White Mountains New Mexico Southwest Naturalist 18(4) 371-382
Hine S Harrington JT Dreesen DR 1997 Seed scarification requirements for Robinia neomexicana In Harrington JTtech coord Proc Third SouthwestemContainer Growers Conf Feb 12-13 1997 Ft Collins CO
Hopkinson JM 1993 The strophiole of leguminous seeds Newsletter- IntI Herbage Seed Production Res Grp 18 7-8
---- 1997 Differences in hardseededness between Cunningham and Tarramba leucaena In Leucnet News The Newsletter of the International LeucaenaRes and Devlt Netwk No4
ISTA (International Seed Testing Association) 1999 International rules for seed testing rules Seed Sci amp Technol27 Supplement
Kelly KM van Staden J 1987 The lens as the site of permeability in the papilionoid seed Aspalathus linearis J Plant Physiol 128(45) 395-404
73
Keresztesi B 1988 Black locust the tree of agriculture Outlook on Agr 17(2) 77~85
Kigel 1 and Galili G 1995 Seed development and germination Marcel Dekker New York 853 p
Klemmedson 10 1994 New Mexico locust and parent material Influence on macronutrients of forest floor and soil Soil Sci Soc Am 1 58 974~980
Kuo WH Tam AW 1988 The pathway of water absorption of mung bean seeds Seed Sci amp Technol16(1) 139~144
Leadem CL 1997 Dormancy--unlocking seed secrets In Landis TD Thompson lR tech coords National Proceedings Forestand Conservation Nursery Assocs Gen Tech Rep PNW-G TR-419 Portland OR USDA For Ser PNW Res Sta 43-52
Lin Y Wagner MR Seweyestewa A Honahni MD DeGomez M McMillin JD 1996 Influence of cultural treatments on seed germination and seedling growth of Robinia neomexicana Forest Farm and Community Tree Res Reports Volume 1
Marunda CT 1990 Effects of seed pretreatment on the development ofAcacia auriculforinis and A holosericea seedlings ACIAR Proceedings Series No 28
Meginnis HG 1937 Sulphuric acid treatment to increase germination of black locust seed USDA Circ 453 34 p
Miklas PN Towensend CE Ladd SL 1987 Seedcoat anatomy and the scarification ofcicer milkvetch seed Crop Sci 27 766-772
Monsen SB 1984 Use of shrubs on mine spoils In The Challenge ofProducing Native Plants for the Intermountain Area Proceedings Intermountain Nurserymans Association 1983 conference Las Vegas NV compo PM Murphy 26-31 Ogden UT USDA For Ser Gen Tech Report INT-l68
Olson DF 1974 Robinia L Locust In Seeds of Woody Plants in the US tech coord CS Schopmeyer 728-731 USDA For Ser Agr Handbk No450
Rehman S Loescher RNl Harris P1C 1998 Dormancy breaking and germination ofAcacia salicina Lindl seeds Seed Sci amp Techno127 553-557
Rolston MP 1978 Water impermeable seed dormancy Bot Rev 44 365-396
74
SAS Institute Inc 1989 SASSTAT Users Guide Version 6 4th Edition Volume 1 Cary NC SAS Institute Inc 943 p
SerratoValenti G Conara L Ferrando M Modenesi P 1993 Structural and histochemical features ofStylosanthes scabra (Leguminosae Papilionoidae) seed coat as related to water entry Can J Bot 71 834-840
Simpson BJ 1988 A field guide to Texas trees Austin TX Texas Monthly Press 372p
Tran VN Cavanagh AK 1984 Structural aspects ofdormancy In Seed Physiology Vol 2 Murray DR (ed) p1-44 Academic Press Australia
USDA Forest Service 1948 Woody-plant seed manual USDA Misc Pub1654 416p
Vines RA 1960 Trees Shrubs and Woody Vines of the Southwest Austin Univ ofTexas Press 1104 p
Wagner MR Larson RE Craig TP 1992 Ecology and silvics ofNew Mexico locust (Robinia neomexicana) with an emphasis on the arboreal form p53-57 In JW Hanover K Miller and S Plesko (eds) Proc Intemat Confon Black Locust Biology Culture and Utilization Mich State Univ USA
Wilson JK 1944 Immersing seeds of species of Robinia in boiling water hastens germination J For 42 453-454
Zimmerman RW Carpenter SB 1980 First-year coppice production from a 5shyyear old black locust stand on surface mine spoil p309-314 Centr Hardwd For ConfProc University ofMissouri Columbia
natural log of the ratio of germinated to non-germinated seeds for each treatment Pshy
values less than 005 were considered significant A limited set of pairwiseshy
comparisons was conducted to compare treatment mean percents using a
conservative alpha value of 005 divided by the number of comparisons
Results
Percussion maximized germination for both New Mexico locust and black
locust There were three germination response levels for New Mexico locust (Figure
A3) First there was a low response in the control indicating a dormant seed lot
The hot water treatment and I-minute duration of percussion share a second
intermediate response Germination was highest at percussion levels from 2 to 10
minutes
A selected percussion treatment intermediate in time (5 minutes) produced the
steepest germination curve indicating the fastest germination rate (Figure A4) The
hot water treatment produced an intermediate curve or relatively slower germination
rate The control curve was very flat indicating the slowest germination rate for New
Mexico locust
Control seeds germinated poorly for black locust as well (Figure A5) Hot
water scarification improved germination over the control but all levels of percussion
improved germination over hot water The optimal durations of percussion treatment
were bracketed with a significant increase from the 1 to 2 minute percussion level a
high germination response from 2 to 5 minutes of percussion and finally a significant
54
~ d 25 CL)
p t1 0 ~ t1
g Vl ~ Vl
100~---------------------------------------------------------------
80
60
40
20
o Control Hot Water Percussion
I min Percussion
2 min Percussion
3 min
Scarification
Percussion 4 min
Figure A3 Effect of scarification on germination response for New Mexico locust
Percussion 5 min
=significant difference between adjacent treatment bars ns = no significant difference
Percussion 10 min
~ ~ () u agt
Il-t d o ~
~ ~ c3
100middotlr~~~------------------------------------e- Percussion 5min middot 0middot Hot Water -- Control
80-J=middot======
6b-middot-lmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middot--middotmiddot-middot~-middotmiddot -middotmiddot middot middotmiddot-middot 1
40
20
-----~-----------
0 0 0 middotmiddotmiddotmiddotmiddot0middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot0
0 --~-----y-----~------_---~--- T-----~
o shy -TDay 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A4 Effect of selected scarification treatments on germination speed for New Mexico locust First seven days of fourteen-day experiment are shown
Day 7
80
~ cd
5 60 ()
[) Cl d 0 1t1 d
40sect dUl
-J
20
o
ns
Scarification
Figure A5 Effect of scarification on germination response for black locust = significant difference between adjacent treatment bars ns = no significant difference
decrease at the 10-minute level This drop-off suggests that 10 minutes of percussion
over-treats seeds from this lot of-black locust
A selected percussion treatment intermediate in time (5 minutes) also had the
fastest germination rate for black locust (Figure A6)_ The hot water curve is
somewhat flatter indicating a slower germination rate The control curve is very flat
indicating a slow germination rate
A qualitative difference between hot water and percussion-treated seeds was
also observed Along with healthy germinants the hot water treatment produced
over-treated seeds (Figure A 7a) Examples of damaged germinants include seeds
where the radicle did not elongate (Figure A 7b) seeds where the radicle elongated
but was delayed in freeing itself from the seed coat (Figure A7c) and seeds where the
radicle broke off completely from the embryo (Figure A7d) In addition to these
examples of over-treatment there were numerous small dark seeds that failed to
imbibe water For percussion treatment nearly all seeds took up water (Figure A8a)
Germination was rapid and uniform with little damage evident (Figure A8b)
To summarize both New Mexico locust and black locust seed lots were
dormant The standard hot water scarification treatment improved germination but
only to 56 and 41 for New Mexico locust and black locust respectively Nearly
all percussion durations improved total germination as well as germination rate over
the hot water treatment Germination was greater than 90 for percussion durations
of 4 5 and 10 minutes for New Mexico locust and 3 4 and 5 minutes for black locust
58
CI)
sgt t CI) 0
igt 0
sect ttl t E
Vl 0
c3
100r==~==~~=r------------------------------------~ ___ Percussion 5 min
middotmiddotmiddot0middotmiddotmiddot Hot Water -lIf- Control
80r=========~--~~----------------------------~
60
40
20 -~-~---~---------- ---~------------I
omiddot
O
_~_----------lIf-----~bull -----_ shy---shy I
Day 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A6 Effect of selected scarification treatments on germination speed for black locust First seven days of fourteen-day experiment are shown
Day 7
a
b c d
Figure A7 Damage to New Mexico locust seeds observed during germination counts following hot water scarification A7a Hot water replicate including damaged seedlings and numerous unimbibed seeds A7b Close-up ofseed where radicle failed to elongate A7c Close-up ofseed where radicle elongated but was delayed in freeing itself from the seed coat A7d Close-up ofseed where radicle completely broke offfrom embryo
60
a
b
Figure A8 New Mexico locust seeds following percussion treatment at time of germination counts A8a Percussion replicate with uniformly healthy seedlings and near complete imbibition of seeds A8b Close-up ofpercussion-treated seedling
61
Discussion
Papilionoid seeds have a specific anatomical feature known as the strophiole
or lens-the natural site of water entry to the seed (Hamly 1932) The strophiole is
located on the cotyledonary lobe of a locust seed (Figure A9) When a papilionoid
legume seed is percussed for the appropriate amount of time repeated hits on the
integrated seed coat loosen the constrained cells of the strophiolar region without
excessively damaging the rest of the seed coat (Ballard 1976)
When a percussed seed is soaked water enters exclusively through the
strophiole in a controlled manner (Kelly and Staden 1987) This regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues This
contrasts with seeds that have been hot water or acid scarified treatments that can
randomly degrade the seed coat Unlocalized cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rehman et al 1998 Marunda 1990)
Papilionoids are the largest subfamily of legumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust and black locust these legumes often play an integral role in the
revegetation ofdisturbed lands Developing superior scarification methods to the
standard hot water and acid treatments should fadlitate the use ofthese legumes in
restoration projects
62
Figure A9 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (bc)
63
APPENDIXB
GERMINATION PERCENTS AND STANDARD ERRORS FOR SOURCE
BLOCK AND TREATMENT FOR NEW MEXICO LOCUST
Table B 1 Gennination percents and standard errors for source block and treatment for New Mexico locust
Source Block Treatment Germination Percentage Standard Error
Capitan 1 Immersion 74 437 Capitan 2 Immersion 69 462 Capitan 3 Immersion 71 454 Capitan 4 Immersion 67 470 Capitan Boiling 30 sec 79 407 Capitan 2 Boiling 30 sec 76 427 Capitan 3 Boiling 30 sec 72 449 Capitan 4 Boiling 30 sec 76 427 Capitan Boiling 60 sec 71 454 Capitan 2 Boiling 60 sec 54 498 Capitan 3 Boiling 60 sec 67 470 Capitan 4 Boiling 60 sec 76 427 Capitan I Control 5 218 Capitan 2 Control 7 255 Capitan 3 Control 4 196 Capitan 4 Control 5 218 Capitan 1 Percussion I min 47 499 Capitan 2 Percussion I min
470
Capitan Percussion I min 39 488
Capitan 4 Percussion I min 38 485 Capitan I Percussion 2 min 55 497 Capitan Capitan
2
Percussion 2 min Percussion 2 min
71 65
454 477
Capitan 4 Percussion 2 min 56 496 Capitan I Percussion 4 min 86 347 Capitan 2 Percussion 4 min 79 407 Capitan 3 Percussion 4 min 81 392 Capitan 4 Percussion 4 min 82 384 Capitan I Percussion 8 min 90 300 Capitan 2 Percussion 8 min 88 325 Capitan 3 Percussion 8 min 90 300 Capitan 4 Percussion 8 min 90 300 Gila I Immersion 71 454 Gila 2 Immersion 77 421 Gila
Immersion 75 433
Gila 4 Immersion 72 449
Gila I Boiling 30 sec 68 466
Gila 2 Boiling 30 sec 81 392
Gila 3 Boiling 30 sec 54 498
Gila 4 Boiling 30 sec 63 483
Gila I Boiling 60 sec 55 497
Gila 2 Boiling 60 sec 68 466
Gila 3 Boiling 60 sec 57 495
Gila 4 Boiling 60 sec 70 458
65
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Gila 2 Control 13 336 Gila 3 Control 13 336 Gila 4 Control 20 400 Gila 1 Percussion 1 min 66 474 Gila 2 Percussion 1 min 57 495 Gila 3 Percussion 1 min 73 444 Gila 4 Percussion 1 min 82 384 Gila 1 Percussion 2 min 81 392 Gila 2 Percussion 2 min 86 347 Gila 3 Percussion 2 min 85 357 Gila 4 Percussion 2 min 54 498 Gila 1 Percussion 4 min 79 407 Gila 2 Percussion 4 min 83 376 Gila 3 Percussion 4 min 84 367 Gila 4 Percussion 4 min 79 407 Gila 1 Percussion 8 min 51 500 Gila 2 Percussion 8 min 76 427 Gila 3 Percussion 8 min 52 500 Gila 4 Percussion 8 min 74 439 Holman 1 Immersion 83 376 Holman 2 Immersion 86 347 Holman 3 Immersion 81 392 Holman 4 Immersion 90 300 Holman 1 Boiling 30 sec 90 300 Holman 2 Boiling 30 sec 85 357 Holman 3 Boiling 30 sec 87 336 Holman 4 Boiling 30 sec 83 376 Holman 1 Boiling 60 sec 79 407 Holman 2 Boiling 60 sec 80 400 Holman 3 Boiling 60 sec 86 347 Holman 4 Boiling 60 sec 93 255 Holman Control 25 433 Holman 2 Control 19 392 Holman Control 21 407
Holman 4 Control 24 427
Holman Percussion 1 min 77 421
Holman 2 Percussion 1 min 85 357
Holman 3 Percussion 1 min 80 400
Holman 4 Percussion 1 min 89 313
Holman 1 Percussion 2 min 88 325
Holman 2 Percussion 2 min 96 196
Holman 3 Percussion 2 min 93 255
Holman 4 Percussion 2 min 96 196
Holman 1 Percussion 4 min 92 271
Holman 2 Percussion 4 min 97 171
66
I
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Holman 3 Percussion 4 min 91 286 Holman 4 Percussion 4 min 96 196 Holman 1 Percussion 8 min 88 325 Holman 2 Percussion 8 min 85 357 Holman 3 Percussion 8 min 83 376 Holman 4 Percussion 8 min 82 384 Jemez 1 Immersion 53 499 Jemez 2 Immersion 44 496 Jemez 3 Immersion 52 500 Jemez 4 Immersion 70 458 Jemez 1 Boiling 30 sec 58 494 Jemez 2 Boiling 30 sec 55 497 Jemez 3 Boiling 30 sec 48 500 Jemez 4 Boiling 30 sec 65 477 Jemez 1 Boiling 60 sec 68 466 Jemez 2 Boiling 60 sec 63 483 Jemez 3 Boiling 60 sec 73 444 Jemez 4 Boiling 60 sec 89 313 Jemez 1 Control 2 140 Jemez 2 Control 9 286 Jemez 3 Control 6 237 Jemez 4 Control 8 271 Jemez 1 Percussion 1 min 58 494 Jemez 2 Percussion 1 min 63 483 Jemez 3 Percussion 1 min 77 421 Jemez 4 Percussion 1 min 89 313 Jemez 1 Percussion 2 min 68 466 Jemez 2 Percussion 2 min 64 480 Jemez 3 Percussion 2 min 80 400 Jemez 4 Percussion 2 min 83 376 Jemez Percussion 4 min 68 466 Jemez 2 Percussion 4 min 79 407 Jemez 3 Percussion 4 min 70 458 Jemez 4 Percussion 4 min 83 376 Jemez 1 Percussion 8 min 52 500 Jemez 2 Percussion 8 min 59 492 Jemez 3 Percussion 8 min 61 488 Jemez 4 Percussion 8 min 70 458 Magdalena 1 Immersion 69 462 Magdalena 2 Immersion 69 462 Magdalena J Immersion 70 458 Magdalena 4 Immersion 85 357 Magdalena 1 Boiling 30 sec 65 477 Magdalena 2 Boiling 30 sec 71 454 Magdalena 3 Boiling 30 sec 68 466
67
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Magdalena 4 Boiling 30 sec 83 376 Magdalena 1 Boiling 60 sec 63 483 Magdalena 2 Boiling 60 sec 68 466 Magdalena 3 Boiling 60 sec 63 483 Magdalena 4 Boiling 60 sec 82 384 Magdalena 1 Control 11 313 Magdalena 2 Control 6 237 Magdalena 3 Control 5 218 Magdalena 4 Control 4 196 Magdalena Percussion 1 min 32 466 Magdalena 2 Percussion 1 min 45 497 Magdalena 3 Percussion 1 min 46 498 Magdalena 4 Percussion I min 36 480 Magdalena 1 Percussion 2 min 67 470 Magdalena 2 Percussion 2 min 68 466 Magdalena 3 Percussion 2 min 65 477 Magdalena 4 Percussion 2 min 81 392 Magdalena 1 Percussion 4 min 87 336 Magdalena 2 Percussion 4 min 97 171 Magdalena 3 Percussion 4 min 83 376 Magdalena 4 Percussion 4 min 97 171 Magdalena 1 Percussion 8 min 98 lAO Magdalena 2 Percussion 8 min 100 000 Magdalena 3 Percussion 8 min 96 196 Magdalena 4 Percussion 8 min 98 lAO Manzano 1 Immersion 67 470 Manzano 2 Immersion 57 495 Manzano 3 Immersion 72 4049 Manzano 4 Immersion 73 4044 Manzano 1 Boiling 30 sec 70 458 Manzano 2 Boiling 30 sec 64 480 Manzano 3 Boiling 30 sec 71 454 Manzano 4 Boiling 30 sec 80 400 Manzano I Boiling 60 sec 50 500 Manzano 2 Boiling 60 sec 53 499 Manzano 3 Boiling 60 sec 59 492 Manzano 4 Boiling 60 sec 74 439 Manzano I Control 4 196 Manzano 2 Control 8 271 Manzano 3 Control 1 099 Manzano 4 Control 6 237 Manzano 1 Percussion 1 min 52 500 Manzano 2 Percussion 1 min 39 488 Manzano 3 Percussion 1 min 36 480 Manzano 4 Percussion 1 min 51 500
68
Table B 1 Continued
Source Block Treatment Gerrni~ation Percentage Standard Error
Manzano Percussion 2 min 53 499 Manzano 2 Percussion 2 min 58 494 Manzano 3 Percussion 2 min 61 488 Manzano 4 Percussion 2 min 62 485 Manzano 1 Percussion 4 min 76 427 Manzano 2 Percussion 4 min 85 357 Manzano 3 Percussion 4 min 79 407 Manzano 4 Percussion 4 min 83 376 Manzano 1 Percussion 8 min 87 336 Manzano 2 Percussion 8 min 83 376 Manzano 3 Percussion 8 min 83 376 Manzano 4 Percussion 8 min 75 433 Raton 1 Immersion 54 498 Raton 2 Immersion 37 483 Raton 3 Immersion 39 488 Raton 4 Immersion 69 462 Raton 1 Boiling 30 sec 36 480 Raton 2 Boiling 30 sec 20 400 Raton 3 Boiling 30 sec 28 449 Raton 4 Boiling 30 sec 60 490 Raton 1 Boiling 60 sec 15 357 Raton 2 Boiling 60 sec 171 Raton 3 Boiling 60 sec 4 196 Raton 4 Boiling 60 sec 35 477 Raton 1 Control 4 196 Raton 2 Control 12 325 Raton 3 Control 2 140 Raton 4 Control 6 237 Raton 1 Percussion 1 min 52 500 Raton 2 Percussion I min 29 454 Raton 3 Percussion I min 35 477 Raton 4 Percussion 1 min 31 462 Raton 1 Percussion 2 min 55 497 Raton 2 Percussion 2 min 35 477 Raton 3 Percussion 2 min 33 470 Raton 4 Percussion 2 min 42 494 Raton 1 Percussion 4 min 65 477 Raton 2 Percussion 4 min 52 500 Raton Percussion 4 min 51 500 Raton 4 Percussion 4 min 53 499 Raton 1 Percussion 8 min 88 325 Raton 2 Percussion 8 min 80 400 Raton 3 Percussion 8 min 73 444 Raton 4 Percussion 8 min 74 439 Sacramento 1 Immersion 43 495
69
Table B 1 Continued
-Source Block Treatment Germination Percentage Standard Error
Sacramento 2 Immersion 30 458 Sacramento 3 Immersion 38 485 Sacramento 4 Immersion 56 496 Sacramento 1 Boiling 30 sec 47 499 Sacramento 2 Boiling 30 sec 48 500 Sacramento 3 Boiling 30 sec 48 500 Sacramento 4 Boiling 30 sec 56 496 Sacramento 1 Boiling 60 sec 52 500 Sacramento 2 Boiling 60 sec 67 470 Sacramento 3 Boiling 60 sec 43 495 Sacramento 4 Boiling 60 sec 48 500 Sacramento 1 Control 0 000 Sacramento 2 Control 3 171 Sacramento 3 Control 3 171 Sacramento 4 Control 099 Sacramento 1 Percussion 1 min 57 495 Sacramento 2 Percussion 1 min 61 488 Sacramento 3 Percussion 1 min 62 485 Sacramento 4 Percussion 1 min 55 497 Sacramento 1 Percussion 2 min 84 367 Sacramento 2 Percussion 2 min 70 458 Sacramento 3 Percussion 2 min 81 392 Sacramento 4 Percussion 2 min 69 462 Sacramento 1 Percussion 4 min 91 286 Sacramento 2 Percussion 4 min 84 367 Sacramento 3 Percussion 4 min 84 367 Sacramento 4 Percussion 4 min 94 237 Sacramento 1 Percussion 8 min 82 384 Sacramento 2 Percussion 8 min 81 392 Sacramento 3 Percussion 8 min 82 384 Sacramento 4 Percussion 8 min 81 392 Sandia 1 Immersion 76 427 Sandia 2 Immersion 70 458 Sandia 3 Immersion 70 458 Sandia 4 Immersion 81 392 Sandia 1 Boiling 30 sec 78 414 Sandia 2 Boiling 30 sec 71 454 Sandia 3 Boiling 30 sec 63 483 Sandia 4 Boiling 30 sec 78 414 Sandia 1 Boiling 60 sec 67 470 Sandia 2 Boiling 60 sec 65 477 Sandia 3 Boiling 60 sec 74 439 Sandia 4 Boiling 60 sec 66 474 Sandia 1 Control 16 367 Sandia 2 Control 7 255
70
Table B 1 Continued
Source Block Treatment Gennination Percentage Standard Error
Sandia 3 Control 8 271 Sandia 4 Control 16 367 Sandia 1 Percussion 1 min 67 470 Sandia 2 Percussion 1 min 61 488 Sandia 3 Percussion 1 min 66 474 Sandia 4 Percussion 1 min 59 492 Sandia I Percussion 2 min 82 384 Sandia 2 Percussion 2 min 83 376 Sandia 3 Percussion 2 min 81 392 Sandia 4 Percussion 2 min 84 367 Sandia 1 Percussion 4 min 89 313 Sandia 2 Percussion 4 min 86 347 Sandia 3 Percussion 4 min 89 313 Sandia 4 Percussion 4 min 85 357 Sandia 1 Percussion 8 min 80 400 Sandia 2 Percussion 8 min 77 421 Sandia 3 Percussion 8 min 69 462 Sandia 4 Percussion 8 min 66 474
LITERATURE CITED
Ashby WC Vogel WG Rogers NF 1985 Black locust in the reclamation equation General Technical Report Northeastern Forest Experiment Station USDA Forest Service NE-I05 12 p
Ballard LAT 1973 Physical barriers to gennination Seed Sci Technoll 285-303
___ 1976 Strophiolar water conduction in the seeds ofTrifolieae induced by action on the testa at nonstrophiolar sites Austr J Plant Physiol3 465-469
Barton LV 1947 Special studies on seed coat impenneability Contrib Boyce Thompson Inst 14 355-362
Baskin CC Baskin JM 1998 Seeds Ecology biogeography and evolution of donnancy and gennination Academic Press London UK 666 p
Bewley JD 1997 Seed germination and donnancy Plant Ce119 1055-1066
Bewley JD Black M 1994 Seeds physiology of development and germination 2nd ed Plenum Press New York NY 445 p
Briscoe CB 1996 Growing Nitrogen Fixing Trees in Acid Soils In MH Powell (ed) Nitrogen Fixing Trees for Acid Soils- A Field ManuaL Winrock International Morrilton AR 22 p
Copeland LO McDonald MB 1995 Principles of Seed Science and Technology 3rded Chapman amp Hall New York NY 409 p
Cox RE Klett JE 1984 Seed gennination requirements of native Colorado plants for use in the landscape Plant Propag 30(2) 6-10
Dell B 1980 Structure and function ofthe strophiolar plug in seeds ofAlbizia Lophantha Am J Bot 67 556-563
Dick-Peddie WA 1993 New Mexico Vegetation Past Present and Future University ofNew Mexico Press Albuquerque NM 244 p
Dreesen DR 2001 Propagation protocol for production ofcontainer Robinia neomexicana plants Los Lunas Plant Materials Center Los Lunas New Mexico In Native Plant Network URL httpwwwnativeplantnetworkorg (accessed 9 January 2002) Moscow (ID) University ofIdaho CoIl Natural Resources For Res Nursery
72
Dreesen DR Harrington JT 1997 Propagation of native plants for restoration projects in the Southwestern US- Preliminary Investigations In Landis TD Thompson JR tech coords National Proceedings Forest and Conservation Nursery Assocs Gen Tech Rep PNW-GTR-419 Portland OR USDA For Ser PNW Res Sta 77-88
Gottfried GJ 1980 Control ofNew Mexican locust and the effect on planted ponderosa pine in central Arizona US For Ser Res Note RM-386 Rocky Mountain For Range ExpStn Fort Collins CO
Gosling PG Samuel YK Jones SK 1995 A systematic examination of germination temperature chipping and water temperaturesoak duration pretreatments on the seeds ofLeucaena leucocephala Seed Sci amp Technol 23521-532
Hamly DH 1932 Softening of the seeds of Melilotus alba Bot Gazette 93 345shy375
Hanna PJ 1984 Anatomical features of the seed coat ofAcacia kempeana (Mueller) which relate to increased germination rate induced by heat treatment New Phytol 96 23-29
Hanks JP Dick-Peddie WA 1974 Vegetation patterns of the White Mountains New Mexico Southwest Naturalist 18(4) 371-382
Hine S Harrington JT Dreesen DR 1997 Seed scarification requirements for Robinia neomexicana In Harrington JTtech coord Proc Third SouthwestemContainer Growers Conf Feb 12-13 1997 Ft Collins CO
Hopkinson JM 1993 The strophiole of leguminous seeds Newsletter- IntI Herbage Seed Production Res Grp 18 7-8
---- 1997 Differences in hardseededness between Cunningham and Tarramba leucaena In Leucnet News The Newsletter of the International LeucaenaRes and Devlt Netwk No4
ISTA (International Seed Testing Association) 1999 International rules for seed testing rules Seed Sci amp Technol27 Supplement
Kelly KM van Staden J 1987 The lens as the site of permeability in the papilionoid seed Aspalathus linearis J Plant Physiol 128(45) 395-404
73
Keresztesi B 1988 Black locust the tree of agriculture Outlook on Agr 17(2) 77~85
Kigel 1 and Galili G 1995 Seed development and germination Marcel Dekker New York 853 p
Klemmedson 10 1994 New Mexico locust and parent material Influence on macronutrients of forest floor and soil Soil Sci Soc Am 1 58 974~980
Kuo WH Tam AW 1988 The pathway of water absorption of mung bean seeds Seed Sci amp Technol16(1) 139~144
Leadem CL 1997 Dormancy--unlocking seed secrets In Landis TD Thompson lR tech coords National Proceedings Forestand Conservation Nursery Assocs Gen Tech Rep PNW-G TR-419 Portland OR USDA For Ser PNW Res Sta 43-52
Lin Y Wagner MR Seweyestewa A Honahni MD DeGomez M McMillin JD 1996 Influence of cultural treatments on seed germination and seedling growth of Robinia neomexicana Forest Farm and Community Tree Res Reports Volume 1
Marunda CT 1990 Effects of seed pretreatment on the development ofAcacia auriculforinis and A holosericea seedlings ACIAR Proceedings Series No 28
Meginnis HG 1937 Sulphuric acid treatment to increase germination of black locust seed USDA Circ 453 34 p
Miklas PN Towensend CE Ladd SL 1987 Seedcoat anatomy and the scarification ofcicer milkvetch seed Crop Sci 27 766-772
Monsen SB 1984 Use of shrubs on mine spoils In The Challenge ofProducing Native Plants for the Intermountain Area Proceedings Intermountain Nurserymans Association 1983 conference Las Vegas NV compo PM Murphy 26-31 Ogden UT USDA For Ser Gen Tech Report INT-l68
Olson DF 1974 Robinia L Locust In Seeds of Woody Plants in the US tech coord CS Schopmeyer 728-731 USDA For Ser Agr Handbk No450
Rehman S Loescher RNl Harris P1C 1998 Dormancy breaking and germination ofAcacia salicina Lindl seeds Seed Sci amp Techno127 553-557
Rolston MP 1978 Water impermeable seed dormancy Bot Rev 44 365-396
74
SAS Institute Inc 1989 SASSTAT Users Guide Version 6 4th Edition Volume 1 Cary NC SAS Institute Inc 943 p
SerratoValenti G Conara L Ferrando M Modenesi P 1993 Structural and histochemical features ofStylosanthes scabra (Leguminosae Papilionoidae) seed coat as related to water entry Can J Bot 71 834-840
Simpson BJ 1988 A field guide to Texas trees Austin TX Texas Monthly Press 372p
Tran VN Cavanagh AK 1984 Structural aspects ofdormancy In Seed Physiology Vol 2 Murray DR (ed) p1-44 Academic Press Australia
USDA Forest Service 1948 Woody-plant seed manual USDA Misc Pub1654 416p
Vines RA 1960 Trees Shrubs and Woody Vines of the Southwest Austin Univ ofTexas Press 1104 p
Wagner MR Larson RE Craig TP 1992 Ecology and silvics ofNew Mexico locust (Robinia neomexicana) with an emphasis on the arboreal form p53-57 In JW Hanover K Miller and S Plesko (eds) Proc Intemat Confon Black Locust Biology Culture and Utilization Mich State Univ USA
Wilson JK 1944 Immersing seeds of species of Robinia in boiling water hastens germination J For 42 453-454
Zimmerman RW Carpenter SB 1980 First-year coppice production from a 5shyyear old black locust stand on surface mine spoil p309-314 Centr Hardwd For ConfProc University ofMissouri Columbia
~ d 25 CL)
p t1 0 ~ t1
g Vl ~ Vl
100~---------------------------------------------------------------
80
60
40
20
o Control Hot Water Percussion
I min Percussion
2 min Percussion
3 min
Scarification
Percussion 4 min
Figure A3 Effect of scarification on germination response for New Mexico locust
Percussion 5 min
=significant difference between adjacent treatment bars ns = no significant difference
Percussion 10 min
~ ~ () u agt
Il-t d o ~
~ ~ c3
100middotlr~~~------------------------------------e- Percussion 5min middot 0middot Hot Water -- Control
80-J=middot======
6b-middot-lmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middot--middotmiddot-middot~-middotmiddot -middotmiddot middot middotmiddot-middot 1
40
20
-----~-----------
0 0 0 middotmiddotmiddotmiddotmiddot0middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot0
0 --~-----y-----~------_---~--- T-----~
o shy -TDay 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A4 Effect of selected scarification treatments on germination speed for New Mexico locust First seven days of fourteen-day experiment are shown
Day 7
80
~ cd
5 60 ()
[) Cl d 0 1t1 d
40sect dUl
-J
20
o
ns
Scarification
Figure A5 Effect of scarification on germination response for black locust = significant difference between adjacent treatment bars ns = no significant difference
decrease at the 10-minute level This drop-off suggests that 10 minutes of percussion
over-treats seeds from this lot of-black locust
A selected percussion treatment intermediate in time (5 minutes) also had the
fastest germination rate for black locust (Figure A6)_ The hot water curve is
somewhat flatter indicating a slower germination rate The control curve is very flat
indicating a slow germination rate
A qualitative difference between hot water and percussion-treated seeds was
also observed Along with healthy germinants the hot water treatment produced
over-treated seeds (Figure A 7a) Examples of damaged germinants include seeds
where the radicle did not elongate (Figure A 7b) seeds where the radicle elongated
but was delayed in freeing itself from the seed coat (Figure A7c) and seeds where the
radicle broke off completely from the embryo (Figure A7d) In addition to these
examples of over-treatment there were numerous small dark seeds that failed to
imbibe water For percussion treatment nearly all seeds took up water (Figure A8a)
Germination was rapid and uniform with little damage evident (Figure A8b)
To summarize both New Mexico locust and black locust seed lots were
dormant The standard hot water scarification treatment improved germination but
only to 56 and 41 for New Mexico locust and black locust respectively Nearly
all percussion durations improved total germination as well as germination rate over
the hot water treatment Germination was greater than 90 for percussion durations
of 4 5 and 10 minutes for New Mexico locust and 3 4 and 5 minutes for black locust
58
CI)
sgt t CI) 0
igt 0
sect ttl t E
Vl 0
c3
100r==~==~~=r------------------------------------~ ___ Percussion 5 min
middotmiddotmiddot0middotmiddotmiddot Hot Water -lIf- Control
80r=========~--~~----------------------------~
60
40
20 -~-~---~---------- ---~------------I
omiddot
O
_~_----------lIf-----~bull -----_ shy---shy I
Day 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A6 Effect of selected scarification treatments on germination speed for black locust First seven days of fourteen-day experiment are shown
Day 7
a
b c d
Figure A7 Damage to New Mexico locust seeds observed during germination counts following hot water scarification A7a Hot water replicate including damaged seedlings and numerous unimbibed seeds A7b Close-up ofseed where radicle failed to elongate A7c Close-up ofseed where radicle elongated but was delayed in freeing itself from the seed coat A7d Close-up ofseed where radicle completely broke offfrom embryo
60
a
b
Figure A8 New Mexico locust seeds following percussion treatment at time of germination counts A8a Percussion replicate with uniformly healthy seedlings and near complete imbibition of seeds A8b Close-up ofpercussion-treated seedling
61
Discussion
Papilionoid seeds have a specific anatomical feature known as the strophiole
or lens-the natural site of water entry to the seed (Hamly 1932) The strophiole is
located on the cotyledonary lobe of a locust seed (Figure A9) When a papilionoid
legume seed is percussed for the appropriate amount of time repeated hits on the
integrated seed coat loosen the constrained cells of the strophiolar region without
excessively damaging the rest of the seed coat (Ballard 1976)
When a percussed seed is soaked water enters exclusively through the
strophiole in a controlled manner (Kelly and Staden 1987) This regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues This
contrasts with seeds that have been hot water or acid scarified treatments that can
randomly degrade the seed coat Unlocalized cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rehman et al 1998 Marunda 1990)
Papilionoids are the largest subfamily of legumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust and black locust these legumes often play an integral role in the
revegetation ofdisturbed lands Developing superior scarification methods to the
standard hot water and acid treatments should fadlitate the use ofthese legumes in
restoration projects
62
Figure A9 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (bc)
63
APPENDIXB
GERMINATION PERCENTS AND STANDARD ERRORS FOR SOURCE
BLOCK AND TREATMENT FOR NEW MEXICO LOCUST
Table B 1 Gennination percents and standard errors for source block and treatment for New Mexico locust
Source Block Treatment Germination Percentage Standard Error
Capitan 1 Immersion 74 437 Capitan 2 Immersion 69 462 Capitan 3 Immersion 71 454 Capitan 4 Immersion 67 470 Capitan Boiling 30 sec 79 407 Capitan 2 Boiling 30 sec 76 427 Capitan 3 Boiling 30 sec 72 449 Capitan 4 Boiling 30 sec 76 427 Capitan Boiling 60 sec 71 454 Capitan 2 Boiling 60 sec 54 498 Capitan 3 Boiling 60 sec 67 470 Capitan 4 Boiling 60 sec 76 427 Capitan I Control 5 218 Capitan 2 Control 7 255 Capitan 3 Control 4 196 Capitan 4 Control 5 218 Capitan 1 Percussion I min 47 499 Capitan 2 Percussion I min
470
Capitan Percussion I min 39 488
Capitan 4 Percussion I min 38 485 Capitan I Percussion 2 min 55 497 Capitan Capitan
2
Percussion 2 min Percussion 2 min
71 65
454 477
Capitan 4 Percussion 2 min 56 496 Capitan I Percussion 4 min 86 347 Capitan 2 Percussion 4 min 79 407 Capitan 3 Percussion 4 min 81 392 Capitan 4 Percussion 4 min 82 384 Capitan I Percussion 8 min 90 300 Capitan 2 Percussion 8 min 88 325 Capitan 3 Percussion 8 min 90 300 Capitan 4 Percussion 8 min 90 300 Gila I Immersion 71 454 Gila 2 Immersion 77 421 Gila
Immersion 75 433
Gila 4 Immersion 72 449
Gila I Boiling 30 sec 68 466
Gila 2 Boiling 30 sec 81 392
Gila 3 Boiling 30 sec 54 498
Gila 4 Boiling 30 sec 63 483
Gila I Boiling 60 sec 55 497
Gila 2 Boiling 60 sec 68 466
Gila 3 Boiling 60 sec 57 495
Gila 4 Boiling 60 sec 70 458
65
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Gila 2 Control 13 336 Gila 3 Control 13 336 Gila 4 Control 20 400 Gila 1 Percussion 1 min 66 474 Gila 2 Percussion 1 min 57 495 Gila 3 Percussion 1 min 73 444 Gila 4 Percussion 1 min 82 384 Gila 1 Percussion 2 min 81 392 Gila 2 Percussion 2 min 86 347 Gila 3 Percussion 2 min 85 357 Gila 4 Percussion 2 min 54 498 Gila 1 Percussion 4 min 79 407 Gila 2 Percussion 4 min 83 376 Gila 3 Percussion 4 min 84 367 Gila 4 Percussion 4 min 79 407 Gila 1 Percussion 8 min 51 500 Gila 2 Percussion 8 min 76 427 Gila 3 Percussion 8 min 52 500 Gila 4 Percussion 8 min 74 439 Holman 1 Immersion 83 376 Holman 2 Immersion 86 347 Holman 3 Immersion 81 392 Holman 4 Immersion 90 300 Holman 1 Boiling 30 sec 90 300 Holman 2 Boiling 30 sec 85 357 Holman 3 Boiling 30 sec 87 336 Holman 4 Boiling 30 sec 83 376 Holman 1 Boiling 60 sec 79 407 Holman 2 Boiling 60 sec 80 400 Holman 3 Boiling 60 sec 86 347 Holman 4 Boiling 60 sec 93 255 Holman Control 25 433 Holman 2 Control 19 392 Holman Control 21 407
Holman 4 Control 24 427
Holman Percussion 1 min 77 421
Holman 2 Percussion 1 min 85 357
Holman 3 Percussion 1 min 80 400
Holman 4 Percussion 1 min 89 313
Holman 1 Percussion 2 min 88 325
Holman 2 Percussion 2 min 96 196
Holman 3 Percussion 2 min 93 255
Holman 4 Percussion 2 min 96 196
Holman 1 Percussion 4 min 92 271
Holman 2 Percussion 4 min 97 171
66
I
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Holman 3 Percussion 4 min 91 286 Holman 4 Percussion 4 min 96 196 Holman 1 Percussion 8 min 88 325 Holman 2 Percussion 8 min 85 357 Holman 3 Percussion 8 min 83 376 Holman 4 Percussion 8 min 82 384 Jemez 1 Immersion 53 499 Jemez 2 Immersion 44 496 Jemez 3 Immersion 52 500 Jemez 4 Immersion 70 458 Jemez 1 Boiling 30 sec 58 494 Jemez 2 Boiling 30 sec 55 497 Jemez 3 Boiling 30 sec 48 500 Jemez 4 Boiling 30 sec 65 477 Jemez 1 Boiling 60 sec 68 466 Jemez 2 Boiling 60 sec 63 483 Jemez 3 Boiling 60 sec 73 444 Jemez 4 Boiling 60 sec 89 313 Jemez 1 Control 2 140 Jemez 2 Control 9 286 Jemez 3 Control 6 237 Jemez 4 Control 8 271 Jemez 1 Percussion 1 min 58 494 Jemez 2 Percussion 1 min 63 483 Jemez 3 Percussion 1 min 77 421 Jemez 4 Percussion 1 min 89 313 Jemez 1 Percussion 2 min 68 466 Jemez 2 Percussion 2 min 64 480 Jemez 3 Percussion 2 min 80 400 Jemez 4 Percussion 2 min 83 376 Jemez Percussion 4 min 68 466 Jemez 2 Percussion 4 min 79 407 Jemez 3 Percussion 4 min 70 458 Jemez 4 Percussion 4 min 83 376 Jemez 1 Percussion 8 min 52 500 Jemez 2 Percussion 8 min 59 492 Jemez 3 Percussion 8 min 61 488 Jemez 4 Percussion 8 min 70 458 Magdalena 1 Immersion 69 462 Magdalena 2 Immersion 69 462 Magdalena J Immersion 70 458 Magdalena 4 Immersion 85 357 Magdalena 1 Boiling 30 sec 65 477 Magdalena 2 Boiling 30 sec 71 454 Magdalena 3 Boiling 30 sec 68 466
67
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Magdalena 4 Boiling 30 sec 83 376 Magdalena 1 Boiling 60 sec 63 483 Magdalena 2 Boiling 60 sec 68 466 Magdalena 3 Boiling 60 sec 63 483 Magdalena 4 Boiling 60 sec 82 384 Magdalena 1 Control 11 313 Magdalena 2 Control 6 237 Magdalena 3 Control 5 218 Magdalena 4 Control 4 196 Magdalena Percussion 1 min 32 466 Magdalena 2 Percussion 1 min 45 497 Magdalena 3 Percussion 1 min 46 498 Magdalena 4 Percussion I min 36 480 Magdalena 1 Percussion 2 min 67 470 Magdalena 2 Percussion 2 min 68 466 Magdalena 3 Percussion 2 min 65 477 Magdalena 4 Percussion 2 min 81 392 Magdalena 1 Percussion 4 min 87 336 Magdalena 2 Percussion 4 min 97 171 Magdalena 3 Percussion 4 min 83 376 Magdalena 4 Percussion 4 min 97 171 Magdalena 1 Percussion 8 min 98 lAO Magdalena 2 Percussion 8 min 100 000 Magdalena 3 Percussion 8 min 96 196 Magdalena 4 Percussion 8 min 98 lAO Manzano 1 Immersion 67 470 Manzano 2 Immersion 57 495 Manzano 3 Immersion 72 4049 Manzano 4 Immersion 73 4044 Manzano 1 Boiling 30 sec 70 458 Manzano 2 Boiling 30 sec 64 480 Manzano 3 Boiling 30 sec 71 454 Manzano 4 Boiling 30 sec 80 400 Manzano I Boiling 60 sec 50 500 Manzano 2 Boiling 60 sec 53 499 Manzano 3 Boiling 60 sec 59 492 Manzano 4 Boiling 60 sec 74 439 Manzano I Control 4 196 Manzano 2 Control 8 271 Manzano 3 Control 1 099 Manzano 4 Control 6 237 Manzano 1 Percussion 1 min 52 500 Manzano 2 Percussion 1 min 39 488 Manzano 3 Percussion 1 min 36 480 Manzano 4 Percussion 1 min 51 500
68
Table B 1 Continued
Source Block Treatment Gerrni~ation Percentage Standard Error
Manzano Percussion 2 min 53 499 Manzano 2 Percussion 2 min 58 494 Manzano 3 Percussion 2 min 61 488 Manzano 4 Percussion 2 min 62 485 Manzano 1 Percussion 4 min 76 427 Manzano 2 Percussion 4 min 85 357 Manzano 3 Percussion 4 min 79 407 Manzano 4 Percussion 4 min 83 376 Manzano 1 Percussion 8 min 87 336 Manzano 2 Percussion 8 min 83 376 Manzano 3 Percussion 8 min 83 376 Manzano 4 Percussion 8 min 75 433 Raton 1 Immersion 54 498 Raton 2 Immersion 37 483 Raton 3 Immersion 39 488 Raton 4 Immersion 69 462 Raton 1 Boiling 30 sec 36 480 Raton 2 Boiling 30 sec 20 400 Raton 3 Boiling 30 sec 28 449 Raton 4 Boiling 30 sec 60 490 Raton 1 Boiling 60 sec 15 357 Raton 2 Boiling 60 sec 171 Raton 3 Boiling 60 sec 4 196 Raton 4 Boiling 60 sec 35 477 Raton 1 Control 4 196 Raton 2 Control 12 325 Raton 3 Control 2 140 Raton 4 Control 6 237 Raton 1 Percussion 1 min 52 500 Raton 2 Percussion I min 29 454 Raton 3 Percussion I min 35 477 Raton 4 Percussion 1 min 31 462 Raton 1 Percussion 2 min 55 497 Raton 2 Percussion 2 min 35 477 Raton 3 Percussion 2 min 33 470 Raton 4 Percussion 2 min 42 494 Raton 1 Percussion 4 min 65 477 Raton 2 Percussion 4 min 52 500 Raton Percussion 4 min 51 500 Raton 4 Percussion 4 min 53 499 Raton 1 Percussion 8 min 88 325 Raton 2 Percussion 8 min 80 400 Raton 3 Percussion 8 min 73 444 Raton 4 Percussion 8 min 74 439 Sacramento 1 Immersion 43 495
69
Table B 1 Continued
-Source Block Treatment Germination Percentage Standard Error
Sacramento 2 Immersion 30 458 Sacramento 3 Immersion 38 485 Sacramento 4 Immersion 56 496 Sacramento 1 Boiling 30 sec 47 499 Sacramento 2 Boiling 30 sec 48 500 Sacramento 3 Boiling 30 sec 48 500 Sacramento 4 Boiling 30 sec 56 496 Sacramento 1 Boiling 60 sec 52 500 Sacramento 2 Boiling 60 sec 67 470 Sacramento 3 Boiling 60 sec 43 495 Sacramento 4 Boiling 60 sec 48 500 Sacramento 1 Control 0 000 Sacramento 2 Control 3 171 Sacramento 3 Control 3 171 Sacramento 4 Control 099 Sacramento 1 Percussion 1 min 57 495 Sacramento 2 Percussion 1 min 61 488 Sacramento 3 Percussion 1 min 62 485 Sacramento 4 Percussion 1 min 55 497 Sacramento 1 Percussion 2 min 84 367 Sacramento 2 Percussion 2 min 70 458 Sacramento 3 Percussion 2 min 81 392 Sacramento 4 Percussion 2 min 69 462 Sacramento 1 Percussion 4 min 91 286 Sacramento 2 Percussion 4 min 84 367 Sacramento 3 Percussion 4 min 84 367 Sacramento 4 Percussion 4 min 94 237 Sacramento 1 Percussion 8 min 82 384 Sacramento 2 Percussion 8 min 81 392 Sacramento 3 Percussion 8 min 82 384 Sacramento 4 Percussion 8 min 81 392 Sandia 1 Immersion 76 427 Sandia 2 Immersion 70 458 Sandia 3 Immersion 70 458 Sandia 4 Immersion 81 392 Sandia 1 Boiling 30 sec 78 414 Sandia 2 Boiling 30 sec 71 454 Sandia 3 Boiling 30 sec 63 483 Sandia 4 Boiling 30 sec 78 414 Sandia 1 Boiling 60 sec 67 470 Sandia 2 Boiling 60 sec 65 477 Sandia 3 Boiling 60 sec 74 439 Sandia 4 Boiling 60 sec 66 474 Sandia 1 Control 16 367 Sandia 2 Control 7 255
70
Table B 1 Continued
Source Block Treatment Gennination Percentage Standard Error
Sandia 3 Control 8 271 Sandia 4 Control 16 367 Sandia 1 Percussion 1 min 67 470 Sandia 2 Percussion 1 min 61 488 Sandia 3 Percussion 1 min 66 474 Sandia 4 Percussion 1 min 59 492 Sandia I Percussion 2 min 82 384 Sandia 2 Percussion 2 min 83 376 Sandia 3 Percussion 2 min 81 392 Sandia 4 Percussion 2 min 84 367 Sandia 1 Percussion 4 min 89 313 Sandia 2 Percussion 4 min 86 347 Sandia 3 Percussion 4 min 89 313 Sandia 4 Percussion 4 min 85 357 Sandia 1 Percussion 8 min 80 400 Sandia 2 Percussion 8 min 77 421 Sandia 3 Percussion 8 min 69 462 Sandia 4 Percussion 8 min 66 474
LITERATURE CITED
Ashby WC Vogel WG Rogers NF 1985 Black locust in the reclamation equation General Technical Report Northeastern Forest Experiment Station USDA Forest Service NE-I05 12 p
Ballard LAT 1973 Physical barriers to gennination Seed Sci Technoll 285-303
___ 1976 Strophiolar water conduction in the seeds ofTrifolieae induced by action on the testa at nonstrophiolar sites Austr J Plant Physiol3 465-469
Barton LV 1947 Special studies on seed coat impenneability Contrib Boyce Thompson Inst 14 355-362
Baskin CC Baskin JM 1998 Seeds Ecology biogeography and evolution of donnancy and gennination Academic Press London UK 666 p
Bewley JD 1997 Seed germination and donnancy Plant Ce119 1055-1066
Bewley JD Black M 1994 Seeds physiology of development and germination 2nd ed Plenum Press New York NY 445 p
Briscoe CB 1996 Growing Nitrogen Fixing Trees in Acid Soils In MH Powell (ed) Nitrogen Fixing Trees for Acid Soils- A Field ManuaL Winrock International Morrilton AR 22 p
Copeland LO McDonald MB 1995 Principles of Seed Science and Technology 3rded Chapman amp Hall New York NY 409 p
Cox RE Klett JE 1984 Seed gennination requirements of native Colorado plants for use in the landscape Plant Propag 30(2) 6-10
Dell B 1980 Structure and function ofthe strophiolar plug in seeds ofAlbizia Lophantha Am J Bot 67 556-563
Dick-Peddie WA 1993 New Mexico Vegetation Past Present and Future University ofNew Mexico Press Albuquerque NM 244 p
Dreesen DR 2001 Propagation protocol for production ofcontainer Robinia neomexicana plants Los Lunas Plant Materials Center Los Lunas New Mexico In Native Plant Network URL httpwwwnativeplantnetworkorg (accessed 9 January 2002) Moscow (ID) University ofIdaho CoIl Natural Resources For Res Nursery
72
Dreesen DR Harrington JT 1997 Propagation of native plants for restoration projects in the Southwestern US- Preliminary Investigations In Landis TD Thompson JR tech coords National Proceedings Forest and Conservation Nursery Assocs Gen Tech Rep PNW-GTR-419 Portland OR USDA For Ser PNW Res Sta 77-88
Gottfried GJ 1980 Control ofNew Mexican locust and the effect on planted ponderosa pine in central Arizona US For Ser Res Note RM-386 Rocky Mountain For Range ExpStn Fort Collins CO
Gosling PG Samuel YK Jones SK 1995 A systematic examination of germination temperature chipping and water temperaturesoak duration pretreatments on the seeds ofLeucaena leucocephala Seed Sci amp Technol 23521-532
Hamly DH 1932 Softening of the seeds of Melilotus alba Bot Gazette 93 345shy375
Hanna PJ 1984 Anatomical features of the seed coat ofAcacia kempeana (Mueller) which relate to increased germination rate induced by heat treatment New Phytol 96 23-29
Hanks JP Dick-Peddie WA 1974 Vegetation patterns of the White Mountains New Mexico Southwest Naturalist 18(4) 371-382
Hine S Harrington JT Dreesen DR 1997 Seed scarification requirements for Robinia neomexicana In Harrington JTtech coord Proc Third SouthwestemContainer Growers Conf Feb 12-13 1997 Ft Collins CO
Hopkinson JM 1993 The strophiole of leguminous seeds Newsletter- IntI Herbage Seed Production Res Grp 18 7-8
---- 1997 Differences in hardseededness between Cunningham and Tarramba leucaena In Leucnet News The Newsletter of the International LeucaenaRes and Devlt Netwk No4
ISTA (International Seed Testing Association) 1999 International rules for seed testing rules Seed Sci amp Technol27 Supplement
Kelly KM van Staden J 1987 The lens as the site of permeability in the papilionoid seed Aspalathus linearis J Plant Physiol 128(45) 395-404
73
Keresztesi B 1988 Black locust the tree of agriculture Outlook on Agr 17(2) 77~85
Kigel 1 and Galili G 1995 Seed development and germination Marcel Dekker New York 853 p
Klemmedson 10 1994 New Mexico locust and parent material Influence on macronutrients of forest floor and soil Soil Sci Soc Am 1 58 974~980
Kuo WH Tam AW 1988 The pathway of water absorption of mung bean seeds Seed Sci amp Technol16(1) 139~144
Leadem CL 1997 Dormancy--unlocking seed secrets In Landis TD Thompson lR tech coords National Proceedings Forestand Conservation Nursery Assocs Gen Tech Rep PNW-G TR-419 Portland OR USDA For Ser PNW Res Sta 43-52
Lin Y Wagner MR Seweyestewa A Honahni MD DeGomez M McMillin JD 1996 Influence of cultural treatments on seed germination and seedling growth of Robinia neomexicana Forest Farm and Community Tree Res Reports Volume 1
Marunda CT 1990 Effects of seed pretreatment on the development ofAcacia auriculforinis and A holosericea seedlings ACIAR Proceedings Series No 28
Meginnis HG 1937 Sulphuric acid treatment to increase germination of black locust seed USDA Circ 453 34 p
Miklas PN Towensend CE Ladd SL 1987 Seedcoat anatomy and the scarification ofcicer milkvetch seed Crop Sci 27 766-772
Monsen SB 1984 Use of shrubs on mine spoils In The Challenge ofProducing Native Plants for the Intermountain Area Proceedings Intermountain Nurserymans Association 1983 conference Las Vegas NV compo PM Murphy 26-31 Ogden UT USDA For Ser Gen Tech Report INT-l68
Olson DF 1974 Robinia L Locust In Seeds of Woody Plants in the US tech coord CS Schopmeyer 728-731 USDA For Ser Agr Handbk No450
Rehman S Loescher RNl Harris P1C 1998 Dormancy breaking and germination ofAcacia salicina Lindl seeds Seed Sci amp Techno127 553-557
Rolston MP 1978 Water impermeable seed dormancy Bot Rev 44 365-396
74
SAS Institute Inc 1989 SASSTAT Users Guide Version 6 4th Edition Volume 1 Cary NC SAS Institute Inc 943 p
SerratoValenti G Conara L Ferrando M Modenesi P 1993 Structural and histochemical features ofStylosanthes scabra (Leguminosae Papilionoidae) seed coat as related to water entry Can J Bot 71 834-840
Simpson BJ 1988 A field guide to Texas trees Austin TX Texas Monthly Press 372p
Tran VN Cavanagh AK 1984 Structural aspects ofdormancy In Seed Physiology Vol 2 Murray DR (ed) p1-44 Academic Press Australia
USDA Forest Service 1948 Woody-plant seed manual USDA Misc Pub1654 416p
Vines RA 1960 Trees Shrubs and Woody Vines of the Southwest Austin Univ ofTexas Press 1104 p
Wagner MR Larson RE Craig TP 1992 Ecology and silvics ofNew Mexico locust (Robinia neomexicana) with an emphasis on the arboreal form p53-57 In JW Hanover K Miller and S Plesko (eds) Proc Intemat Confon Black Locust Biology Culture and Utilization Mich State Univ USA
Wilson JK 1944 Immersing seeds of species of Robinia in boiling water hastens germination J For 42 453-454
Zimmerman RW Carpenter SB 1980 First-year coppice production from a 5shyyear old black locust stand on surface mine spoil p309-314 Centr Hardwd For ConfProc University ofMissouri Columbia
~ ~ () u agt
Il-t d o ~
~ ~ c3
100middotlr~~~------------------------------------e- Percussion 5min middot 0middot Hot Water -- Control
80-J=middot======
6b-middot-lmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot-middot--middotmiddot-middot~-middotmiddot -middotmiddot middot middotmiddot-middot 1
40
20
-----~-----------
0 0 0 middotmiddotmiddotmiddotmiddot0middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot0
0 --~-----y-----~------_---~--- T-----~
o shy -TDay 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A4 Effect of selected scarification treatments on germination speed for New Mexico locust First seven days of fourteen-day experiment are shown
Day 7
80
~ cd
5 60 ()
[) Cl d 0 1t1 d
40sect dUl
-J
20
o
ns
Scarification
Figure A5 Effect of scarification on germination response for black locust = significant difference between adjacent treatment bars ns = no significant difference
decrease at the 10-minute level This drop-off suggests that 10 minutes of percussion
over-treats seeds from this lot of-black locust
A selected percussion treatment intermediate in time (5 minutes) also had the
fastest germination rate for black locust (Figure A6)_ The hot water curve is
somewhat flatter indicating a slower germination rate The control curve is very flat
indicating a slow germination rate
A qualitative difference between hot water and percussion-treated seeds was
also observed Along with healthy germinants the hot water treatment produced
over-treated seeds (Figure A 7a) Examples of damaged germinants include seeds
where the radicle did not elongate (Figure A 7b) seeds where the radicle elongated
but was delayed in freeing itself from the seed coat (Figure A7c) and seeds where the
radicle broke off completely from the embryo (Figure A7d) In addition to these
examples of over-treatment there were numerous small dark seeds that failed to
imbibe water For percussion treatment nearly all seeds took up water (Figure A8a)
Germination was rapid and uniform with little damage evident (Figure A8b)
To summarize both New Mexico locust and black locust seed lots were
dormant The standard hot water scarification treatment improved germination but
only to 56 and 41 for New Mexico locust and black locust respectively Nearly
all percussion durations improved total germination as well as germination rate over
the hot water treatment Germination was greater than 90 for percussion durations
of 4 5 and 10 minutes for New Mexico locust and 3 4 and 5 minutes for black locust
58
CI)
sgt t CI) 0
igt 0
sect ttl t E
Vl 0
c3
100r==~==~~=r------------------------------------~ ___ Percussion 5 min
middotmiddotmiddot0middotmiddotmiddot Hot Water -lIf- Control
80r=========~--~~----------------------------~
60
40
20 -~-~---~---------- ---~------------I
omiddot
O
_~_----------lIf-----~bull -----_ shy---shy I
Day 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A6 Effect of selected scarification treatments on germination speed for black locust First seven days of fourteen-day experiment are shown
Day 7
a
b c d
Figure A7 Damage to New Mexico locust seeds observed during germination counts following hot water scarification A7a Hot water replicate including damaged seedlings and numerous unimbibed seeds A7b Close-up ofseed where radicle failed to elongate A7c Close-up ofseed where radicle elongated but was delayed in freeing itself from the seed coat A7d Close-up ofseed where radicle completely broke offfrom embryo
60
a
b
Figure A8 New Mexico locust seeds following percussion treatment at time of germination counts A8a Percussion replicate with uniformly healthy seedlings and near complete imbibition of seeds A8b Close-up ofpercussion-treated seedling
61
Discussion
Papilionoid seeds have a specific anatomical feature known as the strophiole
or lens-the natural site of water entry to the seed (Hamly 1932) The strophiole is
located on the cotyledonary lobe of a locust seed (Figure A9) When a papilionoid
legume seed is percussed for the appropriate amount of time repeated hits on the
integrated seed coat loosen the constrained cells of the strophiolar region without
excessively damaging the rest of the seed coat (Ballard 1976)
When a percussed seed is soaked water enters exclusively through the
strophiole in a controlled manner (Kelly and Staden 1987) This regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues This
contrasts with seeds that have been hot water or acid scarified treatments that can
randomly degrade the seed coat Unlocalized cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rehman et al 1998 Marunda 1990)
Papilionoids are the largest subfamily of legumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust and black locust these legumes often play an integral role in the
revegetation ofdisturbed lands Developing superior scarification methods to the
standard hot water and acid treatments should fadlitate the use ofthese legumes in
restoration projects
62
Figure A9 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (bc)
63
APPENDIXB
GERMINATION PERCENTS AND STANDARD ERRORS FOR SOURCE
BLOCK AND TREATMENT FOR NEW MEXICO LOCUST
Table B 1 Gennination percents and standard errors for source block and treatment for New Mexico locust
Source Block Treatment Germination Percentage Standard Error
Capitan 1 Immersion 74 437 Capitan 2 Immersion 69 462 Capitan 3 Immersion 71 454 Capitan 4 Immersion 67 470 Capitan Boiling 30 sec 79 407 Capitan 2 Boiling 30 sec 76 427 Capitan 3 Boiling 30 sec 72 449 Capitan 4 Boiling 30 sec 76 427 Capitan Boiling 60 sec 71 454 Capitan 2 Boiling 60 sec 54 498 Capitan 3 Boiling 60 sec 67 470 Capitan 4 Boiling 60 sec 76 427 Capitan I Control 5 218 Capitan 2 Control 7 255 Capitan 3 Control 4 196 Capitan 4 Control 5 218 Capitan 1 Percussion I min 47 499 Capitan 2 Percussion I min
470
Capitan Percussion I min 39 488
Capitan 4 Percussion I min 38 485 Capitan I Percussion 2 min 55 497 Capitan Capitan
2
Percussion 2 min Percussion 2 min
71 65
454 477
Capitan 4 Percussion 2 min 56 496 Capitan I Percussion 4 min 86 347 Capitan 2 Percussion 4 min 79 407 Capitan 3 Percussion 4 min 81 392 Capitan 4 Percussion 4 min 82 384 Capitan I Percussion 8 min 90 300 Capitan 2 Percussion 8 min 88 325 Capitan 3 Percussion 8 min 90 300 Capitan 4 Percussion 8 min 90 300 Gila I Immersion 71 454 Gila 2 Immersion 77 421 Gila
Immersion 75 433
Gila 4 Immersion 72 449
Gila I Boiling 30 sec 68 466
Gila 2 Boiling 30 sec 81 392
Gila 3 Boiling 30 sec 54 498
Gila 4 Boiling 30 sec 63 483
Gila I Boiling 60 sec 55 497
Gila 2 Boiling 60 sec 68 466
Gila 3 Boiling 60 sec 57 495
Gila 4 Boiling 60 sec 70 458
65
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Gila 2 Control 13 336 Gila 3 Control 13 336 Gila 4 Control 20 400 Gila 1 Percussion 1 min 66 474 Gila 2 Percussion 1 min 57 495 Gila 3 Percussion 1 min 73 444 Gila 4 Percussion 1 min 82 384 Gila 1 Percussion 2 min 81 392 Gila 2 Percussion 2 min 86 347 Gila 3 Percussion 2 min 85 357 Gila 4 Percussion 2 min 54 498 Gila 1 Percussion 4 min 79 407 Gila 2 Percussion 4 min 83 376 Gila 3 Percussion 4 min 84 367 Gila 4 Percussion 4 min 79 407 Gila 1 Percussion 8 min 51 500 Gila 2 Percussion 8 min 76 427 Gila 3 Percussion 8 min 52 500 Gila 4 Percussion 8 min 74 439 Holman 1 Immersion 83 376 Holman 2 Immersion 86 347 Holman 3 Immersion 81 392 Holman 4 Immersion 90 300 Holman 1 Boiling 30 sec 90 300 Holman 2 Boiling 30 sec 85 357 Holman 3 Boiling 30 sec 87 336 Holman 4 Boiling 30 sec 83 376 Holman 1 Boiling 60 sec 79 407 Holman 2 Boiling 60 sec 80 400 Holman 3 Boiling 60 sec 86 347 Holman 4 Boiling 60 sec 93 255 Holman Control 25 433 Holman 2 Control 19 392 Holman Control 21 407
Holman 4 Control 24 427
Holman Percussion 1 min 77 421
Holman 2 Percussion 1 min 85 357
Holman 3 Percussion 1 min 80 400
Holman 4 Percussion 1 min 89 313
Holman 1 Percussion 2 min 88 325
Holman 2 Percussion 2 min 96 196
Holman 3 Percussion 2 min 93 255
Holman 4 Percussion 2 min 96 196
Holman 1 Percussion 4 min 92 271
Holman 2 Percussion 4 min 97 171
66
I
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Holman 3 Percussion 4 min 91 286 Holman 4 Percussion 4 min 96 196 Holman 1 Percussion 8 min 88 325 Holman 2 Percussion 8 min 85 357 Holman 3 Percussion 8 min 83 376 Holman 4 Percussion 8 min 82 384 Jemez 1 Immersion 53 499 Jemez 2 Immersion 44 496 Jemez 3 Immersion 52 500 Jemez 4 Immersion 70 458 Jemez 1 Boiling 30 sec 58 494 Jemez 2 Boiling 30 sec 55 497 Jemez 3 Boiling 30 sec 48 500 Jemez 4 Boiling 30 sec 65 477 Jemez 1 Boiling 60 sec 68 466 Jemez 2 Boiling 60 sec 63 483 Jemez 3 Boiling 60 sec 73 444 Jemez 4 Boiling 60 sec 89 313 Jemez 1 Control 2 140 Jemez 2 Control 9 286 Jemez 3 Control 6 237 Jemez 4 Control 8 271 Jemez 1 Percussion 1 min 58 494 Jemez 2 Percussion 1 min 63 483 Jemez 3 Percussion 1 min 77 421 Jemez 4 Percussion 1 min 89 313 Jemez 1 Percussion 2 min 68 466 Jemez 2 Percussion 2 min 64 480 Jemez 3 Percussion 2 min 80 400 Jemez 4 Percussion 2 min 83 376 Jemez Percussion 4 min 68 466 Jemez 2 Percussion 4 min 79 407 Jemez 3 Percussion 4 min 70 458 Jemez 4 Percussion 4 min 83 376 Jemez 1 Percussion 8 min 52 500 Jemez 2 Percussion 8 min 59 492 Jemez 3 Percussion 8 min 61 488 Jemez 4 Percussion 8 min 70 458 Magdalena 1 Immersion 69 462 Magdalena 2 Immersion 69 462 Magdalena J Immersion 70 458 Magdalena 4 Immersion 85 357 Magdalena 1 Boiling 30 sec 65 477 Magdalena 2 Boiling 30 sec 71 454 Magdalena 3 Boiling 30 sec 68 466
67
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Magdalena 4 Boiling 30 sec 83 376 Magdalena 1 Boiling 60 sec 63 483 Magdalena 2 Boiling 60 sec 68 466 Magdalena 3 Boiling 60 sec 63 483 Magdalena 4 Boiling 60 sec 82 384 Magdalena 1 Control 11 313 Magdalena 2 Control 6 237 Magdalena 3 Control 5 218 Magdalena 4 Control 4 196 Magdalena Percussion 1 min 32 466 Magdalena 2 Percussion 1 min 45 497 Magdalena 3 Percussion 1 min 46 498 Magdalena 4 Percussion I min 36 480 Magdalena 1 Percussion 2 min 67 470 Magdalena 2 Percussion 2 min 68 466 Magdalena 3 Percussion 2 min 65 477 Magdalena 4 Percussion 2 min 81 392 Magdalena 1 Percussion 4 min 87 336 Magdalena 2 Percussion 4 min 97 171 Magdalena 3 Percussion 4 min 83 376 Magdalena 4 Percussion 4 min 97 171 Magdalena 1 Percussion 8 min 98 lAO Magdalena 2 Percussion 8 min 100 000 Magdalena 3 Percussion 8 min 96 196 Magdalena 4 Percussion 8 min 98 lAO Manzano 1 Immersion 67 470 Manzano 2 Immersion 57 495 Manzano 3 Immersion 72 4049 Manzano 4 Immersion 73 4044 Manzano 1 Boiling 30 sec 70 458 Manzano 2 Boiling 30 sec 64 480 Manzano 3 Boiling 30 sec 71 454 Manzano 4 Boiling 30 sec 80 400 Manzano I Boiling 60 sec 50 500 Manzano 2 Boiling 60 sec 53 499 Manzano 3 Boiling 60 sec 59 492 Manzano 4 Boiling 60 sec 74 439 Manzano I Control 4 196 Manzano 2 Control 8 271 Manzano 3 Control 1 099 Manzano 4 Control 6 237 Manzano 1 Percussion 1 min 52 500 Manzano 2 Percussion 1 min 39 488 Manzano 3 Percussion 1 min 36 480 Manzano 4 Percussion 1 min 51 500
68
Table B 1 Continued
Source Block Treatment Gerrni~ation Percentage Standard Error
Manzano Percussion 2 min 53 499 Manzano 2 Percussion 2 min 58 494 Manzano 3 Percussion 2 min 61 488 Manzano 4 Percussion 2 min 62 485 Manzano 1 Percussion 4 min 76 427 Manzano 2 Percussion 4 min 85 357 Manzano 3 Percussion 4 min 79 407 Manzano 4 Percussion 4 min 83 376 Manzano 1 Percussion 8 min 87 336 Manzano 2 Percussion 8 min 83 376 Manzano 3 Percussion 8 min 83 376 Manzano 4 Percussion 8 min 75 433 Raton 1 Immersion 54 498 Raton 2 Immersion 37 483 Raton 3 Immersion 39 488 Raton 4 Immersion 69 462 Raton 1 Boiling 30 sec 36 480 Raton 2 Boiling 30 sec 20 400 Raton 3 Boiling 30 sec 28 449 Raton 4 Boiling 30 sec 60 490 Raton 1 Boiling 60 sec 15 357 Raton 2 Boiling 60 sec 171 Raton 3 Boiling 60 sec 4 196 Raton 4 Boiling 60 sec 35 477 Raton 1 Control 4 196 Raton 2 Control 12 325 Raton 3 Control 2 140 Raton 4 Control 6 237 Raton 1 Percussion 1 min 52 500 Raton 2 Percussion I min 29 454 Raton 3 Percussion I min 35 477 Raton 4 Percussion 1 min 31 462 Raton 1 Percussion 2 min 55 497 Raton 2 Percussion 2 min 35 477 Raton 3 Percussion 2 min 33 470 Raton 4 Percussion 2 min 42 494 Raton 1 Percussion 4 min 65 477 Raton 2 Percussion 4 min 52 500 Raton Percussion 4 min 51 500 Raton 4 Percussion 4 min 53 499 Raton 1 Percussion 8 min 88 325 Raton 2 Percussion 8 min 80 400 Raton 3 Percussion 8 min 73 444 Raton 4 Percussion 8 min 74 439 Sacramento 1 Immersion 43 495
69
Table B 1 Continued
-Source Block Treatment Germination Percentage Standard Error
Sacramento 2 Immersion 30 458 Sacramento 3 Immersion 38 485 Sacramento 4 Immersion 56 496 Sacramento 1 Boiling 30 sec 47 499 Sacramento 2 Boiling 30 sec 48 500 Sacramento 3 Boiling 30 sec 48 500 Sacramento 4 Boiling 30 sec 56 496 Sacramento 1 Boiling 60 sec 52 500 Sacramento 2 Boiling 60 sec 67 470 Sacramento 3 Boiling 60 sec 43 495 Sacramento 4 Boiling 60 sec 48 500 Sacramento 1 Control 0 000 Sacramento 2 Control 3 171 Sacramento 3 Control 3 171 Sacramento 4 Control 099 Sacramento 1 Percussion 1 min 57 495 Sacramento 2 Percussion 1 min 61 488 Sacramento 3 Percussion 1 min 62 485 Sacramento 4 Percussion 1 min 55 497 Sacramento 1 Percussion 2 min 84 367 Sacramento 2 Percussion 2 min 70 458 Sacramento 3 Percussion 2 min 81 392 Sacramento 4 Percussion 2 min 69 462 Sacramento 1 Percussion 4 min 91 286 Sacramento 2 Percussion 4 min 84 367 Sacramento 3 Percussion 4 min 84 367 Sacramento 4 Percussion 4 min 94 237 Sacramento 1 Percussion 8 min 82 384 Sacramento 2 Percussion 8 min 81 392 Sacramento 3 Percussion 8 min 82 384 Sacramento 4 Percussion 8 min 81 392 Sandia 1 Immersion 76 427 Sandia 2 Immersion 70 458 Sandia 3 Immersion 70 458 Sandia 4 Immersion 81 392 Sandia 1 Boiling 30 sec 78 414 Sandia 2 Boiling 30 sec 71 454 Sandia 3 Boiling 30 sec 63 483 Sandia 4 Boiling 30 sec 78 414 Sandia 1 Boiling 60 sec 67 470 Sandia 2 Boiling 60 sec 65 477 Sandia 3 Boiling 60 sec 74 439 Sandia 4 Boiling 60 sec 66 474 Sandia 1 Control 16 367 Sandia 2 Control 7 255
70
Table B 1 Continued
Source Block Treatment Gennination Percentage Standard Error
Sandia 3 Control 8 271 Sandia 4 Control 16 367 Sandia 1 Percussion 1 min 67 470 Sandia 2 Percussion 1 min 61 488 Sandia 3 Percussion 1 min 66 474 Sandia 4 Percussion 1 min 59 492 Sandia I Percussion 2 min 82 384 Sandia 2 Percussion 2 min 83 376 Sandia 3 Percussion 2 min 81 392 Sandia 4 Percussion 2 min 84 367 Sandia 1 Percussion 4 min 89 313 Sandia 2 Percussion 4 min 86 347 Sandia 3 Percussion 4 min 89 313 Sandia 4 Percussion 4 min 85 357 Sandia 1 Percussion 8 min 80 400 Sandia 2 Percussion 8 min 77 421 Sandia 3 Percussion 8 min 69 462 Sandia 4 Percussion 8 min 66 474
LITERATURE CITED
Ashby WC Vogel WG Rogers NF 1985 Black locust in the reclamation equation General Technical Report Northeastern Forest Experiment Station USDA Forest Service NE-I05 12 p
Ballard LAT 1973 Physical barriers to gennination Seed Sci Technoll 285-303
___ 1976 Strophiolar water conduction in the seeds ofTrifolieae induced by action on the testa at nonstrophiolar sites Austr J Plant Physiol3 465-469
Barton LV 1947 Special studies on seed coat impenneability Contrib Boyce Thompson Inst 14 355-362
Baskin CC Baskin JM 1998 Seeds Ecology biogeography and evolution of donnancy and gennination Academic Press London UK 666 p
Bewley JD 1997 Seed germination and donnancy Plant Ce119 1055-1066
Bewley JD Black M 1994 Seeds physiology of development and germination 2nd ed Plenum Press New York NY 445 p
Briscoe CB 1996 Growing Nitrogen Fixing Trees in Acid Soils In MH Powell (ed) Nitrogen Fixing Trees for Acid Soils- A Field ManuaL Winrock International Morrilton AR 22 p
Copeland LO McDonald MB 1995 Principles of Seed Science and Technology 3rded Chapman amp Hall New York NY 409 p
Cox RE Klett JE 1984 Seed gennination requirements of native Colorado plants for use in the landscape Plant Propag 30(2) 6-10
Dell B 1980 Structure and function ofthe strophiolar plug in seeds ofAlbizia Lophantha Am J Bot 67 556-563
Dick-Peddie WA 1993 New Mexico Vegetation Past Present and Future University ofNew Mexico Press Albuquerque NM 244 p
Dreesen DR 2001 Propagation protocol for production ofcontainer Robinia neomexicana plants Los Lunas Plant Materials Center Los Lunas New Mexico In Native Plant Network URL httpwwwnativeplantnetworkorg (accessed 9 January 2002) Moscow (ID) University ofIdaho CoIl Natural Resources For Res Nursery
72
Dreesen DR Harrington JT 1997 Propagation of native plants for restoration projects in the Southwestern US- Preliminary Investigations In Landis TD Thompson JR tech coords National Proceedings Forest and Conservation Nursery Assocs Gen Tech Rep PNW-GTR-419 Portland OR USDA For Ser PNW Res Sta 77-88
Gottfried GJ 1980 Control ofNew Mexican locust and the effect on planted ponderosa pine in central Arizona US For Ser Res Note RM-386 Rocky Mountain For Range ExpStn Fort Collins CO
Gosling PG Samuel YK Jones SK 1995 A systematic examination of germination temperature chipping and water temperaturesoak duration pretreatments on the seeds ofLeucaena leucocephala Seed Sci amp Technol 23521-532
Hamly DH 1932 Softening of the seeds of Melilotus alba Bot Gazette 93 345shy375
Hanna PJ 1984 Anatomical features of the seed coat ofAcacia kempeana (Mueller) which relate to increased germination rate induced by heat treatment New Phytol 96 23-29
Hanks JP Dick-Peddie WA 1974 Vegetation patterns of the White Mountains New Mexico Southwest Naturalist 18(4) 371-382
Hine S Harrington JT Dreesen DR 1997 Seed scarification requirements for Robinia neomexicana In Harrington JTtech coord Proc Third SouthwestemContainer Growers Conf Feb 12-13 1997 Ft Collins CO
Hopkinson JM 1993 The strophiole of leguminous seeds Newsletter- IntI Herbage Seed Production Res Grp 18 7-8
---- 1997 Differences in hardseededness between Cunningham and Tarramba leucaena In Leucnet News The Newsletter of the International LeucaenaRes and Devlt Netwk No4
ISTA (International Seed Testing Association) 1999 International rules for seed testing rules Seed Sci amp Technol27 Supplement
Kelly KM van Staden J 1987 The lens as the site of permeability in the papilionoid seed Aspalathus linearis J Plant Physiol 128(45) 395-404
73
Keresztesi B 1988 Black locust the tree of agriculture Outlook on Agr 17(2) 77~85
Kigel 1 and Galili G 1995 Seed development and germination Marcel Dekker New York 853 p
Klemmedson 10 1994 New Mexico locust and parent material Influence on macronutrients of forest floor and soil Soil Sci Soc Am 1 58 974~980
Kuo WH Tam AW 1988 The pathway of water absorption of mung bean seeds Seed Sci amp Technol16(1) 139~144
Leadem CL 1997 Dormancy--unlocking seed secrets In Landis TD Thompson lR tech coords National Proceedings Forestand Conservation Nursery Assocs Gen Tech Rep PNW-G TR-419 Portland OR USDA For Ser PNW Res Sta 43-52
Lin Y Wagner MR Seweyestewa A Honahni MD DeGomez M McMillin JD 1996 Influence of cultural treatments on seed germination and seedling growth of Robinia neomexicana Forest Farm and Community Tree Res Reports Volume 1
Marunda CT 1990 Effects of seed pretreatment on the development ofAcacia auriculforinis and A holosericea seedlings ACIAR Proceedings Series No 28
Meginnis HG 1937 Sulphuric acid treatment to increase germination of black locust seed USDA Circ 453 34 p
Miklas PN Towensend CE Ladd SL 1987 Seedcoat anatomy and the scarification ofcicer milkvetch seed Crop Sci 27 766-772
Monsen SB 1984 Use of shrubs on mine spoils In The Challenge ofProducing Native Plants for the Intermountain Area Proceedings Intermountain Nurserymans Association 1983 conference Las Vegas NV compo PM Murphy 26-31 Ogden UT USDA For Ser Gen Tech Report INT-l68
Olson DF 1974 Robinia L Locust In Seeds of Woody Plants in the US tech coord CS Schopmeyer 728-731 USDA For Ser Agr Handbk No450
Rehman S Loescher RNl Harris P1C 1998 Dormancy breaking and germination ofAcacia salicina Lindl seeds Seed Sci amp Techno127 553-557
Rolston MP 1978 Water impermeable seed dormancy Bot Rev 44 365-396
74
SAS Institute Inc 1989 SASSTAT Users Guide Version 6 4th Edition Volume 1 Cary NC SAS Institute Inc 943 p
SerratoValenti G Conara L Ferrando M Modenesi P 1993 Structural and histochemical features ofStylosanthes scabra (Leguminosae Papilionoidae) seed coat as related to water entry Can J Bot 71 834-840
Simpson BJ 1988 A field guide to Texas trees Austin TX Texas Monthly Press 372p
Tran VN Cavanagh AK 1984 Structural aspects ofdormancy In Seed Physiology Vol 2 Murray DR (ed) p1-44 Academic Press Australia
USDA Forest Service 1948 Woody-plant seed manual USDA Misc Pub1654 416p
Vines RA 1960 Trees Shrubs and Woody Vines of the Southwest Austin Univ ofTexas Press 1104 p
Wagner MR Larson RE Craig TP 1992 Ecology and silvics ofNew Mexico locust (Robinia neomexicana) with an emphasis on the arboreal form p53-57 In JW Hanover K Miller and S Plesko (eds) Proc Intemat Confon Black Locust Biology Culture and Utilization Mich State Univ USA
Wilson JK 1944 Immersing seeds of species of Robinia in boiling water hastens germination J For 42 453-454
Zimmerman RW Carpenter SB 1980 First-year coppice production from a 5shyyear old black locust stand on surface mine spoil p309-314 Centr Hardwd For ConfProc University ofMissouri Columbia
80
~ cd
5 60 ()
[) Cl d 0 1t1 d
40sect dUl
-J
20
o
ns
Scarification
Figure A5 Effect of scarification on germination response for black locust = significant difference between adjacent treatment bars ns = no significant difference
decrease at the 10-minute level This drop-off suggests that 10 minutes of percussion
over-treats seeds from this lot of-black locust
A selected percussion treatment intermediate in time (5 minutes) also had the
fastest germination rate for black locust (Figure A6)_ The hot water curve is
somewhat flatter indicating a slower germination rate The control curve is very flat
indicating a slow germination rate
A qualitative difference between hot water and percussion-treated seeds was
also observed Along with healthy germinants the hot water treatment produced
over-treated seeds (Figure A 7a) Examples of damaged germinants include seeds
where the radicle did not elongate (Figure A 7b) seeds where the radicle elongated
but was delayed in freeing itself from the seed coat (Figure A7c) and seeds where the
radicle broke off completely from the embryo (Figure A7d) In addition to these
examples of over-treatment there were numerous small dark seeds that failed to
imbibe water For percussion treatment nearly all seeds took up water (Figure A8a)
Germination was rapid and uniform with little damage evident (Figure A8b)
To summarize both New Mexico locust and black locust seed lots were
dormant The standard hot water scarification treatment improved germination but
only to 56 and 41 for New Mexico locust and black locust respectively Nearly
all percussion durations improved total germination as well as germination rate over
the hot water treatment Germination was greater than 90 for percussion durations
of 4 5 and 10 minutes for New Mexico locust and 3 4 and 5 minutes for black locust
58
CI)
sgt t CI) 0
igt 0
sect ttl t E
Vl 0
c3
100r==~==~~=r------------------------------------~ ___ Percussion 5 min
middotmiddotmiddot0middotmiddotmiddot Hot Water -lIf- Control
80r=========~--~~----------------------------~
60
40
20 -~-~---~---------- ---~------------I
omiddot
O
_~_----------lIf-----~bull -----_ shy---shy I
Day 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A6 Effect of selected scarification treatments on germination speed for black locust First seven days of fourteen-day experiment are shown
Day 7
a
b c d
Figure A7 Damage to New Mexico locust seeds observed during germination counts following hot water scarification A7a Hot water replicate including damaged seedlings and numerous unimbibed seeds A7b Close-up ofseed where radicle failed to elongate A7c Close-up ofseed where radicle elongated but was delayed in freeing itself from the seed coat A7d Close-up ofseed where radicle completely broke offfrom embryo
60
a
b
Figure A8 New Mexico locust seeds following percussion treatment at time of germination counts A8a Percussion replicate with uniformly healthy seedlings and near complete imbibition of seeds A8b Close-up ofpercussion-treated seedling
61
Discussion
Papilionoid seeds have a specific anatomical feature known as the strophiole
or lens-the natural site of water entry to the seed (Hamly 1932) The strophiole is
located on the cotyledonary lobe of a locust seed (Figure A9) When a papilionoid
legume seed is percussed for the appropriate amount of time repeated hits on the
integrated seed coat loosen the constrained cells of the strophiolar region without
excessively damaging the rest of the seed coat (Ballard 1976)
When a percussed seed is soaked water enters exclusively through the
strophiole in a controlled manner (Kelly and Staden 1987) This regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues This
contrasts with seeds that have been hot water or acid scarified treatments that can
randomly degrade the seed coat Unlocalized cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rehman et al 1998 Marunda 1990)
Papilionoids are the largest subfamily of legumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust and black locust these legumes often play an integral role in the
revegetation ofdisturbed lands Developing superior scarification methods to the
standard hot water and acid treatments should fadlitate the use ofthese legumes in
restoration projects
62
Figure A9 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (bc)
63
APPENDIXB
GERMINATION PERCENTS AND STANDARD ERRORS FOR SOURCE
BLOCK AND TREATMENT FOR NEW MEXICO LOCUST
Table B 1 Gennination percents and standard errors for source block and treatment for New Mexico locust
Source Block Treatment Germination Percentage Standard Error
Capitan 1 Immersion 74 437 Capitan 2 Immersion 69 462 Capitan 3 Immersion 71 454 Capitan 4 Immersion 67 470 Capitan Boiling 30 sec 79 407 Capitan 2 Boiling 30 sec 76 427 Capitan 3 Boiling 30 sec 72 449 Capitan 4 Boiling 30 sec 76 427 Capitan Boiling 60 sec 71 454 Capitan 2 Boiling 60 sec 54 498 Capitan 3 Boiling 60 sec 67 470 Capitan 4 Boiling 60 sec 76 427 Capitan I Control 5 218 Capitan 2 Control 7 255 Capitan 3 Control 4 196 Capitan 4 Control 5 218 Capitan 1 Percussion I min 47 499 Capitan 2 Percussion I min
470
Capitan Percussion I min 39 488
Capitan 4 Percussion I min 38 485 Capitan I Percussion 2 min 55 497 Capitan Capitan
2
Percussion 2 min Percussion 2 min
71 65
454 477
Capitan 4 Percussion 2 min 56 496 Capitan I Percussion 4 min 86 347 Capitan 2 Percussion 4 min 79 407 Capitan 3 Percussion 4 min 81 392 Capitan 4 Percussion 4 min 82 384 Capitan I Percussion 8 min 90 300 Capitan 2 Percussion 8 min 88 325 Capitan 3 Percussion 8 min 90 300 Capitan 4 Percussion 8 min 90 300 Gila I Immersion 71 454 Gila 2 Immersion 77 421 Gila
Immersion 75 433
Gila 4 Immersion 72 449
Gila I Boiling 30 sec 68 466
Gila 2 Boiling 30 sec 81 392
Gila 3 Boiling 30 sec 54 498
Gila 4 Boiling 30 sec 63 483
Gila I Boiling 60 sec 55 497
Gila 2 Boiling 60 sec 68 466
Gila 3 Boiling 60 sec 57 495
Gila 4 Boiling 60 sec 70 458
65
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Gila 2 Control 13 336 Gila 3 Control 13 336 Gila 4 Control 20 400 Gila 1 Percussion 1 min 66 474 Gila 2 Percussion 1 min 57 495 Gila 3 Percussion 1 min 73 444 Gila 4 Percussion 1 min 82 384 Gila 1 Percussion 2 min 81 392 Gila 2 Percussion 2 min 86 347 Gila 3 Percussion 2 min 85 357 Gila 4 Percussion 2 min 54 498 Gila 1 Percussion 4 min 79 407 Gila 2 Percussion 4 min 83 376 Gila 3 Percussion 4 min 84 367 Gila 4 Percussion 4 min 79 407 Gila 1 Percussion 8 min 51 500 Gila 2 Percussion 8 min 76 427 Gila 3 Percussion 8 min 52 500 Gila 4 Percussion 8 min 74 439 Holman 1 Immersion 83 376 Holman 2 Immersion 86 347 Holman 3 Immersion 81 392 Holman 4 Immersion 90 300 Holman 1 Boiling 30 sec 90 300 Holman 2 Boiling 30 sec 85 357 Holman 3 Boiling 30 sec 87 336 Holman 4 Boiling 30 sec 83 376 Holman 1 Boiling 60 sec 79 407 Holman 2 Boiling 60 sec 80 400 Holman 3 Boiling 60 sec 86 347 Holman 4 Boiling 60 sec 93 255 Holman Control 25 433 Holman 2 Control 19 392 Holman Control 21 407
Holman 4 Control 24 427
Holman Percussion 1 min 77 421
Holman 2 Percussion 1 min 85 357
Holman 3 Percussion 1 min 80 400
Holman 4 Percussion 1 min 89 313
Holman 1 Percussion 2 min 88 325
Holman 2 Percussion 2 min 96 196
Holman 3 Percussion 2 min 93 255
Holman 4 Percussion 2 min 96 196
Holman 1 Percussion 4 min 92 271
Holman 2 Percussion 4 min 97 171
66
I
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Holman 3 Percussion 4 min 91 286 Holman 4 Percussion 4 min 96 196 Holman 1 Percussion 8 min 88 325 Holman 2 Percussion 8 min 85 357 Holman 3 Percussion 8 min 83 376 Holman 4 Percussion 8 min 82 384 Jemez 1 Immersion 53 499 Jemez 2 Immersion 44 496 Jemez 3 Immersion 52 500 Jemez 4 Immersion 70 458 Jemez 1 Boiling 30 sec 58 494 Jemez 2 Boiling 30 sec 55 497 Jemez 3 Boiling 30 sec 48 500 Jemez 4 Boiling 30 sec 65 477 Jemez 1 Boiling 60 sec 68 466 Jemez 2 Boiling 60 sec 63 483 Jemez 3 Boiling 60 sec 73 444 Jemez 4 Boiling 60 sec 89 313 Jemez 1 Control 2 140 Jemez 2 Control 9 286 Jemez 3 Control 6 237 Jemez 4 Control 8 271 Jemez 1 Percussion 1 min 58 494 Jemez 2 Percussion 1 min 63 483 Jemez 3 Percussion 1 min 77 421 Jemez 4 Percussion 1 min 89 313 Jemez 1 Percussion 2 min 68 466 Jemez 2 Percussion 2 min 64 480 Jemez 3 Percussion 2 min 80 400 Jemez 4 Percussion 2 min 83 376 Jemez Percussion 4 min 68 466 Jemez 2 Percussion 4 min 79 407 Jemez 3 Percussion 4 min 70 458 Jemez 4 Percussion 4 min 83 376 Jemez 1 Percussion 8 min 52 500 Jemez 2 Percussion 8 min 59 492 Jemez 3 Percussion 8 min 61 488 Jemez 4 Percussion 8 min 70 458 Magdalena 1 Immersion 69 462 Magdalena 2 Immersion 69 462 Magdalena J Immersion 70 458 Magdalena 4 Immersion 85 357 Magdalena 1 Boiling 30 sec 65 477 Magdalena 2 Boiling 30 sec 71 454 Magdalena 3 Boiling 30 sec 68 466
67
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Magdalena 4 Boiling 30 sec 83 376 Magdalena 1 Boiling 60 sec 63 483 Magdalena 2 Boiling 60 sec 68 466 Magdalena 3 Boiling 60 sec 63 483 Magdalena 4 Boiling 60 sec 82 384 Magdalena 1 Control 11 313 Magdalena 2 Control 6 237 Magdalena 3 Control 5 218 Magdalena 4 Control 4 196 Magdalena Percussion 1 min 32 466 Magdalena 2 Percussion 1 min 45 497 Magdalena 3 Percussion 1 min 46 498 Magdalena 4 Percussion I min 36 480 Magdalena 1 Percussion 2 min 67 470 Magdalena 2 Percussion 2 min 68 466 Magdalena 3 Percussion 2 min 65 477 Magdalena 4 Percussion 2 min 81 392 Magdalena 1 Percussion 4 min 87 336 Magdalena 2 Percussion 4 min 97 171 Magdalena 3 Percussion 4 min 83 376 Magdalena 4 Percussion 4 min 97 171 Magdalena 1 Percussion 8 min 98 lAO Magdalena 2 Percussion 8 min 100 000 Magdalena 3 Percussion 8 min 96 196 Magdalena 4 Percussion 8 min 98 lAO Manzano 1 Immersion 67 470 Manzano 2 Immersion 57 495 Manzano 3 Immersion 72 4049 Manzano 4 Immersion 73 4044 Manzano 1 Boiling 30 sec 70 458 Manzano 2 Boiling 30 sec 64 480 Manzano 3 Boiling 30 sec 71 454 Manzano 4 Boiling 30 sec 80 400 Manzano I Boiling 60 sec 50 500 Manzano 2 Boiling 60 sec 53 499 Manzano 3 Boiling 60 sec 59 492 Manzano 4 Boiling 60 sec 74 439 Manzano I Control 4 196 Manzano 2 Control 8 271 Manzano 3 Control 1 099 Manzano 4 Control 6 237 Manzano 1 Percussion 1 min 52 500 Manzano 2 Percussion 1 min 39 488 Manzano 3 Percussion 1 min 36 480 Manzano 4 Percussion 1 min 51 500
68
Table B 1 Continued
Source Block Treatment Gerrni~ation Percentage Standard Error
Manzano Percussion 2 min 53 499 Manzano 2 Percussion 2 min 58 494 Manzano 3 Percussion 2 min 61 488 Manzano 4 Percussion 2 min 62 485 Manzano 1 Percussion 4 min 76 427 Manzano 2 Percussion 4 min 85 357 Manzano 3 Percussion 4 min 79 407 Manzano 4 Percussion 4 min 83 376 Manzano 1 Percussion 8 min 87 336 Manzano 2 Percussion 8 min 83 376 Manzano 3 Percussion 8 min 83 376 Manzano 4 Percussion 8 min 75 433 Raton 1 Immersion 54 498 Raton 2 Immersion 37 483 Raton 3 Immersion 39 488 Raton 4 Immersion 69 462 Raton 1 Boiling 30 sec 36 480 Raton 2 Boiling 30 sec 20 400 Raton 3 Boiling 30 sec 28 449 Raton 4 Boiling 30 sec 60 490 Raton 1 Boiling 60 sec 15 357 Raton 2 Boiling 60 sec 171 Raton 3 Boiling 60 sec 4 196 Raton 4 Boiling 60 sec 35 477 Raton 1 Control 4 196 Raton 2 Control 12 325 Raton 3 Control 2 140 Raton 4 Control 6 237 Raton 1 Percussion 1 min 52 500 Raton 2 Percussion I min 29 454 Raton 3 Percussion I min 35 477 Raton 4 Percussion 1 min 31 462 Raton 1 Percussion 2 min 55 497 Raton 2 Percussion 2 min 35 477 Raton 3 Percussion 2 min 33 470 Raton 4 Percussion 2 min 42 494 Raton 1 Percussion 4 min 65 477 Raton 2 Percussion 4 min 52 500 Raton Percussion 4 min 51 500 Raton 4 Percussion 4 min 53 499 Raton 1 Percussion 8 min 88 325 Raton 2 Percussion 8 min 80 400 Raton 3 Percussion 8 min 73 444 Raton 4 Percussion 8 min 74 439 Sacramento 1 Immersion 43 495
69
Table B 1 Continued
-Source Block Treatment Germination Percentage Standard Error
Sacramento 2 Immersion 30 458 Sacramento 3 Immersion 38 485 Sacramento 4 Immersion 56 496 Sacramento 1 Boiling 30 sec 47 499 Sacramento 2 Boiling 30 sec 48 500 Sacramento 3 Boiling 30 sec 48 500 Sacramento 4 Boiling 30 sec 56 496 Sacramento 1 Boiling 60 sec 52 500 Sacramento 2 Boiling 60 sec 67 470 Sacramento 3 Boiling 60 sec 43 495 Sacramento 4 Boiling 60 sec 48 500 Sacramento 1 Control 0 000 Sacramento 2 Control 3 171 Sacramento 3 Control 3 171 Sacramento 4 Control 099 Sacramento 1 Percussion 1 min 57 495 Sacramento 2 Percussion 1 min 61 488 Sacramento 3 Percussion 1 min 62 485 Sacramento 4 Percussion 1 min 55 497 Sacramento 1 Percussion 2 min 84 367 Sacramento 2 Percussion 2 min 70 458 Sacramento 3 Percussion 2 min 81 392 Sacramento 4 Percussion 2 min 69 462 Sacramento 1 Percussion 4 min 91 286 Sacramento 2 Percussion 4 min 84 367 Sacramento 3 Percussion 4 min 84 367 Sacramento 4 Percussion 4 min 94 237 Sacramento 1 Percussion 8 min 82 384 Sacramento 2 Percussion 8 min 81 392 Sacramento 3 Percussion 8 min 82 384 Sacramento 4 Percussion 8 min 81 392 Sandia 1 Immersion 76 427 Sandia 2 Immersion 70 458 Sandia 3 Immersion 70 458 Sandia 4 Immersion 81 392 Sandia 1 Boiling 30 sec 78 414 Sandia 2 Boiling 30 sec 71 454 Sandia 3 Boiling 30 sec 63 483 Sandia 4 Boiling 30 sec 78 414 Sandia 1 Boiling 60 sec 67 470 Sandia 2 Boiling 60 sec 65 477 Sandia 3 Boiling 60 sec 74 439 Sandia 4 Boiling 60 sec 66 474 Sandia 1 Control 16 367 Sandia 2 Control 7 255
70
Table B 1 Continued
Source Block Treatment Gennination Percentage Standard Error
Sandia 3 Control 8 271 Sandia 4 Control 16 367 Sandia 1 Percussion 1 min 67 470 Sandia 2 Percussion 1 min 61 488 Sandia 3 Percussion 1 min 66 474 Sandia 4 Percussion 1 min 59 492 Sandia I Percussion 2 min 82 384 Sandia 2 Percussion 2 min 83 376 Sandia 3 Percussion 2 min 81 392 Sandia 4 Percussion 2 min 84 367 Sandia 1 Percussion 4 min 89 313 Sandia 2 Percussion 4 min 86 347 Sandia 3 Percussion 4 min 89 313 Sandia 4 Percussion 4 min 85 357 Sandia 1 Percussion 8 min 80 400 Sandia 2 Percussion 8 min 77 421 Sandia 3 Percussion 8 min 69 462 Sandia 4 Percussion 8 min 66 474
LITERATURE CITED
Ashby WC Vogel WG Rogers NF 1985 Black locust in the reclamation equation General Technical Report Northeastern Forest Experiment Station USDA Forest Service NE-I05 12 p
Ballard LAT 1973 Physical barriers to gennination Seed Sci Technoll 285-303
___ 1976 Strophiolar water conduction in the seeds ofTrifolieae induced by action on the testa at nonstrophiolar sites Austr J Plant Physiol3 465-469
Barton LV 1947 Special studies on seed coat impenneability Contrib Boyce Thompson Inst 14 355-362
Baskin CC Baskin JM 1998 Seeds Ecology biogeography and evolution of donnancy and gennination Academic Press London UK 666 p
Bewley JD 1997 Seed germination and donnancy Plant Ce119 1055-1066
Bewley JD Black M 1994 Seeds physiology of development and germination 2nd ed Plenum Press New York NY 445 p
Briscoe CB 1996 Growing Nitrogen Fixing Trees in Acid Soils In MH Powell (ed) Nitrogen Fixing Trees for Acid Soils- A Field ManuaL Winrock International Morrilton AR 22 p
Copeland LO McDonald MB 1995 Principles of Seed Science and Technology 3rded Chapman amp Hall New York NY 409 p
Cox RE Klett JE 1984 Seed gennination requirements of native Colorado plants for use in the landscape Plant Propag 30(2) 6-10
Dell B 1980 Structure and function ofthe strophiolar plug in seeds ofAlbizia Lophantha Am J Bot 67 556-563
Dick-Peddie WA 1993 New Mexico Vegetation Past Present and Future University ofNew Mexico Press Albuquerque NM 244 p
Dreesen DR 2001 Propagation protocol for production ofcontainer Robinia neomexicana plants Los Lunas Plant Materials Center Los Lunas New Mexico In Native Plant Network URL httpwwwnativeplantnetworkorg (accessed 9 January 2002) Moscow (ID) University ofIdaho CoIl Natural Resources For Res Nursery
72
Dreesen DR Harrington JT 1997 Propagation of native plants for restoration projects in the Southwestern US- Preliminary Investigations In Landis TD Thompson JR tech coords National Proceedings Forest and Conservation Nursery Assocs Gen Tech Rep PNW-GTR-419 Portland OR USDA For Ser PNW Res Sta 77-88
Gottfried GJ 1980 Control ofNew Mexican locust and the effect on planted ponderosa pine in central Arizona US For Ser Res Note RM-386 Rocky Mountain For Range ExpStn Fort Collins CO
Gosling PG Samuel YK Jones SK 1995 A systematic examination of germination temperature chipping and water temperaturesoak duration pretreatments on the seeds ofLeucaena leucocephala Seed Sci amp Technol 23521-532
Hamly DH 1932 Softening of the seeds of Melilotus alba Bot Gazette 93 345shy375
Hanna PJ 1984 Anatomical features of the seed coat ofAcacia kempeana (Mueller) which relate to increased germination rate induced by heat treatment New Phytol 96 23-29
Hanks JP Dick-Peddie WA 1974 Vegetation patterns of the White Mountains New Mexico Southwest Naturalist 18(4) 371-382
Hine S Harrington JT Dreesen DR 1997 Seed scarification requirements for Robinia neomexicana In Harrington JTtech coord Proc Third SouthwestemContainer Growers Conf Feb 12-13 1997 Ft Collins CO
Hopkinson JM 1993 The strophiole of leguminous seeds Newsletter- IntI Herbage Seed Production Res Grp 18 7-8
---- 1997 Differences in hardseededness between Cunningham and Tarramba leucaena In Leucnet News The Newsletter of the International LeucaenaRes and Devlt Netwk No4
ISTA (International Seed Testing Association) 1999 International rules for seed testing rules Seed Sci amp Technol27 Supplement
Kelly KM van Staden J 1987 The lens as the site of permeability in the papilionoid seed Aspalathus linearis J Plant Physiol 128(45) 395-404
73
Keresztesi B 1988 Black locust the tree of agriculture Outlook on Agr 17(2) 77~85
Kigel 1 and Galili G 1995 Seed development and germination Marcel Dekker New York 853 p
Klemmedson 10 1994 New Mexico locust and parent material Influence on macronutrients of forest floor and soil Soil Sci Soc Am 1 58 974~980
Kuo WH Tam AW 1988 The pathway of water absorption of mung bean seeds Seed Sci amp Technol16(1) 139~144
Leadem CL 1997 Dormancy--unlocking seed secrets In Landis TD Thompson lR tech coords National Proceedings Forestand Conservation Nursery Assocs Gen Tech Rep PNW-G TR-419 Portland OR USDA For Ser PNW Res Sta 43-52
Lin Y Wagner MR Seweyestewa A Honahni MD DeGomez M McMillin JD 1996 Influence of cultural treatments on seed germination and seedling growth of Robinia neomexicana Forest Farm and Community Tree Res Reports Volume 1
Marunda CT 1990 Effects of seed pretreatment on the development ofAcacia auriculforinis and A holosericea seedlings ACIAR Proceedings Series No 28
Meginnis HG 1937 Sulphuric acid treatment to increase germination of black locust seed USDA Circ 453 34 p
Miklas PN Towensend CE Ladd SL 1987 Seedcoat anatomy and the scarification ofcicer milkvetch seed Crop Sci 27 766-772
Monsen SB 1984 Use of shrubs on mine spoils In The Challenge ofProducing Native Plants for the Intermountain Area Proceedings Intermountain Nurserymans Association 1983 conference Las Vegas NV compo PM Murphy 26-31 Ogden UT USDA For Ser Gen Tech Report INT-l68
Olson DF 1974 Robinia L Locust In Seeds of Woody Plants in the US tech coord CS Schopmeyer 728-731 USDA For Ser Agr Handbk No450
Rehman S Loescher RNl Harris P1C 1998 Dormancy breaking and germination ofAcacia salicina Lindl seeds Seed Sci amp Techno127 553-557
Rolston MP 1978 Water impermeable seed dormancy Bot Rev 44 365-396
74
SAS Institute Inc 1989 SASSTAT Users Guide Version 6 4th Edition Volume 1 Cary NC SAS Institute Inc 943 p
SerratoValenti G Conara L Ferrando M Modenesi P 1993 Structural and histochemical features ofStylosanthes scabra (Leguminosae Papilionoidae) seed coat as related to water entry Can J Bot 71 834-840
Simpson BJ 1988 A field guide to Texas trees Austin TX Texas Monthly Press 372p
Tran VN Cavanagh AK 1984 Structural aspects ofdormancy In Seed Physiology Vol 2 Murray DR (ed) p1-44 Academic Press Australia
USDA Forest Service 1948 Woody-plant seed manual USDA Misc Pub1654 416p
Vines RA 1960 Trees Shrubs and Woody Vines of the Southwest Austin Univ ofTexas Press 1104 p
Wagner MR Larson RE Craig TP 1992 Ecology and silvics ofNew Mexico locust (Robinia neomexicana) with an emphasis on the arboreal form p53-57 In JW Hanover K Miller and S Plesko (eds) Proc Intemat Confon Black Locust Biology Culture and Utilization Mich State Univ USA
Wilson JK 1944 Immersing seeds of species of Robinia in boiling water hastens germination J For 42 453-454
Zimmerman RW Carpenter SB 1980 First-year coppice production from a 5shyyear old black locust stand on surface mine spoil p309-314 Centr Hardwd For ConfProc University ofMissouri Columbia
decrease at the 10-minute level This drop-off suggests that 10 minutes of percussion
over-treats seeds from this lot of-black locust
A selected percussion treatment intermediate in time (5 minutes) also had the
fastest germination rate for black locust (Figure A6)_ The hot water curve is
somewhat flatter indicating a slower germination rate The control curve is very flat
indicating a slow germination rate
A qualitative difference between hot water and percussion-treated seeds was
also observed Along with healthy germinants the hot water treatment produced
over-treated seeds (Figure A 7a) Examples of damaged germinants include seeds
where the radicle did not elongate (Figure A 7b) seeds where the radicle elongated
but was delayed in freeing itself from the seed coat (Figure A7c) and seeds where the
radicle broke off completely from the embryo (Figure A7d) In addition to these
examples of over-treatment there were numerous small dark seeds that failed to
imbibe water For percussion treatment nearly all seeds took up water (Figure A8a)
Germination was rapid and uniform with little damage evident (Figure A8b)
To summarize both New Mexico locust and black locust seed lots were
dormant The standard hot water scarification treatment improved germination but
only to 56 and 41 for New Mexico locust and black locust respectively Nearly
all percussion durations improved total germination as well as germination rate over
the hot water treatment Germination was greater than 90 for percussion durations
of 4 5 and 10 minutes for New Mexico locust and 3 4 and 5 minutes for black locust
58
CI)
sgt t CI) 0
igt 0
sect ttl t E
Vl 0
c3
100r==~==~~=r------------------------------------~ ___ Percussion 5 min
middotmiddotmiddot0middotmiddotmiddot Hot Water -lIf- Control
80r=========~--~~----------------------------~
60
40
20 -~-~---~---------- ---~------------I
omiddot
O
_~_----------lIf-----~bull -----_ shy---shy I
Day 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A6 Effect of selected scarification treatments on germination speed for black locust First seven days of fourteen-day experiment are shown
Day 7
a
b c d
Figure A7 Damage to New Mexico locust seeds observed during germination counts following hot water scarification A7a Hot water replicate including damaged seedlings and numerous unimbibed seeds A7b Close-up ofseed where radicle failed to elongate A7c Close-up ofseed where radicle elongated but was delayed in freeing itself from the seed coat A7d Close-up ofseed where radicle completely broke offfrom embryo
60
a
b
Figure A8 New Mexico locust seeds following percussion treatment at time of germination counts A8a Percussion replicate with uniformly healthy seedlings and near complete imbibition of seeds A8b Close-up ofpercussion-treated seedling
61
Discussion
Papilionoid seeds have a specific anatomical feature known as the strophiole
or lens-the natural site of water entry to the seed (Hamly 1932) The strophiole is
located on the cotyledonary lobe of a locust seed (Figure A9) When a papilionoid
legume seed is percussed for the appropriate amount of time repeated hits on the
integrated seed coat loosen the constrained cells of the strophiolar region without
excessively damaging the rest of the seed coat (Ballard 1976)
When a percussed seed is soaked water enters exclusively through the
strophiole in a controlled manner (Kelly and Staden 1987) This regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues This
contrasts with seeds that have been hot water or acid scarified treatments that can
randomly degrade the seed coat Unlocalized cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rehman et al 1998 Marunda 1990)
Papilionoids are the largest subfamily of legumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust and black locust these legumes often play an integral role in the
revegetation ofdisturbed lands Developing superior scarification methods to the
standard hot water and acid treatments should fadlitate the use ofthese legumes in
restoration projects
62
Figure A9 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (bc)
63
APPENDIXB
GERMINATION PERCENTS AND STANDARD ERRORS FOR SOURCE
BLOCK AND TREATMENT FOR NEW MEXICO LOCUST
Table B 1 Gennination percents and standard errors for source block and treatment for New Mexico locust
Source Block Treatment Germination Percentage Standard Error
Capitan 1 Immersion 74 437 Capitan 2 Immersion 69 462 Capitan 3 Immersion 71 454 Capitan 4 Immersion 67 470 Capitan Boiling 30 sec 79 407 Capitan 2 Boiling 30 sec 76 427 Capitan 3 Boiling 30 sec 72 449 Capitan 4 Boiling 30 sec 76 427 Capitan Boiling 60 sec 71 454 Capitan 2 Boiling 60 sec 54 498 Capitan 3 Boiling 60 sec 67 470 Capitan 4 Boiling 60 sec 76 427 Capitan I Control 5 218 Capitan 2 Control 7 255 Capitan 3 Control 4 196 Capitan 4 Control 5 218 Capitan 1 Percussion I min 47 499 Capitan 2 Percussion I min
470
Capitan Percussion I min 39 488
Capitan 4 Percussion I min 38 485 Capitan I Percussion 2 min 55 497 Capitan Capitan
2
Percussion 2 min Percussion 2 min
71 65
454 477
Capitan 4 Percussion 2 min 56 496 Capitan I Percussion 4 min 86 347 Capitan 2 Percussion 4 min 79 407 Capitan 3 Percussion 4 min 81 392 Capitan 4 Percussion 4 min 82 384 Capitan I Percussion 8 min 90 300 Capitan 2 Percussion 8 min 88 325 Capitan 3 Percussion 8 min 90 300 Capitan 4 Percussion 8 min 90 300 Gila I Immersion 71 454 Gila 2 Immersion 77 421 Gila
Immersion 75 433
Gila 4 Immersion 72 449
Gila I Boiling 30 sec 68 466
Gila 2 Boiling 30 sec 81 392
Gila 3 Boiling 30 sec 54 498
Gila 4 Boiling 30 sec 63 483
Gila I Boiling 60 sec 55 497
Gila 2 Boiling 60 sec 68 466
Gila 3 Boiling 60 sec 57 495
Gila 4 Boiling 60 sec 70 458
65
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Gila 2 Control 13 336 Gila 3 Control 13 336 Gila 4 Control 20 400 Gila 1 Percussion 1 min 66 474 Gila 2 Percussion 1 min 57 495 Gila 3 Percussion 1 min 73 444 Gila 4 Percussion 1 min 82 384 Gila 1 Percussion 2 min 81 392 Gila 2 Percussion 2 min 86 347 Gila 3 Percussion 2 min 85 357 Gila 4 Percussion 2 min 54 498 Gila 1 Percussion 4 min 79 407 Gila 2 Percussion 4 min 83 376 Gila 3 Percussion 4 min 84 367 Gila 4 Percussion 4 min 79 407 Gila 1 Percussion 8 min 51 500 Gila 2 Percussion 8 min 76 427 Gila 3 Percussion 8 min 52 500 Gila 4 Percussion 8 min 74 439 Holman 1 Immersion 83 376 Holman 2 Immersion 86 347 Holman 3 Immersion 81 392 Holman 4 Immersion 90 300 Holman 1 Boiling 30 sec 90 300 Holman 2 Boiling 30 sec 85 357 Holman 3 Boiling 30 sec 87 336 Holman 4 Boiling 30 sec 83 376 Holman 1 Boiling 60 sec 79 407 Holman 2 Boiling 60 sec 80 400 Holman 3 Boiling 60 sec 86 347 Holman 4 Boiling 60 sec 93 255 Holman Control 25 433 Holman 2 Control 19 392 Holman Control 21 407
Holman 4 Control 24 427
Holman Percussion 1 min 77 421
Holman 2 Percussion 1 min 85 357
Holman 3 Percussion 1 min 80 400
Holman 4 Percussion 1 min 89 313
Holman 1 Percussion 2 min 88 325
Holman 2 Percussion 2 min 96 196
Holman 3 Percussion 2 min 93 255
Holman 4 Percussion 2 min 96 196
Holman 1 Percussion 4 min 92 271
Holman 2 Percussion 4 min 97 171
66
I
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Holman 3 Percussion 4 min 91 286 Holman 4 Percussion 4 min 96 196 Holman 1 Percussion 8 min 88 325 Holman 2 Percussion 8 min 85 357 Holman 3 Percussion 8 min 83 376 Holman 4 Percussion 8 min 82 384 Jemez 1 Immersion 53 499 Jemez 2 Immersion 44 496 Jemez 3 Immersion 52 500 Jemez 4 Immersion 70 458 Jemez 1 Boiling 30 sec 58 494 Jemez 2 Boiling 30 sec 55 497 Jemez 3 Boiling 30 sec 48 500 Jemez 4 Boiling 30 sec 65 477 Jemez 1 Boiling 60 sec 68 466 Jemez 2 Boiling 60 sec 63 483 Jemez 3 Boiling 60 sec 73 444 Jemez 4 Boiling 60 sec 89 313 Jemez 1 Control 2 140 Jemez 2 Control 9 286 Jemez 3 Control 6 237 Jemez 4 Control 8 271 Jemez 1 Percussion 1 min 58 494 Jemez 2 Percussion 1 min 63 483 Jemez 3 Percussion 1 min 77 421 Jemez 4 Percussion 1 min 89 313 Jemez 1 Percussion 2 min 68 466 Jemez 2 Percussion 2 min 64 480 Jemez 3 Percussion 2 min 80 400 Jemez 4 Percussion 2 min 83 376 Jemez Percussion 4 min 68 466 Jemez 2 Percussion 4 min 79 407 Jemez 3 Percussion 4 min 70 458 Jemez 4 Percussion 4 min 83 376 Jemez 1 Percussion 8 min 52 500 Jemez 2 Percussion 8 min 59 492 Jemez 3 Percussion 8 min 61 488 Jemez 4 Percussion 8 min 70 458 Magdalena 1 Immersion 69 462 Magdalena 2 Immersion 69 462 Magdalena J Immersion 70 458 Magdalena 4 Immersion 85 357 Magdalena 1 Boiling 30 sec 65 477 Magdalena 2 Boiling 30 sec 71 454 Magdalena 3 Boiling 30 sec 68 466
67
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Magdalena 4 Boiling 30 sec 83 376 Magdalena 1 Boiling 60 sec 63 483 Magdalena 2 Boiling 60 sec 68 466 Magdalena 3 Boiling 60 sec 63 483 Magdalena 4 Boiling 60 sec 82 384 Magdalena 1 Control 11 313 Magdalena 2 Control 6 237 Magdalena 3 Control 5 218 Magdalena 4 Control 4 196 Magdalena Percussion 1 min 32 466 Magdalena 2 Percussion 1 min 45 497 Magdalena 3 Percussion 1 min 46 498 Magdalena 4 Percussion I min 36 480 Magdalena 1 Percussion 2 min 67 470 Magdalena 2 Percussion 2 min 68 466 Magdalena 3 Percussion 2 min 65 477 Magdalena 4 Percussion 2 min 81 392 Magdalena 1 Percussion 4 min 87 336 Magdalena 2 Percussion 4 min 97 171 Magdalena 3 Percussion 4 min 83 376 Magdalena 4 Percussion 4 min 97 171 Magdalena 1 Percussion 8 min 98 lAO Magdalena 2 Percussion 8 min 100 000 Magdalena 3 Percussion 8 min 96 196 Magdalena 4 Percussion 8 min 98 lAO Manzano 1 Immersion 67 470 Manzano 2 Immersion 57 495 Manzano 3 Immersion 72 4049 Manzano 4 Immersion 73 4044 Manzano 1 Boiling 30 sec 70 458 Manzano 2 Boiling 30 sec 64 480 Manzano 3 Boiling 30 sec 71 454 Manzano 4 Boiling 30 sec 80 400 Manzano I Boiling 60 sec 50 500 Manzano 2 Boiling 60 sec 53 499 Manzano 3 Boiling 60 sec 59 492 Manzano 4 Boiling 60 sec 74 439 Manzano I Control 4 196 Manzano 2 Control 8 271 Manzano 3 Control 1 099 Manzano 4 Control 6 237 Manzano 1 Percussion 1 min 52 500 Manzano 2 Percussion 1 min 39 488 Manzano 3 Percussion 1 min 36 480 Manzano 4 Percussion 1 min 51 500
68
Table B 1 Continued
Source Block Treatment Gerrni~ation Percentage Standard Error
Manzano Percussion 2 min 53 499 Manzano 2 Percussion 2 min 58 494 Manzano 3 Percussion 2 min 61 488 Manzano 4 Percussion 2 min 62 485 Manzano 1 Percussion 4 min 76 427 Manzano 2 Percussion 4 min 85 357 Manzano 3 Percussion 4 min 79 407 Manzano 4 Percussion 4 min 83 376 Manzano 1 Percussion 8 min 87 336 Manzano 2 Percussion 8 min 83 376 Manzano 3 Percussion 8 min 83 376 Manzano 4 Percussion 8 min 75 433 Raton 1 Immersion 54 498 Raton 2 Immersion 37 483 Raton 3 Immersion 39 488 Raton 4 Immersion 69 462 Raton 1 Boiling 30 sec 36 480 Raton 2 Boiling 30 sec 20 400 Raton 3 Boiling 30 sec 28 449 Raton 4 Boiling 30 sec 60 490 Raton 1 Boiling 60 sec 15 357 Raton 2 Boiling 60 sec 171 Raton 3 Boiling 60 sec 4 196 Raton 4 Boiling 60 sec 35 477 Raton 1 Control 4 196 Raton 2 Control 12 325 Raton 3 Control 2 140 Raton 4 Control 6 237 Raton 1 Percussion 1 min 52 500 Raton 2 Percussion I min 29 454 Raton 3 Percussion I min 35 477 Raton 4 Percussion 1 min 31 462 Raton 1 Percussion 2 min 55 497 Raton 2 Percussion 2 min 35 477 Raton 3 Percussion 2 min 33 470 Raton 4 Percussion 2 min 42 494 Raton 1 Percussion 4 min 65 477 Raton 2 Percussion 4 min 52 500 Raton Percussion 4 min 51 500 Raton 4 Percussion 4 min 53 499 Raton 1 Percussion 8 min 88 325 Raton 2 Percussion 8 min 80 400 Raton 3 Percussion 8 min 73 444 Raton 4 Percussion 8 min 74 439 Sacramento 1 Immersion 43 495
69
Table B 1 Continued
-Source Block Treatment Germination Percentage Standard Error
Sacramento 2 Immersion 30 458 Sacramento 3 Immersion 38 485 Sacramento 4 Immersion 56 496 Sacramento 1 Boiling 30 sec 47 499 Sacramento 2 Boiling 30 sec 48 500 Sacramento 3 Boiling 30 sec 48 500 Sacramento 4 Boiling 30 sec 56 496 Sacramento 1 Boiling 60 sec 52 500 Sacramento 2 Boiling 60 sec 67 470 Sacramento 3 Boiling 60 sec 43 495 Sacramento 4 Boiling 60 sec 48 500 Sacramento 1 Control 0 000 Sacramento 2 Control 3 171 Sacramento 3 Control 3 171 Sacramento 4 Control 099 Sacramento 1 Percussion 1 min 57 495 Sacramento 2 Percussion 1 min 61 488 Sacramento 3 Percussion 1 min 62 485 Sacramento 4 Percussion 1 min 55 497 Sacramento 1 Percussion 2 min 84 367 Sacramento 2 Percussion 2 min 70 458 Sacramento 3 Percussion 2 min 81 392 Sacramento 4 Percussion 2 min 69 462 Sacramento 1 Percussion 4 min 91 286 Sacramento 2 Percussion 4 min 84 367 Sacramento 3 Percussion 4 min 84 367 Sacramento 4 Percussion 4 min 94 237 Sacramento 1 Percussion 8 min 82 384 Sacramento 2 Percussion 8 min 81 392 Sacramento 3 Percussion 8 min 82 384 Sacramento 4 Percussion 8 min 81 392 Sandia 1 Immersion 76 427 Sandia 2 Immersion 70 458 Sandia 3 Immersion 70 458 Sandia 4 Immersion 81 392 Sandia 1 Boiling 30 sec 78 414 Sandia 2 Boiling 30 sec 71 454 Sandia 3 Boiling 30 sec 63 483 Sandia 4 Boiling 30 sec 78 414 Sandia 1 Boiling 60 sec 67 470 Sandia 2 Boiling 60 sec 65 477 Sandia 3 Boiling 60 sec 74 439 Sandia 4 Boiling 60 sec 66 474 Sandia 1 Control 16 367 Sandia 2 Control 7 255
70
Table B 1 Continued
Source Block Treatment Gennination Percentage Standard Error
Sandia 3 Control 8 271 Sandia 4 Control 16 367 Sandia 1 Percussion 1 min 67 470 Sandia 2 Percussion 1 min 61 488 Sandia 3 Percussion 1 min 66 474 Sandia 4 Percussion 1 min 59 492 Sandia I Percussion 2 min 82 384 Sandia 2 Percussion 2 min 83 376 Sandia 3 Percussion 2 min 81 392 Sandia 4 Percussion 2 min 84 367 Sandia 1 Percussion 4 min 89 313 Sandia 2 Percussion 4 min 86 347 Sandia 3 Percussion 4 min 89 313 Sandia 4 Percussion 4 min 85 357 Sandia 1 Percussion 8 min 80 400 Sandia 2 Percussion 8 min 77 421 Sandia 3 Percussion 8 min 69 462 Sandia 4 Percussion 8 min 66 474
LITERATURE CITED
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Ballard LAT 1973 Physical barriers to gennination Seed Sci Technoll 285-303
___ 1976 Strophiolar water conduction in the seeds ofTrifolieae induced by action on the testa at nonstrophiolar sites Austr J Plant Physiol3 465-469
Barton LV 1947 Special studies on seed coat impenneability Contrib Boyce Thompson Inst 14 355-362
Baskin CC Baskin JM 1998 Seeds Ecology biogeography and evolution of donnancy and gennination Academic Press London UK 666 p
Bewley JD 1997 Seed germination and donnancy Plant Ce119 1055-1066
Bewley JD Black M 1994 Seeds physiology of development and germination 2nd ed Plenum Press New York NY 445 p
Briscoe CB 1996 Growing Nitrogen Fixing Trees in Acid Soils In MH Powell (ed) Nitrogen Fixing Trees for Acid Soils- A Field ManuaL Winrock International Morrilton AR 22 p
Copeland LO McDonald MB 1995 Principles of Seed Science and Technology 3rded Chapman amp Hall New York NY 409 p
Cox RE Klett JE 1984 Seed gennination requirements of native Colorado plants for use in the landscape Plant Propag 30(2) 6-10
Dell B 1980 Structure and function ofthe strophiolar plug in seeds ofAlbizia Lophantha Am J Bot 67 556-563
Dick-Peddie WA 1993 New Mexico Vegetation Past Present and Future University ofNew Mexico Press Albuquerque NM 244 p
Dreesen DR 2001 Propagation protocol for production ofcontainer Robinia neomexicana plants Los Lunas Plant Materials Center Los Lunas New Mexico In Native Plant Network URL httpwwwnativeplantnetworkorg (accessed 9 January 2002) Moscow (ID) University ofIdaho CoIl Natural Resources For Res Nursery
72
Dreesen DR Harrington JT 1997 Propagation of native plants for restoration projects in the Southwestern US- Preliminary Investigations In Landis TD Thompson JR tech coords National Proceedings Forest and Conservation Nursery Assocs Gen Tech Rep PNW-GTR-419 Portland OR USDA For Ser PNW Res Sta 77-88
Gottfried GJ 1980 Control ofNew Mexican locust and the effect on planted ponderosa pine in central Arizona US For Ser Res Note RM-386 Rocky Mountain For Range ExpStn Fort Collins CO
Gosling PG Samuel YK Jones SK 1995 A systematic examination of germination temperature chipping and water temperaturesoak duration pretreatments on the seeds ofLeucaena leucocephala Seed Sci amp Technol 23521-532
Hamly DH 1932 Softening of the seeds of Melilotus alba Bot Gazette 93 345shy375
Hanna PJ 1984 Anatomical features of the seed coat ofAcacia kempeana (Mueller) which relate to increased germination rate induced by heat treatment New Phytol 96 23-29
Hanks JP Dick-Peddie WA 1974 Vegetation patterns of the White Mountains New Mexico Southwest Naturalist 18(4) 371-382
Hine S Harrington JT Dreesen DR 1997 Seed scarification requirements for Robinia neomexicana In Harrington JTtech coord Proc Third SouthwestemContainer Growers Conf Feb 12-13 1997 Ft Collins CO
Hopkinson JM 1993 The strophiole of leguminous seeds Newsletter- IntI Herbage Seed Production Res Grp 18 7-8
---- 1997 Differences in hardseededness between Cunningham and Tarramba leucaena In Leucnet News The Newsletter of the International LeucaenaRes and Devlt Netwk No4
ISTA (International Seed Testing Association) 1999 International rules for seed testing rules Seed Sci amp Technol27 Supplement
Kelly KM van Staden J 1987 The lens as the site of permeability in the papilionoid seed Aspalathus linearis J Plant Physiol 128(45) 395-404
73
Keresztesi B 1988 Black locust the tree of agriculture Outlook on Agr 17(2) 77~85
Kigel 1 and Galili G 1995 Seed development and germination Marcel Dekker New York 853 p
Klemmedson 10 1994 New Mexico locust and parent material Influence on macronutrients of forest floor and soil Soil Sci Soc Am 1 58 974~980
Kuo WH Tam AW 1988 The pathway of water absorption of mung bean seeds Seed Sci amp Technol16(1) 139~144
Leadem CL 1997 Dormancy--unlocking seed secrets In Landis TD Thompson lR tech coords National Proceedings Forestand Conservation Nursery Assocs Gen Tech Rep PNW-G TR-419 Portland OR USDA For Ser PNW Res Sta 43-52
Lin Y Wagner MR Seweyestewa A Honahni MD DeGomez M McMillin JD 1996 Influence of cultural treatments on seed germination and seedling growth of Robinia neomexicana Forest Farm and Community Tree Res Reports Volume 1
Marunda CT 1990 Effects of seed pretreatment on the development ofAcacia auriculforinis and A holosericea seedlings ACIAR Proceedings Series No 28
Meginnis HG 1937 Sulphuric acid treatment to increase germination of black locust seed USDA Circ 453 34 p
Miklas PN Towensend CE Ladd SL 1987 Seedcoat anatomy and the scarification ofcicer milkvetch seed Crop Sci 27 766-772
Monsen SB 1984 Use of shrubs on mine spoils In The Challenge ofProducing Native Plants for the Intermountain Area Proceedings Intermountain Nurserymans Association 1983 conference Las Vegas NV compo PM Murphy 26-31 Ogden UT USDA For Ser Gen Tech Report INT-l68
Olson DF 1974 Robinia L Locust In Seeds of Woody Plants in the US tech coord CS Schopmeyer 728-731 USDA For Ser Agr Handbk No450
Rehman S Loescher RNl Harris P1C 1998 Dormancy breaking and germination ofAcacia salicina Lindl seeds Seed Sci amp Techno127 553-557
Rolston MP 1978 Water impermeable seed dormancy Bot Rev 44 365-396
74
SAS Institute Inc 1989 SASSTAT Users Guide Version 6 4th Edition Volume 1 Cary NC SAS Institute Inc 943 p
SerratoValenti G Conara L Ferrando M Modenesi P 1993 Structural and histochemical features ofStylosanthes scabra (Leguminosae Papilionoidae) seed coat as related to water entry Can J Bot 71 834-840
Simpson BJ 1988 A field guide to Texas trees Austin TX Texas Monthly Press 372p
Tran VN Cavanagh AK 1984 Structural aspects ofdormancy In Seed Physiology Vol 2 Murray DR (ed) p1-44 Academic Press Australia
USDA Forest Service 1948 Woody-plant seed manual USDA Misc Pub1654 416p
Vines RA 1960 Trees Shrubs and Woody Vines of the Southwest Austin Univ ofTexas Press 1104 p
Wagner MR Larson RE Craig TP 1992 Ecology and silvics ofNew Mexico locust (Robinia neomexicana) with an emphasis on the arboreal form p53-57 In JW Hanover K Miller and S Plesko (eds) Proc Intemat Confon Black Locust Biology Culture and Utilization Mich State Univ USA
Wilson JK 1944 Immersing seeds of species of Robinia in boiling water hastens germination J For 42 453-454
Zimmerman RW Carpenter SB 1980 First-year coppice production from a 5shyyear old black locust stand on surface mine spoil p309-314 Centr Hardwd For ConfProc University ofMissouri Columbia
CI)
sgt t CI) 0
igt 0
sect ttl t E
Vl 0
c3
100r==~==~~=r------------------------------------~ ___ Percussion 5 min
middotmiddotmiddot0middotmiddotmiddot Hot Water -lIf- Control
80r=========~--~~----------------------------~
60
40
20 -~-~---~---------- ---~------------I
omiddot
O
_~_----------lIf-----~bull -----_ shy---shy I
Day 0 Day I Day 2 Day 3 Day 4 Day 5 Day 6
Time
Figure A6 Effect of selected scarification treatments on germination speed for black locust First seven days of fourteen-day experiment are shown
Day 7
a
b c d
Figure A7 Damage to New Mexico locust seeds observed during germination counts following hot water scarification A7a Hot water replicate including damaged seedlings and numerous unimbibed seeds A7b Close-up ofseed where radicle failed to elongate A7c Close-up ofseed where radicle elongated but was delayed in freeing itself from the seed coat A7d Close-up ofseed where radicle completely broke offfrom embryo
60
a
b
Figure A8 New Mexico locust seeds following percussion treatment at time of germination counts A8a Percussion replicate with uniformly healthy seedlings and near complete imbibition of seeds A8b Close-up ofpercussion-treated seedling
61
Discussion
Papilionoid seeds have a specific anatomical feature known as the strophiole
or lens-the natural site of water entry to the seed (Hamly 1932) The strophiole is
located on the cotyledonary lobe of a locust seed (Figure A9) When a papilionoid
legume seed is percussed for the appropriate amount of time repeated hits on the
integrated seed coat loosen the constrained cells of the strophiolar region without
excessively damaging the rest of the seed coat (Ballard 1976)
When a percussed seed is soaked water enters exclusively through the
strophiole in a controlled manner (Kelly and Staden 1987) This regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues This
contrasts with seeds that have been hot water or acid scarified treatments that can
randomly degrade the seed coat Unlocalized cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rehman et al 1998 Marunda 1990)
Papilionoids are the largest subfamily of legumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust and black locust these legumes often play an integral role in the
revegetation ofdisturbed lands Developing superior scarification methods to the
standard hot water and acid treatments should fadlitate the use ofthese legumes in
restoration projects
62
Figure A9 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (bc)
63
APPENDIXB
GERMINATION PERCENTS AND STANDARD ERRORS FOR SOURCE
BLOCK AND TREATMENT FOR NEW MEXICO LOCUST
Table B 1 Gennination percents and standard errors for source block and treatment for New Mexico locust
Source Block Treatment Germination Percentage Standard Error
Capitan 1 Immersion 74 437 Capitan 2 Immersion 69 462 Capitan 3 Immersion 71 454 Capitan 4 Immersion 67 470 Capitan Boiling 30 sec 79 407 Capitan 2 Boiling 30 sec 76 427 Capitan 3 Boiling 30 sec 72 449 Capitan 4 Boiling 30 sec 76 427 Capitan Boiling 60 sec 71 454 Capitan 2 Boiling 60 sec 54 498 Capitan 3 Boiling 60 sec 67 470 Capitan 4 Boiling 60 sec 76 427 Capitan I Control 5 218 Capitan 2 Control 7 255 Capitan 3 Control 4 196 Capitan 4 Control 5 218 Capitan 1 Percussion I min 47 499 Capitan 2 Percussion I min
470
Capitan Percussion I min 39 488
Capitan 4 Percussion I min 38 485 Capitan I Percussion 2 min 55 497 Capitan Capitan
2
Percussion 2 min Percussion 2 min
71 65
454 477
Capitan 4 Percussion 2 min 56 496 Capitan I Percussion 4 min 86 347 Capitan 2 Percussion 4 min 79 407 Capitan 3 Percussion 4 min 81 392 Capitan 4 Percussion 4 min 82 384 Capitan I Percussion 8 min 90 300 Capitan 2 Percussion 8 min 88 325 Capitan 3 Percussion 8 min 90 300 Capitan 4 Percussion 8 min 90 300 Gila I Immersion 71 454 Gila 2 Immersion 77 421 Gila
Immersion 75 433
Gila 4 Immersion 72 449
Gila I Boiling 30 sec 68 466
Gila 2 Boiling 30 sec 81 392
Gila 3 Boiling 30 sec 54 498
Gila 4 Boiling 30 sec 63 483
Gila I Boiling 60 sec 55 497
Gila 2 Boiling 60 sec 68 466
Gila 3 Boiling 60 sec 57 495
Gila 4 Boiling 60 sec 70 458
65
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Gila 2 Control 13 336 Gila 3 Control 13 336 Gila 4 Control 20 400 Gila 1 Percussion 1 min 66 474 Gila 2 Percussion 1 min 57 495 Gila 3 Percussion 1 min 73 444 Gila 4 Percussion 1 min 82 384 Gila 1 Percussion 2 min 81 392 Gila 2 Percussion 2 min 86 347 Gila 3 Percussion 2 min 85 357 Gila 4 Percussion 2 min 54 498 Gila 1 Percussion 4 min 79 407 Gila 2 Percussion 4 min 83 376 Gila 3 Percussion 4 min 84 367 Gila 4 Percussion 4 min 79 407 Gila 1 Percussion 8 min 51 500 Gila 2 Percussion 8 min 76 427 Gila 3 Percussion 8 min 52 500 Gila 4 Percussion 8 min 74 439 Holman 1 Immersion 83 376 Holman 2 Immersion 86 347 Holman 3 Immersion 81 392 Holman 4 Immersion 90 300 Holman 1 Boiling 30 sec 90 300 Holman 2 Boiling 30 sec 85 357 Holman 3 Boiling 30 sec 87 336 Holman 4 Boiling 30 sec 83 376 Holman 1 Boiling 60 sec 79 407 Holman 2 Boiling 60 sec 80 400 Holman 3 Boiling 60 sec 86 347 Holman 4 Boiling 60 sec 93 255 Holman Control 25 433 Holman 2 Control 19 392 Holman Control 21 407
Holman 4 Control 24 427
Holman Percussion 1 min 77 421
Holman 2 Percussion 1 min 85 357
Holman 3 Percussion 1 min 80 400
Holman 4 Percussion 1 min 89 313
Holman 1 Percussion 2 min 88 325
Holman 2 Percussion 2 min 96 196
Holman 3 Percussion 2 min 93 255
Holman 4 Percussion 2 min 96 196
Holman 1 Percussion 4 min 92 271
Holman 2 Percussion 4 min 97 171
66
I
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Holman 3 Percussion 4 min 91 286 Holman 4 Percussion 4 min 96 196 Holman 1 Percussion 8 min 88 325 Holman 2 Percussion 8 min 85 357 Holman 3 Percussion 8 min 83 376 Holman 4 Percussion 8 min 82 384 Jemez 1 Immersion 53 499 Jemez 2 Immersion 44 496 Jemez 3 Immersion 52 500 Jemez 4 Immersion 70 458 Jemez 1 Boiling 30 sec 58 494 Jemez 2 Boiling 30 sec 55 497 Jemez 3 Boiling 30 sec 48 500 Jemez 4 Boiling 30 sec 65 477 Jemez 1 Boiling 60 sec 68 466 Jemez 2 Boiling 60 sec 63 483 Jemez 3 Boiling 60 sec 73 444 Jemez 4 Boiling 60 sec 89 313 Jemez 1 Control 2 140 Jemez 2 Control 9 286 Jemez 3 Control 6 237 Jemez 4 Control 8 271 Jemez 1 Percussion 1 min 58 494 Jemez 2 Percussion 1 min 63 483 Jemez 3 Percussion 1 min 77 421 Jemez 4 Percussion 1 min 89 313 Jemez 1 Percussion 2 min 68 466 Jemez 2 Percussion 2 min 64 480 Jemez 3 Percussion 2 min 80 400 Jemez 4 Percussion 2 min 83 376 Jemez Percussion 4 min 68 466 Jemez 2 Percussion 4 min 79 407 Jemez 3 Percussion 4 min 70 458 Jemez 4 Percussion 4 min 83 376 Jemez 1 Percussion 8 min 52 500 Jemez 2 Percussion 8 min 59 492 Jemez 3 Percussion 8 min 61 488 Jemez 4 Percussion 8 min 70 458 Magdalena 1 Immersion 69 462 Magdalena 2 Immersion 69 462 Magdalena J Immersion 70 458 Magdalena 4 Immersion 85 357 Magdalena 1 Boiling 30 sec 65 477 Magdalena 2 Boiling 30 sec 71 454 Magdalena 3 Boiling 30 sec 68 466
67
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Magdalena 4 Boiling 30 sec 83 376 Magdalena 1 Boiling 60 sec 63 483 Magdalena 2 Boiling 60 sec 68 466 Magdalena 3 Boiling 60 sec 63 483 Magdalena 4 Boiling 60 sec 82 384 Magdalena 1 Control 11 313 Magdalena 2 Control 6 237 Magdalena 3 Control 5 218 Magdalena 4 Control 4 196 Magdalena Percussion 1 min 32 466 Magdalena 2 Percussion 1 min 45 497 Magdalena 3 Percussion 1 min 46 498 Magdalena 4 Percussion I min 36 480 Magdalena 1 Percussion 2 min 67 470 Magdalena 2 Percussion 2 min 68 466 Magdalena 3 Percussion 2 min 65 477 Magdalena 4 Percussion 2 min 81 392 Magdalena 1 Percussion 4 min 87 336 Magdalena 2 Percussion 4 min 97 171 Magdalena 3 Percussion 4 min 83 376 Magdalena 4 Percussion 4 min 97 171 Magdalena 1 Percussion 8 min 98 lAO Magdalena 2 Percussion 8 min 100 000 Magdalena 3 Percussion 8 min 96 196 Magdalena 4 Percussion 8 min 98 lAO Manzano 1 Immersion 67 470 Manzano 2 Immersion 57 495 Manzano 3 Immersion 72 4049 Manzano 4 Immersion 73 4044 Manzano 1 Boiling 30 sec 70 458 Manzano 2 Boiling 30 sec 64 480 Manzano 3 Boiling 30 sec 71 454 Manzano 4 Boiling 30 sec 80 400 Manzano I Boiling 60 sec 50 500 Manzano 2 Boiling 60 sec 53 499 Manzano 3 Boiling 60 sec 59 492 Manzano 4 Boiling 60 sec 74 439 Manzano I Control 4 196 Manzano 2 Control 8 271 Manzano 3 Control 1 099 Manzano 4 Control 6 237 Manzano 1 Percussion 1 min 52 500 Manzano 2 Percussion 1 min 39 488 Manzano 3 Percussion 1 min 36 480 Manzano 4 Percussion 1 min 51 500
68
Table B 1 Continued
Source Block Treatment Gerrni~ation Percentage Standard Error
Manzano Percussion 2 min 53 499 Manzano 2 Percussion 2 min 58 494 Manzano 3 Percussion 2 min 61 488 Manzano 4 Percussion 2 min 62 485 Manzano 1 Percussion 4 min 76 427 Manzano 2 Percussion 4 min 85 357 Manzano 3 Percussion 4 min 79 407 Manzano 4 Percussion 4 min 83 376 Manzano 1 Percussion 8 min 87 336 Manzano 2 Percussion 8 min 83 376 Manzano 3 Percussion 8 min 83 376 Manzano 4 Percussion 8 min 75 433 Raton 1 Immersion 54 498 Raton 2 Immersion 37 483 Raton 3 Immersion 39 488 Raton 4 Immersion 69 462 Raton 1 Boiling 30 sec 36 480 Raton 2 Boiling 30 sec 20 400 Raton 3 Boiling 30 sec 28 449 Raton 4 Boiling 30 sec 60 490 Raton 1 Boiling 60 sec 15 357 Raton 2 Boiling 60 sec 171 Raton 3 Boiling 60 sec 4 196 Raton 4 Boiling 60 sec 35 477 Raton 1 Control 4 196 Raton 2 Control 12 325 Raton 3 Control 2 140 Raton 4 Control 6 237 Raton 1 Percussion 1 min 52 500 Raton 2 Percussion I min 29 454 Raton 3 Percussion I min 35 477 Raton 4 Percussion 1 min 31 462 Raton 1 Percussion 2 min 55 497 Raton 2 Percussion 2 min 35 477 Raton 3 Percussion 2 min 33 470 Raton 4 Percussion 2 min 42 494 Raton 1 Percussion 4 min 65 477 Raton 2 Percussion 4 min 52 500 Raton Percussion 4 min 51 500 Raton 4 Percussion 4 min 53 499 Raton 1 Percussion 8 min 88 325 Raton 2 Percussion 8 min 80 400 Raton 3 Percussion 8 min 73 444 Raton 4 Percussion 8 min 74 439 Sacramento 1 Immersion 43 495
69
Table B 1 Continued
-Source Block Treatment Germination Percentage Standard Error
Sacramento 2 Immersion 30 458 Sacramento 3 Immersion 38 485 Sacramento 4 Immersion 56 496 Sacramento 1 Boiling 30 sec 47 499 Sacramento 2 Boiling 30 sec 48 500 Sacramento 3 Boiling 30 sec 48 500 Sacramento 4 Boiling 30 sec 56 496 Sacramento 1 Boiling 60 sec 52 500 Sacramento 2 Boiling 60 sec 67 470 Sacramento 3 Boiling 60 sec 43 495 Sacramento 4 Boiling 60 sec 48 500 Sacramento 1 Control 0 000 Sacramento 2 Control 3 171 Sacramento 3 Control 3 171 Sacramento 4 Control 099 Sacramento 1 Percussion 1 min 57 495 Sacramento 2 Percussion 1 min 61 488 Sacramento 3 Percussion 1 min 62 485 Sacramento 4 Percussion 1 min 55 497 Sacramento 1 Percussion 2 min 84 367 Sacramento 2 Percussion 2 min 70 458 Sacramento 3 Percussion 2 min 81 392 Sacramento 4 Percussion 2 min 69 462 Sacramento 1 Percussion 4 min 91 286 Sacramento 2 Percussion 4 min 84 367 Sacramento 3 Percussion 4 min 84 367 Sacramento 4 Percussion 4 min 94 237 Sacramento 1 Percussion 8 min 82 384 Sacramento 2 Percussion 8 min 81 392 Sacramento 3 Percussion 8 min 82 384 Sacramento 4 Percussion 8 min 81 392 Sandia 1 Immersion 76 427 Sandia 2 Immersion 70 458 Sandia 3 Immersion 70 458 Sandia 4 Immersion 81 392 Sandia 1 Boiling 30 sec 78 414 Sandia 2 Boiling 30 sec 71 454 Sandia 3 Boiling 30 sec 63 483 Sandia 4 Boiling 30 sec 78 414 Sandia 1 Boiling 60 sec 67 470 Sandia 2 Boiling 60 sec 65 477 Sandia 3 Boiling 60 sec 74 439 Sandia 4 Boiling 60 sec 66 474 Sandia 1 Control 16 367 Sandia 2 Control 7 255
70
Table B 1 Continued
Source Block Treatment Gennination Percentage Standard Error
Sandia 3 Control 8 271 Sandia 4 Control 16 367 Sandia 1 Percussion 1 min 67 470 Sandia 2 Percussion 1 min 61 488 Sandia 3 Percussion 1 min 66 474 Sandia 4 Percussion 1 min 59 492 Sandia I Percussion 2 min 82 384 Sandia 2 Percussion 2 min 83 376 Sandia 3 Percussion 2 min 81 392 Sandia 4 Percussion 2 min 84 367 Sandia 1 Percussion 4 min 89 313 Sandia 2 Percussion 4 min 86 347 Sandia 3 Percussion 4 min 89 313 Sandia 4 Percussion 4 min 85 357 Sandia 1 Percussion 8 min 80 400 Sandia 2 Percussion 8 min 77 421 Sandia 3 Percussion 8 min 69 462 Sandia 4 Percussion 8 min 66 474
LITERATURE CITED
Ashby WC Vogel WG Rogers NF 1985 Black locust in the reclamation equation General Technical Report Northeastern Forest Experiment Station USDA Forest Service NE-I05 12 p
Ballard LAT 1973 Physical barriers to gennination Seed Sci Technoll 285-303
___ 1976 Strophiolar water conduction in the seeds ofTrifolieae induced by action on the testa at nonstrophiolar sites Austr J Plant Physiol3 465-469
Barton LV 1947 Special studies on seed coat impenneability Contrib Boyce Thompson Inst 14 355-362
Baskin CC Baskin JM 1998 Seeds Ecology biogeography and evolution of donnancy and gennination Academic Press London UK 666 p
Bewley JD 1997 Seed germination and donnancy Plant Ce119 1055-1066
Bewley JD Black M 1994 Seeds physiology of development and germination 2nd ed Plenum Press New York NY 445 p
Briscoe CB 1996 Growing Nitrogen Fixing Trees in Acid Soils In MH Powell (ed) Nitrogen Fixing Trees for Acid Soils- A Field ManuaL Winrock International Morrilton AR 22 p
Copeland LO McDonald MB 1995 Principles of Seed Science and Technology 3rded Chapman amp Hall New York NY 409 p
Cox RE Klett JE 1984 Seed gennination requirements of native Colorado plants for use in the landscape Plant Propag 30(2) 6-10
Dell B 1980 Structure and function ofthe strophiolar plug in seeds ofAlbizia Lophantha Am J Bot 67 556-563
Dick-Peddie WA 1993 New Mexico Vegetation Past Present and Future University ofNew Mexico Press Albuquerque NM 244 p
Dreesen DR 2001 Propagation protocol for production ofcontainer Robinia neomexicana plants Los Lunas Plant Materials Center Los Lunas New Mexico In Native Plant Network URL httpwwwnativeplantnetworkorg (accessed 9 January 2002) Moscow (ID) University ofIdaho CoIl Natural Resources For Res Nursery
72
Dreesen DR Harrington JT 1997 Propagation of native plants for restoration projects in the Southwestern US- Preliminary Investigations In Landis TD Thompson JR tech coords National Proceedings Forest and Conservation Nursery Assocs Gen Tech Rep PNW-GTR-419 Portland OR USDA For Ser PNW Res Sta 77-88
Gottfried GJ 1980 Control ofNew Mexican locust and the effect on planted ponderosa pine in central Arizona US For Ser Res Note RM-386 Rocky Mountain For Range ExpStn Fort Collins CO
Gosling PG Samuel YK Jones SK 1995 A systematic examination of germination temperature chipping and water temperaturesoak duration pretreatments on the seeds ofLeucaena leucocephala Seed Sci amp Technol 23521-532
Hamly DH 1932 Softening of the seeds of Melilotus alba Bot Gazette 93 345shy375
Hanna PJ 1984 Anatomical features of the seed coat ofAcacia kempeana (Mueller) which relate to increased germination rate induced by heat treatment New Phytol 96 23-29
Hanks JP Dick-Peddie WA 1974 Vegetation patterns of the White Mountains New Mexico Southwest Naturalist 18(4) 371-382
Hine S Harrington JT Dreesen DR 1997 Seed scarification requirements for Robinia neomexicana In Harrington JTtech coord Proc Third SouthwestemContainer Growers Conf Feb 12-13 1997 Ft Collins CO
Hopkinson JM 1993 The strophiole of leguminous seeds Newsletter- IntI Herbage Seed Production Res Grp 18 7-8
---- 1997 Differences in hardseededness between Cunningham and Tarramba leucaena In Leucnet News The Newsletter of the International LeucaenaRes and Devlt Netwk No4
ISTA (International Seed Testing Association) 1999 International rules for seed testing rules Seed Sci amp Technol27 Supplement
Kelly KM van Staden J 1987 The lens as the site of permeability in the papilionoid seed Aspalathus linearis J Plant Physiol 128(45) 395-404
73
Keresztesi B 1988 Black locust the tree of agriculture Outlook on Agr 17(2) 77~85
Kigel 1 and Galili G 1995 Seed development and germination Marcel Dekker New York 853 p
Klemmedson 10 1994 New Mexico locust and parent material Influence on macronutrients of forest floor and soil Soil Sci Soc Am 1 58 974~980
Kuo WH Tam AW 1988 The pathway of water absorption of mung bean seeds Seed Sci amp Technol16(1) 139~144
Leadem CL 1997 Dormancy--unlocking seed secrets In Landis TD Thompson lR tech coords National Proceedings Forestand Conservation Nursery Assocs Gen Tech Rep PNW-G TR-419 Portland OR USDA For Ser PNW Res Sta 43-52
Lin Y Wagner MR Seweyestewa A Honahni MD DeGomez M McMillin JD 1996 Influence of cultural treatments on seed germination and seedling growth of Robinia neomexicana Forest Farm and Community Tree Res Reports Volume 1
Marunda CT 1990 Effects of seed pretreatment on the development ofAcacia auriculforinis and A holosericea seedlings ACIAR Proceedings Series No 28
Meginnis HG 1937 Sulphuric acid treatment to increase germination of black locust seed USDA Circ 453 34 p
Miklas PN Towensend CE Ladd SL 1987 Seedcoat anatomy and the scarification ofcicer milkvetch seed Crop Sci 27 766-772
Monsen SB 1984 Use of shrubs on mine spoils In The Challenge ofProducing Native Plants for the Intermountain Area Proceedings Intermountain Nurserymans Association 1983 conference Las Vegas NV compo PM Murphy 26-31 Ogden UT USDA For Ser Gen Tech Report INT-l68
Olson DF 1974 Robinia L Locust In Seeds of Woody Plants in the US tech coord CS Schopmeyer 728-731 USDA For Ser Agr Handbk No450
Rehman S Loescher RNl Harris P1C 1998 Dormancy breaking and germination ofAcacia salicina Lindl seeds Seed Sci amp Techno127 553-557
Rolston MP 1978 Water impermeable seed dormancy Bot Rev 44 365-396
74
SAS Institute Inc 1989 SASSTAT Users Guide Version 6 4th Edition Volume 1 Cary NC SAS Institute Inc 943 p
SerratoValenti G Conara L Ferrando M Modenesi P 1993 Structural and histochemical features ofStylosanthes scabra (Leguminosae Papilionoidae) seed coat as related to water entry Can J Bot 71 834-840
Simpson BJ 1988 A field guide to Texas trees Austin TX Texas Monthly Press 372p
Tran VN Cavanagh AK 1984 Structural aspects ofdormancy In Seed Physiology Vol 2 Murray DR (ed) p1-44 Academic Press Australia
USDA Forest Service 1948 Woody-plant seed manual USDA Misc Pub1654 416p
Vines RA 1960 Trees Shrubs and Woody Vines of the Southwest Austin Univ ofTexas Press 1104 p
Wagner MR Larson RE Craig TP 1992 Ecology and silvics ofNew Mexico locust (Robinia neomexicana) with an emphasis on the arboreal form p53-57 In JW Hanover K Miller and S Plesko (eds) Proc Intemat Confon Black Locust Biology Culture and Utilization Mich State Univ USA
Wilson JK 1944 Immersing seeds of species of Robinia in boiling water hastens germination J For 42 453-454
Zimmerman RW Carpenter SB 1980 First-year coppice production from a 5shyyear old black locust stand on surface mine spoil p309-314 Centr Hardwd For ConfProc University ofMissouri Columbia
a
b c d
Figure A7 Damage to New Mexico locust seeds observed during germination counts following hot water scarification A7a Hot water replicate including damaged seedlings and numerous unimbibed seeds A7b Close-up ofseed where radicle failed to elongate A7c Close-up ofseed where radicle elongated but was delayed in freeing itself from the seed coat A7d Close-up ofseed where radicle completely broke offfrom embryo
60
a
b
Figure A8 New Mexico locust seeds following percussion treatment at time of germination counts A8a Percussion replicate with uniformly healthy seedlings and near complete imbibition of seeds A8b Close-up ofpercussion-treated seedling
61
Discussion
Papilionoid seeds have a specific anatomical feature known as the strophiole
or lens-the natural site of water entry to the seed (Hamly 1932) The strophiole is
located on the cotyledonary lobe of a locust seed (Figure A9) When a papilionoid
legume seed is percussed for the appropriate amount of time repeated hits on the
integrated seed coat loosen the constrained cells of the strophiolar region without
excessively damaging the rest of the seed coat (Ballard 1976)
When a percussed seed is soaked water enters exclusively through the
strophiole in a controlled manner (Kelly and Staden 1987) This regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues This
contrasts with seeds that have been hot water or acid scarified treatments that can
randomly degrade the seed coat Unlocalized cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rehman et al 1998 Marunda 1990)
Papilionoids are the largest subfamily of legumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust and black locust these legumes often play an integral role in the
revegetation ofdisturbed lands Developing superior scarification methods to the
standard hot water and acid treatments should fadlitate the use ofthese legumes in
restoration projects
62
Figure A9 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (bc)
63
APPENDIXB
GERMINATION PERCENTS AND STANDARD ERRORS FOR SOURCE
BLOCK AND TREATMENT FOR NEW MEXICO LOCUST
Table B 1 Gennination percents and standard errors for source block and treatment for New Mexico locust
Source Block Treatment Germination Percentage Standard Error
Capitan 1 Immersion 74 437 Capitan 2 Immersion 69 462 Capitan 3 Immersion 71 454 Capitan 4 Immersion 67 470 Capitan Boiling 30 sec 79 407 Capitan 2 Boiling 30 sec 76 427 Capitan 3 Boiling 30 sec 72 449 Capitan 4 Boiling 30 sec 76 427 Capitan Boiling 60 sec 71 454 Capitan 2 Boiling 60 sec 54 498 Capitan 3 Boiling 60 sec 67 470 Capitan 4 Boiling 60 sec 76 427 Capitan I Control 5 218 Capitan 2 Control 7 255 Capitan 3 Control 4 196 Capitan 4 Control 5 218 Capitan 1 Percussion I min 47 499 Capitan 2 Percussion I min
470
Capitan Percussion I min 39 488
Capitan 4 Percussion I min 38 485 Capitan I Percussion 2 min 55 497 Capitan Capitan
2
Percussion 2 min Percussion 2 min
71 65
454 477
Capitan 4 Percussion 2 min 56 496 Capitan I Percussion 4 min 86 347 Capitan 2 Percussion 4 min 79 407 Capitan 3 Percussion 4 min 81 392 Capitan 4 Percussion 4 min 82 384 Capitan I Percussion 8 min 90 300 Capitan 2 Percussion 8 min 88 325 Capitan 3 Percussion 8 min 90 300 Capitan 4 Percussion 8 min 90 300 Gila I Immersion 71 454 Gila 2 Immersion 77 421 Gila
Immersion 75 433
Gila 4 Immersion 72 449
Gila I Boiling 30 sec 68 466
Gila 2 Boiling 30 sec 81 392
Gila 3 Boiling 30 sec 54 498
Gila 4 Boiling 30 sec 63 483
Gila I Boiling 60 sec 55 497
Gila 2 Boiling 60 sec 68 466
Gila 3 Boiling 60 sec 57 495
Gila 4 Boiling 60 sec 70 458
65
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Gila 2 Control 13 336 Gila 3 Control 13 336 Gila 4 Control 20 400 Gila 1 Percussion 1 min 66 474 Gila 2 Percussion 1 min 57 495 Gila 3 Percussion 1 min 73 444 Gila 4 Percussion 1 min 82 384 Gila 1 Percussion 2 min 81 392 Gila 2 Percussion 2 min 86 347 Gila 3 Percussion 2 min 85 357 Gila 4 Percussion 2 min 54 498 Gila 1 Percussion 4 min 79 407 Gila 2 Percussion 4 min 83 376 Gila 3 Percussion 4 min 84 367 Gila 4 Percussion 4 min 79 407 Gila 1 Percussion 8 min 51 500 Gila 2 Percussion 8 min 76 427 Gila 3 Percussion 8 min 52 500 Gila 4 Percussion 8 min 74 439 Holman 1 Immersion 83 376 Holman 2 Immersion 86 347 Holman 3 Immersion 81 392 Holman 4 Immersion 90 300 Holman 1 Boiling 30 sec 90 300 Holman 2 Boiling 30 sec 85 357 Holman 3 Boiling 30 sec 87 336 Holman 4 Boiling 30 sec 83 376 Holman 1 Boiling 60 sec 79 407 Holman 2 Boiling 60 sec 80 400 Holman 3 Boiling 60 sec 86 347 Holman 4 Boiling 60 sec 93 255 Holman Control 25 433 Holman 2 Control 19 392 Holman Control 21 407
Holman 4 Control 24 427
Holman Percussion 1 min 77 421
Holman 2 Percussion 1 min 85 357
Holman 3 Percussion 1 min 80 400
Holman 4 Percussion 1 min 89 313
Holman 1 Percussion 2 min 88 325
Holman 2 Percussion 2 min 96 196
Holman 3 Percussion 2 min 93 255
Holman 4 Percussion 2 min 96 196
Holman 1 Percussion 4 min 92 271
Holman 2 Percussion 4 min 97 171
66
I
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Holman 3 Percussion 4 min 91 286 Holman 4 Percussion 4 min 96 196 Holman 1 Percussion 8 min 88 325 Holman 2 Percussion 8 min 85 357 Holman 3 Percussion 8 min 83 376 Holman 4 Percussion 8 min 82 384 Jemez 1 Immersion 53 499 Jemez 2 Immersion 44 496 Jemez 3 Immersion 52 500 Jemez 4 Immersion 70 458 Jemez 1 Boiling 30 sec 58 494 Jemez 2 Boiling 30 sec 55 497 Jemez 3 Boiling 30 sec 48 500 Jemez 4 Boiling 30 sec 65 477 Jemez 1 Boiling 60 sec 68 466 Jemez 2 Boiling 60 sec 63 483 Jemez 3 Boiling 60 sec 73 444 Jemez 4 Boiling 60 sec 89 313 Jemez 1 Control 2 140 Jemez 2 Control 9 286 Jemez 3 Control 6 237 Jemez 4 Control 8 271 Jemez 1 Percussion 1 min 58 494 Jemez 2 Percussion 1 min 63 483 Jemez 3 Percussion 1 min 77 421 Jemez 4 Percussion 1 min 89 313 Jemez 1 Percussion 2 min 68 466 Jemez 2 Percussion 2 min 64 480 Jemez 3 Percussion 2 min 80 400 Jemez 4 Percussion 2 min 83 376 Jemez Percussion 4 min 68 466 Jemez 2 Percussion 4 min 79 407 Jemez 3 Percussion 4 min 70 458 Jemez 4 Percussion 4 min 83 376 Jemez 1 Percussion 8 min 52 500 Jemez 2 Percussion 8 min 59 492 Jemez 3 Percussion 8 min 61 488 Jemez 4 Percussion 8 min 70 458 Magdalena 1 Immersion 69 462 Magdalena 2 Immersion 69 462 Magdalena J Immersion 70 458 Magdalena 4 Immersion 85 357 Magdalena 1 Boiling 30 sec 65 477 Magdalena 2 Boiling 30 sec 71 454 Magdalena 3 Boiling 30 sec 68 466
67
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Magdalena 4 Boiling 30 sec 83 376 Magdalena 1 Boiling 60 sec 63 483 Magdalena 2 Boiling 60 sec 68 466 Magdalena 3 Boiling 60 sec 63 483 Magdalena 4 Boiling 60 sec 82 384 Magdalena 1 Control 11 313 Magdalena 2 Control 6 237 Magdalena 3 Control 5 218 Magdalena 4 Control 4 196 Magdalena Percussion 1 min 32 466 Magdalena 2 Percussion 1 min 45 497 Magdalena 3 Percussion 1 min 46 498 Magdalena 4 Percussion I min 36 480 Magdalena 1 Percussion 2 min 67 470 Magdalena 2 Percussion 2 min 68 466 Magdalena 3 Percussion 2 min 65 477 Magdalena 4 Percussion 2 min 81 392 Magdalena 1 Percussion 4 min 87 336 Magdalena 2 Percussion 4 min 97 171 Magdalena 3 Percussion 4 min 83 376 Magdalena 4 Percussion 4 min 97 171 Magdalena 1 Percussion 8 min 98 lAO Magdalena 2 Percussion 8 min 100 000 Magdalena 3 Percussion 8 min 96 196 Magdalena 4 Percussion 8 min 98 lAO Manzano 1 Immersion 67 470 Manzano 2 Immersion 57 495 Manzano 3 Immersion 72 4049 Manzano 4 Immersion 73 4044 Manzano 1 Boiling 30 sec 70 458 Manzano 2 Boiling 30 sec 64 480 Manzano 3 Boiling 30 sec 71 454 Manzano 4 Boiling 30 sec 80 400 Manzano I Boiling 60 sec 50 500 Manzano 2 Boiling 60 sec 53 499 Manzano 3 Boiling 60 sec 59 492 Manzano 4 Boiling 60 sec 74 439 Manzano I Control 4 196 Manzano 2 Control 8 271 Manzano 3 Control 1 099 Manzano 4 Control 6 237 Manzano 1 Percussion 1 min 52 500 Manzano 2 Percussion 1 min 39 488 Manzano 3 Percussion 1 min 36 480 Manzano 4 Percussion 1 min 51 500
68
Table B 1 Continued
Source Block Treatment Gerrni~ation Percentage Standard Error
Manzano Percussion 2 min 53 499 Manzano 2 Percussion 2 min 58 494 Manzano 3 Percussion 2 min 61 488 Manzano 4 Percussion 2 min 62 485 Manzano 1 Percussion 4 min 76 427 Manzano 2 Percussion 4 min 85 357 Manzano 3 Percussion 4 min 79 407 Manzano 4 Percussion 4 min 83 376 Manzano 1 Percussion 8 min 87 336 Manzano 2 Percussion 8 min 83 376 Manzano 3 Percussion 8 min 83 376 Manzano 4 Percussion 8 min 75 433 Raton 1 Immersion 54 498 Raton 2 Immersion 37 483 Raton 3 Immersion 39 488 Raton 4 Immersion 69 462 Raton 1 Boiling 30 sec 36 480 Raton 2 Boiling 30 sec 20 400 Raton 3 Boiling 30 sec 28 449 Raton 4 Boiling 30 sec 60 490 Raton 1 Boiling 60 sec 15 357 Raton 2 Boiling 60 sec 171 Raton 3 Boiling 60 sec 4 196 Raton 4 Boiling 60 sec 35 477 Raton 1 Control 4 196 Raton 2 Control 12 325 Raton 3 Control 2 140 Raton 4 Control 6 237 Raton 1 Percussion 1 min 52 500 Raton 2 Percussion I min 29 454 Raton 3 Percussion I min 35 477 Raton 4 Percussion 1 min 31 462 Raton 1 Percussion 2 min 55 497 Raton 2 Percussion 2 min 35 477 Raton 3 Percussion 2 min 33 470 Raton 4 Percussion 2 min 42 494 Raton 1 Percussion 4 min 65 477 Raton 2 Percussion 4 min 52 500 Raton Percussion 4 min 51 500 Raton 4 Percussion 4 min 53 499 Raton 1 Percussion 8 min 88 325 Raton 2 Percussion 8 min 80 400 Raton 3 Percussion 8 min 73 444 Raton 4 Percussion 8 min 74 439 Sacramento 1 Immersion 43 495
69
Table B 1 Continued
-Source Block Treatment Germination Percentage Standard Error
Sacramento 2 Immersion 30 458 Sacramento 3 Immersion 38 485 Sacramento 4 Immersion 56 496 Sacramento 1 Boiling 30 sec 47 499 Sacramento 2 Boiling 30 sec 48 500 Sacramento 3 Boiling 30 sec 48 500 Sacramento 4 Boiling 30 sec 56 496 Sacramento 1 Boiling 60 sec 52 500 Sacramento 2 Boiling 60 sec 67 470 Sacramento 3 Boiling 60 sec 43 495 Sacramento 4 Boiling 60 sec 48 500 Sacramento 1 Control 0 000 Sacramento 2 Control 3 171 Sacramento 3 Control 3 171 Sacramento 4 Control 099 Sacramento 1 Percussion 1 min 57 495 Sacramento 2 Percussion 1 min 61 488 Sacramento 3 Percussion 1 min 62 485 Sacramento 4 Percussion 1 min 55 497 Sacramento 1 Percussion 2 min 84 367 Sacramento 2 Percussion 2 min 70 458 Sacramento 3 Percussion 2 min 81 392 Sacramento 4 Percussion 2 min 69 462 Sacramento 1 Percussion 4 min 91 286 Sacramento 2 Percussion 4 min 84 367 Sacramento 3 Percussion 4 min 84 367 Sacramento 4 Percussion 4 min 94 237 Sacramento 1 Percussion 8 min 82 384 Sacramento 2 Percussion 8 min 81 392 Sacramento 3 Percussion 8 min 82 384 Sacramento 4 Percussion 8 min 81 392 Sandia 1 Immersion 76 427 Sandia 2 Immersion 70 458 Sandia 3 Immersion 70 458 Sandia 4 Immersion 81 392 Sandia 1 Boiling 30 sec 78 414 Sandia 2 Boiling 30 sec 71 454 Sandia 3 Boiling 30 sec 63 483 Sandia 4 Boiling 30 sec 78 414 Sandia 1 Boiling 60 sec 67 470 Sandia 2 Boiling 60 sec 65 477 Sandia 3 Boiling 60 sec 74 439 Sandia 4 Boiling 60 sec 66 474 Sandia 1 Control 16 367 Sandia 2 Control 7 255
70
Table B 1 Continued
Source Block Treatment Gennination Percentage Standard Error
Sandia 3 Control 8 271 Sandia 4 Control 16 367 Sandia 1 Percussion 1 min 67 470 Sandia 2 Percussion 1 min 61 488 Sandia 3 Percussion 1 min 66 474 Sandia 4 Percussion 1 min 59 492 Sandia I Percussion 2 min 82 384 Sandia 2 Percussion 2 min 83 376 Sandia 3 Percussion 2 min 81 392 Sandia 4 Percussion 2 min 84 367 Sandia 1 Percussion 4 min 89 313 Sandia 2 Percussion 4 min 86 347 Sandia 3 Percussion 4 min 89 313 Sandia 4 Percussion 4 min 85 357 Sandia 1 Percussion 8 min 80 400 Sandia 2 Percussion 8 min 77 421 Sandia 3 Percussion 8 min 69 462 Sandia 4 Percussion 8 min 66 474
LITERATURE CITED
Ashby WC Vogel WG Rogers NF 1985 Black locust in the reclamation equation General Technical Report Northeastern Forest Experiment Station USDA Forest Service NE-I05 12 p
Ballard LAT 1973 Physical barriers to gennination Seed Sci Technoll 285-303
___ 1976 Strophiolar water conduction in the seeds ofTrifolieae induced by action on the testa at nonstrophiolar sites Austr J Plant Physiol3 465-469
Barton LV 1947 Special studies on seed coat impenneability Contrib Boyce Thompson Inst 14 355-362
Baskin CC Baskin JM 1998 Seeds Ecology biogeography and evolution of donnancy and gennination Academic Press London UK 666 p
Bewley JD 1997 Seed germination and donnancy Plant Ce119 1055-1066
Bewley JD Black M 1994 Seeds physiology of development and germination 2nd ed Plenum Press New York NY 445 p
Briscoe CB 1996 Growing Nitrogen Fixing Trees in Acid Soils In MH Powell (ed) Nitrogen Fixing Trees for Acid Soils- A Field ManuaL Winrock International Morrilton AR 22 p
Copeland LO McDonald MB 1995 Principles of Seed Science and Technology 3rded Chapman amp Hall New York NY 409 p
Cox RE Klett JE 1984 Seed gennination requirements of native Colorado plants for use in the landscape Plant Propag 30(2) 6-10
Dell B 1980 Structure and function ofthe strophiolar plug in seeds ofAlbizia Lophantha Am J Bot 67 556-563
Dick-Peddie WA 1993 New Mexico Vegetation Past Present and Future University ofNew Mexico Press Albuquerque NM 244 p
Dreesen DR 2001 Propagation protocol for production ofcontainer Robinia neomexicana plants Los Lunas Plant Materials Center Los Lunas New Mexico In Native Plant Network URL httpwwwnativeplantnetworkorg (accessed 9 January 2002) Moscow (ID) University ofIdaho CoIl Natural Resources For Res Nursery
72
Dreesen DR Harrington JT 1997 Propagation of native plants for restoration projects in the Southwestern US- Preliminary Investigations In Landis TD Thompson JR tech coords National Proceedings Forest and Conservation Nursery Assocs Gen Tech Rep PNW-GTR-419 Portland OR USDA For Ser PNW Res Sta 77-88
Gottfried GJ 1980 Control ofNew Mexican locust and the effect on planted ponderosa pine in central Arizona US For Ser Res Note RM-386 Rocky Mountain For Range ExpStn Fort Collins CO
Gosling PG Samuel YK Jones SK 1995 A systematic examination of germination temperature chipping and water temperaturesoak duration pretreatments on the seeds ofLeucaena leucocephala Seed Sci amp Technol 23521-532
Hamly DH 1932 Softening of the seeds of Melilotus alba Bot Gazette 93 345shy375
Hanna PJ 1984 Anatomical features of the seed coat ofAcacia kempeana (Mueller) which relate to increased germination rate induced by heat treatment New Phytol 96 23-29
Hanks JP Dick-Peddie WA 1974 Vegetation patterns of the White Mountains New Mexico Southwest Naturalist 18(4) 371-382
Hine S Harrington JT Dreesen DR 1997 Seed scarification requirements for Robinia neomexicana In Harrington JTtech coord Proc Third SouthwestemContainer Growers Conf Feb 12-13 1997 Ft Collins CO
Hopkinson JM 1993 The strophiole of leguminous seeds Newsletter- IntI Herbage Seed Production Res Grp 18 7-8
---- 1997 Differences in hardseededness between Cunningham and Tarramba leucaena In Leucnet News The Newsletter of the International LeucaenaRes and Devlt Netwk No4
ISTA (International Seed Testing Association) 1999 International rules for seed testing rules Seed Sci amp Technol27 Supplement
Kelly KM van Staden J 1987 The lens as the site of permeability in the papilionoid seed Aspalathus linearis J Plant Physiol 128(45) 395-404
73
Keresztesi B 1988 Black locust the tree of agriculture Outlook on Agr 17(2) 77~85
Kigel 1 and Galili G 1995 Seed development and germination Marcel Dekker New York 853 p
Klemmedson 10 1994 New Mexico locust and parent material Influence on macronutrients of forest floor and soil Soil Sci Soc Am 1 58 974~980
Kuo WH Tam AW 1988 The pathway of water absorption of mung bean seeds Seed Sci amp Technol16(1) 139~144
Leadem CL 1997 Dormancy--unlocking seed secrets In Landis TD Thompson lR tech coords National Proceedings Forestand Conservation Nursery Assocs Gen Tech Rep PNW-G TR-419 Portland OR USDA For Ser PNW Res Sta 43-52
Lin Y Wagner MR Seweyestewa A Honahni MD DeGomez M McMillin JD 1996 Influence of cultural treatments on seed germination and seedling growth of Robinia neomexicana Forest Farm and Community Tree Res Reports Volume 1
Marunda CT 1990 Effects of seed pretreatment on the development ofAcacia auriculforinis and A holosericea seedlings ACIAR Proceedings Series No 28
Meginnis HG 1937 Sulphuric acid treatment to increase germination of black locust seed USDA Circ 453 34 p
Miklas PN Towensend CE Ladd SL 1987 Seedcoat anatomy and the scarification ofcicer milkvetch seed Crop Sci 27 766-772
Monsen SB 1984 Use of shrubs on mine spoils In The Challenge ofProducing Native Plants for the Intermountain Area Proceedings Intermountain Nurserymans Association 1983 conference Las Vegas NV compo PM Murphy 26-31 Ogden UT USDA For Ser Gen Tech Report INT-l68
Olson DF 1974 Robinia L Locust In Seeds of Woody Plants in the US tech coord CS Schopmeyer 728-731 USDA For Ser Agr Handbk No450
Rehman S Loescher RNl Harris P1C 1998 Dormancy breaking and germination ofAcacia salicina Lindl seeds Seed Sci amp Techno127 553-557
Rolston MP 1978 Water impermeable seed dormancy Bot Rev 44 365-396
74
SAS Institute Inc 1989 SASSTAT Users Guide Version 6 4th Edition Volume 1 Cary NC SAS Institute Inc 943 p
SerratoValenti G Conara L Ferrando M Modenesi P 1993 Structural and histochemical features ofStylosanthes scabra (Leguminosae Papilionoidae) seed coat as related to water entry Can J Bot 71 834-840
Simpson BJ 1988 A field guide to Texas trees Austin TX Texas Monthly Press 372p
Tran VN Cavanagh AK 1984 Structural aspects ofdormancy In Seed Physiology Vol 2 Murray DR (ed) p1-44 Academic Press Australia
USDA Forest Service 1948 Woody-plant seed manual USDA Misc Pub1654 416p
Vines RA 1960 Trees Shrubs and Woody Vines of the Southwest Austin Univ ofTexas Press 1104 p
Wagner MR Larson RE Craig TP 1992 Ecology and silvics ofNew Mexico locust (Robinia neomexicana) with an emphasis on the arboreal form p53-57 In JW Hanover K Miller and S Plesko (eds) Proc Intemat Confon Black Locust Biology Culture and Utilization Mich State Univ USA
Wilson JK 1944 Immersing seeds of species of Robinia in boiling water hastens germination J For 42 453-454
Zimmerman RW Carpenter SB 1980 First-year coppice production from a 5shyyear old black locust stand on surface mine spoil p309-314 Centr Hardwd For ConfProc University ofMissouri Columbia
a
b
Figure A8 New Mexico locust seeds following percussion treatment at time of germination counts A8a Percussion replicate with uniformly healthy seedlings and near complete imbibition of seeds A8b Close-up ofpercussion-treated seedling
61
Discussion
Papilionoid seeds have a specific anatomical feature known as the strophiole
or lens-the natural site of water entry to the seed (Hamly 1932) The strophiole is
located on the cotyledonary lobe of a locust seed (Figure A9) When a papilionoid
legume seed is percussed for the appropriate amount of time repeated hits on the
integrated seed coat loosen the constrained cells of the strophiolar region without
excessively damaging the rest of the seed coat (Ballard 1976)
When a percussed seed is soaked water enters exclusively through the
strophiole in a controlled manner (Kelly and Staden 1987) This regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues This
contrasts with seeds that have been hot water or acid scarified treatments that can
randomly degrade the seed coat Unlocalized cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rehman et al 1998 Marunda 1990)
Papilionoids are the largest subfamily of legumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust and black locust these legumes often play an integral role in the
revegetation ofdisturbed lands Developing superior scarification methods to the
standard hot water and acid treatments should fadlitate the use ofthese legumes in
restoration projects
62
Figure A9 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (bc)
63
APPENDIXB
GERMINATION PERCENTS AND STANDARD ERRORS FOR SOURCE
BLOCK AND TREATMENT FOR NEW MEXICO LOCUST
Table B 1 Gennination percents and standard errors for source block and treatment for New Mexico locust
Source Block Treatment Germination Percentage Standard Error
Capitan 1 Immersion 74 437 Capitan 2 Immersion 69 462 Capitan 3 Immersion 71 454 Capitan 4 Immersion 67 470 Capitan Boiling 30 sec 79 407 Capitan 2 Boiling 30 sec 76 427 Capitan 3 Boiling 30 sec 72 449 Capitan 4 Boiling 30 sec 76 427 Capitan Boiling 60 sec 71 454 Capitan 2 Boiling 60 sec 54 498 Capitan 3 Boiling 60 sec 67 470 Capitan 4 Boiling 60 sec 76 427 Capitan I Control 5 218 Capitan 2 Control 7 255 Capitan 3 Control 4 196 Capitan 4 Control 5 218 Capitan 1 Percussion I min 47 499 Capitan 2 Percussion I min
470
Capitan Percussion I min 39 488
Capitan 4 Percussion I min 38 485 Capitan I Percussion 2 min 55 497 Capitan Capitan
2
Percussion 2 min Percussion 2 min
71 65
454 477
Capitan 4 Percussion 2 min 56 496 Capitan I Percussion 4 min 86 347 Capitan 2 Percussion 4 min 79 407 Capitan 3 Percussion 4 min 81 392 Capitan 4 Percussion 4 min 82 384 Capitan I Percussion 8 min 90 300 Capitan 2 Percussion 8 min 88 325 Capitan 3 Percussion 8 min 90 300 Capitan 4 Percussion 8 min 90 300 Gila I Immersion 71 454 Gila 2 Immersion 77 421 Gila
Immersion 75 433
Gila 4 Immersion 72 449
Gila I Boiling 30 sec 68 466
Gila 2 Boiling 30 sec 81 392
Gila 3 Boiling 30 sec 54 498
Gila 4 Boiling 30 sec 63 483
Gila I Boiling 60 sec 55 497
Gila 2 Boiling 60 sec 68 466
Gila 3 Boiling 60 sec 57 495
Gila 4 Boiling 60 sec 70 458
65
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Gila 2 Control 13 336 Gila 3 Control 13 336 Gila 4 Control 20 400 Gila 1 Percussion 1 min 66 474 Gila 2 Percussion 1 min 57 495 Gila 3 Percussion 1 min 73 444 Gila 4 Percussion 1 min 82 384 Gila 1 Percussion 2 min 81 392 Gila 2 Percussion 2 min 86 347 Gila 3 Percussion 2 min 85 357 Gila 4 Percussion 2 min 54 498 Gila 1 Percussion 4 min 79 407 Gila 2 Percussion 4 min 83 376 Gila 3 Percussion 4 min 84 367 Gila 4 Percussion 4 min 79 407 Gila 1 Percussion 8 min 51 500 Gila 2 Percussion 8 min 76 427 Gila 3 Percussion 8 min 52 500 Gila 4 Percussion 8 min 74 439 Holman 1 Immersion 83 376 Holman 2 Immersion 86 347 Holman 3 Immersion 81 392 Holman 4 Immersion 90 300 Holman 1 Boiling 30 sec 90 300 Holman 2 Boiling 30 sec 85 357 Holman 3 Boiling 30 sec 87 336 Holman 4 Boiling 30 sec 83 376 Holman 1 Boiling 60 sec 79 407 Holman 2 Boiling 60 sec 80 400 Holman 3 Boiling 60 sec 86 347 Holman 4 Boiling 60 sec 93 255 Holman Control 25 433 Holman 2 Control 19 392 Holman Control 21 407
Holman 4 Control 24 427
Holman Percussion 1 min 77 421
Holman 2 Percussion 1 min 85 357
Holman 3 Percussion 1 min 80 400
Holman 4 Percussion 1 min 89 313
Holman 1 Percussion 2 min 88 325
Holman 2 Percussion 2 min 96 196
Holman 3 Percussion 2 min 93 255
Holman 4 Percussion 2 min 96 196
Holman 1 Percussion 4 min 92 271
Holman 2 Percussion 4 min 97 171
66
I
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Holman 3 Percussion 4 min 91 286 Holman 4 Percussion 4 min 96 196 Holman 1 Percussion 8 min 88 325 Holman 2 Percussion 8 min 85 357 Holman 3 Percussion 8 min 83 376 Holman 4 Percussion 8 min 82 384 Jemez 1 Immersion 53 499 Jemez 2 Immersion 44 496 Jemez 3 Immersion 52 500 Jemez 4 Immersion 70 458 Jemez 1 Boiling 30 sec 58 494 Jemez 2 Boiling 30 sec 55 497 Jemez 3 Boiling 30 sec 48 500 Jemez 4 Boiling 30 sec 65 477 Jemez 1 Boiling 60 sec 68 466 Jemez 2 Boiling 60 sec 63 483 Jemez 3 Boiling 60 sec 73 444 Jemez 4 Boiling 60 sec 89 313 Jemez 1 Control 2 140 Jemez 2 Control 9 286 Jemez 3 Control 6 237 Jemez 4 Control 8 271 Jemez 1 Percussion 1 min 58 494 Jemez 2 Percussion 1 min 63 483 Jemez 3 Percussion 1 min 77 421 Jemez 4 Percussion 1 min 89 313 Jemez 1 Percussion 2 min 68 466 Jemez 2 Percussion 2 min 64 480 Jemez 3 Percussion 2 min 80 400 Jemez 4 Percussion 2 min 83 376 Jemez Percussion 4 min 68 466 Jemez 2 Percussion 4 min 79 407 Jemez 3 Percussion 4 min 70 458 Jemez 4 Percussion 4 min 83 376 Jemez 1 Percussion 8 min 52 500 Jemez 2 Percussion 8 min 59 492 Jemez 3 Percussion 8 min 61 488 Jemez 4 Percussion 8 min 70 458 Magdalena 1 Immersion 69 462 Magdalena 2 Immersion 69 462 Magdalena J Immersion 70 458 Magdalena 4 Immersion 85 357 Magdalena 1 Boiling 30 sec 65 477 Magdalena 2 Boiling 30 sec 71 454 Magdalena 3 Boiling 30 sec 68 466
67
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Magdalena 4 Boiling 30 sec 83 376 Magdalena 1 Boiling 60 sec 63 483 Magdalena 2 Boiling 60 sec 68 466 Magdalena 3 Boiling 60 sec 63 483 Magdalena 4 Boiling 60 sec 82 384 Magdalena 1 Control 11 313 Magdalena 2 Control 6 237 Magdalena 3 Control 5 218 Magdalena 4 Control 4 196 Magdalena Percussion 1 min 32 466 Magdalena 2 Percussion 1 min 45 497 Magdalena 3 Percussion 1 min 46 498 Magdalena 4 Percussion I min 36 480 Magdalena 1 Percussion 2 min 67 470 Magdalena 2 Percussion 2 min 68 466 Magdalena 3 Percussion 2 min 65 477 Magdalena 4 Percussion 2 min 81 392 Magdalena 1 Percussion 4 min 87 336 Magdalena 2 Percussion 4 min 97 171 Magdalena 3 Percussion 4 min 83 376 Magdalena 4 Percussion 4 min 97 171 Magdalena 1 Percussion 8 min 98 lAO Magdalena 2 Percussion 8 min 100 000 Magdalena 3 Percussion 8 min 96 196 Magdalena 4 Percussion 8 min 98 lAO Manzano 1 Immersion 67 470 Manzano 2 Immersion 57 495 Manzano 3 Immersion 72 4049 Manzano 4 Immersion 73 4044 Manzano 1 Boiling 30 sec 70 458 Manzano 2 Boiling 30 sec 64 480 Manzano 3 Boiling 30 sec 71 454 Manzano 4 Boiling 30 sec 80 400 Manzano I Boiling 60 sec 50 500 Manzano 2 Boiling 60 sec 53 499 Manzano 3 Boiling 60 sec 59 492 Manzano 4 Boiling 60 sec 74 439 Manzano I Control 4 196 Manzano 2 Control 8 271 Manzano 3 Control 1 099 Manzano 4 Control 6 237 Manzano 1 Percussion 1 min 52 500 Manzano 2 Percussion 1 min 39 488 Manzano 3 Percussion 1 min 36 480 Manzano 4 Percussion 1 min 51 500
68
Table B 1 Continued
Source Block Treatment Gerrni~ation Percentage Standard Error
Manzano Percussion 2 min 53 499 Manzano 2 Percussion 2 min 58 494 Manzano 3 Percussion 2 min 61 488 Manzano 4 Percussion 2 min 62 485 Manzano 1 Percussion 4 min 76 427 Manzano 2 Percussion 4 min 85 357 Manzano 3 Percussion 4 min 79 407 Manzano 4 Percussion 4 min 83 376 Manzano 1 Percussion 8 min 87 336 Manzano 2 Percussion 8 min 83 376 Manzano 3 Percussion 8 min 83 376 Manzano 4 Percussion 8 min 75 433 Raton 1 Immersion 54 498 Raton 2 Immersion 37 483 Raton 3 Immersion 39 488 Raton 4 Immersion 69 462 Raton 1 Boiling 30 sec 36 480 Raton 2 Boiling 30 sec 20 400 Raton 3 Boiling 30 sec 28 449 Raton 4 Boiling 30 sec 60 490 Raton 1 Boiling 60 sec 15 357 Raton 2 Boiling 60 sec 171 Raton 3 Boiling 60 sec 4 196 Raton 4 Boiling 60 sec 35 477 Raton 1 Control 4 196 Raton 2 Control 12 325 Raton 3 Control 2 140 Raton 4 Control 6 237 Raton 1 Percussion 1 min 52 500 Raton 2 Percussion I min 29 454 Raton 3 Percussion I min 35 477 Raton 4 Percussion 1 min 31 462 Raton 1 Percussion 2 min 55 497 Raton 2 Percussion 2 min 35 477 Raton 3 Percussion 2 min 33 470 Raton 4 Percussion 2 min 42 494 Raton 1 Percussion 4 min 65 477 Raton 2 Percussion 4 min 52 500 Raton Percussion 4 min 51 500 Raton 4 Percussion 4 min 53 499 Raton 1 Percussion 8 min 88 325 Raton 2 Percussion 8 min 80 400 Raton 3 Percussion 8 min 73 444 Raton 4 Percussion 8 min 74 439 Sacramento 1 Immersion 43 495
69
Table B 1 Continued
-Source Block Treatment Germination Percentage Standard Error
Sacramento 2 Immersion 30 458 Sacramento 3 Immersion 38 485 Sacramento 4 Immersion 56 496 Sacramento 1 Boiling 30 sec 47 499 Sacramento 2 Boiling 30 sec 48 500 Sacramento 3 Boiling 30 sec 48 500 Sacramento 4 Boiling 30 sec 56 496 Sacramento 1 Boiling 60 sec 52 500 Sacramento 2 Boiling 60 sec 67 470 Sacramento 3 Boiling 60 sec 43 495 Sacramento 4 Boiling 60 sec 48 500 Sacramento 1 Control 0 000 Sacramento 2 Control 3 171 Sacramento 3 Control 3 171 Sacramento 4 Control 099 Sacramento 1 Percussion 1 min 57 495 Sacramento 2 Percussion 1 min 61 488 Sacramento 3 Percussion 1 min 62 485 Sacramento 4 Percussion 1 min 55 497 Sacramento 1 Percussion 2 min 84 367 Sacramento 2 Percussion 2 min 70 458 Sacramento 3 Percussion 2 min 81 392 Sacramento 4 Percussion 2 min 69 462 Sacramento 1 Percussion 4 min 91 286 Sacramento 2 Percussion 4 min 84 367 Sacramento 3 Percussion 4 min 84 367 Sacramento 4 Percussion 4 min 94 237 Sacramento 1 Percussion 8 min 82 384 Sacramento 2 Percussion 8 min 81 392 Sacramento 3 Percussion 8 min 82 384 Sacramento 4 Percussion 8 min 81 392 Sandia 1 Immersion 76 427 Sandia 2 Immersion 70 458 Sandia 3 Immersion 70 458 Sandia 4 Immersion 81 392 Sandia 1 Boiling 30 sec 78 414 Sandia 2 Boiling 30 sec 71 454 Sandia 3 Boiling 30 sec 63 483 Sandia 4 Boiling 30 sec 78 414 Sandia 1 Boiling 60 sec 67 470 Sandia 2 Boiling 60 sec 65 477 Sandia 3 Boiling 60 sec 74 439 Sandia 4 Boiling 60 sec 66 474 Sandia 1 Control 16 367 Sandia 2 Control 7 255
70
Table B 1 Continued
Source Block Treatment Gennination Percentage Standard Error
Sandia 3 Control 8 271 Sandia 4 Control 16 367 Sandia 1 Percussion 1 min 67 470 Sandia 2 Percussion 1 min 61 488 Sandia 3 Percussion 1 min 66 474 Sandia 4 Percussion 1 min 59 492 Sandia I Percussion 2 min 82 384 Sandia 2 Percussion 2 min 83 376 Sandia 3 Percussion 2 min 81 392 Sandia 4 Percussion 2 min 84 367 Sandia 1 Percussion 4 min 89 313 Sandia 2 Percussion 4 min 86 347 Sandia 3 Percussion 4 min 89 313 Sandia 4 Percussion 4 min 85 357 Sandia 1 Percussion 8 min 80 400 Sandia 2 Percussion 8 min 77 421 Sandia 3 Percussion 8 min 69 462 Sandia 4 Percussion 8 min 66 474
LITERATURE CITED
Ashby WC Vogel WG Rogers NF 1985 Black locust in the reclamation equation General Technical Report Northeastern Forest Experiment Station USDA Forest Service NE-I05 12 p
Ballard LAT 1973 Physical barriers to gennination Seed Sci Technoll 285-303
___ 1976 Strophiolar water conduction in the seeds ofTrifolieae induced by action on the testa at nonstrophiolar sites Austr J Plant Physiol3 465-469
Barton LV 1947 Special studies on seed coat impenneability Contrib Boyce Thompson Inst 14 355-362
Baskin CC Baskin JM 1998 Seeds Ecology biogeography and evolution of donnancy and gennination Academic Press London UK 666 p
Bewley JD 1997 Seed germination and donnancy Plant Ce119 1055-1066
Bewley JD Black M 1994 Seeds physiology of development and germination 2nd ed Plenum Press New York NY 445 p
Briscoe CB 1996 Growing Nitrogen Fixing Trees in Acid Soils In MH Powell (ed) Nitrogen Fixing Trees for Acid Soils- A Field ManuaL Winrock International Morrilton AR 22 p
Copeland LO McDonald MB 1995 Principles of Seed Science and Technology 3rded Chapman amp Hall New York NY 409 p
Cox RE Klett JE 1984 Seed gennination requirements of native Colorado plants for use in the landscape Plant Propag 30(2) 6-10
Dell B 1980 Structure and function ofthe strophiolar plug in seeds ofAlbizia Lophantha Am J Bot 67 556-563
Dick-Peddie WA 1993 New Mexico Vegetation Past Present and Future University ofNew Mexico Press Albuquerque NM 244 p
Dreesen DR 2001 Propagation protocol for production ofcontainer Robinia neomexicana plants Los Lunas Plant Materials Center Los Lunas New Mexico In Native Plant Network URL httpwwwnativeplantnetworkorg (accessed 9 January 2002) Moscow (ID) University ofIdaho CoIl Natural Resources For Res Nursery
72
Dreesen DR Harrington JT 1997 Propagation of native plants for restoration projects in the Southwestern US- Preliminary Investigations In Landis TD Thompson JR tech coords National Proceedings Forest and Conservation Nursery Assocs Gen Tech Rep PNW-GTR-419 Portland OR USDA For Ser PNW Res Sta 77-88
Gottfried GJ 1980 Control ofNew Mexican locust and the effect on planted ponderosa pine in central Arizona US For Ser Res Note RM-386 Rocky Mountain For Range ExpStn Fort Collins CO
Gosling PG Samuel YK Jones SK 1995 A systematic examination of germination temperature chipping and water temperaturesoak duration pretreatments on the seeds ofLeucaena leucocephala Seed Sci amp Technol 23521-532
Hamly DH 1932 Softening of the seeds of Melilotus alba Bot Gazette 93 345shy375
Hanna PJ 1984 Anatomical features of the seed coat ofAcacia kempeana (Mueller) which relate to increased germination rate induced by heat treatment New Phytol 96 23-29
Hanks JP Dick-Peddie WA 1974 Vegetation patterns of the White Mountains New Mexico Southwest Naturalist 18(4) 371-382
Hine S Harrington JT Dreesen DR 1997 Seed scarification requirements for Robinia neomexicana In Harrington JTtech coord Proc Third SouthwestemContainer Growers Conf Feb 12-13 1997 Ft Collins CO
Hopkinson JM 1993 The strophiole of leguminous seeds Newsletter- IntI Herbage Seed Production Res Grp 18 7-8
---- 1997 Differences in hardseededness between Cunningham and Tarramba leucaena In Leucnet News The Newsletter of the International LeucaenaRes and Devlt Netwk No4
ISTA (International Seed Testing Association) 1999 International rules for seed testing rules Seed Sci amp Technol27 Supplement
Kelly KM van Staden J 1987 The lens as the site of permeability in the papilionoid seed Aspalathus linearis J Plant Physiol 128(45) 395-404
73
Keresztesi B 1988 Black locust the tree of agriculture Outlook on Agr 17(2) 77~85
Kigel 1 and Galili G 1995 Seed development and germination Marcel Dekker New York 853 p
Klemmedson 10 1994 New Mexico locust and parent material Influence on macronutrients of forest floor and soil Soil Sci Soc Am 1 58 974~980
Kuo WH Tam AW 1988 The pathway of water absorption of mung bean seeds Seed Sci amp Technol16(1) 139~144
Leadem CL 1997 Dormancy--unlocking seed secrets In Landis TD Thompson lR tech coords National Proceedings Forestand Conservation Nursery Assocs Gen Tech Rep PNW-G TR-419 Portland OR USDA For Ser PNW Res Sta 43-52
Lin Y Wagner MR Seweyestewa A Honahni MD DeGomez M McMillin JD 1996 Influence of cultural treatments on seed germination and seedling growth of Robinia neomexicana Forest Farm and Community Tree Res Reports Volume 1
Marunda CT 1990 Effects of seed pretreatment on the development ofAcacia auriculforinis and A holosericea seedlings ACIAR Proceedings Series No 28
Meginnis HG 1937 Sulphuric acid treatment to increase germination of black locust seed USDA Circ 453 34 p
Miklas PN Towensend CE Ladd SL 1987 Seedcoat anatomy and the scarification ofcicer milkvetch seed Crop Sci 27 766-772
Monsen SB 1984 Use of shrubs on mine spoils In The Challenge ofProducing Native Plants for the Intermountain Area Proceedings Intermountain Nurserymans Association 1983 conference Las Vegas NV compo PM Murphy 26-31 Ogden UT USDA For Ser Gen Tech Report INT-l68
Olson DF 1974 Robinia L Locust In Seeds of Woody Plants in the US tech coord CS Schopmeyer 728-731 USDA For Ser Agr Handbk No450
Rehman S Loescher RNl Harris P1C 1998 Dormancy breaking and germination ofAcacia salicina Lindl seeds Seed Sci amp Techno127 553-557
Rolston MP 1978 Water impermeable seed dormancy Bot Rev 44 365-396
74
SAS Institute Inc 1989 SASSTAT Users Guide Version 6 4th Edition Volume 1 Cary NC SAS Institute Inc 943 p
SerratoValenti G Conara L Ferrando M Modenesi P 1993 Structural and histochemical features ofStylosanthes scabra (Leguminosae Papilionoidae) seed coat as related to water entry Can J Bot 71 834-840
Simpson BJ 1988 A field guide to Texas trees Austin TX Texas Monthly Press 372p
Tran VN Cavanagh AK 1984 Structural aspects ofdormancy In Seed Physiology Vol 2 Murray DR (ed) p1-44 Academic Press Australia
USDA Forest Service 1948 Woody-plant seed manual USDA Misc Pub1654 416p
Vines RA 1960 Trees Shrubs and Woody Vines of the Southwest Austin Univ ofTexas Press 1104 p
Wagner MR Larson RE Craig TP 1992 Ecology and silvics ofNew Mexico locust (Robinia neomexicana) with an emphasis on the arboreal form p53-57 In JW Hanover K Miller and S Plesko (eds) Proc Intemat Confon Black Locust Biology Culture and Utilization Mich State Univ USA
Wilson JK 1944 Immersing seeds of species of Robinia in boiling water hastens germination J For 42 453-454
Zimmerman RW Carpenter SB 1980 First-year coppice production from a 5shyyear old black locust stand on surface mine spoil p309-314 Centr Hardwd For ConfProc University ofMissouri Columbia
Discussion
Papilionoid seeds have a specific anatomical feature known as the strophiole
or lens-the natural site of water entry to the seed (Hamly 1932) The strophiole is
located on the cotyledonary lobe of a locust seed (Figure A9) When a papilionoid
legume seed is percussed for the appropriate amount of time repeated hits on the
integrated seed coat loosen the constrained cells of the strophiolar region without
excessively damaging the rest of the seed coat (Ballard 1976)
When a percussed seed is soaked water enters exclusively through the
strophiole in a controlled manner (Kelly and Staden 1987) This regulated entry of
water to the embryo is associated with even pressure on underlying seed tissues This
contrasts with seeds that have been hot water or acid scarified treatments that can
randomly degrade the seed coat Unlocalized cracks in the seed coat can promote
irregular water uptake associated with uneven pressure on underlying seed tissues and
subsequent seed damage (Rehman et al 1998 Marunda 1990)
Papilionoids are the largest subfamily of legumes covering almost all
legumes occurring in temperate climates (Baskin and Baskin 1998) As with New
Mexico locust and black locust these legumes often play an integral role in the
revegetation ofdisturbed lands Developing superior scarification methods to the
standard hot water and acid treatments should fadlitate the use ofthese legumes in
restoration projects
62
Figure A9 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (bc)
63
APPENDIXB
GERMINATION PERCENTS AND STANDARD ERRORS FOR SOURCE
BLOCK AND TREATMENT FOR NEW MEXICO LOCUST
Table B 1 Gennination percents and standard errors for source block and treatment for New Mexico locust
Source Block Treatment Germination Percentage Standard Error
Capitan 1 Immersion 74 437 Capitan 2 Immersion 69 462 Capitan 3 Immersion 71 454 Capitan 4 Immersion 67 470 Capitan Boiling 30 sec 79 407 Capitan 2 Boiling 30 sec 76 427 Capitan 3 Boiling 30 sec 72 449 Capitan 4 Boiling 30 sec 76 427 Capitan Boiling 60 sec 71 454 Capitan 2 Boiling 60 sec 54 498 Capitan 3 Boiling 60 sec 67 470 Capitan 4 Boiling 60 sec 76 427 Capitan I Control 5 218 Capitan 2 Control 7 255 Capitan 3 Control 4 196 Capitan 4 Control 5 218 Capitan 1 Percussion I min 47 499 Capitan 2 Percussion I min
470
Capitan Percussion I min 39 488
Capitan 4 Percussion I min 38 485 Capitan I Percussion 2 min 55 497 Capitan Capitan
2
Percussion 2 min Percussion 2 min
71 65
454 477
Capitan 4 Percussion 2 min 56 496 Capitan I Percussion 4 min 86 347 Capitan 2 Percussion 4 min 79 407 Capitan 3 Percussion 4 min 81 392 Capitan 4 Percussion 4 min 82 384 Capitan I Percussion 8 min 90 300 Capitan 2 Percussion 8 min 88 325 Capitan 3 Percussion 8 min 90 300 Capitan 4 Percussion 8 min 90 300 Gila I Immersion 71 454 Gila 2 Immersion 77 421 Gila
Immersion 75 433
Gila 4 Immersion 72 449
Gila I Boiling 30 sec 68 466
Gila 2 Boiling 30 sec 81 392
Gila 3 Boiling 30 sec 54 498
Gila 4 Boiling 30 sec 63 483
Gila I Boiling 60 sec 55 497
Gila 2 Boiling 60 sec 68 466
Gila 3 Boiling 60 sec 57 495
Gila 4 Boiling 60 sec 70 458
65
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Gila 2 Control 13 336 Gila 3 Control 13 336 Gila 4 Control 20 400 Gila 1 Percussion 1 min 66 474 Gila 2 Percussion 1 min 57 495 Gila 3 Percussion 1 min 73 444 Gila 4 Percussion 1 min 82 384 Gila 1 Percussion 2 min 81 392 Gila 2 Percussion 2 min 86 347 Gila 3 Percussion 2 min 85 357 Gila 4 Percussion 2 min 54 498 Gila 1 Percussion 4 min 79 407 Gila 2 Percussion 4 min 83 376 Gila 3 Percussion 4 min 84 367 Gila 4 Percussion 4 min 79 407 Gila 1 Percussion 8 min 51 500 Gila 2 Percussion 8 min 76 427 Gila 3 Percussion 8 min 52 500 Gila 4 Percussion 8 min 74 439 Holman 1 Immersion 83 376 Holman 2 Immersion 86 347 Holman 3 Immersion 81 392 Holman 4 Immersion 90 300 Holman 1 Boiling 30 sec 90 300 Holman 2 Boiling 30 sec 85 357 Holman 3 Boiling 30 sec 87 336 Holman 4 Boiling 30 sec 83 376 Holman 1 Boiling 60 sec 79 407 Holman 2 Boiling 60 sec 80 400 Holman 3 Boiling 60 sec 86 347 Holman 4 Boiling 60 sec 93 255 Holman Control 25 433 Holman 2 Control 19 392 Holman Control 21 407
Holman 4 Control 24 427
Holman Percussion 1 min 77 421
Holman 2 Percussion 1 min 85 357
Holman 3 Percussion 1 min 80 400
Holman 4 Percussion 1 min 89 313
Holman 1 Percussion 2 min 88 325
Holman 2 Percussion 2 min 96 196
Holman 3 Percussion 2 min 93 255
Holman 4 Percussion 2 min 96 196
Holman 1 Percussion 4 min 92 271
Holman 2 Percussion 4 min 97 171
66
I
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Holman 3 Percussion 4 min 91 286 Holman 4 Percussion 4 min 96 196 Holman 1 Percussion 8 min 88 325 Holman 2 Percussion 8 min 85 357 Holman 3 Percussion 8 min 83 376 Holman 4 Percussion 8 min 82 384 Jemez 1 Immersion 53 499 Jemez 2 Immersion 44 496 Jemez 3 Immersion 52 500 Jemez 4 Immersion 70 458 Jemez 1 Boiling 30 sec 58 494 Jemez 2 Boiling 30 sec 55 497 Jemez 3 Boiling 30 sec 48 500 Jemez 4 Boiling 30 sec 65 477 Jemez 1 Boiling 60 sec 68 466 Jemez 2 Boiling 60 sec 63 483 Jemez 3 Boiling 60 sec 73 444 Jemez 4 Boiling 60 sec 89 313 Jemez 1 Control 2 140 Jemez 2 Control 9 286 Jemez 3 Control 6 237 Jemez 4 Control 8 271 Jemez 1 Percussion 1 min 58 494 Jemez 2 Percussion 1 min 63 483 Jemez 3 Percussion 1 min 77 421 Jemez 4 Percussion 1 min 89 313 Jemez 1 Percussion 2 min 68 466 Jemez 2 Percussion 2 min 64 480 Jemez 3 Percussion 2 min 80 400 Jemez 4 Percussion 2 min 83 376 Jemez Percussion 4 min 68 466 Jemez 2 Percussion 4 min 79 407 Jemez 3 Percussion 4 min 70 458 Jemez 4 Percussion 4 min 83 376 Jemez 1 Percussion 8 min 52 500 Jemez 2 Percussion 8 min 59 492 Jemez 3 Percussion 8 min 61 488 Jemez 4 Percussion 8 min 70 458 Magdalena 1 Immersion 69 462 Magdalena 2 Immersion 69 462 Magdalena J Immersion 70 458 Magdalena 4 Immersion 85 357 Magdalena 1 Boiling 30 sec 65 477 Magdalena 2 Boiling 30 sec 71 454 Magdalena 3 Boiling 30 sec 68 466
67
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Magdalena 4 Boiling 30 sec 83 376 Magdalena 1 Boiling 60 sec 63 483 Magdalena 2 Boiling 60 sec 68 466 Magdalena 3 Boiling 60 sec 63 483 Magdalena 4 Boiling 60 sec 82 384 Magdalena 1 Control 11 313 Magdalena 2 Control 6 237 Magdalena 3 Control 5 218 Magdalena 4 Control 4 196 Magdalena Percussion 1 min 32 466 Magdalena 2 Percussion 1 min 45 497 Magdalena 3 Percussion 1 min 46 498 Magdalena 4 Percussion I min 36 480 Magdalena 1 Percussion 2 min 67 470 Magdalena 2 Percussion 2 min 68 466 Magdalena 3 Percussion 2 min 65 477 Magdalena 4 Percussion 2 min 81 392 Magdalena 1 Percussion 4 min 87 336 Magdalena 2 Percussion 4 min 97 171 Magdalena 3 Percussion 4 min 83 376 Magdalena 4 Percussion 4 min 97 171 Magdalena 1 Percussion 8 min 98 lAO Magdalena 2 Percussion 8 min 100 000 Magdalena 3 Percussion 8 min 96 196 Magdalena 4 Percussion 8 min 98 lAO Manzano 1 Immersion 67 470 Manzano 2 Immersion 57 495 Manzano 3 Immersion 72 4049 Manzano 4 Immersion 73 4044 Manzano 1 Boiling 30 sec 70 458 Manzano 2 Boiling 30 sec 64 480 Manzano 3 Boiling 30 sec 71 454 Manzano 4 Boiling 30 sec 80 400 Manzano I Boiling 60 sec 50 500 Manzano 2 Boiling 60 sec 53 499 Manzano 3 Boiling 60 sec 59 492 Manzano 4 Boiling 60 sec 74 439 Manzano I Control 4 196 Manzano 2 Control 8 271 Manzano 3 Control 1 099 Manzano 4 Control 6 237 Manzano 1 Percussion 1 min 52 500 Manzano 2 Percussion 1 min 39 488 Manzano 3 Percussion 1 min 36 480 Manzano 4 Percussion 1 min 51 500
68
Table B 1 Continued
Source Block Treatment Gerrni~ation Percentage Standard Error
Manzano Percussion 2 min 53 499 Manzano 2 Percussion 2 min 58 494 Manzano 3 Percussion 2 min 61 488 Manzano 4 Percussion 2 min 62 485 Manzano 1 Percussion 4 min 76 427 Manzano 2 Percussion 4 min 85 357 Manzano 3 Percussion 4 min 79 407 Manzano 4 Percussion 4 min 83 376 Manzano 1 Percussion 8 min 87 336 Manzano 2 Percussion 8 min 83 376 Manzano 3 Percussion 8 min 83 376 Manzano 4 Percussion 8 min 75 433 Raton 1 Immersion 54 498 Raton 2 Immersion 37 483 Raton 3 Immersion 39 488 Raton 4 Immersion 69 462 Raton 1 Boiling 30 sec 36 480 Raton 2 Boiling 30 sec 20 400 Raton 3 Boiling 30 sec 28 449 Raton 4 Boiling 30 sec 60 490 Raton 1 Boiling 60 sec 15 357 Raton 2 Boiling 60 sec 171 Raton 3 Boiling 60 sec 4 196 Raton 4 Boiling 60 sec 35 477 Raton 1 Control 4 196 Raton 2 Control 12 325 Raton 3 Control 2 140 Raton 4 Control 6 237 Raton 1 Percussion 1 min 52 500 Raton 2 Percussion I min 29 454 Raton 3 Percussion I min 35 477 Raton 4 Percussion 1 min 31 462 Raton 1 Percussion 2 min 55 497 Raton 2 Percussion 2 min 35 477 Raton 3 Percussion 2 min 33 470 Raton 4 Percussion 2 min 42 494 Raton 1 Percussion 4 min 65 477 Raton 2 Percussion 4 min 52 500 Raton Percussion 4 min 51 500 Raton 4 Percussion 4 min 53 499 Raton 1 Percussion 8 min 88 325 Raton 2 Percussion 8 min 80 400 Raton 3 Percussion 8 min 73 444 Raton 4 Percussion 8 min 74 439 Sacramento 1 Immersion 43 495
69
Table B 1 Continued
-Source Block Treatment Germination Percentage Standard Error
Sacramento 2 Immersion 30 458 Sacramento 3 Immersion 38 485 Sacramento 4 Immersion 56 496 Sacramento 1 Boiling 30 sec 47 499 Sacramento 2 Boiling 30 sec 48 500 Sacramento 3 Boiling 30 sec 48 500 Sacramento 4 Boiling 30 sec 56 496 Sacramento 1 Boiling 60 sec 52 500 Sacramento 2 Boiling 60 sec 67 470 Sacramento 3 Boiling 60 sec 43 495 Sacramento 4 Boiling 60 sec 48 500 Sacramento 1 Control 0 000 Sacramento 2 Control 3 171 Sacramento 3 Control 3 171 Sacramento 4 Control 099 Sacramento 1 Percussion 1 min 57 495 Sacramento 2 Percussion 1 min 61 488 Sacramento 3 Percussion 1 min 62 485 Sacramento 4 Percussion 1 min 55 497 Sacramento 1 Percussion 2 min 84 367 Sacramento 2 Percussion 2 min 70 458 Sacramento 3 Percussion 2 min 81 392 Sacramento 4 Percussion 2 min 69 462 Sacramento 1 Percussion 4 min 91 286 Sacramento 2 Percussion 4 min 84 367 Sacramento 3 Percussion 4 min 84 367 Sacramento 4 Percussion 4 min 94 237 Sacramento 1 Percussion 8 min 82 384 Sacramento 2 Percussion 8 min 81 392 Sacramento 3 Percussion 8 min 82 384 Sacramento 4 Percussion 8 min 81 392 Sandia 1 Immersion 76 427 Sandia 2 Immersion 70 458 Sandia 3 Immersion 70 458 Sandia 4 Immersion 81 392 Sandia 1 Boiling 30 sec 78 414 Sandia 2 Boiling 30 sec 71 454 Sandia 3 Boiling 30 sec 63 483 Sandia 4 Boiling 30 sec 78 414 Sandia 1 Boiling 60 sec 67 470 Sandia 2 Boiling 60 sec 65 477 Sandia 3 Boiling 60 sec 74 439 Sandia 4 Boiling 60 sec 66 474 Sandia 1 Control 16 367 Sandia 2 Control 7 255
70
Table B 1 Continued
Source Block Treatment Gennination Percentage Standard Error
Sandia 3 Control 8 271 Sandia 4 Control 16 367 Sandia 1 Percussion 1 min 67 470 Sandia 2 Percussion 1 min 61 488 Sandia 3 Percussion 1 min 66 474 Sandia 4 Percussion 1 min 59 492 Sandia I Percussion 2 min 82 384 Sandia 2 Percussion 2 min 83 376 Sandia 3 Percussion 2 min 81 392 Sandia 4 Percussion 2 min 84 367 Sandia 1 Percussion 4 min 89 313 Sandia 2 Percussion 4 min 86 347 Sandia 3 Percussion 4 min 89 313 Sandia 4 Percussion 4 min 85 357 Sandia 1 Percussion 8 min 80 400 Sandia 2 Percussion 8 min 77 421 Sandia 3 Percussion 8 min 69 462 Sandia 4 Percussion 8 min 66 474
LITERATURE CITED
Ashby WC Vogel WG Rogers NF 1985 Black locust in the reclamation equation General Technical Report Northeastern Forest Experiment Station USDA Forest Service NE-I05 12 p
Ballard LAT 1973 Physical barriers to gennination Seed Sci Technoll 285-303
___ 1976 Strophiolar water conduction in the seeds ofTrifolieae induced by action on the testa at nonstrophiolar sites Austr J Plant Physiol3 465-469
Barton LV 1947 Special studies on seed coat impenneability Contrib Boyce Thompson Inst 14 355-362
Baskin CC Baskin JM 1998 Seeds Ecology biogeography and evolution of donnancy and gennination Academic Press London UK 666 p
Bewley JD 1997 Seed germination and donnancy Plant Ce119 1055-1066
Bewley JD Black M 1994 Seeds physiology of development and germination 2nd ed Plenum Press New York NY 445 p
Briscoe CB 1996 Growing Nitrogen Fixing Trees in Acid Soils In MH Powell (ed) Nitrogen Fixing Trees for Acid Soils- A Field ManuaL Winrock International Morrilton AR 22 p
Copeland LO McDonald MB 1995 Principles of Seed Science and Technology 3rded Chapman amp Hall New York NY 409 p
Cox RE Klett JE 1984 Seed gennination requirements of native Colorado plants for use in the landscape Plant Propag 30(2) 6-10
Dell B 1980 Structure and function ofthe strophiolar plug in seeds ofAlbizia Lophantha Am J Bot 67 556-563
Dick-Peddie WA 1993 New Mexico Vegetation Past Present and Future University ofNew Mexico Press Albuquerque NM 244 p
Dreesen DR 2001 Propagation protocol for production ofcontainer Robinia neomexicana plants Los Lunas Plant Materials Center Los Lunas New Mexico In Native Plant Network URL httpwwwnativeplantnetworkorg (accessed 9 January 2002) Moscow (ID) University ofIdaho CoIl Natural Resources For Res Nursery
72
Dreesen DR Harrington JT 1997 Propagation of native plants for restoration projects in the Southwestern US- Preliminary Investigations In Landis TD Thompson JR tech coords National Proceedings Forest and Conservation Nursery Assocs Gen Tech Rep PNW-GTR-419 Portland OR USDA For Ser PNW Res Sta 77-88
Gottfried GJ 1980 Control ofNew Mexican locust and the effect on planted ponderosa pine in central Arizona US For Ser Res Note RM-386 Rocky Mountain For Range ExpStn Fort Collins CO
Gosling PG Samuel YK Jones SK 1995 A systematic examination of germination temperature chipping and water temperaturesoak duration pretreatments on the seeds ofLeucaena leucocephala Seed Sci amp Technol 23521-532
Hamly DH 1932 Softening of the seeds of Melilotus alba Bot Gazette 93 345shy375
Hanna PJ 1984 Anatomical features of the seed coat ofAcacia kempeana (Mueller) which relate to increased germination rate induced by heat treatment New Phytol 96 23-29
Hanks JP Dick-Peddie WA 1974 Vegetation patterns of the White Mountains New Mexico Southwest Naturalist 18(4) 371-382
Hine S Harrington JT Dreesen DR 1997 Seed scarification requirements for Robinia neomexicana In Harrington JTtech coord Proc Third SouthwestemContainer Growers Conf Feb 12-13 1997 Ft Collins CO
Hopkinson JM 1993 The strophiole of leguminous seeds Newsletter- IntI Herbage Seed Production Res Grp 18 7-8
---- 1997 Differences in hardseededness between Cunningham and Tarramba leucaena In Leucnet News The Newsletter of the International LeucaenaRes and Devlt Netwk No4
ISTA (International Seed Testing Association) 1999 International rules for seed testing rules Seed Sci amp Technol27 Supplement
Kelly KM van Staden J 1987 The lens as the site of permeability in the papilionoid seed Aspalathus linearis J Plant Physiol 128(45) 395-404
73
Keresztesi B 1988 Black locust the tree of agriculture Outlook on Agr 17(2) 77~85
Kigel 1 and Galili G 1995 Seed development and germination Marcel Dekker New York 853 p
Klemmedson 10 1994 New Mexico locust and parent material Influence on macronutrients of forest floor and soil Soil Sci Soc Am 1 58 974~980
Kuo WH Tam AW 1988 The pathway of water absorption of mung bean seeds Seed Sci amp Technol16(1) 139~144
Leadem CL 1997 Dormancy--unlocking seed secrets In Landis TD Thompson lR tech coords National Proceedings Forestand Conservation Nursery Assocs Gen Tech Rep PNW-G TR-419 Portland OR USDA For Ser PNW Res Sta 43-52
Lin Y Wagner MR Seweyestewa A Honahni MD DeGomez M McMillin JD 1996 Influence of cultural treatments on seed germination and seedling growth of Robinia neomexicana Forest Farm and Community Tree Res Reports Volume 1
Marunda CT 1990 Effects of seed pretreatment on the development ofAcacia auriculforinis and A holosericea seedlings ACIAR Proceedings Series No 28
Meginnis HG 1937 Sulphuric acid treatment to increase germination of black locust seed USDA Circ 453 34 p
Miklas PN Towensend CE Ladd SL 1987 Seedcoat anatomy and the scarification ofcicer milkvetch seed Crop Sci 27 766-772
Monsen SB 1984 Use of shrubs on mine spoils In The Challenge ofProducing Native Plants for the Intermountain Area Proceedings Intermountain Nurserymans Association 1983 conference Las Vegas NV compo PM Murphy 26-31 Ogden UT USDA For Ser Gen Tech Report INT-l68
Olson DF 1974 Robinia L Locust In Seeds of Woody Plants in the US tech coord CS Schopmeyer 728-731 USDA For Ser Agr Handbk No450
Rehman S Loescher RNl Harris P1C 1998 Dormancy breaking and germination ofAcacia salicina Lindl seeds Seed Sci amp Techno127 553-557
Rolston MP 1978 Water impermeable seed dormancy Bot Rev 44 365-396
74
SAS Institute Inc 1989 SASSTAT Users Guide Version 6 4th Edition Volume 1 Cary NC SAS Institute Inc 943 p
SerratoValenti G Conara L Ferrando M Modenesi P 1993 Structural and histochemical features ofStylosanthes scabra (Leguminosae Papilionoidae) seed coat as related to water entry Can J Bot 71 834-840
Simpson BJ 1988 A field guide to Texas trees Austin TX Texas Monthly Press 372p
Tran VN Cavanagh AK 1984 Structural aspects ofdormancy In Seed Physiology Vol 2 Murray DR (ed) p1-44 Academic Press Australia
USDA Forest Service 1948 Woody-plant seed manual USDA Misc Pub1654 416p
Vines RA 1960 Trees Shrubs and Woody Vines of the Southwest Austin Univ ofTexas Press 1104 p
Wagner MR Larson RE Craig TP 1992 Ecology and silvics ofNew Mexico locust (Robinia neomexicana) with an emphasis on the arboreal form p53-57 In JW Hanover K Miller and S Plesko (eds) Proc Intemat Confon Black Locust Biology Culture and Utilization Mich State Univ USA
Wilson JK 1944 Immersing seeds of species of Robinia in boiling water hastens germination J For 42 453-454
Zimmerman RW Carpenter SB 1980 First-year coppice production from a 5shyyear old black locust stand on surface mine spoil p309-314 Centr Hardwd For ConfProc University ofMissouri Columbia
Figure A9 Strophiole ofunimbibed New Mexico locust seed (a) and following water uptake (bc)
63
APPENDIXB
GERMINATION PERCENTS AND STANDARD ERRORS FOR SOURCE
BLOCK AND TREATMENT FOR NEW MEXICO LOCUST
Table B 1 Gennination percents and standard errors for source block and treatment for New Mexico locust
Source Block Treatment Germination Percentage Standard Error
Capitan 1 Immersion 74 437 Capitan 2 Immersion 69 462 Capitan 3 Immersion 71 454 Capitan 4 Immersion 67 470 Capitan Boiling 30 sec 79 407 Capitan 2 Boiling 30 sec 76 427 Capitan 3 Boiling 30 sec 72 449 Capitan 4 Boiling 30 sec 76 427 Capitan Boiling 60 sec 71 454 Capitan 2 Boiling 60 sec 54 498 Capitan 3 Boiling 60 sec 67 470 Capitan 4 Boiling 60 sec 76 427 Capitan I Control 5 218 Capitan 2 Control 7 255 Capitan 3 Control 4 196 Capitan 4 Control 5 218 Capitan 1 Percussion I min 47 499 Capitan 2 Percussion I min
470
Capitan Percussion I min 39 488
Capitan 4 Percussion I min 38 485 Capitan I Percussion 2 min 55 497 Capitan Capitan
2
Percussion 2 min Percussion 2 min
71 65
454 477
Capitan 4 Percussion 2 min 56 496 Capitan I Percussion 4 min 86 347 Capitan 2 Percussion 4 min 79 407 Capitan 3 Percussion 4 min 81 392 Capitan 4 Percussion 4 min 82 384 Capitan I Percussion 8 min 90 300 Capitan 2 Percussion 8 min 88 325 Capitan 3 Percussion 8 min 90 300 Capitan 4 Percussion 8 min 90 300 Gila I Immersion 71 454 Gila 2 Immersion 77 421 Gila
Immersion 75 433
Gila 4 Immersion 72 449
Gila I Boiling 30 sec 68 466
Gila 2 Boiling 30 sec 81 392
Gila 3 Boiling 30 sec 54 498
Gila 4 Boiling 30 sec 63 483
Gila I Boiling 60 sec 55 497
Gila 2 Boiling 60 sec 68 466
Gila 3 Boiling 60 sec 57 495
Gila 4 Boiling 60 sec 70 458
65
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Gila 2 Control 13 336 Gila 3 Control 13 336 Gila 4 Control 20 400 Gila 1 Percussion 1 min 66 474 Gila 2 Percussion 1 min 57 495 Gila 3 Percussion 1 min 73 444 Gila 4 Percussion 1 min 82 384 Gila 1 Percussion 2 min 81 392 Gila 2 Percussion 2 min 86 347 Gila 3 Percussion 2 min 85 357 Gila 4 Percussion 2 min 54 498 Gila 1 Percussion 4 min 79 407 Gila 2 Percussion 4 min 83 376 Gila 3 Percussion 4 min 84 367 Gila 4 Percussion 4 min 79 407 Gila 1 Percussion 8 min 51 500 Gila 2 Percussion 8 min 76 427 Gila 3 Percussion 8 min 52 500 Gila 4 Percussion 8 min 74 439 Holman 1 Immersion 83 376 Holman 2 Immersion 86 347 Holman 3 Immersion 81 392 Holman 4 Immersion 90 300 Holman 1 Boiling 30 sec 90 300 Holman 2 Boiling 30 sec 85 357 Holman 3 Boiling 30 sec 87 336 Holman 4 Boiling 30 sec 83 376 Holman 1 Boiling 60 sec 79 407 Holman 2 Boiling 60 sec 80 400 Holman 3 Boiling 60 sec 86 347 Holman 4 Boiling 60 sec 93 255 Holman Control 25 433 Holman 2 Control 19 392 Holman Control 21 407
Holman 4 Control 24 427
Holman Percussion 1 min 77 421
Holman 2 Percussion 1 min 85 357
Holman 3 Percussion 1 min 80 400
Holman 4 Percussion 1 min 89 313
Holman 1 Percussion 2 min 88 325
Holman 2 Percussion 2 min 96 196
Holman 3 Percussion 2 min 93 255
Holman 4 Percussion 2 min 96 196
Holman 1 Percussion 4 min 92 271
Holman 2 Percussion 4 min 97 171
66
I
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Holman 3 Percussion 4 min 91 286 Holman 4 Percussion 4 min 96 196 Holman 1 Percussion 8 min 88 325 Holman 2 Percussion 8 min 85 357 Holman 3 Percussion 8 min 83 376 Holman 4 Percussion 8 min 82 384 Jemez 1 Immersion 53 499 Jemez 2 Immersion 44 496 Jemez 3 Immersion 52 500 Jemez 4 Immersion 70 458 Jemez 1 Boiling 30 sec 58 494 Jemez 2 Boiling 30 sec 55 497 Jemez 3 Boiling 30 sec 48 500 Jemez 4 Boiling 30 sec 65 477 Jemez 1 Boiling 60 sec 68 466 Jemez 2 Boiling 60 sec 63 483 Jemez 3 Boiling 60 sec 73 444 Jemez 4 Boiling 60 sec 89 313 Jemez 1 Control 2 140 Jemez 2 Control 9 286 Jemez 3 Control 6 237 Jemez 4 Control 8 271 Jemez 1 Percussion 1 min 58 494 Jemez 2 Percussion 1 min 63 483 Jemez 3 Percussion 1 min 77 421 Jemez 4 Percussion 1 min 89 313 Jemez 1 Percussion 2 min 68 466 Jemez 2 Percussion 2 min 64 480 Jemez 3 Percussion 2 min 80 400 Jemez 4 Percussion 2 min 83 376 Jemez Percussion 4 min 68 466 Jemez 2 Percussion 4 min 79 407 Jemez 3 Percussion 4 min 70 458 Jemez 4 Percussion 4 min 83 376 Jemez 1 Percussion 8 min 52 500 Jemez 2 Percussion 8 min 59 492 Jemez 3 Percussion 8 min 61 488 Jemez 4 Percussion 8 min 70 458 Magdalena 1 Immersion 69 462 Magdalena 2 Immersion 69 462 Magdalena J Immersion 70 458 Magdalena 4 Immersion 85 357 Magdalena 1 Boiling 30 sec 65 477 Magdalena 2 Boiling 30 sec 71 454 Magdalena 3 Boiling 30 sec 68 466
67
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Magdalena 4 Boiling 30 sec 83 376 Magdalena 1 Boiling 60 sec 63 483 Magdalena 2 Boiling 60 sec 68 466 Magdalena 3 Boiling 60 sec 63 483 Magdalena 4 Boiling 60 sec 82 384 Magdalena 1 Control 11 313 Magdalena 2 Control 6 237 Magdalena 3 Control 5 218 Magdalena 4 Control 4 196 Magdalena Percussion 1 min 32 466 Magdalena 2 Percussion 1 min 45 497 Magdalena 3 Percussion 1 min 46 498 Magdalena 4 Percussion I min 36 480 Magdalena 1 Percussion 2 min 67 470 Magdalena 2 Percussion 2 min 68 466 Magdalena 3 Percussion 2 min 65 477 Magdalena 4 Percussion 2 min 81 392 Magdalena 1 Percussion 4 min 87 336 Magdalena 2 Percussion 4 min 97 171 Magdalena 3 Percussion 4 min 83 376 Magdalena 4 Percussion 4 min 97 171 Magdalena 1 Percussion 8 min 98 lAO Magdalena 2 Percussion 8 min 100 000 Magdalena 3 Percussion 8 min 96 196 Magdalena 4 Percussion 8 min 98 lAO Manzano 1 Immersion 67 470 Manzano 2 Immersion 57 495 Manzano 3 Immersion 72 4049 Manzano 4 Immersion 73 4044 Manzano 1 Boiling 30 sec 70 458 Manzano 2 Boiling 30 sec 64 480 Manzano 3 Boiling 30 sec 71 454 Manzano 4 Boiling 30 sec 80 400 Manzano I Boiling 60 sec 50 500 Manzano 2 Boiling 60 sec 53 499 Manzano 3 Boiling 60 sec 59 492 Manzano 4 Boiling 60 sec 74 439 Manzano I Control 4 196 Manzano 2 Control 8 271 Manzano 3 Control 1 099 Manzano 4 Control 6 237 Manzano 1 Percussion 1 min 52 500 Manzano 2 Percussion 1 min 39 488 Manzano 3 Percussion 1 min 36 480 Manzano 4 Percussion 1 min 51 500
68
Table B 1 Continued
Source Block Treatment Gerrni~ation Percentage Standard Error
Manzano Percussion 2 min 53 499 Manzano 2 Percussion 2 min 58 494 Manzano 3 Percussion 2 min 61 488 Manzano 4 Percussion 2 min 62 485 Manzano 1 Percussion 4 min 76 427 Manzano 2 Percussion 4 min 85 357 Manzano 3 Percussion 4 min 79 407 Manzano 4 Percussion 4 min 83 376 Manzano 1 Percussion 8 min 87 336 Manzano 2 Percussion 8 min 83 376 Manzano 3 Percussion 8 min 83 376 Manzano 4 Percussion 8 min 75 433 Raton 1 Immersion 54 498 Raton 2 Immersion 37 483 Raton 3 Immersion 39 488 Raton 4 Immersion 69 462 Raton 1 Boiling 30 sec 36 480 Raton 2 Boiling 30 sec 20 400 Raton 3 Boiling 30 sec 28 449 Raton 4 Boiling 30 sec 60 490 Raton 1 Boiling 60 sec 15 357 Raton 2 Boiling 60 sec 171 Raton 3 Boiling 60 sec 4 196 Raton 4 Boiling 60 sec 35 477 Raton 1 Control 4 196 Raton 2 Control 12 325 Raton 3 Control 2 140 Raton 4 Control 6 237 Raton 1 Percussion 1 min 52 500 Raton 2 Percussion I min 29 454 Raton 3 Percussion I min 35 477 Raton 4 Percussion 1 min 31 462 Raton 1 Percussion 2 min 55 497 Raton 2 Percussion 2 min 35 477 Raton 3 Percussion 2 min 33 470 Raton 4 Percussion 2 min 42 494 Raton 1 Percussion 4 min 65 477 Raton 2 Percussion 4 min 52 500 Raton Percussion 4 min 51 500 Raton 4 Percussion 4 min 53 499 Raton 1 Percussion 8 min 88 325 Raton 2 Percussion 8 min 80 400 Raton 3 Percussion 8 min 73 444 Raton 4 Percussion 8 min 74 439 Sacramento 1 Immersion 43 495
69
Table B 1 Continued
-Source Block Treatment Germination Percentage Standard Error
Sacramento 2 Immersion 30 458 Sacramento 3 Immersion 38 485 Sacramento 4 Immersion 56 496 Sacramento 1 Boiling 30 sec 47 499 Sacramento 2 Boiling 30 sec 48 500 Sacramento 3 Boiling 30 sec 48 500 Sacramento 4 Boiling 30 sec 56 496 Sacramento 1 Boiling 60 sec 52 500 Sacramento 2 Boiling 60 sec 67 470 Sacramento 3 Boiling 60 sec 43 495 Sacramento 4 Boiling 60 sec 48 500 Sacramento 1 Control 0 000 Sacramento 2 Control 3 171 Sacramento 3 Control 3 171 Sacramento 4 Control 099 Sacramento 1 Percussion 1 min 57 495 Sacramento 2 Percussion 1 min 61 488 Sacramento 3 Percussion 1 min 62 485 Sacramento 4 Percussion 1 min 55 497 Sacramento 1 Percussion 2 min 84 367 Sacramento 2 Percussion 2 min 70 458 Sacramento 3 Percussion 2 min 81 392 Sacramento 4 Percussion 2 min 69 462 Sacramento 1 Percussion 4 min 91 286 Sacramento 2 Percussion 4 min 84 367 Sacramento 3 Percussion 4 min 84 367 Sacramento 4 Percussion 4 min 94 237 Sacramento 1 Percussion 8 min 82 384 Sacramento 2 Percussion 8 min 81 392 Sacramento 3 Percussion 8 min 82 384 Sacramento 4 Percussion 8 min 81 392 Sandia 1 Immersion 76 427 Sandia 2 Immersion 70 458 Sandia 3 Immersion 70 458 Sandia 4 Immersion 81 392 Sandia 1 Boiling 30 sec 78 414 Sandia 2 Boiling 30 sec 71 454 Sandia 3 Boiling 30 sec 63 483 Sandia 4 Boiling 30 sec 78 414 Sandia 1 Boiling 60 sec 67 470 Sandia 2 Boiling 60 sec 65 477 Sandia 3 Boiling 60 sec 74 439 Sandia 4 Boiling 60 sec 66 474 Sandia 1 Control 16 367 Sandia 2 Control 7 255
70
Table B 1 Continued
Source Block Treatment Gennination Percentage Standard Error
Sandia 3 Control 8 271 Sandia 4 Control 16 367 Sandia 1 Percussion 1 min 67 470 Sandia 2 Percussion 1 min 61 488 Sandia 3 Percussion 1 min 66 474 Sandia 4 Percussion 1 min 59 492 Sandia I Percussion 2 min 82 384 Sandia 2 Percussion 2 min 83 376 Sandia 3 Percussion 2 min 81 392 Sandia 4 Percussion 2 min 84 367 Sandia 1 Percussion 4 min 89 313 Sandia 2 Percussion 4 min 86 347 Sandia 3 Percussion 4 min 89 313 Sandia 4 Percussion 4 min 85 357 Sandia 1 Percussion 8 min 80 400 Sandia 2 Percussion 8 min 77 421 Sandia 3 Percussion 8 min 69 462 Sandia 4 Percussion 8 min 66 474
LITERATURE CITED
Ashby WC Vogel WG Rogers NF 1985 Black locust in the reclamation equation General Technical Report Northeastern Forest Experiment Station USDA Forest Service NE-I05 12 p
Ballard LAT 1973 Physical barriers to gennination Seed Sci Technoll 285-303
___ 1976 Strophiolar water conduction in the seeds ofTrifolieae induced by action on the testa at nonstrophiolar sites Austr J Plant Physiol3 465-469
Barton LV 1947 Special studies on seed coat impenneability Contrib Boyce Thompson Inst 14 355-362
Baskin CC Baskin JM 1998 Seeds Ecology biogeography and evolution of donnancy and gennination Academic Press London UK 666 p
Bewley JD 1997 Seed germination and donnancy Plant Ce119 1055-1066
Bewley JD Black M 1994 Seeds physiology of development and germination 2nd ed Plenum Press New York NY 445 p
Briscoe CB 1996 Growing Nitrogen Fixing Trees in Acid Soils In MH Powell (ed) Nitrogen Fixing Trees for Acid Soils- A Field ManuaL Winrock International Morrilton AR 22 p
Copeland LO McDonald MB 1995 Principles of Seed Science and Technology 3rded Chapman amp Hall New York NY 409 p
Cox RE Klett JE 1984 Seed gennination requirements of native Colorado plants for use in the landscape Plant Propag 30(2) 6-10
Dell B 1980 Structure and function ofthe strophiolar plug in seeds ofAlbizia Lophantha Am J Bot 67 556-563
Dick-Peddie WA 1993 New Mexico Vegetation Past Present and Future University ofNew Mexico Press Albuquerque NM 244 p
Dreesen DR 2001 Propagation protocol for production ofcontainer Robinia neomexicana plants Los Lunas Plant Materials Center Los Lunas New Mexico In Native Plant Network URL httpwwwnativeplantnetworkorg (accessed 9 January 2002) Moscow (ID) University ofIdaho CoIl Natural Resources For Res Nursery
72
Dreesen DR Harrington JT 1997 Propagation of native plants for restoration projects in the Southwestern US- Preliminary Investigations In Landis TD Thompson JR tech coords National Proceedings Forest and Conservation Nursery Assocs Gen Tech Rep PNW-GTR-419 Portland OR USDA For Ser PNW Res Sta 77-88
Gottfried GJ 1980 Control ofNew Mexican locust and the effect on planted ponderosa pine in central Arizona US For Ser Res Note RM-386 Rocky Mountain For Range ExpStn Fort Collins CO
Gosling PG Samuel YK Jones SK 1995 A systematic examination of germination temperature chipping and water temperaturesoak duration pretreatments on the seeds ofLeucaena leucocephala Seed Sci amp Technol 23521-532
Hamly DH 1932 Softening of the seeds of Melilotus alba Bot Gazette 93 345shy375
Hanna PJ 1984 Anatomical features of the seed coat ofAcacia kempeana (Mueller) which relate to increased germination rate induced by heat treatment New Phytol 96 23-29
Hanks JP Dick-Peddie WA 1974 Vegetation patterns of the White Mountains New Mexico Southwest Naturalist 18(4) 371-382
Hine S Harrington JT Dreesen DR 1997 Seed scarification requirements for Robinia neomexicana In Harrington JTtech coord Proc Third SouthwestemContainer Growers Conf Feb 12-13 1997 Ft Collins CO
Hopkinson JM 1993 The strophiole of leguminous seeds Newsletter- IntI Herbage Seed Production Res Grp 18 7-8
---- 1997 Differences in hardseededness between Cunningham and Tarramba leucaena In Leucnet News The Newsletter of the International LeucaenaRes and Devlt Netwk No4
ISTA (International Seed Testing Association) 1999 International rules for seed testing rules Seed Sci amp Technol27 Supplement
Kelly KM van Staden J 1987 The lens as the site of permeability in the papilionoid seed Aspalathus linearis J Plant Physiol 128(45) 395-404
73
Keresztesi B 1988 Black locust the tree of agriculture Outlook on Agr 17(2) 77~85
Kigel 1 and Galili G 1995 Seed development and germination Marcel Dekker New York 853 p
Klemmedson 10 1994 New Mexico locust and parent material Influence on macronutrients of forest floor and soil Soil Sci Soc Am 1 58 974~980
Kuo WH Tam AW 1988 The pathway of water absorption of mung bean seeds Seed Sci amp Technol16(1) 139~144
Leadem CL 1997 Dormancy--unlocking seed secrets In Landis TD Thompson lR tech coords National Proceedings Forestand Conservation Nursery Assocs Gen Tech Rep PNW-G TR-419 Portland OR USDA For Ser PNW Res Sta 43-52
Lin Y Wagner MR Seweyestewa A Honahni MD DeGomez M McMillin JD 1996 Influence of cultural treatments on seed germination and seedling growth of Robinia neomexicana Forest Farm and Community Tree Res Reports Volume 1
Marunda CT 1990 Effects of seed pretreatment on the development ofAcacia auriculforinis and A holosericea seedlings ACIAR Proceedings Series No 28
Meginnis HG 1937 Sulphuric acid treatment to increase germination of black locust seed USDA Circ 453 34 p
Miklas PN Towensend CE Ladd SL 1987 Seedcoat anatomy and the scarification ofcicer milkvetch seed Crop Sci 27 766-772
Monsen SB 1984 Use of shrubs on mine spoils In The Challenge ofProducing Native Plants for the Intermountain Area Proceedings Intermountain Nurserymans Association 1983 conference Las Vegas NV compo PM Murphy 26-31 Ogden UT USDA For Ser Gen Tech Report INT-l68
Olson DF 1974 Robinia L Locust In Seeds of Woody Plants in the US tech coord CS Schopmeyer 728-731 USDA For Ser Agr Handbk No450
Rehman S Loescher RNl Harris P1C 1998 Dormancy breaking and germination ofAcacia salicina Lindl seeds Seed Sci amp Techno127 553-557
Rolston MP 1978 Water impermeable seed dormancy Bot Rev 44 365-396
74
SAS Institute Inc 1989 SASSTAT Users Guide Version 6 4th Edition Volume 1 Cary NC SAS Institute Inc 943 p
SerratoValenti G Conara L Ferrando M Modenesi P 1993 Structural and histochemical features ofStylosanthes scabra (Leguminosae Papilionoidae) seed coat as related to water entry Can J Bot 71 834-840
Simpson BJ 1988 A field guide to Texas trees Austin TX Texas Monthly Press 372p
Tran VN Cavanagh AK 1984 Structural aspects ofdormancy In Seed Physiology Vol 2 Murray DR (ed) p1-44 Academic Press Australia
USDA Forest Service 1948 Woody-plant seed manual USDA Misc Pub1654 416p
Vines RA 1960 Trees Shrubs and Woody Vines of the Southwest Austin Univ ofTexas Press 1104 p
Wagner MR Larson RE Craig TP 1992 Ecology and silvics ofNew Mexico locust (Robinia neomexicana) with an emphasis on the arboreal form p53-57 In JW Hanover K Miller and S Plesko (eds) Proc Intemat Confon Black Locust Biology Culture and Utilization Mich State Univ USA
Wilson JK 1944 Immersing seeds of species of Robinia in boiling water hastens germination J For 42 453-454
Zimmerman RW Carpenter SB 1980 First-year coppice production from a 5shyyear old black locust stand on surface mine spoil p309-314 Centr Hardwd For ConfProc University ofMissouri Columbia
APPENDIXB
GERMINATION PERCENTS AND STANDARD ERRORS FOR SOURCE
BLOCK AND TREATMENT FOR NEW MEXICO LOCUST
Table B 1 Gennination percents and standard errors for source block and treatment for New Mexico locust
Source Block Treatment Germination Percentage Standard Error
Capitan 1 Immersion 74 437 Capitan 2 Immersion 69 462 Capitan 3 Immersion 71 454 Capitan 4 Immersion 67 470 Capitan Boiling 30 sec 79 407 Capitan 2 Boiling 30 sec 76 427 Capitan 3 Boiling 30 sec 72 449 Capitan 4 Boiling 30 sec 76 427 Capitan Boiling 60 sec 71 454 Capitan 2 Boiling 60 sec 54 498 Capitan 3 Boiling 60 sec 67 470 Capitan 4 Boiling 60 sec 76 427 Capitan I Control 5 218 Capitan 2 Control 7 255 Capitan 3 Control 4 196 Capitan 4 Control 5 218 Capitan 1 Percussion I min 47 499 Capitan 2 Percussion I min
470
Capitan Percussion I min 39 488
Capitan 4 Percussion I min 38 485 Capitan I Percussion 2 min 55 497 Capitan Capitan
2
Percussion 2 min Percussion 2 min
71 65
454 477
Capitan 4 Percussion 2 min 56 496 Capitan I Percussion 4 min 86 347 Capitan 2 Percussion 4 min 79 407 Capitan 3 Percussion 4 min 81 392 Capitan 4 Percussion 4 min 82 384 Capitan I Percussion 8 min 90 300 Capitan 2 Percussion 8 min 88 325 Capitan 3 Percussion 8 min 90 300 Capitan 4 Percussion 8 min 90 300 Gila I Immersion 71 454 Gila 2 Immersion 77 421 Gila
Immersion 75 433
Gila 4 Immersion 72 449
Gila I Boiling 30 sec 68 466
Gila 2 Boiling 30 sec 81 392
Gila 3 Boiling 30 sec 54 498
Gila 4 Boiling 30 sec 63 483
Gila I Boiling 60 sec 55 497
Gila 2 Boiling 60 sec 68 466
Gila 3 Boiling 60 sec 57 495
Gila 4 Boiling 60 sec 70 458
65
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Gila 2 Control 13 336 Gila 3 Control 13 336 Gila 4 Control 20 400 Gila 1 Percussion 1 min 66 474 Gila 2 Percussion 1 min 57 495 Gila 3 Percussion 1 min 73 444 Gila 4 Percussion 1 min 82 384 Gila 1 Percussion 2 min 81 392 Gila 2 Percussion 2 min 86 347 Gila 3 Percussion 2 min 85 357 Gila 4 Percussion 2 min 54 498 Gila 1 Percussion 4 min 79 407 Gila 2 Percussion 4 min 83 376 Gila 3 Percussion 4 min 84 367 Gila 4 Percussion 4 min 79 407 Gila 1 Percussion 8 min 51 500 Gila 2 Percussion 8 min 76 427 Gila 3 Percussion 8 min 52 500 Gila 4 Percussion 8 min 74 439 Holman 1 Immersion 83 376 Holman 2 Immersion 86 347 Holman 3 Immersion 81 392 Holman 4 Immersion 90 300 Holman 1 Boiling 30 sec 90 300 Holman 2 Boiling 30 sec 85 357 Holman 3 Boiling 30 sec 87 336 Holman 4 Boiling 30 sec 83 376 Holman 1 Boiling 60 sec 79 407 Holman 2 Boiling 60 sec 80 400 Holman 3 Boiling 60 sec 86 347 Holman 4 Boiling 60 sec 93 255 Holman Control 25 433 Holman 2 Control 19 392 Holman Control 21 407
Holman 4 Control 24 427
Holman Percussion 1 min 77 421
Holman 2 Percussion 1 min 85 357
Holman 3 Percussion 1 min 80 400
Holman 4 Percussion 1 min 89 313
Holman 1 Percussion 2 min 88 325
Holman 2 Percussion 2 min 96 196
Holman 3 Percussion 2 min 93 255
Holman 4 Percussion 2 min 96 196
Holman 1 Percussion 4 min 92 271
Holman 2 Percussion 4 min 97 171
66
I
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Holman 3 Percussion 4 min 91 286 Holman 4 Percussion 4 min 96 196 Holman 1 Percussion 8 min 88 325 Holman 2 Percussion 8 min 85 357 Holman 3 Percussion 8 min 83 376 Holman 4 Percussion 8 min 82 384 Jemez 1 Immersion 53 499 Jemez 2 Immersion 44 496 Jemez 3 Immersion 52 500 Jemez 4 Immersion 70 458 Jemez 1 Boiling 30 sec 58 494 Jemez 2 Boiling 30 sec 55 497 Jemez 3 Boiling 30 sec 48 500 Jemez 4 Boiling 30 sec 65 477 Jemez 1 Boiling 60 sec 68 466 Jemez 2 Boiling 60 sec 63 483 Jemez 3 Boiling 60 sec 73 444 Jemez 4 Boiling 60 sec 89 313 Jemez 1 Control 2 140 Jemez 2 Control 9 286 Jemez 3 Control 6 237 Jemez 4 Control 8 271 Jemez 1 Percussion 1 min 58 494 Jemez 2 Percussion 1 min 63 483 Jemez 3 Percussion 1 min 77 421 Jemez 4 Percussion 1 min 89 313 Jemez 1 Percussion 2 min 68 466 Jemez 2 Percussion 2 min 64 480 Jemez 3 Percussion 2 min 80 400 Jemez 4 Percussion 2 min 83 376 Jemez Percussion 4 min 68 466 Jemez 2 Percussion 4 min 79 407 Jemez 3 Percussion 4 min 70 458 Jemez 4 Percussion 4 min 83 376 Jemez 1 Percussion 8 min 52 500 Jemez 2 Percussion 8 min 59 492 Jemez 3 Percussion 8 min 61 488 Jemez 4 Percussion 8 min 70 458 Magdalena 1 Immersion 69 462 Magdalena 2 Immersion 69 462 Magdalena J Immersion 70 458 Magdalena 4 Immersion 85 357 Magdalena 1 Boiling 30 sec 65 477 Magdalena 2 Boiling 30 sec 71 454 Magdalena 3 Boiling 30 sec 68 466
67
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Magdalena 4 Boiling 30 sec 83 376 Magdalena 1 Boiling 60 sec 63 483 Magdalena 2 Boiling 60 sec 68 466 Magdalena 3 Boiling 60 sec 63 483 Magdalena 4 Boiling 60 sec 82 384 Magdalena 1 Control 11 313 Magdalena 2 Control 6 237 Magdalena 3 Control 5 218 Magdalena 4 Control 4 196 Magdalena Percussion 1 min 32 466 Magdalena 2 Percussion 1 min 45 497 Magdalena 3 Percussion 1 min 46 498 Magdalena 4 Percussion I min 36 480 Magdalena 1 Percussion 2 min 67 470 Magdalena 2 Percussion 2 min 68 466 Magdalena 3 Percussion 2 min 65 477 Magdalena 4 Percussion 2 min 81 392 Magdalena 1 Percussion 4 min 87 336 Magdalena 2 Percussion 4 min 97 171 Magdalena 3 Percussion 4 min 83 376 Magdalena 4 Percussion 4 min 97 171 Magdalena 1 Percussion 8 min 98 lAO Magdalena 2 Percussion 8 min 100 000 Magdalena 3 Percussion 8 min 96 196 Magdalena 4 Percussion 8 min 98 lAO Manzano 1 Immersion 67 470 Manzano 2 Immersion 57 495 Manzano 3 Immersion 72 4049 Manzano 4 Immersion 73 4044 Manzano 1 Boiling 30 sec 70 458 Manzano 2 Boiling 30 sec 64 480 Manzano 3 Boiling 30 sec 71 454 Manzano 4 Boiling 30 sec 80 400 Manzano I Boiling 60 sec 50 500 Manzano 2 Boiling 60 sec 53 499 Manzano 3 Boiling 60 sec 59 492 Manzano 4 Boiling 60 sec 74 439 Manzano I Control 4 196 Manzano 2 Control 8 271 Manzano 3 Control 1 099 Manzano 4 Control 6 237 Manzano 1 Percussion 1 min 52 500 Manzano 2 Percussion 1 min 39 488 Manzano 3 Percussion 1 min 36 480 Manzano 4 Percussion 1 min 51 500
68
Table B 1 Continued
Source Block Treatment Gerrni~ation Percentage Standard Error
Manzano Percussion 2 min 53 499 Manzano 2 Percussion 2 min 58 494 Manzano 3 Percussion 2 min 61 488 Manzano 4 Percussion 2 min 62 485 Manzano 1 Percussion 4 min 76 427 Manzano 2 Percussion 4 min 85 357 Manzano 3 Percussion 4 min 79 407 Manzano 4 Percussion 4 min 83 376 Manzano 1 Percussion 8 min 87 336 Manzano 2 Percussion 8 min 83 376 Manzano 3 Percussion 8 min 83 376 Manzano 4 Percussion 8 min 75 433 Raton 1 Immersion 54 498 Raton 2 Immersion 37 483 Raton 3 Immersion 39 488 Raton 4 Immersion 69 462 Raton 1 Boiling 30 sec 36 480 Raton 2 Boiling 30 sec 20 400 Raton 3 Boiling 30 sec 28 449 Raton 4 Boiling 30 sec 60 490 Raton 1 Boiling 60 sec 15 357 Raton 2 Boiling 60 sec 171 Raton 3 Boiling 60 sec 4 196 Raton 4 Boiling 60 sec 35 477 Raton 1 Control 4 196 Raton 2 Control 12 325 Raton 3 Control 2 140 Raton 4 Control 6 237 Raton 1 Percussion 1 min 52 500 Raton 2 Percussion I min 29 454 Raton 3 Percussion I min 35 477 Raton 4 Percussion 1 min 31 462 Raton 1 Percussion 2 min 55 497 Raton 2 Percussion 2 min 35 477 Raton 3 Percussion 2 min 33 470 Raton 4 Percussion 2 min 42 494 Raton 1 Percussion 4 min 65 477 Raton 2 Percussion 4 min 52 500 Raton Percussion 4 min 51 500 Raton 4 Percussion 4 min 53 499 Raton 1 Percussion 8 min 88 325 Raton 2 Percussion 8 min 80 400 Raton 3 Percussion 8 min 73 444 Raton 4 Percussion 8 min 74 439 Sacramento 1 Immersion 43 495
69
Table B 1 Continued
-Source Block Treatment Germination Percentage Standard Error
Sacramento 2 Immersion 30 458 Sacramento 3 Immersion 38 485 Sacramento 4 Immersion 56 496 Sacramento 1 Boiling 30 sec 47 499 Sacramento 2 Boiling 30 sec 48 500 Sacramento 3 Boiling 30 sec 48 500 Sacramento 4 Boiling 30 sec 56 496 Sacramento 1 Boiling 60 sec 52 500 Sacramento 2 Boiling 60 sec 67 470 Sacramento 3 Boiling 60 sec 43 495 Sacramento 4 Boiling 60 sec 48 500 Sacramento 1 Control 0 000 Sacramento 2 Control 3 171 Sacramento 3 Control 3 171 Sacramento 4 Control 099 Sacramento 1 Percussion 1 min 57 495 Sacramento 2 Percussion 1 min 61 488 Sacramento 3 Percussion 1 min 62 485 Sacramento 4 Percussion 1 min 55 497 Sacramento 1 Percussion 2 min 84 367 Sacramento 2 Percussion 2 min 70 458 Sacramento 3 Percussion 2 min 81 392 Sacramento 4 Percussion 2 min 69 462 Sacramento 1 Percussion 4 min 91 286 Sacramento 2 Percussion 4 min 84 367 Sacramento 3 Percussion 4 min 84 367 Sacramento 4 Percussion 4 min 94 237 Sacramento 1 Percussion 8 min 82 384 Sacramento 2 Percussion 8 min 81 392 Sacramento 3 Percussion 8 min 82 384 Sacramento 4 Percussion 8 min 81 392 Sandia 1 Immersion 76 427 Sandia 2 Immersion 70 458 Sandia 3 Immersion 70 458 Sandia 4 Immersion 81 392 Sandia 1 Boiling 30 sec 78 414 Sandia 2 Boiling 30 sec 71 454 Sandia 3 Boiling 30 sec 63 483 Sandia 4 Boiling 30 sec 78 414 Sandia 1 Boiling 60 sec 67 470 Sandia 2 Boiling 60 sec 65 477 Sandia 3 Boiling 60 sec 74 439 Sandia 4 Boiling 60 sec 66 474 Sandia 1 Control 16 367 Sandia 2 Control 7 255
70
Table B 1 Continued
Source Block Treatment Gennination Percentage Standard Error
Sandia 3 Control 8 271 Sandia 4 Control 16 367 Sandia 1 Percussion 1 min 67 470 Sandia 2 Percussion 1 min 61 488 Sandia 3 Percussion 1 min 66 474 Sandia 4 Percussion 1 min 59 492 Sandia I Percussion 2 min 82 384 Sandia 2 Percussion 2 min 83 376 Sandia 3 Percussion 2 min 81 392 Sandia 4 Percussion 2 min 84 367 Sandia 1 Percussion 4 min 89 313 Sandia 2 Percussion 4 min 86 347 Sandia 3 Percussion 4 min 89 313 Sandia 4 Percussion 4 min 85 357 Sandia 1 Percussion 8 min 80 400 Sandia 2 Percussion 8 min 77 421 Sandia 3 Percussion 8 min 69 462 Sandia 4 Percussion 8 min 66 474
LITERATURE CITED
Ashby WC Vogel WG Rogers NF 1985 Black locust in the reclamation equation General Technical Report Northeastern Forest Experiment Station USDA Forest Service NE-I05 12 p
Ballard LAT 1973 Physical barriers to gennination Seed Sci Technoll 285-303
___ 1976 Strophiolar water conduction in the seeds ofTrifolieae induced by action on the testa at nonstrophiolar sites Austr J Plant Physiol3 465-469
Barton LV 1947 Special studies on seed coat impenneability Contrib Boyce Thompson Inst 14 355-362
Baskin CC Baskin JM 1998 Seeds Ecology biogeography and evolution of donnancy and gennination Academic Press London UK 666 p
Bewley JD 1997 Seed germination and donnancy Plant Ce119 1055-1066
Bewley JD Black M 1994 Seeds physiology of development and germination 2nd ed Plenum Press New York NY 445 p
Briscoe CB 1996 Growing Nitrogen Fixing Trees in Acid Soils In MH Powell (ed) Nitrogen Fixing Trees for Acid Soils- A Field ManuaL Winrock International Morrilton AR 22 p
Copeland LO McDonald MB 1995 Principles of Seed Science and Technology 3rded Chapman amp Hall New York NY 409 p
Cox RE Klett JE 1984 Seed gennination requirements of native Colorado plants for use in the landscape Plant Propag 30(2) 6-10
Dell B 1980 Structure and function ofthe strophiolar plug in seeds ofAlbizia Lophantha Am J Bot 67 556-563
Dick-Peddie WA 1993 New Mexico Vegetation Past Present and Future University ofNew Mexico Press Albuquerque NM 244 p
Dreesen DR 2001 Propagation protocol for production ofcontainer Robinia neomexicana plants Los Lunas Plant Materials Center Los Lunas New Mexico In Native Plant Network URL httpwwwnativeplantnetworkorg (accessed 9 January 2002) Moscow (ID) University ofIdaho CoIl Natural Resources For Res Nursery
72
Dreesen DR Harrington JT 1997 Propagation of native plants for restoration projects in the Southwestern US- Preliminary Investigations In Landis TD Thompson JR tech coords National Proceedings Forest and Conservation Nursery Assocs Gen Tech Rep PNW-GTR-419 Portland OR USDA For Ser PNW Res Sta 77-88
Gottfried GJ 1980 Control ofNew Mexican locust and the effect on planted ponderosa pine in central Arizona US For Ser Res Note RM-386 Rocky Mountain For Range ExpStn Fort Collins CO
Gosling PG Samuel YK Jones SK 1995 A systematic examination of germination temperature chipping and water temperaturesoak duration pretreatments on the seeds ofLeucaena leucocephala Seed Sci amp Technol 23521-532
Hamly DH 1932 Softening of the seeds of Melilotus alba Bot Gazette 93 345shy375
Hanna PJ 1984 Anatomical features of the seed coat ofAcacia kempeana (Mueller) which relate to increased germination rate induced by heat treatment New Phytol 96 23-29
Hanks JP Dick-Peddie WA 1974 Vegetation patterns of the White Mountains New Mexico Southwest Naturalist 18(4) 371-382
Hine S Harrington JT Dreesen DR 1997 Seed scarification requirements for Robinia neomexicana In Harrington JTtech coord Proc Third SouthwestemContainer Growers Conf Feb 12-13 1997 Ft Collins CO
Hopkinson JM 1993 The strophiole of leguminous seeds Newsletter- IntI Herbage Seed Production Res Grp 18 7-8
---- 1997 Differences in hardseededness between Cunningham and Tarramba leucaena In Leucnet News The Newsletter of the International LeucaenaRes and Devlt Netwk No4
ISTA (International Seed Testing Association) 1999 International rules for seed testing rules Seed Sci amp Technol27 Supplement
Kelly KM van Staden J 1987 The lens as the site of permeability in the papilionoid seed Aspalathus linearis J Plant Physiol 128(45) 395-404
73
Keresztesi B 1988 Black locust the tree of agriculture Outlook on Agr 17(2) 77~85
Kigel 1 and Galili G 1995 Seed development and germination Marcel Dekker New York 853 p
Klemmedson 10 1994 New Mexico locust and parent material Influence on macronutrients of forest floor and soil Soil Sci Soc Am 1 58 974~980
Kuo WH Tam AW 1988 The pathway of water absorption of mung bean seeds Seed Sci amp Technol16(1) 139~144
Leadem CL 1997 Dormancy--unlocking seed secrets In Landis TD Thompson lR tech coords National Proceedings Forestand Conservation Nursery Assocs Gen Tech Rep PNW-G TR-419 Portland OR USDA For Ser PNW Res Sta 43-52
Lin Y Wagner MR Seweyestewa A Honahni MD DeGomez M McMillin JD 1996 Influence of cultural treatments on seed germination and seedling growth of Robinia neomexicana Forest Farm and Community Tree Res Reports Volume 1
Marunda CT 1990 Effects of seed pretreatment on the development ofAcacia auriculforinis and A holosericea seedlings ACIAR Proceedings Series No 28
Meginnis HG 1937 Sulphuric acid treatment to increase germination of black locust seed USDA Circ 453 34 p
Miklas PN Towensend CE Ladd SL 1987 Seedcoat anatomy and the scarification ofcicer milkvetch seed Crop Sci 27 766-772
Monsen SB 1984 Use of shrubs on mine spoils In The Challenge ofProducing Native Plants for the Intermountain Area Proceedings Intermountain Nurserymans Association 1983 conference Las Vegas NV compo PM Murphy 26-31 Ogden UT USDA For Ser Gen Tech Report INT-l68
Olson DF 1974 Robinia L Locust In Seeds of Woody Plants in the US tech coord CS Schopmeyer 728-731 USDA For Ser Agr Handbk No450
Rehman S Loescher RNl Harris P1C 1998 Dormancy breaking and germination ofAcacia salicina Lindl seeds Seed Sci amp Techno127 553-557
Rolston MP 1978 Water impermeable seed dormancy Bot Rev 44 365-396
74
SAS Institute Inc 1989 SASSTAT Users Guide Version 6 4th Edition Volume 1 Cary NC SAS Institute Inc 943 p
SerratoValenti G Conara L Ferrando M Modenesi P 1993 Structural and histochemical features ofStylosanthes scabra (Leguminosae Papilionoidae) seed coat as related to water entry Can J Bot 71 834-840
Simpson BJ 1988 A field guide to Texas trees Austin TX Texas Monthly Press 372p
Tran VN Cavanagh AK 1984 Structural aspects ofdormancy In Seed Physiology Vol 2 Murray DR (ed) p1-44 Academic Press Australia
USDA Forest Service 1948 Woody-plant seed manual USDA Misc Pub1654 416p
Vines RA 1960 Trees Shrubs and Woody Vines of the Southwest Austin Univ ofTexas Press 1104 p
Wagner MR Larson RE Craig TP 1992 Ecology and silvics ofNew Mexico locust (Robinia neomexicana) with an emphasis on the arboreal form p53-57 In JW Hanover K Miller and S Plesko (eds) Proc Intemat Confon Black Locust Biology Culture and Utilization Mich State Univ USA
Wilson JK 1944 Immersing seeds of species of Robinia in boiling water hastens germination J For 42 453-454
Zimmerman RW Carpenter SB 1980 First-year coppice production from a 5shyyear old black locust stand on surface mine spoil p309-314 Centr Hardwd For ConfProc University ofMissouri Columbia
Table B 1 Gennination percents and standard errors for source block and treatment for New Mexico locust
Source Block Treatment Germination Percentage Standard Error
Capitan 1 Immersion 74 437 Capitan 2 Immersion 69 462 Capitan 3 Immersion 71 454 Capitan 4 Immersion 67 470 Capitan Boiling 30 sec 79 407 Capitan 2 Boiling 30 sec 76 427 Capitan 3 Boiling 30 sec 72 449 Capitan 4 Boiling 30 sec 76 427 Capitan Boiling 60 sec 71 454 Capitan 2 Boiling 60 sec 54 498 Capitan 3 Boiling 60 sec 67 470 Capitan 4 Boiling 60 sec 76 427 Capitan I Control 5 218 Capitan 2 Control 7 255 Capitan 3 Control 4 196 Capitan 4 Control 5 218 Capitan 1 Percussion I min 47 499 Capitan 2 Percussion I min
470
Capitan Percussion I min 39 488
Capitan 4 Percussion I min 38 485 Capitan I Percussion 2 min 55 497 Capitan Capitan
2
Percussion 2 min Percussion 2 min
71 65
454 477
Capitan 4 Percussion 2 min 56 496 Capitan I Percussion 4 min 86 347 Capitan 2 Percussion 4 min 79 407 Capitan 3 Percussion 4 min 81 392 Capitan 4 Percussion 4 min 82 384 Capitan I Percussion 8 min 90 300 Capitan 2 Percussion 8 min 88 325 Capitan 3 Percussion 8 min 90 300 Capitan 4 Percussion 8 min 90 300 Gila I Immersion 71 454 Gila 2 Immersion 77 421 Gila
Immersion 75 433
Gila 4 Immersion 72 449
Gila I Boiling 30 sec 68 466
Gila 2 Boiling 30 sec 81 392
Gila 3 Boiling 30 sec 54 498
Gila 4 Boiling 30 sec 63 483
Gila I Boiling 60 sec 55 497
Gila 2 Boiling 60 sec 68 466
Gila 3 Boiling 60 sec 57 495
Gila 4 Boiling 60 sec 70 458
65
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Gila 2 Control 13 336 Gila 3 Control 13 336 Gila 4 Control 20 400 Gila 1 Percussion 1 min 66 474 Gila 2 Percussion 1 min 57 495 Gila 3 Percussion 1 min 73 444 Gila 4 Percussion 1 min 82 384 Gila 1 Percussion 2 min 81 392 Gila 2 Percussion 2 min 86 347 Gila 3 Percussion 2 min 85 357 Gila 4 Percussion 2 min 54 498 Gila 1 Percussion 4 min 79 407 Gila 2 Percussion 4 min 83 376 Gila 3 Percussion 4 min 84 367 Gila 4 Percussion 4 min 79 407 Gila 1 Percussion 8 min 51 500 Gila 2 Percussion 8 min 76 427 Gila 3 Percussion 8 min 52 500 Gila 4 Percussion 8 min 74 439 Holman 1 Immersion 83 376 Holman 2 Immersion 86 347 Holman 3 Immersion 81 392 Holman 4 Immersion 90 300 Holman 1 Boiling 30 sec 90 300 Holman 2 Boiling 30 sec 85 357 Holman 3 Boiling 30 sec 87 336 Holman 4 Boiling 30 sec 83 376 Holman 1 Boiling 60 sec 79 407 Holman 2 Boiling 60 sec 80 400 Holman 3 Boiling 60 sec 86 347 Holman 4 Boiling 60 sec 93 255 Holman Control 25 433 Holman 2 Control 19 392 Holman Control 21 407
Holman 4 Control 24 427
Holman Percussion 1 min 77 421
Holman 2 Percussion 1 min 85 357
Holman 3 Percussion 1 min 80 400
Holman 4 Percussion 1 min 89 313
Holman 1 Percussion 2 min 88 325
Holman 2 Percussion 2 min 96 196
Holman 3 Percussion 2 min 93 255
Holman 4 Percussion 2 min 96 196
Holman 1 Percussion 4 min 92 271
Holman 2 Percussion 4 min 97 171
66
I
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Holman 3 Percussion 4 min 91 286 Holman 4 Percussion 4 min 96 196 Holman 1 Percussion 8 min 88 325 Holman 2 Percussion 8 min 85 357 Holman 3 Percussion 8 min 83 376 Holman 4 Percussion 8 min 82 384 Jemez 1 Immersion 53 499 Jemez 2 Immersion 44 496 Jemez 3 Immersion 52 500 Jemez 4 Immersion 70 458 Jemez 1 Boiling 30 sec 58 494 Jemez 2 Boiling 30 sec 55 497 Jemez 3 Boiling 30 sec 48 500 Jemez 4 Boiling 30 sec 65 477 Jemez 1 Boiling 60 sec 68 466 Jemez 2 Boiling 60 sec 63 483 Jemez 3 Boiling 60 sec 73 444 Jemez 4 Boiling 60 sec 89 313 Jemez 1 Control 2 140 Jemez 2 Control 9 286 Jemez 3 Control 6 237 Jemez 4 Control 8 271 Jemez 1 Percussion 1 min 58 494 Jemez 2 Percussion 1 min 63 483 Jemez 3 Percussion 1 min 77 421 Jemez 4 Percussion 1 min 89 313 Jemez 1 Percussion 2 min 68 466 Jemez 2 Percussion 2 min 64 480 Jemez 3 Percussion 2 min 80 400 Jemez 4 Percussion 2 min 83 376 Jemez Percussion 4 min 68 466 Jemez 2 Percussion 4 min 79 407 Jemez 3 Percussion 4 min 70 458 Jemez 4 Percussion 4 min 83 376 Jemez 1 Percussion 8 min 52 500 Jemez 2 Percussion 8 min 59 492 Jemez 3 Percussion 8 min 61 488 Jemez 4 Percussion 8 min 70 458 Magdalena 1 Immersion 69 462 Magdalena 2 Immersion 69 462 Magdalena J Immersion 70 458 Magdalena 4 Immersion 85 357 Magdalena 1 Boiling 30 sec 65 477 Magdalena 2 Boiling 30 sec 71 454 Magdalena 3 Boiling 30 sec 68 466
67
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Magdalena 4 Boiling 30 sec 83 376 Magdalena 1 Boiling 60 sec 63 483 Magdalena 2 Boiling 60 sec 68 466 Magdalena 3 Boiling 60 sec 63 483 Magdalena 4 Boiling 60 sec 82 384 Magdalena 1 Control 11 313 Magdalena 2 Control 6 237 Magdalena 3 Control 5 218 Magdalena 4 Control 4 196 Magdalena Percussion 1 min 32 466 Magdalena 2 Percussion 1 min 45 497 Magdalena 3 Percussion 1 min 46 498 Magdalena 4 Percussion I min 36 480 Magdalena 1 Percussion 2 min 67 470 Magdalena 2 Percussion 2 min 68 466 Magdalena 3 Percussion 2 min 65 477 Magdalena 4 Percussion 2 min 81 392 Magdalena 1 Percussion 4 min 87 336 Magdalena 2 Percussion 4 min 97 171 Magdalena 3 Percussion 4 min 83 376 Magdalena 4 Percussion 4 min 97 171 Magdalena 1 Percussion 8 min 98 lAO Magdalena 2 Percussion 8 min 100 000 Magdalena 3 Percussion 8 min 96 196 Magdalena 4 Percussion 8 min 98 lAO Manzano 1 Immersion 67 470 Manzano 2 Immersion 57 495 Manzano 3 Immersion 72 4049 Manzano 4 Immersion 73 4044 Manzano 1 Boiling 30 sec 70 458 Manzano 2 Boiling 30 sec 64 480 Manzano 3 Boiling 30 sec 71 454 Manzano 4 Boiling 30 sec 80 400 Manzano I Boiling 60 sec 50 500 Manzano 2 Boiling 60 sec 53 499 Manzano 3 Boiling 60 sec 59 492 Manzano 4 Boiling 60 sec 74 439 Manzano I Control 4 196 Manzano 2 Control 8 271 Manzano 3 Control 1 099 Manzano 4 Control 6 237 Manzano 1 Percussion 1 min 52 500 Manzano 2 Percussion 1 min 39 488 Manzano 3 Percussion 1 min 36 480 Manzano 4 Percussion 1 min 51 500
68
Table B 1 Continued
Source Block Treatment Gerrni~ation Percentage Standard Error
Manzano Percussion 2 min 53 499 Manzano 2 Percussion 2 min 58 494 Manzano 3 Percussion 2 min 61 488 Manzano 4 Percussion 2 min 62 485 Manzano 1 Percussion 4 min 76 427 Manzano 2 Percussion 4 min 85 357 Manzano 3 Percussion 4 min 79 407 Manzano 4 Percussion 4 min 83 376 Manzano 1 Percussion 8 min 87 336 Manzano 2 Percussion 8 min 83 376 Manzano 3 Percussion 8 min 83 376 Manzano 4 Percussion 8 min 75 433 Raton 1 Immersion 54 498 Raton 2 Immersion 37 483 Raton 3 Immersion 39 488 Raton 4 Immersion 69 462 Raton 1 Boiling 30 sec 36 480 Raton 2 Boiling 30 sec 20 400 Raton 3 Boiling 30 sec 28 449 Raton 4 Boiling 30 sec 60 490 Raton 1 Boiling 60 sec 15 357 Raton 2 Boiling 60 sec 171 Raton 3 Boiling 60 sec 4 196 Raton 4 Boiling 60 sec 35 477 Raton 1 Control 4 196 Raton 2 Control 12 325 Raton 3 Control 2 140 Raton 4 Control 6 237 Raton 1 Percussion 1 min 52 500 Raton 2 Percussion I min 29 454 Raton 3 Percussion I min 35 477 Raton 4 Percussion 1 min 31 462 Raton 1 Percussion 2 min 55 497 Raton 2 Percussion 2 min 35 477 Raton 3 Percussion 2 min 33 470 Raton 4 Percussion 2 min 42 494 Raton 1 Percussion 4 min 65 477 Raton 2 Percussion 4 min 52 500 Raton Percussion 4 min 51 500 Raton 4 Percussion 4 min 53 499 Raton 1 Percussion 8 min 88 325 Raton 2 Percussion 8 min 80 400 Raton 3 Percussion 8 min 73 444 Raton 4 Percussion 8 min 74 439 Sacramento 1 Immersion 43 495
69
Table B 1 Continued
-Source Block Treatment Germination Percentage Standard Error
Sacramento 2 Immersion 30 458 Sacramento 3 Immersion 38 485 Sacramento 4 Immersion 56 496 Sacramento 1 Boiling 30 sec 47 499 Sacramento 2 Boiling 30 sec 48 500 Sacramento 3 Boiling 30 sec 48 500 Sacramento 4 Boiling 30 sec 56 496 Sacramento 1 Boiling 60 sec 52 500 Sacramento 2 Boiling 60 sec 67 470 Sacramento 3 Boiling 60 sec 43 495 Sacramento 4 Boiling 60 sec 48 500 Sacramento 1 Control 0 000 Sacramento 2 Control 3 171 Sacramento 3 Control 3 171 Sacramento 4 Control 099 Sacramento 1 Percussion 1 min 57 495 Sacramento 2 Percussion 1 min 61 488 Sacramento 3 Percussion 1 min 62 485 Sacramento 4 Percussion 1 min 55 497 Sacramento 1 Percussion 2 min 84 367 Sacramento 2 Percussion 2 min 70 458 Sacramento 3 Percussion 2 min 81 392 Sacramento 4 Percussion 2 min 69 462 Sacramento 1 Percussion 4 min 91 286 Sacramento 2 Percussion 4 min 84 367 Sacramento 3 Percussion 4 min 84 367 Sacramento 4 Percussion 4 min 94 237 Sacramento 1 Percussion 8 min 82 384 Sacramento 2 Percussion 8 min 81 392 Sacramento 3 Percussion 8 min 82 384 Sacramento 4 Percussion 8 min 81 392 Sandia 1 Immersion 76 427 Sandia 2 Immersion 70 458 Sandia 3 Immersion 70 458 Sandia 4 Immersion 81 392 Sandia 1 Boiling 30 sec 78 414 Sandia 2 Boiling 30 sec 71 454 Sandia 3 Boiling 30 sec 63 483 Sandia 4 Boiling 30 sec 78 414 Sandia 1 Boiling 60 sec 67 470 Sandia 2 Boiling 60 sec 65 477 Sandia 3 Boiling 60 sec 74 439 Sandia 4 Boiling 60 sec 66 474 Sandia 1 Control 16 367 Sandia 2 Control 7 255
70
Table B 1 Continued
Source Block Treatment Gennination Percentage Standard Error
Sandia 3 Control 8 271 Sandia 4 Control 16 367 Sandia 1 Percussion 1 min 67 470 Sandia 2 Percussion 1 min 61 488 Sandia 3 Percussion 1 min 66 474 Sandia 4 Percussion 1 min 59 492 Sandia I Percussion 2 min 82 384 Sandia 2 Percussion 2 min 83 376 Sandia 3 Percussion 2 min 81 392 Sandia 4 Percussion 2 min 84 367 Sandia 1 Percussion 4 min 89 313 Sandia 2 Percussion 4 min 86 347 Sandia 3 Percussion 4 min 89 313 Sandia 4 Percussion 4 min 85 357 Sandia 1 Percussion 8 min 80 400 Sandia 2 Percussion 8 min 77 421 Sandia 3 Percussion 8 min 69 462 Sandia 4 Percussion 8 min 66 474
LITERATURE CITED
Ashby WC Vogel WG Rogers NF 1985 Black locust in the reclamation equation General Technical Report Northeastern Forest Experiment Station USDA Forest Service NE-I05 12 p
Ballard LAT 1973 Physical barriers to gennination Seed Sci Technoll 285-303
___ 1976 Strophiolar water conduction in the seeds ofTrifolieae induced by action on the testa at nonstrophiolar sites Austr J Plant Physiol3 465-469
Barton LV 1947 Special studies on seed coat impenneability Contrib Boyce Thompson Inst 14 355-362
Baskin CC Baskin JM 1998 Seeds Ecology biogeography and evolution of donnancy and gennination Academic Press London UK 666 p
Bewley JD 1997 Seed germination and donnancy Plant Ce119 1055-1066
Bewley JD Black M 1994 Seeds physiology of development and germination 2nd ed Plenum Press New York NY 445 p
Briscoe CB 1996 Growing Nitrogen Fixing Trees in Acid Soils In MH Powell (ed) Nitrogen Fixing Trees for Acid Soils- A Field ManuaL Winrock International Morrilton AR 22 p
Copeland LO McDonald MB 1995 Principles of Seed Science and Technology 3rded Chapman amp Hall New York NY 409 p
Cox RE Klett JE 1984 Seed gennination requirements of native Colorado plants for use in the landscape Plant Propag 30(2) 6-10
Dell B 1980 Structure and function ofthe strophiolar plug in seeds ofAlbizia Lophantha Am J Bot 67 556-563
Dick-Peddie WA 1993 New Mexico Vegetation Past Present and Future University ofNew Mexico Press Albuquerque NM 244 p
Dreesen DR 2001 Propagation protocol for production ofcontainer Robinia neomexicana plants Los Lunas Plant Materials Center Los Lunas New Mexico In Native Plant Network URL httpwwwnativeplantnetworkorg (accessed 9 January 2002) Moscow (ID) University ofIdaho CoIl Natural Resources For Res Nursery
72
Dreesen DR Harrington JT 1997 Propagation of native plants for restoration projects in the Southwestern US- Preliminary Investigations In Landis TD Thompson JR tech coords National Proceedings Forest and Conservation Nursery Assocs Gen Tech Rep PNW-GTR-419 Portland OR USDA For Ser PNW Res Sta 77-88
Gottfried GJ 1980 Control ofNew Mexican locust and the effect on planted ponderosa pine in central Arizona US For Ser Res Note RM-386 Rocky Mountain For Range ExpStn Fort Collins CO
Gosling PG Samuel YK Jones SK 1995 A systematic examination of germination temperature chipping and water temperaturesoak duration pretreatments on the seeds ofLeucaena leucocephala Seed Sci amp Technol 23521-532
Hamly DH 1932 Softening of the seeds of Melilotus alba Bot Gazette 93 345shy375
Hanna PJ 1984 Anatomical features of the seed coat ofAcacia kempeana (Mueller) which relate to increased germination rate induced by heat treatment New Phytol 96 23-29
Hanks JP Dick-Peddie WA 1974 Vegetation patterns of the White Mountains New Mexico Southwest Naturalist 18(4) 371-382
Hine S Harrington JT Dreesen DR 1997 Seed scarification requirements for Robinia neomexicana In Harrington JTtech coord Proc Third SouthwestemContainer Growers Conf Feb 12-13 1997 Ft Collins CO
Hopkinson JM 1993 The strophiole of leguminous seeds Newsletter- IntI Herbage Seed Production Res Grp 18 7-8
---- 1997 Differences in hardseededness between Cunningham and Tarramba leucaena In Leucnet News The Newsletter of the International LeucaenaRes and Devlt Netwk No4
ISTA (International Seed Testing Association) 1999 International rules for seed testing rules Seed Sci amp Technol27 Supplement
Kelly KM van Staden J 1987 The lens as the site of permeability in the papilionoid seed Aspalathus linearis J Plant Physiol 128(45) 395-404
73
Keresztesi B 1988 Black locust the tree of agriculture Outlook on Agr 17(2) 77~85
Kigel 1 and Galili G 1995 Seed development and germination Marcel Dekker New York 853 p
Klemmedson 10 1994 New Mexico locust and parent material Influence on macronutrients of forest floor and soil Soil Sci Soc Am 1 58 974~980
Kuo WH Tam AW 1988 The pathway of water absorption of mung bean seeds Seed Sci amp Technol16(1) 139~144
Leadem CL 1997 Dormancy--unlocking seed secrets In Landis TD Thompson lR tech coords National Proceedings Forestand Conservation Nursery Assocs Gen Tech Rep PNW-G TR-419 Portland OR USDA For Ser PNW Res Sta 43-52
Lin Y Wagner MR Seweyestewa A Honahni MD DeGomez M McMillin JD 1996 Influence of cultural treatments on seed germination and seedling growth of Robinia neomexicana Forest Farm and Community Tree Res Reports Volume 1
Marunda CT 1990 Effects of seed pretreatment on the development ofAcacia auriculforinis and A holosericea seedlings ACIAR Proceedings Series No 28
Meginnis HG 1937 Sulphuric acid treatment to increase germination of black locust seed USDA Circ 453 34 p
Miklas PN Towensend CE Ladd SL 1987 Seedcoat anatomy and the scarification ofcicer milkvetch seed Crop Sci 27 766-772
Monsen SB 1984 Use of shrubs on mine spoils In The Challenge ofProducing Native Plants for the Intermountain Area Proceedings Intermountain Nurserymans Association 1983 conference Las Vegas NV compo PM Murphy 26-31 Ogden UT USDA For Ser Gen Tech Report INT-l68
Olson DF 1974 Robinia L Locust In Seeds of Woody Plants in the US tech coord CS Schopmeyer 728-731 USDA For Ser Agr Handbk No450
Rehman S Loescher RNl Harris P1C 1998 Dormancy breaking and germination ofAcacia salicina Lindl seeds Seed Sci amp Techno127 553-557
Rolston MP 1978 Water impermeable seed dormancy Bot Rev 44 365-396
74
SAS Institute Inc 1989 SASSTAT Users Guide Version 6 4th Edition Volume 1 Cary NC SAS Institute Inc 943 p
SerratoValenti G Conara L Ferrando M Modenesi P 1993 Structural and histochemical features ofStylosanthes scabra (Leguminosae Papilionoidae) seed coat as related to water entry Can J Bot 71 834-840
Simpson BJ 1988 A field guide to Texas trees Austin TX Texas Monthly Press 372p
Tran VN Cavanagh AK 1984 Structural aspects ofdormancy In Seed Physiology Vol 2 Murray DR (ed) p1-44 Academic Press Australia
USDA Forest Service 1948 Woody-plant seed manual USDA Misc Pub1654 416p
Vines RA 1960 Trees Shrubs and Woody Vines of the Southwest Austin Univ ofTexas Press 1104 p
Wagner MR Larson RE Craig TP 1992 Ecology and silvics ofNew Mexico locust (Robinia neomexicana) with an emphasis on the arboreal form p53-57 In JW Hanover K Miller and S Plesko (eds) Proc Intemat Confon Black Locust Biology Culture and Utilization Mich State Univ USA
Wilson JK 1944 Immersing seeds of species of Robinia in boiling water hastens germination J For 42 453-454
Zimmerman RW Carpenter SB 1980 First-year coppice production from a 5shyyear old black locust stand on surface mine spoil p309-314 Centr Hardwd For ConfProc University ofMissouri Columbia
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Gila 2 Control 13 336 Gila 3 Control 13 336 Gila 4 Control 20 400 Gila 1 Percussion 1 min 66 474 Gila 2 Percussion 1 min 57 495 Gila 3 Percussion 1 min 73 444 Gila 4 Percussion 1 min 82 384 Gila 1 Percussion 2 min 81 392 Gila 2 Percussion 2 min 86 347 Gila 3 Percussion 2 min 85 357 Gila 4 Percussion 2 min 54 498 Gila 1 Percussion 4 min 79 407 Gila 2 Percussion 4 min 83 376 Gila 3 Percussion 4 min 84 367 Gila 4 Percussion 4 min 79 407 Gila 1 Percussion 8 min 51 500 Gila 2 Percussion 8 min 76 427 Gila 3 Percussion 8 min 52 500 Gila 4 Percussion 8 min 74 439 Holman 1 Immersion 83 376 Holman 2 Immersion 86 347 Holman 3 Immersion 81 392 Holman 4 Immersion 90 300 Holman 1 Boiling 30 sec 90 300 Holman 2 Boiling 30 sec 85 357 Holman 3 Boiling 30 sec 87 336 Holman 4 Boiling 30 sec 83 376 Holman 1 Boiling 60 sec 79 407 Holman 2 Boiling 60 sec 80 400 Holman 3 Boiling 60 sec 86 347 Holman 4 Boiling 60 sec 93 255 Holman Control 25 433 Holman 2 Control 19 392 Holman Control 21 407
Holman 4 Control 24 427
Holman Percussion 1 min 77 421
Holman 2 Percussion 1 min 85 357
Holman 3 Percussion 1 min 80 400
Holman 4 Percussion 1 min 89 313
Holman 1 Percussion 2 min 88 325
Holman 2 Percussion 2 min 96 196
Holman 3 Percussion 2 min 93 255
Holman 4 Percussion 2 min 96 196
Holman 1 Percussion 4 min 92 271
Holman 2 Percussion 4 min 97 171
66
I
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Holman 3 Percussion 4 min 91 286 Holman 4 Percussion 4 min 96 196 Holman 1 Percussion 8 min 88 325 Holman 2 Percussion 8 min 85 357 Holman 3 Percussion 8 min 83 376 Holman 4 Percussion 8 min 82 384 Jemez 1 Immersion 53 499 Jemez 2 Immersion 44 496 Jemez 3 Immersion 52 500 Jemez 4 Immersion 70 458 Jemez 1 Boiling 30 sec 58 494 Jemez 2 Boiling 30 sec 55 497 Jemez 3 Boiling 30 sec 48 500 Jemez 4 Boiling 30 sec 65 477 Jemez 1 Boiling 60 sec 68 466 Jemez 2 Boiling 60 sec 63 483 Jemez 3 Boiling 60 sec 73 444 Jemez 4 Boiling 60 sec 89 313 Jemez 1 Control 2 140 Jemez 2 Control 9 286 Jemez 3 Control 6 237 Jemez 4 Control 8 271 Jemez 1 Percussion 1 min 58 494 Jemez 2 Percussion 1 min 63 483 Jemez 3 Percussion 1 min 77 421 Jemez 4 Percussion 1 min 89 313 Jemez 1 Percussion 2 min 68 466 Jemez 2 Percussion 2 min 64 480 Jemez 3 Percussion 2 min 80 400 Jemez 4 Percussion 2 min 83 376 Jemez Percussion 4 min 68 466 Jemez 2 Percussion 4 min 79 407 Jemez 3 Percussion 4 min 70 458 Jemez 4 Percussion 4 min 83 376 Jemez 1 Percussion 8 min 52 500 Jemez 2 Percussion 8 min 59 492 Jemez 3 Percussion 8 min 61 488 Jemez 4 Percussion 8 min 70 458 Magdalena 1 Immersion 69 462 Magdalena 2 Immersion 69 462 Magdalena J Immersion 70 458 Magdalena 4 Immersion 85 357 Magdalena 1 Boiling 30 sec 65 477 Magdalena 2 Boiling 30 sec 71 454 Magdalena 3 Boiling 30 sec 68 466
67
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Magdalena 4 Boiling 30 sec 83 376 Magdalena 1 Boiling 60 sec 63 483 Magdalena 2 Boiling 60 sec 68 466 Magdalena 3 Boiling 60 sec 63 483 Magdalena 4 Boiling 60 sec 82 384 Magdalena 1 Control 11 313 Magdalena 2 Control 6 237 Magdalena 3 Control 5 218 Magdalena 4 Control 4 196 Magdalena Percussion 1 min 32 466 Magdalena 2 Percussion 1 min 45 497 Magdalena 3 Percussion 1 min 46 498 Magdalena 4 Percussion I min 36 480 Magdalena 1 Percussion 2 min 67 470 Magdalena 2 Percussion 2 min 68 466 Magdalena 3 Percussion 2 min 65 477 Magdalena 4 Percussion 2 min 81 392 Magdalena 1 Percussion 4 min 87 336 Magdalena 2 Percussion 4 min 97 171 Magdalena 3 Percussion 4 min 83 376 Magdalena 4 Percussion 4 min 97 171 Magdalena 1 Percussion 8 min 98 lAO Magdalena 2 Percussion 8 min 100 000 Magdalena 3 Percussion 8 min 96 196 Magdalena 4 Percussion 8 min 98 lAO Manzano 1 Immersion 67 470 Manzano 2 Immersion 57 495 Manzano 3 Immersion 72 4049 Manzano 4 Immersion 73 4044 Manzano 1 Boiling 30 sec 70 458 Manzano 2 Boiling 30 sec 64 480 Manzano 3 Boiling 30 sec 71 454 Manzano 4 Boiling 30 sec 80 400 Manzano I Boiling 60 sec 50 500 Manzano 2 Boiling 60 sec 53 499 Manzano 3 Boiling 60 sec 59 492 Manzano 4 Boiling 60 sec 74 439 Manzano I Control 4 196 Manzano 2 Control 8 271 Manzano 3 Control 1 099 Manzano 4 Control 6 237 Manzano 1 Percussion 1 min 52 500 Manzano 2 Percussion 1 min 39 488 Manzano 3 Percussion 1 min 36 480 Manzano 4 Percussion 1 min 51 500
68
Table B 1 Continued
Source Block Treatment Gerrni~ation Percentage Standard Error
Manzano Percussion 2 min 53 499 Manzano 2 Percussion 2 min 58 494 Manzano 3 Percussion 2 min 61 488 Manzano 4 Percussion 2 min 62 485 Manzano 1 Percussion 4 min 76 427 Manzano 2 Percussion 4 min 85 357 Manzano 3 Percussion 4 min 79 407 Manzano 4 Percussion 4 min 83 376 Manzano 1 Percussion 8 min 87 336 Manzano 2 Percussion 8 min 83 376 Manzano 3 Percussion 8 min 83 376 Manzano 4 Percussion 8 min 75 433 Raton 1 Immersion 54 498 Raton 2 Immersion 37 483 Raton 3 Immersion 39 488 Raton 4 Immersion 69 462 Raton 1 Boiling 30 sec 36 480 Raton 2 Boiling 30 sec 20 400 Raton 3 Boiling 30 sec 28 449 Raton 4 Boiling 30 sec 60 490 Raton 1 Boiling 60 sec 15 357 Raton 2 Boiling 60 sec 171 Raton 3 Boiling 60 sec 4 196 Raton 4 Boiling 60 sec 35 477 Raton 1 Control 4 196 Raton 2 Control 12 325 Raton 3 Control 2 140 Raton 4 Control 6 237 Raton 1 Percussion 1 min 52 500 Raton 2 Percussion I min 29 454 Raton 3 Percussion I min 35 477 Raton 4 Percussion 1 min 31 462 Raton 1 Percussion 2 min 55 497 Raton 2 Percussion 2 min 35 477 Raton 3 Percussion 2 min 33 470 Raton 4 Percussion 2 min 42 494 Raton 1 Percussion 4 min 65 477 Raton 2 Percussion 4 min 52 500 Raton Percussion 4 min 51 500 Raton 4 Percussion 4 min 53 499 Raton 1 Percussion 8 min 88 325 Raton 2 Percussion 8 min 80 400 Raton 3 Percussion 8 min 73 444 Raton 4 Percussion 8 min 74 439 Sacramento 1 Immersion 43 495
69
Table B 1 Continued
-Source Block Treatment Germination Percentage Standard Error
Sacramento 2 Immersion 30 458 Sacramento 3 Immersion 38 485 Sacramento 4 Immersion 56 496 Sacramento 1 Boiling 30 sec 47 499 Sacramento 2 Boiling 30 sec 48 500 Sacramento 3 Boiling 30 sec 48 500 Sacramento 4 Boiling 30 sec 56 496 Sacramento 1 Boiling 60 sec 52 500 Sacramento 2 Boiling 60 sec 67 470 Sacramento 3 Boiling 60 sec 43 495 Sacramento 4 Boiling 60 sec 48 500 Sacramento 1 Control 0 000 Sacramento 2 Control 3 171 Sacramento 3 Control 3 171 Sacramento 4 Control 099 Sacramento 1 Percussion 1 min 57 495 Sacramento 2 Percussion 1 min 61 488 Sacramento 3 Percussion 1 min 62 485 Sacramento 4 Percussion 1 min 55 497 Sacramento 1 Percussion 2 min 84 367 Sacramento 2 Percussion 2 min 70 458 Sacramento 3 Percussion 2 min 81 392 Sacramento 4 Percussion 2 min 69 462 Sacramento 1 Percussion 4 min 91 286 Sacramento 2 Percussion 4 min 84 367 Sacramento 3 Percussion 4 min 84 367 Sacramento 4 Percussion 4 min 94 237 Sacramento 1 Percussion 8 min 82 384 Sacramento 2 Percussion 8 min 81 392 Sacramento 3 Percussion 8 min 82 384 Sacramento 4 Percussion 8 min 81 392 Sandia 1 Immersion 76 427 Sandia 2 Immersion 70 458 Sandia 3 Immersion 70 458 Sandia 4 Immersion 81 392 Sandia 1 Boiling 30 sec 78 414 Sandia 2 Boiling 30 sec 71 454 Sandia 3 Boiling 30 sec 63 483 Sandia 4 Boiling 30 sec 78 414 Sandia 1 Boiling 60 sec 67 470 Sandia 2 Boiling 60 sec 65 477 Sandia 3 Boiling 60 sec 74 439 Sandia 4 Boiling 60 sec 66 474 Sandia 1 Control 16 367 Sandia 2 Control 7 255
70
Table B 1 Continued
Source Block Treatment Gennination Percentage Standard Error
Sandia 3 Control 8 271 Sandia 4 Control 16 367 Sandia 1 Percussion 1 min 67 470 Sandia 2 Percussion 1 min 61 488 Sandia 3 Percussion 1 min 66 474 Sandia 4 Percussion 1 min 59 492 Sandia I Percussion 2 min 82 384 Sandia 2 Percussion 2 min 83 376 Sandia 3 Percussion 2 min 81 392 Sandia 4 Percussion 2 min 84 367 Sandia 1 Percussion 4 min 89 313 Sandia 2 Percussion 4 min 86 347 Sandia 3 Percussion 4 min 89 313 Sandia 4 Percussion 4 min 85 357 Sandia 1 Percussion 8 min 80 400 Sandia 2 Percussion 8 min 77 421 Sandia 3 Percussion 8 min 69 462 Sandia 4 Percussion 8 min 66 474
LITERATURE CITED
Ashby WC Vogel WG Rogers NF 1985 Black locust in the reclamation equation General Technical Report Northeastern Forest Experiment Station USDA Forest Service NE-I05 12 p
Ballard LAT 1973 Physical barriers to gennination Seed Sci Technoll 285-303
___ 1976 Strophiolar water conduction in the seeds ofTrifolieae induced by action on the testa at nonstrophiolar sites Austr J Plant Physiol3 465-469
Barton LV 1947 Special studies on seed coat impenneability Contrib Boyce Thompson Inst 14 355-362
Baskin CC Baskin JM 1998 Seeds Ecology biogeography and evolution of donnancy and gennination Academic Press London UK 666 p
Bewley JD 1997 Seed germination and donnancy Plant Ce119 1055-1066
Bewley JD Black M 1994 Seeds physiology of development and germination 2nd ed Plenum Press New York NY 445 p
Briscoe CB 1996 Growing Nitrogen Fixing Trees in Acid Soils In MH Powell (ed) Nitrogen Fixing Trees for Acid Soils- A Field ManuaL Winrock International Morrilton AR 22 p
Copeland LO McDonald MB 1995 Principles of Seed Science and Technology 3rded Chapman amp Hall New York NY 409 p
Cox RE Klett JE 1984 Seed gennination requirements of native Colorado plants for use in the landscape Plant Propag 30(2) 6-10
Dell B 1980 Structure and function ofthe strophiolar plug in seeds ofAlbizia Lophantha Am J Bot 67 556-563
Dick-Peddie WA 1993 New Mexico Vegetation Past Present and Future University ofNew Mexico Press Albuquerque NM 244 p
Dreesen DR 2001 Propagation protocol for production ofcontainer Robinia neomexicana plants Los Lunas Plant Materials Center Los Lunas New Mexico In Native Plant Network URL httpwwwnativeplantnetworkorg (accessed 9 January 2002) Moscow (ID) University ofIdaho CoIl Natural Resources For Res Nursery
72
Dreesen DR Harrington JT 1997 Propagation of native plants for restoration projects in the Southwestern US- Preliminary Investigations In Landis TD Thompson JR tech coords National Proceedings Forest and Conservation Nursery Assocs Gen Tech Rep PNW-GTR-419 Portland OR USDA For Ser PNW Res Sta 77-88
Gottfried GJ 1980 Control ofNew Mexican locust and the effect on planted ponderosa pine in central Arizona US For Ser Res Note RM-386 Rocky Mountain For Range ExpStn Fort Collins CO
Gosling PG Samuel YK Jones SK 1995 A systematic examination of germination temperature chipping and water temperaturesoak duration pretreatments on the seeds ofLeucaena leucocephala Seed Sci amp Technol 23521-532
Hamly DH 1932 Softening of the seeds of Melilotus alba Bot Gazette 93 345shy375
Hanna PJ 1984 Anatomical features of the seed coat ofAcacia kempeana (Mueller) which relate to increased germination rate induced by heat treatment New Phytol 96 23-29
Hanks JP Dick-Peddie WA 1974 Vegetation patterns of the White Mountains New Mexico Southwest Naturalist 18(4) 371-382
Hine S Harrington JT Dreesen DR 1997 Seed scarification requirements for Robinia neomexicana In Harrington JTtech coord Proc Third SouthwestemContainer Growers Conf Feb 12-13 1997 Ft Collins CO
Hopkinson JM 1993 The strophiole of leguminous seeds Newsletter- IntI Herbage Seed Production Res Grp 18 7-8
---- 1997 Differences in hardseededness between Cunningham and Tarramba leucaena In Leucnet News The Newsletter of the International LeucaenaRes and Devlt Netwk No4
ISTA (International Seed Testing Association) 1999 International rules for seed testing rules Seed Sci amp Technol27 Supplement
Kelly KM van Staden J 1987 The lens as the site of permeability in the papilionoid seed Aspalathus linearis J Plant Physiol 128(45) 395-404
73
Keresztesi B 1988 Black locust the tree of agriculture Outlook on Agr 17(2) 77~85
Kigel 1 and Galili G 1995 Seed development and germination Marcel Dekker New York 853 p
Klemmedson 10 1994 New Mexico locust and parent material Influence on macronutrients of forest floor and soil Soil Sci Soc Am 1 58 974~980
Kuo WH Tam AW 1988 The pathway of water absorption of mung bean seeds Seed Sci amp Technol16(1) 139~144
Leadem CL 1997 Dormancy--unlocking seed secrets In Landis TD Thompson lR tech coords National Proceedings Forestand Conservation Nursery Assocs Gen Tech Rep PNW-G TR-419 Portland OR USDA For Ser PNW Res Sta 43-52
Lin Y Wagner MR Seweyestewa A Honahni MD DeGomez M McMillin JD 1996 Influence of cultural treatments on seed germination and seedling growth of Robinia neomexicana Forest Farm and Community Tree Res Reports Volume 1
Marunda CT 1990 Effects of seed pretreatment on the development ofAcacia auriculforinis and A holosericea seedlings ACIAR Proceedings Series No 28
Meginnis HG 1937 Sulphuric acid treatment to increase germination of black locust seed USDA Circ 453 34 p
Miklas PN Towensend CE Ladd SL 1987 Seedcoat anatomy and the scarification ofcicer milkvetch seed Crop Sci 27 766-772
Monsen SB 1984 Use of shrubs on mine spoils In The Challenge ofProducing Native Plants for the Intermountain Area Proceedings Intermountain Nurserymans Association 1983 conference Las Vegas NV compo PM Murphy 26-31 Ogden UT USDA For Ser Gen Tech Report INT-l68
Olson DF 1974 Robinia L Locust In Seeds of Woody Plants in the US tech coord CS Schopmeyer 728-731 USDA For Ser Agr Handbk No450
Rehman S Loescher RNl Harris P1C 1998 Dormancy breaking and germination ofAcacia salicina Lindl seeds Seed Sci amp Techno127 553-557
Rolston MP 1978 Water impermeable seed dormancy Bot Rev 44 365-396
74
SAS Institute Inc 1989 SASSTAT Users Guide Version 6 4th Edition Volume 1 Cary NC SAS Institute Inc 943 p
SerratoValenti G Conara L Ferrando M Modenesi P 1993 Structural and histochemical features ofStylosanthes scabra (Leguminosae Papilionoidae) seed coat as related to water entry Can J Bot 71 834-840
Simpson BJ 1988 A field guide to Texas trees Austin TX Texas Monthly Press 372p
Tran VN Cavanagh AK 1984 Structural aspects ofdormancy In Seed Physiology Vol 2 Murray DR (ed) p1-44 Academic Press Australia
USDA Forest Service 1948 Woody-plant seed manual USDA Misc Pub1654 416p
Vines RA 1960 Trees Shrubs and Woody Vines of the Southwest Austin Univ ofTexas Press 1104 p
Wagner MR Larson RE Craig TP 1992 Ecology and silvics ofNew Mexico locust (Robinia neomexicana) with an emphasis on the arboreal form p53-57 In JW Hanover K Miller and S Plesko (eds) Proc Intemat Confon Black Locust Biology Culture and Utilization Mich State Univ USA
Wilson JK 1944 Immersing seeds of species of Robinia in boiling water hastens germination J For 42 453-454
Zimmerman RW Carpenter SB 1980 First-year coppice production from a 5shyyear old black locust stand on surface mine spoil p309-314 Centr Hardwd For ConfProc University ofMissouri Columbia
I
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Holman 3 Percussion 4 min 91 286 Holman 4 Percussion 4 min 96 196 Holman 1 Percussion 8 min 88 325 Holman 2 Percussion 8 min 85 357 Holman 3 Percussion 8 min 83 376 Holman 4 Percussion 8 min 82 384 Jemez 1 Immersion 53 499 Jemez 2 Immersion 44 496 Jemez 3 Immersion 52 500 Jemez 4 Immersion 70 458 Jemez 1 Boiling 30 sec 58 494 Jemez 2 Boiling 30 sec 55 497 Jemez 3 Boiling 30 sec 48 500 Jemez 4 Boiling 30 sec 65 477 Jemez 1 Boiling 60 sec 68 466 Jemez 2 Boiling 60 sec 63 483 Jemez 3 Boiling 60 sec 73 444 Jemez 4 Boiling 60 sec 89 313 Jemez 1 Control 2 140 Jemez 2 Control 9 286 Jemez 3 Control 6 237 Jemez 4 Control 8 271 Jemez 1 Percussion 1 min 58 494 Jemez 2 Percussion 1 min 63 483 Jemez 3 Percussion 1 min 77 421 Jemez 4 Percussion 1 min 89 313 Jemez 1 Percussion 2 min 68 466 Jemez 2 Percussion 2 min 64 480 Jemez 3 Percussion 2 min 80 400 Jemez 4 Percussion 2 min 83 376 Jemez Percussion 4 min 68 466 Jemez 2 Percussion 4 min 79 407 Jemez 3 Percussion 4 min 70 458 Jemez 4 Percussion 4 min 83 376 Jemez 1 Percussion 8 min 52 500 Jemez 2 Percussion 8 min 59 492 Jemez 3 Percussion 8 min 61 488 Jemez 4 Percussion 8 min 70 458 Magdalena 1 Immersion 69 462 Magdalena 2 Immersion 69 462 Magdalena J Immersion 70 458 Magdalena 4 Immersion 85 357 Magdalena 1 Boiling 30 sec 65 477 Magdalena 2 Boiling 30 sec 71 454 Magdalena 3 Boiling 30 sec 68 466
67
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Magdalena 4 Boiling 30 sec 83 376 Magdalena 1 Boiling 60 sec 63 483 Magdalena 2 Boiling 60 sec 68 466 Magdalena 3 Boiling 60 sec 63 483 Magdalena 4 Boiling 60 sec 82 384 Magdalena 1 Control 11 313 Magdalena 2 Control 6 237 Magdalena 3 Control 5 218 Magdalena 4 Control 4 196 Magdalena Percussion 1 min 32 466 Magdalena 2 Percussion 1 min 45 497 Magdalena 3 Percussion 1 min 46 498 Magdalena 4 Percussion I min 36 480 Magdalena 1 Percussion 2 min 67 470 Magdalena 2 Percussion 2 min 68 466 Magdalena 3 Percussion 2 min 65 477 Magdalena 4 Percussion 2 min 81 392 Magdalena 1 Percussion 4 min 87 336 Magdalena 2 Percussion 4 min 97 171 Magdalena 3 Percussion 4 min 83 376 Magdalena 4 Percussion 4 min 97 171 Magdalena 1 Percussion 8 min 98 lAO Magdalena 2 Percussion 8 min 100 000 Magdalena 3 Percussion 8 min 96 196 Magdalena 4 Percussion 8 min 98 lAO Manzano 1 Immersion 67 470 Manzano 2 Immersion 57 495 Manzano 3 Immersion 72 4049 Manzano 4 Immersion 73 4044 Manzano 1 Boiling 30 sec 70 458 Manzano 2 Boiling 30 sec 64 480 Manzano 3 Boiling 30 sec 71 454 Manzano 4 Boiling 30 sec 80 400 Manzano I Boiling 60 sec 50 500 Manzano 2 Boiling 60 sec 53 499 Manzano 3 Boiling 60 sec 59 492 Manzano 4 Boiling 60 sec 74 439 Manzano I Control 4 196 Manzano 2 Control 8 271 Manzano 3 Control 1 099 Manzano 4 Control 6 237 Manzano 1 Percussion 1 min 52 500 Manzano 2 Percussion 1 min 39 488 Manzano 3 Percussion 1 min 36 480 Manzano 4 Percussion 1 min 51 500
68
Table B 1 Continued
Source Block Treatment Gerrni~ation Percentage Standard Error
Manzano Percussion 2 min 53 499 Manzano 2 Percussion 2 min 58 494 Manzano 3 Percussion 2 min 61 488 Manzano 4 Percussion 2 min 62 485 Manzano 1 Percussion 4 min 76 427 Manzano 2 Percussion 4 min 85 357 Manzano 3 Percussion 4 min 79 407 Manzano 4 Percussion 4 min 83 376 Manzano 1 Percussion 8 min 87 336 Manzano 2 Percussion 8 min 83 376 Manzano 3 Percussion 8 min 83 376 Manzano 4 Percussion 8 min 75 433 Raton 1 Immersion 54 498 Raton 2 Immersion 37 483 Raton 3 Immersion 39 488 Raton 4 Immersion 69 462 Raton 1 Boiling 30 sec 36 480 Raton 2 Boiling 30 sec 20 400 Raton 3 Boiling 30 sec 28 449 Raton 4 Boiling 30 sec 60 490 Raton 1 Boiling 60 sec 15 357 Raton 2 Boiling 60 sec 171 Raton 3 Boiling 60 sec 4 196 Raton 4 Boiling 60 sec 35 477 Raton 1 Control 4 196 Raton 2 Control 12 325 Raton 3 Control 2 140 Raton 4 Control 6 237 Raton 1 Percussion 1 min 52 500 Raton 2 Percussion I min 29 454 Raton 3 Percussion I min 35 477 Raton 4 Percussion 1 min 31 462 Raton 1 Percussion 2 min 55 497 Raton 2 Percussion 2 min 35 477 Raton 3 Percussion 2 min 33 470 Raton 4 Percussion 2 min 42 494 Raton 1 Percussion 4 min 65 477 Raton 2 Percussion 4 min 52 500 Raton Percussion 4 min 51 500 Raton 4 Percussion 4 min 53 499 Raton 1 Percussion 8 min 88 325 Raton 2 Percussion 8 min 80 400 Raton 3 Percussion 8 min 73 444 Raton 4 Percussion 8 min 74 439 Sacramento 1 Immersion 43 495
69
Table B 1 Continued
-Source Block Treatment Germination Percentage Standard Error
Sacramento 2 Immersion 30 458 Sacramento 3 Immersion 38 485 Sacramento 4 Immersion 56 496 Sacramento 1 Boiling 30 sec 47 499 Sacramento 2 Boiling 30 sec 48 500 Sacramento 3 Boiling 30 sec 48 500 Sacramento 4 Boiling 30 sec 56 496 Sacramento 1 Boiling 60 sec 52 500 Sacramento 2 Boiling 60 sec 67 470 Sacramento 3 Boiling 60 sec 43 495 Sacramento 4 Boiling 60 sec 48 500 Sacramento 1 Control 0 000 Sacramento 2 Control 3 171 Sacramento 3 Control 3 171 Sacramento 4 Control 099 Sacramento 1 Percussion 1 min 57 495 Sacramento 2 Percussion 1 min 61 488 Sacramento 3 Percussion 1 min 62 485 Sacramento 4 Percussion 1 min 55 497 Sacramento 1 Percussion 2 min 84 367 Sacramento 2 Percussion 2 min 70 458 Sacramento 3 Percussion 2 min 81 392 Sacramento 4 Percussion 2 min 69 462 Sacramento 1 Percussion 4 min 91 286 Sacramento 2 Percussion 4 min 84 367 Sacramento 3 Percussion 4 min 84 367 Sacramento 4 Percussion 4 min 94 237 Sacramento 1 Percussion 8 min 82 384 Sacramento 2 Percussion 8 min 81 392 Sacramento 3 Percussion 8 min 82 384 Sacramento 4 Percussion 8 min 81 392 Sandia 1 Immersion 76 427 Sandia 2 Immersion 70 458 Sandia 3 Immersion 70 458 Sandia 4 Immersion 81 392 Sandia 1 Boiling 30 sec 78 414 Sandia 2 Boiling 30 sec 71 454 Sandia 3 Boiling 30 sec 63 483 Sandia 4 Boiling 30 sec 78 414 Sandia 1 Boiling 60 sec 67 470 Sandia 2 Boiling 60 sec 65 477 Sandia 3 Boiling 60 sec 74 439 Sandia 4 Boiling 60 sec 66 474 Sandia 1 Control 16 367 Sandia 2 Control 7 255
70
Table B 1 Continued
Source Block Treatment Gennination Percentage Standard Error
Sandia 3 Control 8 271 Sandia 4 Control 16 367 Sandia 1 Percussion 1 min 67 470 Sandia 2 Percussion 1 min 61 488 Sandia 3 Percussion 1 min 66 474 Sandia 4 Percussion 1 min 59 492 Sandia I Percussion 2 min 82 384 Sandia 2 Percussion 2 min 83 376 Sandia 3 Percussion 2 min 81 392 Sandia 4 Percussion 2 min 84 367 Sandia 1 Percussion 4 min 89 313 Sandia 2 Percussion 4 min 86 347 Sandia 3 Percussion 4 min 89 313 Sandia 4 Percussion 4 min 85 357 Sandia 1 Percussion 8 min 80 400 Sandia 2 Percussion 8 min 77 421 Sandia 3 Percussion 8 min 69 462 Sandia 4 Percussion 8 min 66 474
LITERATURE CITED
Ashby WC Vogel WG Rogers NF 1985 Black locust in the reclamation equation General Technical Report Northeastern Forest Experiment Station USDA Forest Service NE-I05 12 p
Ballard LAT 1973 Physical barriers to gennination Seed Sci Technoll 285-303
___ 1976 Strophiolar water conduction in the seeds ofTrifolieae induced by action on the testa at nonstrophiolar sites Austr J Plant Physiol3 465-469
Barton LV 1947 Special studies on seed coat impenneability Contrib Boyce Thompson Inst 14 355-362
Baskin CC Baskin JM 1998 Seeds Ecology biogeography and evolution of donnancy and gennination Academic Press London UK 666 p
Bewley JD 1997 Seed germination and donnancy Plant Ce119 1055-1066
Bewley JD Black M 1994 Seeds physiology of development and germination 2nd ed Plenum Press New York NY 445 p
Briscoe CB 1996 Growing Nitrogen Fixing Trees in Acid Soils In MH Powell (ed) Nitrogen Fixing Trees for Acid Soils- A Field ManuaL Winrock International Morrilton AR 22 p
Copeland LO McDonald MB 1995 Principles of Seed Science and Technology 3rded Chapman amp Hall New York NY 409 p
Cox RE Klett JE 1984 Seed gennination requirements of native Colorado plants for use in the landscape Plant Propag 30(2) 6-10
Dell B 1980 Structure and function ofthe strophiolar plug in seeds ofAlbizia Lophantha Am J Bot 67 556-563
Dick-Peddie WA 1993 New Mexico Vegetation Past Present and Future University ofNew Mexico Press Albuquerque NM 244 p
Dreesen DR 2001 Propagation protocol for production ofcontainer Robinia neomexicana plants Los Lunas Plant Materials Center Los Lunas New Mexico In Native Plant Network URL httpwwwnativeplantnetworkorg (accessed 9 January 2002) Moscow (ID) University ofIdaho CoIl Natural Resources For Res Nursery
72
Dreesen DR Harrington JT 1997 Propagation of native plants for restoration projects in the Southwestern US- Preliminary Investigations In Landis TD Thompson JR tech coords National Proceedings Forest and Conservation Nursery Assocs Gen Tech Rep PNW-GTR-419 Portland OR USDA For Ser PNW Res Sta 77-88
Gottfried GJ 1980 Control ofNew Mexican locust and the effect on planted ponderosa pine in central Arizona US For Ser Res Note RM-386 Rocky Mountain For Range ExpStn Fort Collins CO
Gosling PG Samuel YK Jones SK 1995 A systematic examination of germination temperature chipping and water temperaturesoak duration pretreatments on the seeds ofLeucaena leucocephala Seed Sci amp Technol 23521-532
Hamly DH 1932 Softening of the seeds of Melilotus alba Bot Gazette 93 345shy375
Hanna PJ 1984 Anatomical features of the seed coat ofAcacia kempeana (Mueller) which relate to increased germination rate induced by heat treatment New Phytol 96 23-29
Hanks JP Dick-Peddie WA 1974 Vegetation patterns of the White Mountains New Mexico Southwest Naturalist 18(4) 371-382
Hine S Harrington JT Dreesen DR 1997 Seed scarification requirements for Robinia neomexicana In Harrington JTtech coord Proc Third SouthwestemContainer Growers Conf Feb 12-13 1997 Ft Collins CO
Hopkinson JM 1993 The strophiole of leguminous seeds Newsletter- IntI Herbage Seed Production Res Grp 18 7-8
---- 1997 Differences in hardseededness between Cunningham and Tarramba leucaena In Leucnet News The Newsletter of the International LeucaenaRes and Devlt Netwk No4
ISTA (International Seed Testing Association) 1999 International rules for seed testing rules Seed Sci amp Technol27 Supplement
Kelly KM van Staden J 1987 The lens as the site of permeability in the papilionoid seed Aspalathus linearis J Plant Physiol 128(45) 395-404
73
Keresztesi B 1988 Black locust the tree of agriculture Outlook on Agr 17(2) 77~85
Kigel 1 and Galili G 1995 Seed development and germination Marcel Dekker New York 853 p
Klemmedson 10 1994 New Mexico locust and parent material Influence on macronutrients of forest floor and soil Soil Sci Soc Am 1 58 974~980
Kuo WH Tam AW 1988 The pathway of water absorption of mung bean seeds Seed Sci amp Technol16(1) 139~144
Leadem CL 1997 Dormancy--unlocking seed secrets In Landis TD Thompson lR tech coords National Proceedings Forestand Conservation Nursery Assocs Gen Tech Rep PNW-G TR-419 Portland OR USDA For Ser PNW Res Sta 43-52
Lin Y Wagner MR Seweyestewa A Honahni MD DeGomez M McMillin JD 1996 Influence of cultural treatments on seed germination and seedling growth of Robinia neomexicana Forest Farm and Community Tree Res Reports Volume 1
Marunda CT 1990 Effects of seed pretreatment on the development ofAcacia auriculforinis and A holosericea seedlings ACIAR Proceedings Series No 28
Meginnis HG 1937 Sulphuric acid treatment to increase germination of black locust seed USDA Circ 453 34 p
Miklas PN Towensend CE Ladd SL 1987 Seedcoat anatomy and the scarification ofcicer milkvetch seed Crop Sci 27 766-772
Monsen SB 1984 Use of shrubs on mine spoils In The Challenge ofProducing Native Plants for the Intermountain Area Proceedings Intermountain Nurserymans Association 1983 conference Las Vegas NV compo PM Murphy 26-31 Ogden UT USDA For Ser Gen Tech Report INT-l68
Olson DF 1974 Robinia L Locust In Seeds of Woody Plants in the US tech coord CS Schopmeyer 728-731 USDA For Ser Agr Handbk No450
Rehman S Loescher RNl Harris P1C 1998 Dormancy breaking and germination ofAcacia salicina Lindl seeds Seed Sci amp Techno127 553-557
Rolston MP 1978 Water impermeable seed dormancy Bot Rev 44 365-396
74
SAS Institute Inc 1989 SASSTAT Users Guide Version 6 4th Edition Volume 1 Cary NC SAS Institute Inc 943 p
SerratoValenti G Conara L Ferrando M Modenesi P 1993 Structural and histochemical features ofStylosanthes scabra (Leguminosae Papilionoidae) seed coat as related to water entry Can J Bot 71 834-840
Simpson BJ 1988 A field guide to Texas trees Austin TX Texas Monthly Press 372p
Tran VN Cavanagh AK 1984 Structural aspects ofdormancy In Seed Physiology Vol 2 Murray DR (ed) p1-44 Academic Press Australia
USDA Forest Service 1948 Woody-plant seed manual USDA Misc Pub1654 416p
Vines RA 1960 Trees Shrubs and Woody Vines of the Southwest Austin Univ ofTexas Press 1104 p
Wagner MR Larson RE Craig TP 1992 Ecology and silvics ofNew Mexico locust (Robinia neomexicana) with an emphasis on the arboreal form p53-57 In JW Hanover K Miller and S Plesko (eds) Proc Intemat Confon Black Locust Biology Culture and Utilization Mich State Univ USA
Wilson JK 1944 Immersing seeds of species of Robinia in boiling water hastens germination J For 42 453-454
Zimmerman RW Carpenter SB 1980 First-year coppice production from a 5shyyear old black locust stand on surface mine spoil p309-314 Centr Hardwd For ConfProc University ofMissouri Columbia
Table B 1 Continued
Source Block Treatment Germination Percentage Standard Error
Magdalena 4 Boiling 30 sec 83 376 Magdalena 1 Boiling 60 sec 63 483 Magdalena 2 Boiling 60 sec 68 466 Magdalena 3 Boiling 60 sec 63 483 Magdalena 4 Boiling 60 sec 82 384 Magdalena 1 Control 11 313 Magdalena 2 Control 6 237 Magdalena 3 Control 5 218 Magdalena 4 Control 4 196 Magdalena Percussion 1 min 32 466 Magdalena 2 Percussion 1 min 45 497 Magdalena 3 Percussion 1 min 46 498 Magdalena 4 Percussion I min 36 480 Magdalena 1 Percussion 2 min 67 470 Magdalena 2 Percussion 2 min 68 466 Magdalena 3 Percussion 2 min 65 477 Magdalena 4 Percussion 2 min 81 392 Magdalena 1 Percussion 4 min 87 336 Magdalena 2 Percussion 4 min 97 171 Magdalena 3 Percussion 4 min 83 376 Magdalena 4 Percussion 4 min 97 171 Magdalena 1 Percussion 8 min 98 lAO Magdalena 2 Percussion 8 min 100 000 Magdalena 3 Percussion 8 min 96 196 Magdalena 4 Percussion 8 min 98 lAO Manzano 1 Immersion 67 470 Manzano 2 Immersion 57 495 Manzano 3 Immersion 72 4049 Manzano 4 Immersion 73 4044 Manzano 1 Boiling 30 sec 70 458 Manzano 2 Boiling 30 sec 64 480 Manzano 3 Boiling 30 sec 71 454 Manzano 4 Boiling 30 sec 80 400 Manzano I Boiling 60 sec 50 500 Manzano 2 Boiling 60 sec 53 499 Manzano 3 Boiling 60 sec 59 492 Manzano 4 Boiling 60 sec 74 439 Manzano I Control 4 196 Manzano 2 Control 8 271 Manzano 3 Control 1 099 Manzano 4 Control 6 237 Manzano 1 Percussion 1 min 52 500 Manzano 2 Percussion 1 min 39 488 Manzano 3 Percussion 1 min 36 480 Manzano 4 Percussion 1 min 51 500
68
Table B 1 Continued
Source Block Treatment Gerrni~ation Percentage Standard Error
Manzano Percussion 2 min 53 499 Manzano 2 Percussion 2 min 58 494 Manzano 3 Percussion 2 min 61 488 Manzano 4 Percussion 2 min 62 485 Manzano 1 Percussion 4 min 76 427 Manzano 2 Percussion 4 min 85 357 Manzano 3 Percussion 4 min 79 407 Manzano 4 Percussion 4 min 83 376 Manzano 1 Percussion 8 min 87 336 Manzano 2 Percussion 8 min 83 376 Manzano 3 Percussion 8 min 83 376 Manzano 4 Percussion 8 min 75 433 Raton 1 Immersion 54 498 Raton 2 Immersion 37 483 Raton 3 Immersion 39 488 Raton 4 Immersion 69 462 Raton 1 Boiling 30 sec 36 480 Raton 2 Boiling 30 sec 20 400 Raton 3 Boiling 30 sec 28 449 Raton 4 Boiling 30 sec 60 490 Raton 1 Boiling 60 sec 15 357 Raton 2 Boiling 60 sec 171 Raton 3 Boiling 60 sec 4 196 Raton 4 Boiling 60 sec 35 477 Raton 1 Control 4 196 Raton 2 Control 12 325 Raton 3 Control 2 140 Raton 4 Control 6 237 Raton 1 Percussion 1 min 52 500 Raton 2 Percussion I min 29 454 Raton 3 Percussion I min 35 477 Raton 4 Percussion 1 min 31 462 Raton 1 Percussion 2 min 55 497 Raton 2 Percussion 2 min 35 477 Raton 3 Percussion 2 min 33 470 Raton 4 Percussion 2 min 42 494 Raton 1 Percussion 4 min 65 477 Raton 2 Percussion 4 min 52 500 Raton Percussion 4 min 51 500 Raton 4 Percussion 4 min 53 499 Raton 1 Percussion 8 min 88 325 Raton 2 Percussion 8 min 80 400 Raton 3 Percussion 8 min 73 444 Raton 4 Percussion 8 min 74 439 Sacramento 1 Immersion 43 495
69
Table B 1 Continued
-Source Block Treatment Germination Percentage Standard Error
Sacramento 2 Immersion 30 458 Sacramento 3 Immersion 38 485 Sacramento 4 Immersion 56 496 Sacramento 1 Boiling 30 sec 47 499 Sacramento 2 Boiling 30 sec 48 500 Sacramento 3 Boiling 30 sec 48 500 Sacramento 4 Boiling 30 sec 56 496 Sacramento 1 Boiling 60 sec 52 500 Sacramento 2 Boiling 60 sec 67 470 Sacramento 3 Boiling 60 sec 43 495 Sacramento 4 Boiling 60 sec 48 500 Sacramento 1 Control 0 000 Sacramento 2 Control 3 171 Sacramento 3 Control 3 171 Sacramento 4 Control 099 Sacramento 1 Percussion 1 min 57 495 Sacramento 2 Percussion 1 min 61 488 Sacramento 3 Percussion 1 min 62 485 Sacramento 4 Percussion 1 min 55 497 Sacramento 1 Percussion 2 min 84 367 Sacramento 2 Percussion 2 min 70 458 Sacramento 3 Percussion 2 min 81 392 Sacramento 4 Percussion 2 min 69 462 Sacramento 1 Percussion 4 min 91 286 Sacramento 2 Percussion 4 min 84 367 Sacramento 3 Percussion 4 min 84 367 Sacramento 4 Percussion 4 min 94 237 Sacramento 1 Percussion 8 min 82 384 Sacramento 2 Percussion 8 min 81 392 Sacramento 3 Percussion 8 min 82 384 Sacramento 4 Percussion 8 min 81 392 Sandia 1 Immersion 76 427 Sandia 2 Immersion 70 458 Sandia 3 Immersion 70 458 Sandia 4 Immersion 81 392 Sandia 1 Boiling 30 sec 78 414 Sandia 2 Boiling 30 sec 71 454 Sandia 3 Boiling 30 sec 63 483 Sandia 4 Boiling 30 sec 78 414 Sandia 1 Boiling 60 sec 67 470 Sandia 2 Boiling 60 sec 65 477 Sandia 3 Boiling 60 sec 74 439 Sandia 4 Boiling 60 sec 66 474 Sandia 1 Control 16 367 Sandia 2 Control 7 255
70
Table B 1 Continued
Source Block Treatment Gennination Percentage Standard Error
Sandia 3 Control 8 271 Sandia 4 Control 16 367 Sandia 1 Percussion 1 min 67 470 Sandia 2 Percussion 1 min 61 488 Sandia 3 Percussion 1 min 66 474 Sandia 4 Percussion 1 min 59 492 Sandia I Percussion 2 min 82 384 Sandia 2 Percussion 2 min 83 376 Sandia 3 Percussion 2 min 81 392 Sandia 4 Percussion 2 min 84 367 Sandia 1 Percussion 4 min 89 313 Sandia 2 Percussion 4 min 86 347 Sandia 3 Percussion 4 min 89 313 Sandia 4 Percussion 4 min 85 357 Sandia 1 Percussion 8 min 80 400 Sandia 2 Percussion 8 min 77 421 Sandia 3 Percussion 8 min 69 462 Sandia 4 Percussion 8 min 66 474
LITERATURE CITED
Ashby WC Vogel WG Rogers NF 1985 Black locust in the reclamation equation General Technical Report Northeastern Forest Experiment Station USDA Forest Service NE-I05 12 p
Ballard LAT 1973 Physical barriers to gennination Seed Sci Technoll 285-303
___ 1976 Strophiolar water conduction in the seeds ofTrifolieae induced by action on the testa at nonstrophiolar sites Austr J Plant Physiol3 465-469
Barton LV 1947 Special studies on seed coat impenneability Contrib Boyce Thompson Inst 14 355-362
Baskin CC Baskin JM 1998 Seeds Ecology biogeography and evolution of donnancy and gennination Academic Press London UK 666 p
Bewley JD 1997 Seed germination and donnancy Plant Ce119 1055-1066
Bewley JD Black M 1994 Seeds physiology of development and germination 2nd ed Plenum Press New York NY 445 p
Briscoe CB 1996 Growing Nitrogen Fixing Trees in Acid Soils In MH Powell (ed) Nitrogen Fixing Trees for Acid Soils- A Field ManuaL Winrock International Morrilton AR 22 p
Copeland LO McDonald MB 1995 Principles of Seed Science and Technology 3rded Chapman amp Hall New York NY 409 p
Cox RE Klett JE 1984 Seed gennination requirements of native Colorado plants for use in the landscape Plant Propag 30(2) 6-10
Dell B 1980 Structure and function ofthe strophiolar plug in seeds ofAlbizia Lophantha Am J Bot 67 556-563
Dick-Peddie WA 1993 New Mexico Vegetation Past Present and Future University ofNew Mexico Press Albuquerque NM 244 p
Dreesen DR 2001 Propagation protocol for production ofcontainer Robinia neomexicana plants Los Lunas Plant Materials Center Los Lunas New Mexico In Native Plant Network URL httpwwwnativeplantnetworkorg (accessed 9 January 2002) Moscow (ID) University ofIdaho CoIl Natural Resources For Res Nursery
72
Dreesen DR Harrington JT 1997 Propagation of native plants for restoration projects in the Southwestern US- Preliminary Investigations In Landis TD Thompson JR tech coords National Proceedings Forest and Conservation Nursery Assocs Gen Tech Rep PNW-GTR-419 Portland OR USDA For Ser PNW Res Sta 77-88
Gottfried GJ 1980 Control ofNew Mexican locust and the effect on planted ponderosa pine in central Arizona US For Ser Res Note RM-386 Rocky Mountain For Range ExpStn Fort Collins CO
Gosling PG Samuel YK Jones SK 1995 A systematic examination of germination temperature chipping and water temperaturesoak duration pretreatments on the seeds ofLeucaena leucocephala Seed Sci amp Technol 23521-532
Hamly DH 1932 Softening of the seeds of Melilotus alba Bot Gazette 93 345shy375
Hanna PJ 1984 Anatomical features of the seed coat ofAcacia kempeana (Mueller) which relate to increased germination rate induced by heat treatment New Phytol 96 23-29
Hanks JP Dick-Peddie WA 1974 Vegetation patterns of the White Mountains New Mexico Southwest Naturalist 18(4) 371-382
Hine S Harrington JT Dreesen DR 1997 Seed scarification requirements for Robinia neomexicana In Harrington JTtech coord Proc Third SouthwestemContainer Growers Conf Feb 12-13 1997 Ft Collins CO
Hopkinson JM 1993 The strophiole of leguminous seeds Newsletter- IntI Herbage Seed Production Res Grp 18 7-8
---- 1997 Differences in hardseededness between Cunningham and Tarramba leucaena In Leucnet News The Newsletter of the International LeucaenaRes and Devlt Netwk No4
ISTA (International Seed Testing Association) 1999 International rules for seed testing rules Seed Sci amp Technol27 Supplement
Kelly KM van Staden J 1987 The lens as the site of permeability in the papilionoid seed Aspalathus linearis J Plant Physiol 128(45) 395-404
73
Keresztesi B 1988 Black locust the tree of agriculture Outlook on Agr 17(2) 77~85
Kigel 1 and Galili G 1995 Seed development and germination Marcel Dekker New York 853 p
Klemmedson 10 1994 New Mexico locust and parent material Influence on macronutrients of forest floor and soil Soil Sci Soc Am 1 58 974~980
Kuo WH Tam AW 1988 The pathway of water absorption of mung bean seeds Seed Sci amp Technol16(1) 139~144
Leadem CL 1997 Dormancy--unlocking seed secrets In Landis TD Thompson lR tech coords National Proceedings Forestand Conservation Nursery Assocs Gen Tech Rep PNW-G TR-419 Portland OR USDA For Ser PNW Res Sta 43-52
Lin Y Wagner MR Seweyestewa A Honahni MD DeGomez M McMillin JD 1996 Influence of cultural treatments on seed germination and seedling growth of Robinia neomexicana Forest Farm and Community Tree Res Reports Volume 1
Marunda CT 1990 Effects of seed pretreatment on the development ofAcacia auriculforinis and A holosericea seedlings ACIAR Proceedings Series No 28
Meginnis HG 1937 Sulphuric acid treatment to increase germination of black locust seed USDA Circ 453 34 p
Miklas PN Towensend CE Ladd SL 1987 Seedcoat anatomy and the scarification ofcicer milkvetch seed Crop Sci 27 766-772
Monsen SB 1984 Use of shrubs on mine spoils In The Challenge ofProducing Native Plants for the Intermountain Area Proceedings Intermountain Nurserymans Association 1983 conference Las Vegas NV compo PM Murphy 26-31 Ogden UT USDA For Ser Gen Tech Report INT-l68
Olson DF 1974 Robinia L Locust In Seeds of Woody Plants in the US tech coord CS Schopmeyer 728-731 USDA For Ser Agr Handbk No450
Rehman S Loescher RNl Harris P1C 1998 Dormancy breaking and germination ofAcacia salicina Lindl seeds Seed Sci amp Techno127 553-557
Rolston MP 1978 Water impermeable seed dormancy Bot Rev 44 365-396
74
SAS Institute Inc 1989 SASSTAT Users Guide Version 6 4th Edition Volume 1 Cary NC SAS Institute Inc 943 p
SerratoValenti G Conara L Ferrando M Modenesi P 1993 Structural and histochemical features ofStylosanthes scabra (Leguminosae Papilionoidae) seed coat as related to water entry Can J Bot 71 834-840
Simpson BJ 1988 A field guide to Texas trees Austin TX Texas Monthly Press 372p
Tran VN Cavanagh AK 1984 Structural aspects ofdormancy In Seed Physiology Vol 2 Murray DR (ed) p1-44 Academic Press Australia
USDA Forest Service 1948 Woody-plant seed manual USDA Misc Pub1654 416p
Vines RA 1960 Trees Shrubs and Woody Vines of the Southwest Austin Univ ofTexas Press 1104 p
Wagner MR Larson RE Craig TP 1992 Ecology and silvics ofNew Mexico locust (Robinia neomexicana) with an emphasis on the arboreal form p53-57 In JW Hanover K Miller and S Plesko (eds) Proc Intemat Confon Black Locust Biology Culture and Utilization Mich State Univ USA
Wilson JK 1944 Immersing seeds of species of Robinia in boiling water hastens germination J For 42 453-454
Zimmerman RW Carpenter SB 1980 First-year coppice production from a 5shyyear old black locust stand on surface mine spoil p309-314 Centr Hardwd For ConfProc University ofMissouri Columbia
Table B 1 Continued
Source Block Treatment Gerrni~ation Percentage Standard Error
Manzano Percussion 2 min 53 499 Manzano 2 Percussion 2 min 58 494 Manzano 3 Percussion 2 min 61 488 Manzano 4 Percussion 2 min 62 485 Manzano 1 Percussion 4 min 76 427 Manzano 2 Percussion 4 min 85 357 Manzano 3 Percussion 4 min 79 407 Manzano 4 Percussion 4 min 83 376 Manzano 1 Percussion 8 min 87 336 Manzano 2 Percussion 8 min 83 376 Manzano 3 Percussion 8 min 83 376 Manzano 4 Percussion 8 min 75 433 Raton 1 Immersion 54 498 Raton 2 Immersion 37 483 Raton 3 Immersion 39 488 Raton 4 Immersion 69 462 Raton 1 Boiling 30 sec 36 480 Raton 2 Boiling 30 sec 20 400 Raton 3 Boiling 30 sec 28 449 Raton 4 Boiling 30 sec 60 490 Raton 1 Boiling 60 sec 15 357 Raton 2 Boiling 60 sec 171 Raton 3 Boiling 60 sec 4 196 Raton 4 Boiling 60 sec 35 477 Raton 1 Control 4 196 Raton 2 Control 12 325 Raton 3 Control 2 140 Raton 4 Control 6 237 Raton 1 Percussion 1 min 52 500 Raton 2 Percussion I min 29 454 Raton 3 Percussion I min 35 477 Raton 4 Percussion 1 min 31 462 Raton 1 Percussion 2 min 55 497 Raton 2 Percussion 2 min 35 477 Raton 3 Percussion 2 min 33 470 Raton 4 Percussion 2 min 42 494 Raton 1 Percussion 4 min 65 477 Raton 2 Percussion 4 min 52 500 Raton Percussion 4 min 51 500 Raton 4 Percussion 4 min 53 499 Raton 1 Percussion 8 min 88 325 Raton 2 Percussion 8 min 80 400 Raton 3 Percussion 8 min 73 444 Raton 4 Percussion 8 min 74 439 Sacramento 1 Immersion 43 495
69
Table B 1 Continued
-Source Block Treatment Germination Percentage Standard Error
Sacramento 2 Immersion 30 458 Sacramento 3 Immersion 38 485 Sacramento 4 Immersion 56 496 Sacramento 1 Boiling 30 sec 47 499 Sacramento 2 Boiling 30 sec 48 500 Sacramento 3 Boiling 30 sec 48 500 Sacramento 4 Boiling 30 sec 56 496 Sacramento 1 Boiling 60 sec 52 500 Sacramento 2 Boiling 60 sec 67 470 Sacramento 3 Boiling 60 sec 43 495 Sacramento 4 Boiling 60 sec 48 500 Sacramento 1 Control 0 000 Sacramento 2 Control 3 171 Sacramento 3 Control 3 171 Sacramento 4 Control 099 Sacramento 1 Percussion 1 min 57 495 Sacramento 2 Percussion 1 min 61 488 Sacramento 3 Percussion 1 min 62 485 Sacramento 4 Percussion 1 min 55 497 Sacramento 1 Percussion 2 min 84 367 Sacramento 2 Percussion 2 min 70 458 Sacramento 3 Percussion 2 min 81 392 Sacramento 4 Percussion 2 min 69 462 Sacramento 1 Percussion 4 min 91 286 Sacramento 2 Percussion 4 min 84 367 Sacramento 3 Percussion 4 min 84 367 Sacramento 4 Percussion 4 min 94 237 Sacramento 1 Percussion 8 min 82 384 Sacramento 2 Percussion 8 min 81 392 Sacramento 3 Percussion 8 min 82 384 Sacramento 4 Percussion 8 min 81 392 Sandia 1 Immersion 76 427 Sandia 2 Immersion 70 458 Sandia 3 Immersion 70 458 Sandia 4 Immersion 81 392 Sandia 1 Boiling 30 sec 78 414 Sandia 2 Boiling 30 sec 71 454 Sandia 3 Boiling 30 sec 63 483 Sandia 4 Boiling 30 sec 78 414 Sandia 1 Boiling 60 sec 67 470 Sandia 2 Boiling 60 sec 65 477 Sandia 3 Boiling 60 sec 74 439 Sandia 4 Boiling 60 sec 66 474 Sandia 1 Control 16 367 Sandia 2 Control 7 255
70
Table B 1 Continued
Source Block Treatment Gennination Percentage Standard Error
Sandia 3 Control 8 271 Sandia 4 Control 16 367 Sandia 1 Percussion 1 min 67 470 Sandia 2 Percussion 1 min 61 488 Sandia 3 Percussion 1 min 66 474 Sandia 4 Percussion 1 min 59 492 Sandia I Percussion 2 min 82 384 Sandia 2 Percussion 2 min 83 376 Sandia 3 Percussion 2 min 81 392 Sandia 4 Percussion 2 min 84 367 Sandia 1 Percussion 4 min 89 313 Sandia 2 Percussion 4 min 86 347 Sandia 3 Percussion 4 min 89 313 Sandia 4 Percussion 4 min 85 357 Sandia 1 Percussion 8 min 80 400 Sandia 2 Percussion 8 min 77 421 Sandia 3 Percussion 8 min 69 462 Sandia 4 Percussion 8 min 66 474
LITERATURE CITED
Ashby WC Vogel WG Rogers NF 1985 Black locust in the reclamation equation General Technical Report Northeastern Forest Experiment Station USDA Forest Service NE-I05 12 p
Ballard LAT 1973 Physical barriers to gennination Seed Sci Technoll 285-303
___ 1976 Strophiolar water conduction in the seeds ofTrifolieae induced by action on the testa at nonstrophiolar sites Austr J Plant Physiol3 465-469
Barton LV 1947 Special studies on seed coat impenneability Contrib Boyce Thompson Inst 14 355-362
Baskin CC Baskin JM 1998 Seeds Ecology biogeography and evolution of donnancy and gennination Academic Press London UK 666 p
Bewley JD 1997 Seed germination and donnancy Plant Ce119 1055-1066
Bewley JD Black M 1994 Seeds physiology of development and germination 2nd ed Plenum Press New York NY 445 p
Briscoe CB 1996 Growing Nitrogen Fixing Trees in Acid Soils In MH Powell (ed) Nitrogen Fixing Trees for Acid Soils- A Field ManuaL Winrock International Morrilton AR 22 p
Copeland LO McDonald MB 1995 Principles of Seed Science and Technology 3rded Chapman amp Hall New York NY 409 p
Cox RE Klett JE 1984 Seed gennination requirements of native Colorado plants for use in the landscape Plant Propag 30(2) 6-10
Dell B 1980 Structure and function ofthe strophiolar plug in seeds ofAlbizia Lophantha Am J Bot 67 556-563
Dick-Peddie WA 1993 New Mexico Vegetation Past Present and Future University ofNew Mexico Press Albuquerque NM 244 p
Dreesen DR 2001 Propagation protocol for production ofcontainer Robinia neomexicana plants Los Lunas Plant Materials Center Los Lunas New Mexico In Native Plant Network URL httpwwwnativeplantnetworkorg (accessed 9 January 2002) Moscow (ID) University ofIdaho CoIl Natural Resources For Res Nursery
72
Dreesen DR Harrington JT 1997 Propagation of native plants for restoration projects in the Southwestern US- Preliminary Investigations In Landis TD Thompson JR tech coords National Proceedings Forest and Conservation Nursery Assocs Gen Tech Rep PNW-GTR-419 Portland OR USDA For Ser PNW Res Sta 77-88
Gottfried GJ 1980 Control ofNew Mexican locust and the effect on planted ponderosa pine in central Arizona US For Ser Res Note RM-386 Rocky Mountain For Range ExpStn Fort Collins CO
Gosling PG Samuel YK Jones SK 1995 A systematic examination of germination temperature chipping and water temperaturesoak duration pretreatments on the seeds ofLeucaena leucocephala Seed Sci amp Technol 23521-532
Hamly DH 1932 Softening of the seeds of Melilotus alba Bot Gazette 93 345shy375
Hanna PJ 1984 Anatomical features of the seed coat ofAcacia kempeana (Mueller) which relate to increased germination rate induced by heat treatment New Phytol 96 23-29
Hanks JP Dick-Peddie WA 1974 Vegetation patterns of the White Mountains New Mexico Southwest Naturalist 18(4) 371-382
Hine S Harrington JT Dreesen DR 1997 Seed scarification requirements for Robinia neomexicana In Harrington JTtech coord Proc Third SouthwestemContainer Growers Conf Feb 12-13 1997 Ft Collins CO
Hopkinson JM 1993 The strophiole of leguminous seeds Newsletter- IntI Herbage Seed Production Res Grp 18 7-8
---- 1997 Differences in hardseededness between Cunningham and Tarramba leucaena In Leucnet News The Newsletter of the International LeucaenaRes and Devlt Netwk No4
ISTA (International Seed Testing Association) 1999 International rules for seed testing rules Seed Sci amp Technol27 Supplement
Kelly KM van Staden J 1987 The lens as the site of permeability in the papilionoid seed Aspalathus linearis J Plant Physiol 128(45) 395-404
73
Keresztesi B 1988 Black locust the tree of agriculture Outlook on Agr 17(2) 77~85
Kigel 1 and Galili G 1995 Seed development and germination Marcel Dekker New York 853 p
Klemmedson 10 1994 New Mexico locust and parent material Influence on macronutrients of forest floor and soil Soil Sci Soc Am 1 58 974~980
Kuo WH Tam AW 1988 The pathway of water absorption of mung bean seeds Seed Sci amp Technol16(1) 139~144
Leadem CL 1997 Dormancy--unlocking seed secrets In Landis TD Thompson lR tech coords National Proceedings Forestand Conservation Nursery Assocs Gen Tech Rep PNW-G TR-419 Portland OR USDA For Ser PNW Res Sta 43-52
Lin Y Wagner MR Seweyestewa A Honahni MD DeGomez M McMillin JD 1996 Influence of cultural treatments on seed germination and seedling growth of Robinia neomexicana Forest Farm and Community Tree Res Reports Volume 1
Marunda CT 1990 Effects of seed pretreatment on the development ofAcacia auriculforinis and A holosericea seedlings ACIAR Proceedings Series No 28
Meginnis HG 1937 Sulphuric acid treatment to increase germination of black locust seed USDA Circ 453 34 p
Miklas PN Towensend CE Ladd SL 1987 Seedcoat anatomy and the scarification ofcicer milkvetch seed Crop Sci 27 766-772
Monsen SB 1984 Use of shrubs on mine spoils In The Challenge ofProducing Native Plants for the Intermountain Area Proceedings Intermountain Nurserymans Association 1983 conference Las Vegas NV compo PM Murphy 26-31 Ogden UT USDA For Ser Gen Tech Report INT-l68
Olson DF 1974 Robinia L Locust In Seeds of Woody Plants in the US tech coord CS Schopmeyer 728-731 USDA For Ser Agr Handbk No450
Rehman S Loescher RNl Harris P1C 1998 Dormancy breaking and germination ofAcacia salicina Lindl seeds Seed Sci amp Techno127 553-557
Rolston MP 1978 Water impermeable seed dormancy Bot Rev 44 365-396
74
SAS Institute Inc 1989 SASSTAT Users Guide Version 6 4th Edition Volume 1 Cary NC SAS Institute Inc 943 p
SerratoValenti G Conara L Ferrando M Modenesi P 1993 Structural and histochemical features ofStylosanthes scabra (Leguminosae Papilionoidae) seed coat as related to water entry Can J Bot 71 834-840
Simpson BJ 1988 A field guide to Texas trees Austin TX Texas Monthly Press 372p
Tran VN Cavanagh AK 1984 Structural aspects ofdormancy In Seed Physiology Vol 2 Murray DR (ed) p1-44 Academic Press Australia
USDA Forest Service 1948 Woody-plant seed manual USDA Misc Pub1654 416p
Vines RA 1960 Trees Shrubs and Woody Vines of the Southwest Austin Univ ofTexas Press 1104 p
Wagner MR Larson RE Craig TP 1992 Ecology and silvics ofNew Mexico locust (Robinia neomexicana) with an emphasis on the arboreal form p53-57 In JW Hanover K Miller and S Plesko (eds) Proc Intemat Confon Black Locust Biology Culture and Utilization Mich State Univ USA
Wilson JK 1944 Immersing seeds of species of Robinia in boiling water hastens germination J For 42 453-454
Zimmerman RW Carpenter SB 1980 First-year coppice production from a 5shyyear old black locust stand on surface mine spoil p309-314 Centr Hardwd For ConfProc University ofMissouri Columbia
Table B 1 Continued
-Source Block Treatment Germination Percentage Standard Error
Sacramento 2 Immersion 30 458 Sacramento 3 Immersion 38 485 Sacramento 4 Immersion 56 496 Sacramento 1 Boiling 30 sec 47 499 Sacramento 2 Boiling 30 sec 48 500 Sacramento 3 Boiling 30 sec 48 500 Sacramento 4 Boiling 30 sec 56 496 Sacramento 1 Boiling 60 sec 52 500 Sacramento 2 Boiling 60 sec 67 470 Sacramento 3 Boiling 60 sec 43 495 Sacramento 4 Boiling 60 sec 48 500 Sacramento 1 Control 0 000 Sacramento 2 Control 3 171 Sacramento 3 Control 3 171 Sacramento 4 Control 099 Sacramento 1 Percussion 1 min 57 495 Sacramento 2 Percussion 1 min 61 488 Sacramento 3 Percussion 1 min 62 485 Sacramento 4 Percussion 1 min 55 497 Sacramento 1 Percussion 2 min 84 367 Sacramento 2 Percussion 2 min 70 458 Sacramento 3 Percussion 2 min 81 392 Sacramento 4 Percussion 2 min 69 462 Sacramento 1 Percussion 4 min 91 286 Sacramento 2 Percussion 4 min 84 367 Sacramento 3 Percussion 4 min 84 367 Sacramento 4 Percussion 4 min 94 237 Sacramento 1 Percussion 8 min 82 384 Sacramento 2 Percussion 8 min 81 392 Sacramento 3 Percussion 8 min 82 384 Sacramento 4 Percussion 8 min 81 392 Sandia 1 Immersion 76 427 Sandia 2 Immersion 70 458 Sandia 3 Immersion 70 458 Sandia 4 Immersion 81 392 Sandia 1 Boiling 30 sec 78 414 Sandia 2 Boiling 30 sec 71 454 Sandia 3 Boiling 30 sec 63 483 Sandia 4 Boiling 30 sec 78 414 Sandia 1 Boiling 60 sec 67 470 Sandia 2 Boiling 60 sec 65 477 Sandia 3 Boiling 60 sec 74 439 Sandia 4 Boiling 60 sec 66 474 Sandia 1 Control 16 367 Sandia 2 Control 7 255
70
Table B 1 Continued
Source Block Treatment Gennination Percentage Standard Error
Sandia 3 Control 8 271 Sandia 4 Control 16 367 Sandia 1 Percussion 1 min 67 470 Sandia 2 Percussion 1 min 61 488 Sandia 3 Percussion 1 min 66 474 Sandia 4 Percussion 1 min 59 492 Sandia I Percussion 2 min 82 384 Sandia 2 Percussion 2 min 83 376 Sandia 3 Percussion 2 min 81 392 Sandia 4 Percussion 2 min 84 367 Sandia 1 Percussion 4 min 89 313 Sandia 2 Percussion 4 min 86 347 Sandia 3 Percussion 4 min 89 313 Sandia 4 Percussion 4 min 85 357 Sandia 1 Percussion 8 min 80 400 Sandia 2 Percussion 8 min 77 421 Sandia 3 Percussion 8 min 69 462 Sandia 4 Percussion 8 min 66 474
LITERATURE CITED
Ashby WC Vogel WG Rogers NF 1985 Black locust in the reclamation equation General Technical Report Northeastern Forest Experiment Station USDA Forest Service NE-I05 12 p
Ballard LAT 1973 Physical barriers to gennination Seed Sci Technoll 285-303
___ 1976 Strophiolar water conduction in the seeds ofTrifolieae induced by action on the testa at nonstrophiolar sites Austr J Plant Physiol3 465-469
Barton LV 1947 Special studies on seed coat impenneability Contrib Boyce Thompson Inst 14 355-362
Baskin CC Baskin JM 1998 Seeds Ecology biogeography and evolution of donnancy and gennination Academic Press London UK 666 p
Bewley JD 1997 Seed germination and donnancy Plant Ce119 1055-1066
Bewley JD Black M 1994 Seeds physiology of development and germination 2nd ed Plenum Press New York NY 445 p
Briscoe CB 1996 Growing Nitrogen Fixing Trees in Acid Soils In MH Powell (ed) Nitrogen Fixing Trees for Acid Soils- A Field ManuaL Winrock International Morrilton AR 22 p
Copeland LO McDonald MB 1995 Principles of Seed Science and Technology 3rded Chapman amp Hall New York NY 409 p
Cox RE Klett JE 1984 Seed gennination requirements of native Colorado plants for use in the landscape Plant Propag 30(2) 6-10
Dell B 1980 Structure and function ofthe strophiolar plug in seeds ofAlbizia Lophantha Am J Bot 67 556-563
Dick-Peddie WA 1993 New Mexico Vegetation Past Present and Future University ofNew Mexico Press Albuquerque NM 244 p
Dreesen DR 2001 Propagation protocol for production ofcontainer Robinia neomexicana plants Los Lunas Plant Materials Center Los Lunas New Mexico In Native Plant Network URL httpwwwnativeplantnetworkorg (accessed 9 January 2002) Moscow (ID) University ofIdaho CoIl Natural Resources For Res Nursery
72
Dreesen DR Harrington JT 1997 Propagation of native plants for restoration projects in the Southwestern US- Preliminary Investigations In Landis TD Thompson JR tech coords National Proceedings Forest and Conservation Nursery Assocs Gen Tech Rep PNW-GTR-419 Portland OR USDA For Ser PNW Res Sta 77-88
Gottfried GJ 1980 Control ofNew Mexican locust and the effect on planted ponderosa pine in central Arizona US For Ser Res Note RM-386 Rocky Mountain For Range ExpStn Fort Collins CO
Gosling PG Samuel YK Jones SK 1995 A systematic examination of germination temperature chipping and water temperaturesoak duration pretreatments on the seeds ofLeucaena leucocephala Seed Sci amp Technol 23521-532
Hamly DH 1932 Softening of the seeds of Melilotus alba Bot Gazette 93 345shy375
Hanna PJ 1984 Anatomical features of the seed coat ofAcacia kempeana (Mueller) which relate to increased germination rate induced by heat treatment New Phytol 96 23-29
Hanks JP Dick-Peddie WA 1974 Vegetation patterns of the White Mountains New Mexico Southwest Naturalist 18(4) 371-382
Hine S Harrington JT Dreesen DR 1997 Seed scarification requirements for Robinia neomexicana In Harrington JTtech coord Proc Third SouthwestemContainer Growers Conf Feb 12-13 1997 Ft Collins CO
Hopkinson JM 1993 The strophiole of leguminous seeds Newsletter- IntI Herbage Seed Production Res Grp 18 7-8
---- 1997 Differences in hardseededness between Cunningham and Tarramba leucaena In Leucnet News The Newsletter of the International LeucaenaRes and Devlt Netwk No4
ISTA (International Seed Testing Association) 1999 International rules for seed testing rules Seed Sci amp Technol27 Supplement
Kelly KM van Staden J 1987 The lens as the site of permeability in the papilionoid seed Aspalathus linearis J Plant Physiol 128(45) 395-404
73
Keresztesi B 1988 Black locust the tree of agriculture Outlook on Agr 17(2) 77~85
Kigel 1 and Galili G 1995 Seed development and germination Marcel Dekker New York 853 p
Klemmedson 10 1994 New Mexico locust and parent material Influence on macronutrients of forest floor and soil Soil Sci Soc Am 1 58 974~980
Kuo WH Tam AW 1988 The pathway of water absorption of mung bean seeds Seed Sci amp Technol16(1) 139~144
Leadem CL 1997 Dormancy--unlocking seed secrets In Landis TD Thompson lR tech coords National Proceedings Forestand Conservation Nursery Assocs Gen Tech Rep PNW-G TR-419 Portland OR USDA For Ser PNW Res Sta 43-52
Lin Y Wagner MR Seweyestewa A Honahni MD DeGomez M McMillin JD 1996 Influence of cultural treatments on seed germination and seedling growth of Robinia neomexicana Forest Farm and Community Tree Res Reports Volume 1
Marunda CT 1990 Effects of seed pretreatment on the development ofAcacia auriculforinis and A holosericea seedlings ACIAR Proceedings Series No 28
Meginnis HG 1937 Sulphuric acid treatment to increase germination of black locust seed USDA Circ 453 34 p
Miklas PN Towensend CE Ladd SL 1987 Seedcoat anatomy and the scarification ofcicer milkvetch seed Crop Sci 27 766-772
Monsen SB 1984 Use of shrubs on mine spoils In The Challenge ofProducing Native Plants for the Intermountain Area Proceedings Intermountain Nurserymans Association 1983 conference Las Vegas NV compo PM Murphy 26-31 Ogden UT USDA For Ser Gen Tech Report INT-l68
Olson DF 1974 Robinia L Locust In Seeds of Woody Plants in the US tech coord CS Schopmeyer 728-731 USDA For Ser Agr Handbk No450
Rehman S Loescher RNl Harris P1C 1998 Dormancy breaking and germination ofAcacia salicina Lindl seeds Seed Sci amp Techno127 553-557
Rolston MP 1978 Water impermeable seed dormancy Bot Rev 44 365-396
74
SAS Institute Inc 1989 SASSTAT Users Guide Version 6 4th Edition Volume 1 Cary NC SAS Institute Inc 943 p
SerratoValenti G Conara L Ferrando M Modenesi P 1993 Structural and histochemical features ofStylosanthes scabra (Leguminosae Papilionoidae) seed coat as related to water entry Can J Bot 71 834-840
Simpson BJ 1988 A field guide to Texas trees Austin TX Texas Monthly Press 372p
Tran VN Cavanagh AK 1984 Structural aspects ofdormancy In Seed Physiology Vol 2 Murray DR (ed) p1-44 Academic Press Australia
USDA Forest Service 1948 Woody-plant seed manual USDA Misc Pub1654 416p
Vines RA 1960 Trees Shrubs and Woody Vines of the Southwest Austin Univ ofTexas Press 1104 p
Wagner MR Larson RE Craig TP 1992 Ecology and silvics ofNew Mexico locust (Robinia neomexicana) with an emphasis on the arboreal form p53-57 In JW Hanover K Miller and S Plesko (eds) Proc Intemat Confon Black Locust Biology Culture and Utilization Mich State Univ USA
Wilson JK 1944 Immersing seeds of species of Robinia in boiling water hastens germination J For 42 453-454
Zimmerman RW Carpenter SB 1980 First-year coppice production from a 5shyyear old black locust stand on surface mine spoil p309-314 Centr Hardwd For ConfProc University ofMissouri Columbia
Table B 1 Continued
Source Block Treatment Gennination Percentage Standard Error
Sandia 3 Control 8 271 Sandia 4 Control 16 367 Sandia 1 Percussion 1 min 67 470 Sandia 2 Percussion 1 min 61 488 Sandia 3 Percussion 1 min 66 474 Sandia 4 Percussion 1 min 59 492 Sandia I Percussion 2 min 82 384 Sandia 2 Percussion 2 min 83 376 Sandia 3 Percussion 2 min 81 392 Sandia 4 Percussion 2 min 84 367 Sandia 1 Percussion 4 min 89 313 Sandia 2 Percussion 4 min 86 347 Sandia 3 Percussion 4 min 89 313 Sandia 4 Percussion 4 min 85 357 Sandia 1 Percussion 8 min 80 400 Sandia 2 Percussion 8 min 77 421 Sandia 3 Percussion 8 min 69 462 Sandia 4 Percussion 8 min 66 474
LITERATURE CITED
Ashby WC Vogel WG Rogers NF 1985 Black locust in the reclamation equation General Technical Report Northeastern Forest Experiment Station USDA Forest Service NE-I05 12 p
Ballard LAT 1973 Physical barriers to gennination Seed Sci Technoll 285-303
___ 1976 Strophiolar water conduction in the seeds ofTrifolieae induced by action on the testa at nonstrophiolar sites Austr J Plant Physiol3 465-469
Barton LV 1947 Special studies on seed coat impenneability Contrib Boyce Thompson Inst 14 355-362
Baskin CC Baskin JM 1998 Seeds Ecology biogeography and evolution of donnancy and gennination Academic Press London UK 666 p
Bewley JD 1997 Seed germination and donnancy Plant Ce119 1055-1066
Bewley JD Black M 1994 Seeds physiology of development and germination 2nd ed Plenum Press New York NY 445 p
Briscoe CB 1996 Growing Nitrogen Fixing Trees in Acid Soils In MH Powell (ed) Nitrogen Fixing Trees for Acid Soils- A Field ManuaL Winrock International Morrilton AR 22 p
Copeland LO McDonald MB 1995 Principles of Seed Science and Technology 3rded Chapman amp Hall New York NY 409 p
Cox RE Klett JE 1984 Seed gennination requirements of native Colorado plants for use in the landscape Plant Propag 30(2) 6-10
Dell B 1980 Structure and function ofthe strophiolar plug in seeds ofAlbizia Lophantha Am J Bot 67 556-563
Dick-Peddie WA 1993 New Mexico Vegetation Past Present and Future University ofNew Mexico Press Albuquerque NM 244 p
Dreesen DR 2001 Propagation protocol for production ofcontainer Robinia neomexicana plants Los Lunas Plant Materials Center Los Lunas New Mexico In Native Plant Network URL httpwwwnativeplantnetworkorg (accessed 9 January 2002) Moscow (ID) University ofIdaho CoIl Natural Resources For Res Nursery
72
Dreesen DR Harrington JT 1997 Propagation of native plants for restoration projects in the Southwestern US- Preliminary Investigations In Landis TD Thompson JR tech coords National Proceedings Forest and Conservation Nursery Assocs Gen Tech Rep PNW-GTR-419 Portland OR USDA For Ser PNW Res Sta 77-88
Gottfried GJ 1980 Control ofNew Mexican locust and the effect on planted ponderosa pine in central Arizona US For Ser Res Note RM-386 Rocky Mountain For Range ExpStn Fort Collins CO
Gosling PG Samuel YK Jones SK 1995 A systematic examination of germination temperature chipping and water temperaturesoak duration pretreatments on the seeds ofLeucaena leucocephala Seed Sci amp Technol 23521-532
Hamly DH 1932 Softening of the seeds of Melilotus alba Bot Gazette 93 345shy375
Hanna PJ 1984 Anatomical features of the seed coat ofAcacia kempeana (Mueller) which relate to increased germination rate induced by heat treatment New Phytol 96 23-29
Hanks JP Dick-Peddie WA 1974 Vegetation patterns of the White Mountains New Mexico Southwest Naturalist 18(4) 371-382
Hine S Harrington JT Dreesen DR 1997 Seed scarification requirements for Robinia neomexicana In Harrington JTtech coord Proc Third SouthwestemContainer Growers Conf Feb 12-13 1997 Ft Collins CO
Hopkinson JM 1993 The strophiole of leguminous seeds Newsletter- IntI Herbage Seed Production Res Grp 18 7-8
---- 1997 Differences in hardseededness between Cunningham and Tarramba leucaena In Leucnet News The Newsletter of the International LeucaenaRes and Devlt Netwk No4
ISTA (International Seed Testing Association) 1999 International rules for seed testing rules Seed Sci amp Technol27 Supplement
Kelly KM van Staden J 1987 The lens as the site of permeability in the papilionoid seed Aspalathus linearis J Plant Physiol 128(45) 395-404
73
Keresztesi B 1988 Black locust the tree of agriculture Outlook on Agr 17(2) 77~85
Kigel 1 and Galili G 1995 Seed development and germination Marcel Dekker New York 853 p
Klemmedson 10 1994 New Mexico locust and parent material Influence on macronutrients of forest floor and soil Soil Sci Soc Am 1 58 974~980
Kuo WH Tam AW 1988 The pathway of water absorption of mung bean seeds Seed Sci amp Technol16(1) 139~144
Leadem CL 1997 Dormancy--unlocking seed secrets In Landis TD Thompson lR tech coords National Proceedings Forestand Conservation Nursery Assocs Gen Tech Rep PNW-G TR-419 Portland OR USDA For Ser PNW Res Sta 43-52
Lin Y Wagner MR Seweyestewa A Honahni MD DeGomez M McMillin JD 1996 Influence of cultural treatments on seed germination and seedling growth of Robinia neomexicana Forest Farm and Community Tree Res Reports Volume 1
Marunda CT 1990 Effects of seed pretreatment on the development ofAcacia auriculforinis and A holosericea seedlings ACIAR Proceedings Series No 28
Meginnis HG 1937 Sulphuric acid treatment to increase germination of black locust seed USDA Circ 453 34 p
Miklas PN Towensend CE Ladd SL 1987 Seedcoat anatomy and the scarification ofcicer milkvetch seed Crop Sci 27 766-772
Monsen SB 1984 Use of shrubs on mine spoils In The Challenge ofProducing Native Plants for the Intermountain Area Proceedings Intermountain Nurserymans Association 1983 conference Las Vegas NV compo PM Murphy 26-31 Ogden UT USDA For Ser Gen Tech Report INT-l68
Olson DF 1974 Robinia L Locust In Seeds of Woody Plants in the US tech coord CS Schopmeyer 728-731 USDA For Ser Agr Handbk No450
Rehman S Loescher RNl Harris P1C 1998 Dormancy breaking and germination ofAcacia salicina Lindl seeds Seed Sci amp Techno127 553-557
Rolston MP 1978 Water impermeable seed dormancy Bot Rev 44 365-396
74
SAS Institute Inc 1989 SASSTAT Users Guide Version 6 4th Edition Volume 1 Cary NC SAS Institute Inc 943 p
SerratoValenti G Conara L Ferrando M Modenesi P 1993 Structural and histochemical features ofStylosanthes scabra (Leguminosae Papilionoidae) seed coat as related to water entry Can J Bot 71 834-840
Simpson BJ 1988 A field guide to Texas trees Austin TX Texas Monthly Press 372p
Tran VN Cavanagh AK 1984 Structural aspects ofdormancy In Seed Physiology Vol 2 Murray DR (ed) p1-44 Academic Press Australia
USDA Forest Service 1948 Woody-plant seed manual USDA Misc Pub1654 416p
Vines RA 1960 Trees Shrubs and Woody Vines of the Southwest Austin Univ ofTexas Press 1104 p
Wagner MR Larson RE Craig TP 1992 Ecology and silvics ofNew Mexico locust (Robinia neomexicana) with an emphasis on the arboreal form p53-57 In JW Hanover K Miller and S Plesko (eds) Proc Intemat Confon Black Locust Biology Culture and Utilization Mich State Univ USA
Wilson JK 1944 Immersing seeds of species of Robinia in boiling water hastens germination J For 42 453-454
Zimmerman RW Carpenter SB 1980 First-year coppice production from a 5shyyear old black locust stand on surface mine spoil p309-314 Centr Hardwd For ConfProc University ofMissouri Columbia
LITERATURE CITED
Ashby WC Vogel WG Rogers NF 1985 Black locust in the reclamation equation General Technical Report Northeastern Forest Experiment Station USDA Forest Service NE-I05 12 p
Ballard LAT 1973 Physical barriers to gennination Seed Sci Technoll 285-303
___ 1976 Strophiolar water conduction in the seeds ofTrifolieae induced by action on the testa at nonstrophiolar sites Austr J Plant Physiol3 465-469
Barton LV 1947 Special studies on seed coat impenneability Contrib Boyce Thompson Inst 14 355-362
Baskin CC Baskin JM 1998 Seeds Ecology biogeography and evolution of donnancy and gennination Academic Press London UK 666 p
Bewley JD 1997 Seed germination and donnancy Plant Ce119 1055-1066
Bewley JD Black M 1994 Seeds physiology of development and germination 2nd ed Plenum Press New York NY 445 p
Briscoe CB 1996 Growing Nitrogen Fixing Trees in Acid Soils In MH Powell (ed) Nitrogen Fixing Trees for Acid Soils- A Field ManuaL Winrock International Morrilton AR 22 p
Copeland LO McDonald MB 1995 Principles of Seed Science and Technology 3rded Chapman amp Hall New York NY 409 p
Cox RE Klett JE 1984 Seed gennination requirements of native Colorado plants for use in the landscape Plant Propag 30(2) 6-10
Dell B 1980 Structure and function ofthe strophiolar plug in seeds ofAlbizia Lophantha Am J Bot 67 556-563
Dick-Peddie WA 1993 New Mexico Vegetation Past Present and Future University ofNew Mexico Press Albuquerque NM 244 p
Dreesen DR 2001 Propagation protocol for production ofcontainer Robinia neomexicana plants Los Lunas Plant Materials Center Los Lunas New Mexico In Native Plant Network URL httpwwwnativeplantnetworkorg (accessed 9 January 2002) Moscow (ID) University ofIdaho CoIl Natural Resources For Res Nursery
72
Dreesen DR Harrington JT 1997 Propagation of native plants for restoration projects in the Southwestern US- Preliminary Investigations In Landis TD Thompson JR tech coords National Proceedings Forest and Conservation Nursery Assocs Gen Tech Rep PNW-GTR-419 Portland OR USDA For Ser PNW Res Sta 77-88
Gottfried GJ 1980 Control ofNew Mexican locust and the effect on planted ponderosa pine in central Arizona US For Ser Res Note RM-386 Rocky Mountain For Range ExpStn Fort Collins CO
Gosling PG Samuel YK Jones SK 1995 A systematic examination of germination temperature chipping and water temperaturesoak duration pretreatments on the seeds ofLeucaena leucocephala Seed Sci amp Technol 23521-532
Hamly DH 1932 Softening of the seeds of Melilotus alba Bot Gazette 93 345shy375
Hanna PJ 1984 Anatomical features of the seed coat ofAcacia kempeana (Mueller) which relate to increased germination rate induced by heat treatment New Phytol 96 23-29
Hanks JP Dick-Peddie WA 1974 Vegetation patterns of the White Mountains New Mexico Southwest Naturalist 18(4) 371-382
Hine S Harrington JT Dreesen DR 1997 Seed scarification requirements for Robinia neomexicana In Harrington JTtech coord Proc Third SouthwestemContainer Growers Conf Feb 12-13 1997 Ft Collins CO
Hopkinson JM 1993 The strophiole of leguminous seeds Newsletter- IntI Herbage Seed Production Res Grp 18 7-8
---- 1997 Differences in hardseededness between Cunningham and Tarramba leucaena In Leucnet News The Newsletter of the International LeucaenaRes and Devlt Netwk No4
ISTA (International Seed Testing Association) 1999 International rules for seed testing rules Seed Sci amp Technol27 Supplement
Kelly KM van Staden J 1987 The lens as the site of permeability in the papilionoid seed Aspalathus linearis J Plant Physiol 128(45) 395-404
73
Keresztesi B 1988 Black locust the tree of agriculture Outlook on Agr 17(2) 77~85
Kigel 1 and Galili G 1995 Seed development and germination Marcel Dekker New York 853 p
Klemmedson 10 1994 New Mexico locust and parent material Influence on macronutrients of forest floor and soil Soil Sci Soc Am 1 58 974~980
Kuo WH Tam AW 1988 The pathway of water absorption of mung bean seeds Seed Sci amp Technol16(1) 139~144
Leadem CL 1997 Dormancy--unlocking seed secrets In Landis TD Thompson lR tech coords National Proceedings Forestand Conservation Nursery Assocs Gen Tech Rep PNW-G TR-419 Portland OR USDA For Ser PNW Res Sta 43-52
Lin Y Wagner MR Seweyestewa A Honahni MD DeGomez M McMillin JD 1996 Influence of cultural treatments on seed germination and seedling growth of Robinia neomexicana Forest Farm and Community Tree Res Reports Volume 1
Marunda CT 1990 Effects of seed pretreatment on the development ofAcacia auriculforinis and A holosericea seedlings ACIAR Proceedings Series No 28
Meginnis HG 1937 Sulphuric acid treatment to increase germination of black locust seed USDA Circ 453 34 p
Miklas PN Towensend CE Ladd SL 1987 Seedcoat anatomy and the scarification ofcicer milkvetch seed Crop Sci 27 766-772
Monsen SB 1984 Use of shrubs on mine spoils In The Challenge ofProducing Native Plants for the Intermountain Area Proceedings Intermountain Nurserymans Association 1983 conference Las Vegas NV compo PM Murphy 26-31 Ogden UT USDA For Ser Gen Tech Report INT-l68
Olson DF 1974 Robinia L Locust In Seeds of Woody Plants in the US tech coord CS Schopmeyer 728-731 USDA For Ser Agr Handbk No450
Rehman S Loescher RNl Harris P1C 1998 Dormancy breaking and germination ofAcacia salicina Lindl seeds Seed Sci amp Techno127 553-557
Rolston MP 1978 Water impermeable seed dormancy Bot Rev 44 365-396
74
SAS Institute Inc 1989 SASSTAT Users Guide Version 6 4th Edition Volume 1 Cary NC SAS Institute Inc 943 p
SerratoValenti G Conara L Ferrando M Modenesi P 1993 Structural and histochemical features ofStylosanthes scabra (Leguminosae Papilionoidae) seed coat as related to water entry Can J Bot 71 834-840
Simpson BJ 1988 A field guide to Texas trees Austin TX Texas Monthly Press 372p
Tran VN Cavanagh AK 1984 Structural aspects ofdormancy In Seed Physiology Vol 2 Murray DR (ed) p1-44 Academic Press Australia
USDA Forest Service 1948 Woody-plant seed manual USDA Misc Pub1654 416p
Vines RA 1960 Trees Shrubs and Woody Vines of the Southwest Austin Univ ofTexas Press 1104 p
Wagner MR Larson RE Craig TP 1992 Ecology and silvics ofNew Mexico locust (Robinia neomexicana) with an emphasis on the arboreal form p53-57 In JW Hanover K Miller and S Plesko (eds) Proc Intemat Confon Black Locust Biology Culture and Utilization Mich State Univ USA
Wilson JK 1944 Immersing seeds of species of Robinia in boiling water hastens germination J For 42 453-454
Zimmerman RW Carpenter SB 1980 First-year coppice production from a 5shyyear old black locust stand on surface mine spoil p309-314 Centr Hardwd For ConfProc University ofMissouri Columbia
Dreesen DR Harrington JT 1997 Propagation of native plants for restoration projects in the Southwestern US- Preliminary Investigations In Landis TD Thompson JR tech coords National Proceedings Forest and Conservation Nursery Assocs Gen Tech Rep PNW-GTR-419 Portland OR USDA For Ser PNW Res Sta 77-88
Gottfried GJ 1980 Control ofNew Mexican locust and the effect on planted ponderosa pine in central Arizona US For Ser Res Note RM-386 Rocky Mountain For Range ExpStn Fort Collins CO
Gosling PG Samuel YK Jones SK 1995 A systematic examination of germination temperature chipping and water temperaturesoak duration pretreatments on the seeds ofLeucaena leucocephala Seed Sci amp Technol 23521-532
Hamly DH 1932 Softening of the seeds of Melilotus alba Bot Gazette 93 345shy375
Hanna PJ 1984 Anatomical features of the seed coat ofAcacia kempeana (Mueller) which relate to increased germination rate induced by heat treatment New Phytol 96 23-29
Hanks JP Dick-Peddie WA 1974 Vegetation patterns of the White Mountains New Mexico Southwest Naturalist 18(4) 371-382
Hine S Harrington JT Dreesen DR 1997 Seed scarification requirements for Robinia neomexicana In Harrington JTtech coord Proc Third SouthwestemContainer Growers Conf Feb 12-13 1997 Ft Collins CO
Hopkinson JM 1993 The strophiole of leguminous seeds Newsletter- IntI Herbage Seed Production Res Grp 18 7-8
---- 1997 Differences in hardseededness between Cunningham and Tarramba leucaena In Leucnet News The Newsletter of the International LeucaenaRes and Devlt Netwk No4
ISTA (International Seed Testing Association) 1999 International rules for seed testing rules Seed Sci amp Technol27 Supplement
Kelly KM van Staden J 1987 The lens as the site of permeability in the papilionoid seed Aspalathus linearis J Plant Physiol 128(45) 395-404
73
Keresztesi B 1988 Black locust the tree of agriculture Outlook on Agr 17(2) 77~85
Kigel 1 and Galili G 1995 Seed development and germination Marcel Dekker New York 853 p
Klemmedson 10 1994 New Mexico locust and parent material Influence on macronutrients of forest floor and soil Soil Sci Soc Am 1 58 974~980
Kuo WH Tam AW 1988 The pathway of water absorption of mung bean seeds Seed Sci amp Technol16(1) 139~144
Leadem CL 1997 Dormancy--unlocking seed secrets In Landis TD Thompson lR tech coords National Proceedings Forestand Conservation Nursery Assocs Gen Tech Rep PNW-G TR-419 Portland OR USDA For Ser PNW Res Sta 43-52
Lin Y Wagner MR Seweyestewa A Honahni MD DeGomez M McMillin JD 1996 Influence of cultural treatments on seed germination and seedling growth of Robinia neomexicana Forest Farm and Community Tree Res Reports Volume 1
Marunda CT 1990 Effects of seed pretreatment on the development ofAcacia auriculforinis and A holosericea seedlings ACIAR Proceedings Series No 28
Meginnis HG 1937 Sulphuric acid treatment to increase germination of black locust seed USDA Circ 453 34 p
Miklas PN Towensend CE Ladd SL 1987 Seedcoat anatomy and the scarification ofcicer milkvetch seed Crop Sci 27 766-772
Monsen SB 1984 Use of shrubs on mine spoils In The Challenge ofProducing Native Plants for the Intermountain Area Proceedings Intermountain Nurserymans Association 1983 conference Las Vegas NV compo PM Murphy 26-31 Ogden UT USDA For Ser Gen Tech Report INT-l68
Olson DF 1974 Robinia L Locust In Seeds of Woody Plants in the US tech coord CS Schopmeyer 728-731 USDA For Ser Agr Handbk No450
Rehman S Loescher RNl Harris P1C 1998 Dormancy breaking and germination ofAcacia salicina Lindl seeds Seed Sci amp Techno127 553-557
Rolston MP 1978 Water impermeable seed dormancy Bot Rev 44 365-396
74
SAS Institute Inc 1989 SASSTAT Users Guide Version 6 4th Edition Volume 1 Cary NC SAS Institute Inc 943 p
SerratoValenti G Conara L Ferrando M Modenesi P 1993 Structural and histochemical features ofStylosanthes scabra (Leguminosae Papilionoidae) seed coat as related to water entry Can J Bot 71 834-840
Simpson BJ 1988 A field guide to Texas trees Austin TX Texas Monthly Press 372p
Tran VN Cavanagh AK 1984 Structural aspects ofdormancy In Seed Physiology Vol 2 Murray DR (ed) p1-44 Academic Press Australia
USDA Forest Service 1948 Woody-plant seed manual USDA Misc Pub1654 416p
Vines RA 1960 Trees Shrubs and Woody Vines of the Southwest Austin Univ ofTexas Press 1104 p
Wagner MR Larson RE Craig TP 1992 Ecology and silvics ofNew Mexico locust (Robinia neomexicana) with an emphasis on the arboreal form p53-57 In JW Hanover K Miller and S Plesko (eds) Proc Intemat Confon Black Locust Biology Culture and Utilization Mich State Univ USA
Wilson JK 1944 Immersing seeds of species of Robinia in boiling water hastens germination J For 42 453-454
Zimmerman RW Carpenter SB 1980 First-year coppice production from a 5shyyear old black locust stand on surface mine spoil p309-314 Centr Hardwd For ConfProc University ofMissouri Columbia
Keresztesi B 1988 Black locust the tree of agriculture Outlook on Agr 17(2) 77~85
Kigel 1 and Galili G 1995 Seed development and germination Marcel Dekker New York 853 p
Klemmedson 10 1994 New Mexico locust and parent material Influence on macronutrients of forest floor and soil Soil Sci Soc Am 1 58 974~980
Kuo WH Tam AW 1988 The pathway of water absorption of mung bean seeds Seed Sci amp Technol16(1) 139~144
Leadem CL 1997 Dormancy--unlocking seed secrets In Landis TD Thompson lR tech coords National Proceedings Forestand Conservation Nursery Assocs Gen Tech Rep PNW-G TR-419 Portland OR USDA For Ser PNW Res Sta 43-52
Lin Y Wagner MR Seweyestewa A Honahni MD DeGomez M McMillin JD 1996 Influence of cultural treatments on seed germination and seedling growth of Robinia neomexicana Forest Farm and Community Tree Res Reports Volume 1
Marunda CT 1990 Effects of seed pretreatment on the development ofAcacia auriculforinis and A holosericea seedlings ACIAR Proceedings Series No 28
Meginnis HG 1937 Sulphuric acid treatment to increase germination of black locust seed USDA Circ 453 34 p
Miklas PN Towensend CE Ladd SL 1987 Seedcoat anatomy and the scarification ofcicer milkvetch seed Crop Sci 27 766-772
Monsen SB 1984 Use of shrubs on mine spoils In The Challenge ofProducing Native Plants for the Intermountain Area Proceedings Intermountain Nurserymans Association 1983 conference Las Vegas NV compo PM Murphy 26-31 Ogden UT USDA For Ser Gen Tech Report INT-l68
Olson DF 1974 Robinia L Locust In Seeds of Woody Plants in the US tech coord CS Schopmeyer 728-731 USDA For Ser Agr Handbk No450
Rehman S Loescher RNl Harris P1C 1998 Dormancy breaking and germination ofAcacia salicina Lindl seeds Seed Sci amp Techno127 553-557
Rolston MP 1978 Water impermeable seed dormancy Bot Rev 44 365-396
74
SAS Institute Inc 1989 SASSTAT Users Guide Version 6 4th Edition Volume 1 Cary NC SAS Institute Inc 943 p
SerratoValenti G Conara L Ferrando M Modenesi P 1993 Structural and histochemical features ofStylosanthes scabra (Leguminosae Papilionoidae) seed coat as related to water entry Can J Bot 71 834-840
Simpson BJ 1988 A field guide to Texas trees Austin TX Texas Monthly Press 372p
Tran VN Cavanagh AK 1984 Structural aspects ofdormancy In Seed Physiology Vol 2 Murray DR (ed) p1-44 Academic Press Australia
USDA Forest Service 1948 Woody-plant seed manual USDA Misc Pub1654 416p
Vines RA 1960 Trees Shrubs and Woody Vines of the Southwest Austin Univ ofTexas Press 1104 p
Wagner MR Larson RE Craig TP 1992 Ecology and silvics ofNew Mexico locust (Robinia neomexicana) with an emphasis on the arboreal form p53-57 In JW Hanover K Miller and S Plesko (eds) Proc Intemat Confon Black Locust Biology Culture and Utilization Mich State Univ USA
Wilson JK 1944 Immersing seeds of species of Robinia in boiling water hastens germination J For 42 453-454
Zimmerman RW Carpenter SB 1980 First-year coppice production from a 5shyyear old black locust stand on surface mine spoil p309-314 Centr Hardwd For ConfProc University ofMissouri Columbia
SAS Institute Inc 1989 SASSTAT Users Guide Version 6 4th Edition Volume 1 Cary NC SAS Institute Inc 943 p
SerratoValenti G Conara L Ferrando M Modenesi P 1993 Structural and histochemical features ofStylosanthes scabra (Leguminosae Papilionoidae) seed coat as related to water entry Can J Bot 71 834-840
Simpson BJ 1988 A field guide to Texas trees Austin TX Texas Monthly Press 372p
Tran VN Cavanagh AK 1984 Structural aspects ofdormancy In Seed Physiology Vol 2 Murray DR (ed) p1-44 Academic Press Australia
USDA Forest Service 1948 Woody-plant seed manual USDA Misc Pub1654 416p
Vines RA 1960 Trees Shrubs and Woody Vines of the Southwest Austin Univ ofTexas Press 1104 p
Wagner MR Larson RE Craig TP 1992 Ecology and silvics ofNew Mexico locust (Robinia neomexicana) with an emphasis on the arboreal form p53-57 In JW Hanover K Miller and S Plesko (eds) Proc Intemat Confon Black Locust Biology Culture and Utilization Mich State Univ USA
Wilson JK 1944 Immersing seeds of species of Robinia in boiling water hastens germination J For 42 453-454
Zimmerman RW Carpenter SB 1980 First-year coppice production from a 5shyyear old black locust stand on surface mine spoil p309-314 Centr Hardwd For ConfProc University ofMissouri Columbia
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