34 Somatic Embryogenesis in Pinus Halepensis Mill an Important Ecological Species From the...
Transcript of 34 Somatic Embryogenesis in Pinus Halepensis Mill an Important Ecological Species From the...
-
8/18/2019 34 Somatic Embryogenesis in Pinus Halepensis Mill an Important Ecological Species From the Mediterranean Forest
1/13
O R I G I N A L P A P E R
Somatic embryogenesis in Pinus halepensis Mill.: an importantecological species from the Mediterranean forest
I. A. Montalbán • A. Setién-Olarra •
C. L. Hargreaves • P. Moncaleán
Received: 31 January 2013/ Revised: 4 April 2013/ Accepted: 11 April 2013 / Published online: 1 May 2013
Springer-Verlag Berlin Heidelberg 2013
Abstract Pinus halepensis Mill. is a common forest
species in the Mediterranean area and it is important forenvironmental conservation. This study established a
method of regenerating Pinus halepensis Mill. through
somatic embryogenesis. The effect of culture medium
(mineral salts, nitrogen source and plant growth regula-
tors), collection date and seed family on embryogenic tis-
sue initiation and proliferation in Pinus halepensis was
analysed during the first steps of embryogenesis process.
This study showed a marked effect of the culture medium
tested as well as some significant differences among col-
lection dates. Furthermore, the embryogenic tissue initia-
tion was affected by the amino acid mixture in the culture
medium and the proliferation stage was significantly
affected by the combination of plant growth regulators. At
the end of the maturation phase the presence of activated
charcoal was also evaluated. Finally, maturation of
embryogenic tissue was affected by the nitrogen source in
the culture medium and these results were different for
high and low mature embryo producing cell lines. To the
best of our knowledge, this is the first report on Aleppo
pine somatic embryogenesis describing a simple and effi-
cient procedure for large-scale somatic embryo production.
Keywords Aleppo pine Embryogenic tissue
Germination
Initiation
Maturation
Propagation
Abbreviations
A Maturation medium, DCR basal mediumsupplemented with: 75 lM ABA, 9 g L-1 of
Gelrite, 4.5 % (w/v) sucrose, and ED amino
acid mixture
ABA Abscisic acid
AC Activated charcoal
B Maturation medium, DCR medium supplemented
with: 75 lM ABA and 9 g L-1 of Gelrite, 4.5 %
(w/v) sucrose and ED amino acid mixture with
1,650 mg L-1 of glutamine
BA N6-benzyladenine
C Maturation medium, DCR basal medium
supplemented with: 75 lM ABA, 9 g L-1 of
Gelrite, 6 % (w/v) sucrose and ED amino acid
mixture
D Maturation medium, DCR medium supplemented
with: 75 lM ABA and 9 g L-1 of Gelrite, 6 %
(w/v) sucrose and ED amino acid mixture with
1,650 mg L-1 glutamine
2,4-D 2,4-Dichlorophenoxyacetic acid
DBA 9 lM 2,4D and 2.7 lM BA
DCR Gupta and Durzan basal medium (Gupta and
Durzan 1985)
DKI 9 lM 2,4D and 2.7 lM Kinetin
DNB 4.5 lM 1-Naphthaleneacetic acid, 4.5 lM 2,4D
and 2.7 lM BA
ECLs Established cell lines
ED EDM amino acid mixture
EDM Embryo development medium (Walter et al. 1998)
ET Embryogenic tissue
FW Fresh weight
LP Quorin and Lepoivre medium (Quoirin and Lepoivre
1977, modified by Aitken-Christie et al. 1988)
SE Somatic embryogenesis
Communicated by K. Klimaszewska.
I. A. Montalbán A. Setién-Olarra P. Moncaleán (&)
Centro de Arkaute, Neiker-Tecnalia, Apdo. 46,
01080 Vitoria-Gasteiz, Spain
e-mail: [email protected]
C. L. Hargreaves
Scion, Private Bag 3020, Rotorua 3046, New Zealand
1 3
Trees (2013) 27:1339–1351
DOI 10.1007/s00468-013-0882-0
-
8/18/2019 34 Somatic Embryogenesis in Pinus Halepensis Mill an Important Ecological Species From the Mediterranean Forest
2/13
Introduction
Pinus halepensis Mill., commonly named Aleppo pine, is a
species native to the Mediterranean region and is wide-
spread from Spain to Algeria (Botella et al. 2010). Tem-
perature and precipitation requirements generally confine
its distribution to sub-humid areas of the Mediterranean. Its
main area of distribution is Southern Europe (Spain, Italyand Greece) and Northern Africa (Algeria, Tunisia and
Morocco). In these regions, as described by Lambardi et al.
(1993), Aleppo pine is of a great economic importance due
to its adaptability to dry, calcareous and poor soils. In light
of predictions of global drying and warming for this region,
there is some concern about the physiological ability of P.
halepensis to persevere in large afforestations in the future
(Oliveras et al. 2003; Maestre and Cortina 2004). This
species is especially suitable for the reforestation of mar-
ginal and submarginal areas because it is one of the most
drought resistant pine species (Klein et al. 2011). More-
over, in marine stands, P. halepensis forests are importantboth for defence against the saline winds and for landscape
purposes (Lambardi et al. 1993). In Spain, virtually all
natural stands are distributed over the whole Eastern coast,
though due to its important ecological plasticity it has also
been intensively used for afforestation in North-Western
areas of the Iberian Peninsula, very often out of its natural
habitat range (Abelló 1998).
In vitro vegetative propagation from physiologically
juvenile tissue has been successful in a number of conifer
species (Moncaleán et al. 2005). This phenomenon has led
many organizations to focus on production of elite families
through juvenile tissue propagation in the short-term, eitherfor operational use or for clonal tests, while they continue
to research methods for propagation of selected mature
trees (De Diego et al. 2008, 2010).
Conventional seed orchards provide genetically
improved seeds, but, as stated by Park et al. (1998) tradi-
tional breeding strategies combined with in vitro vegetative
propagation have shown advantages. These include addi-
tional genetic gain achieved by capturing non-additive
genetic variation and the capacity of introducing clones to
meet market goals at a higher speed. In this sense, propa-
gation via somatic embryogenesis (SE) from immature
zygotic embryos is an effective method of propagation
when combined with other technologies, such as cryopre-
serving the embryogenic tissue (ET) and selecting elite
clones in field tests (Park 2002). This system offers the
capability to produce large numbers of somatic embryo
derived plantlets (Montalbán et al. 2010) as well as to use
the somatic embryos for organogenesis procedures and thus
provide further amplification (Montalbán et al. 2011a).
However, this technology has a number of bottlenecks in
the Pinus genus, namely, a narrow competence window for
embryogenic tissue (ET) initiation (MacKay et al. 2006),
low maturation frequency of the embryogenic tissue
(Montalbán et al. 2011a) and poor efficiency in the ger-
mination process (Maruyama and Hosoi 2012). Conse-
quently, the number of genotypes that can be candidates for
clonal tests decreases (Davis and Becwar 2007).
To date, as far as we know, Pinus halepensis SE process
has not been studied. Considering the aforementionedaspects, the general aim of this work was to study the
feasibility of SE in Pinus halepensis using immature
embryos as initial explants.
This general objective included the following tasks: (1)
to determine the optimal explant collection time and
induction medium for initiation of ET and (2) to test the
effects of activated charcoal (AC), sucrose concentration
and nitrogen source in the culture medium on somatic
embryo maturation and conversion into plants.
Materials and methods
Initiation and proliferation
Plant material
One-year-old green female cones (Fig. 1a), enclosing
immature zygotic embryos of Pinus halepensis, were col-
lected from open-pollinated trees in Berantevilla (Álava,
Spain).
Intact cones were sprayed with 70 % (v/v) ethanol,
and they were split into quarters and all immature seeds
were removed from the cones. Then, immature seeds
(Fig. 1b) were surface sterilised in 10 % H2O2 (v/v) plus
two drops of Tween 20 for 8 min and then rinsed three
times under sterile distilled H2O in sterile conditions in
the laminar flow unit. Seed coats were removed and
whole megagametophytes containing immature embryos
were excised aseptically and placed horizontally onto the
medium.
Experiment 1
One green cone was sampled weekly, from seven open-
pollinated families, from the 7th of June to the 16th of
August (eleven collection dates). Then, cones were stored
for a maximum of a week in paper bags at 4 C. These
cones were collected from trees: 1, 2, 3, 4, 5, 6 and 7.
Three basal initiation media (macronutrients, micronu-
trients and vitamins of these media) were tested: DCR
medium (Gupta and Durzan 1985), EDM medium (Walter
et al. 1998) and LP medium (Quoirin and Lepoivre 1977)
[modified by Aitken-Christie et al. (1988)]. These media
were supplemented with sucrose at 3 % (w/v) and a
1340 Trees (2013) 27:1339–1351
1 3
-
8/18/2019 34 Somatic Embryogenesis in Pinus Halepensis Mill an Important Ecological Species From the Mediterranean Forest
3/13
combination of 4.5 lM 2,4-dichlorophenoxyacetic acid
(2,4-D) and 2.7 lM benzyladenine (BA). Before auto-
claving, the pH of the media was adjusted to 5.7 and then
3 g L-1 gellan gum (Gelrite) was added. The medium
was autoclaved at 121 C for 20 min. After autoclaving, a
filter-sterilized solution with the pH adjusted to 5.7 con-
taining the ED amino acid mixture (550 mg L-1 L-
glutamine, 525 mg L-1 asparagine, 175 mg L-1 arginine,
19.75 mg L-1 L-citrulline, 19 mg L-1 L-ornithine, 13.75
mg L-1 L-lysine, 10 mg L-1 L-alanine and 8.75 mg L-1 L-
proline) was added to the cooled medium prior, and the
media were dispensed into Petri dishes (90 9 20 mm).
Ten megagametophytes per Petri dish, three Petri dishes
per initiation medium, family and collection date were
Fig. 1 Somatic embryogenesis in Pinus halepensis: a green cone
collected on the 12th of July, bar 25 mm; b immature seed from
green cone collected on the 16th of July, bar 7 mm; c initiation and
d proliferation of embryogenic tissue on DCR medium (Gupta andDurzan 1985), bars 6 mm; e somatic embryo developing on DCR
maturation medium, bar 2 mm; f mature somatic embryo, bar 5 mm;
g somatic embryos germinating on half strength LP medium (Quoirin
and Lepoivre 1977, modified by Aitken-Christie et al. 1988), bar
12 mm; h somatic plant growing in the greenhouse, bar 25 mm
Trees (2013) 27:1339–1351 1341
1 3
-
8/18/2019 34 Somatic Embryogenesis in Pinus Halepensis Mill an Important Ecological Species From the Mediterranean Forest
4/13
cultured, resulting in a total number of 6,930 explants. The
Petri dishes were laid out randomly on the shelves of the
growth chamber. Cultures were maintained in the dark at
218 ± 1 C.
After 4–8 weeks from the beginning of the experiment,
proliferating ET with a size around 3–5 mm in diameter
was separated from the megagametophytes (Fig. 1c). ET
was sub-cultured to maintenance medium every 2 weeks;maintenance media had the same composition of the ini-
tiation media, but 4 g L-1 of Gelrite. The concentration
of gellan gum in the media was increased to maintain the
spiky morphology of the embryogenic cell lines (ECLs)
(Fig. 1d), according to Breton et al. (2005).
At each collection date, ten megagametophytes per seed
family were destructively sampled, and classification of the
stage of zygotic embryo development was made according
to the eight-stage scale developed by Hargreaves et al.
(2009).
Experiment 2
Cones from two collection dates (28th of July and 2nd of
August) from the families described in Experiment 1 were
collected and stored for a maximum of a week in paper
bags at 4 C.
Ten megagametophytes per Petri dish were cultured. Six
to eight Petri dishes per initiation medium were laid out
randomly on the shelves of the growth chamber. Cultures
were maintained in the dark at 21 ± 1 C.
Twelve initiation media were tested. All media had the
DCR (Gupta and Durzan 1985) basal composition and
differed in:
• The amino acid mixture (ED as in experiment 1, or
1 g L-1 casein hydrolysate and 500 mg L-1 L-
glutamine).
• The growth regulators (DKI mixture: 9 lM 2,4D and
2.7 lM Kinetin; or DBA mixture: 9 lM 2,4D and
2.7 lM BA; or DNB mixture: 4.5 lM 1-naphthalene-
acetic acid, 4.5 lM 2,4D and 2.7 lM BA) (Table 1).
• The gellan gum concentration: 2 or 3 g L-1 Gelrite.
After 4–8 weeks, proliferating ET with a size around
3–5 mm in diameter was separated from the megaga-
metophytes. The ET was sub-cultured to maintenance
medium every 2 weeks; maintenance media had the same
composition of the initiation media but with 4 g L-1 of
gellan gum.
Maturation of ECLs
Plant material
The ECLs from initiation experiment 1 were used for
maturation experiment 1 and ECLs from initiation exper-
iment 2 were used for maturation experiment 2.
Experiment 1
A maturation experiment was designed to assess the effect
of the presence of AC in the ET suspension. The ET was
suspended in liquid growth regulator-free EDM medium(Walter et al. 1998), supplemented with 2 g L-1 of acti-
vated charcoal (AC) (Sigma), or without AC, in 50 mL
centrifuge tubes. Then, ET suspension was vigorously
shaken by hand for a few seconds. Thereafter, a 5 mL
aliquot of the suspension containing 50–60 mg fresh
weight (FW) of ET was poured onto a filter paper disc
(Whatman no. 2, 7 cm) in a Büchner funnel. A vacuum
pulse was applied for 10 s, and the filter paper with the
attached ET was transferred to maturation medium. The
maturation medium had the salt formulation of DCR
(Gupta and Durzan 1985) supplemented with 6 % (w/v)
sucrose, 75 lM abscisic acid (ABA), ED amino acid
mixture used for initiation and proliferation of the ET and a
higher concentration of gellan gum (9 g L-1 of Gel-
rite)(medium C). Sixteen ECLs were tested, three to five
Petri dishes per ECL and treatment were laid out randomly
on the shelves of the growth chamber. Cultures were
maintained in the dark at 21 ± 1 C. After 9 weeks in
maturation medium, the embryos reach the torpedo stage
(Fig. 1e) prior to the cotyledonary one (Fig. 1f) which it is
considered to be the end of maturation phase.
Experiment 2
A maturation experiment was designed to assess the effect
of the composition of maturation medium related to
nitrogen and carbon source on the number of somatic
embryos per gram of ET. The ET was first suspended in
liquid growth regulators-free EDM medium (Walter et al.
1998) and a 5 mL aliquot containing 70–80 mg of sus-
pended ET (FW) was filtered and transferred to maturation
medium as described above. Four maturation media were
tested; one of them was described in experiment 1 (medium
Table 1 Different plant growth regulator combinations included in
initiation and proliferation media
DBA
(lM)
DKI
(lM)
DNB
(lM)
Benzyladenine 2.7 – 2.7
Kinetin – 2.7 –
2,4-Dichlorophenoxyacetic
acid
9 9 4.5
1-Naphthaleneacetic acid – – 4.5
1342 Trees (2013) 27:1339–1351
1 3
-
8/18/2019 34 Somatic Embryogenesis in Pinus Halepensis Mill an Important Ecological Species From the Mediterranean Forest
5/13
C). All maturation media had the salt formulation of DCR
(Gupta and Durzan 1985), 75 lM ABA and 9 g L-1 of
Gelrite. These media were supplemented with sucrose at
4.5 % (maturation media A and B) or 6 % (maturation
media C and D), and ED amino acid mixture used for
initiation and proliferation of the ET (maturation media A
and C) or the same amino acid mixture but with a higher
concentration of glutamine (1,650 mg L-1 instead of the550 mg L-1) (maturation media B and D) (Table 2). Three
ECLs were tested; three Petri dishes per ECL and treatment
were laid out randomly on the shelves of the growth
chamber. Cultures were maintained in the dark at
21 ± 1 C.
Germination
Mature somatic embryos from maturation experiments
(Fig. 1f) were collected and cultured on half strength LP
[(Quoirin and Lepoivre 1977) modified by Aitken-Christie
et al. (1988)] supplemented with 2 g L-1 AC and 10 g L-1
Difco agar granulated. Ten to twenty mature somatic
embryos per Petri dish were cultured.
The plants (Fig. 1g) were sub-cultured once onto fresh
medium of the same composition every 6 weeks. Cultures
were maintained at 21 ± 1 C under a 16-h photoperiod at
120 lmol m-2s-1 provided by cool white fluorescent tubes
(TLD 58 W/33; Philips, France).
After 18–24 weeks on germination medium, the plants
were transferred to sterile peat: perlite (7:3, v/v) and
acclimatized in a greenhouse under controlled temperatures
at 21 ± 2 C and progressively decreasing humidity from
90 to 60 % (Fig. 1h).
Data collection and statistical analysis
Initiation and proliferation experiments
After 8–10 weeks from the beginning of the experiments,
the number of proliferating ET calli with a size around
3–5 mm in diameter were recorded and initiation per-
centage per Petri dish was calculated. Following three
subculture periods, actively growing ETs were recorded as
ECLs and proliferation percentage per Petri dish was cal-
culated. Each ECL was named with letter H followed by a
number for experiment 1 and with h followed by a number
for experiment 2.
Maturation
After 18 weeks from the beginning of the experiments, the
number of mature somatic embryos per Petri dish was
recorded and the number of mature somatic embryos pergram was calculated.
Germination
After 18 weeks on germination medium the number of
germinated somatic embryos per gram of ET and the
overall germinated somatic embryos related to the total
number of somatic embryos introduced (conversion, %)
was calculated.
The results for all the experiments carried out were
analysed by ANOVA, and significant differences between
means were determined by the Tukey post hoc test at asignificance level of p \ 0.05.
Results
Initiation and proliferation
Experiment 1
From the 2,310 megagametophytes cultured on EDM
medium, only one initiation of ET was observed and it did
not continue to proliferate. On LP medium, 25 initiations of
ET from the 2,310 megagametophytes cultured were
recorded; 52 % of these initiations were obtained from
families 4 and 5 at collection dates 10 and 11. From the 25
initiations observed, only three of them led to ECLs (data
not shown).
DCR medium gave the best results. The initiation of ET
on DCR medium was significantly affected by the seed
family and the collection date tested (Table 3). There was
also a significant ( p\ 0.01) interaction among the mother
trees and the weeks studied (Table 3). Family 4 produced a
significantly ( p B 0.05) higher initiation percentage
(25.6 %) than family 1 (8.3 %) (Table 4). The rest of the
Table 2 Description of the
different maturation media
tested, giving amino acid
composition and sucrose
percentage
Sucrose (%) Amino acid mixture
Maturation medium A 4.5 ED amino acid mixture (550 mg L-1
glutamine)
Maturation medium B 4.5 ED mixture (1,650 mg L-1
glutamine)
Maturation medium C 6 ED amino acid mixture (550 mg L-1
glutamine)
Maturation medium D 6 ED mixture (1,650 mg L-1
glutamine)
Trees (2013) 27:1339–1351 1343
1 3
-
8/18/2019 34 Somatic Embryogenesis in Pinus Halepensis Mill an Important Ecological Species From the Mediterranean Forest
6/13
families presented initiation percentage means ranging
from 16.1 to 23.9 and did not show significant differences
either with family 4 or with family 1 (Table 4).
When collection dates were considered, the megaga-
metophytes cultured on initiation medium from the 7th of
Junetothe5thofJuly(collectiondates1,2,3,4and5)didnot
initiate ET (data not shown). The best results for initiation of
ET were obtained between collection dates 9 and 10 (27.1
and 28.1 %, respectively) (Table 4); these collection dates
correspond to zygotic embryo developmental stages
between 3 and 4 (early bullet stage embryos). The results
obtained at collection dates 9 and 10 were significantly
( p B 0.05) higher than initiation percentage at collection
date 8 (6.7 %) (Table 4). The other collection dates studied
showed initiation percentages ranging from 14.3 to 19.5 and
did not show significant differences either with collection
dates 9 and 10 or with collection date 8 (Table 4).
In summary, the highest initiation percentage (70 %)
was obtained in family 4 in collection date 10. Taking
into account the interaction among seed families and
collection dates, six from the seven seed families studied
achieved their peak initiation percentages between col-
lection dates 9 and 11 (Table 4). Family 7 showed the
highest initiation of ET at collection date 6, whereas
family 2 had equal initiation percentages at collection
dates 6 and 9 (Table 4).
Regarding ET proliferation, this was significantly
( p\ 0.05) affected by the seed family studied (Table 3). In
this sense, family 3 had significantly ( p\ 0.05) better
values (7.2 %) than families 1, 2 and 5 with proliferation
percentages lower than 1.2 % (Fig. 2). Families 4, 6 and 7
whose proliferation percentages ranged from 4.4 to 6.7 %
did not show significant differences with the other families
tested (Fig. 2).
Table 3 ANOVA for embryogenic tissue initiation and proliferation (%) in Pinus halepensis megagametophytes from seven open-pollinated
trees cultured on DCR medium (Gupta and Durzan 1985) at six collection dates
Source df ET Initiation (%) ET Proliferation (%)
Mean square F test p value Mean square F test p value
Seed family (S) 6 724.07 3.02 \0.01 140.21 2.90 \0.05
Collection date (C) 5 1,441.43 6.01 \0.001 94.13 1.94 n.s.
S 9 C 30 715.50 2.99 \0.01 72.28 1.49 n.s.
Error 84 239.68 239.68
n.s. non-significant
Table 4 Embryogenic tissue (ET) initiation (%) in Pinus halepensis in seven open-pollinated trees cultured on DCR medium (Gupta and Durzan
1985) at six collection dates, different letters show significant differences at p \ 0.01 by Tukey’s post hoc test (M ± SE)
Seed
family
ET initiation (%)
Collection date
6th 7th 8th 9th 10th 11th Mean
12 July 19 July 26 July 2 August 9 August 16 August
1 6.7 ± 6.7cd 10.0 ± 5.8cd 0.0 ± 0.0d 6.7 ± 6.7cd 10.0 ± 5.8cd 16.7 ± 8.8bcd 8.3 ± 2.5b
2 30.0 ± 0.0abcd
6.7 ± 6.7cd
10.0 ± 5.8cd
30.0 ± 5.8abcd
6.7 ± 6.7cd
13.3 ± 6.7bcd
16.1 ± 3.1ab
3 16.7 ± 8.8bcd 26.7 ± 6.7abcd 3.3 ± 3.3cd 26.7 ± 8.8abcd 53.3 ± 8.8abc 16.7 ± 3.3bcd 23.9 ± 4.4ab
4 0.0 ± 0.0d 23.3 ± 3.3abcd 20.0 ± 11.6abcd 26.7 ± 8.8abcd 70.0 ± 0.0a 13.3 ± 8.8bcd 25.6 ± 5.8a
5 0.0 ± 0.0d 10.0 ± 10cd 0.0 ± 0.0d 16.7 ± 16.7bcd 16.7 ± 12.0bcd 30.0 ± 15.3abcd 12.2 ± 4.6ab
6 0.0 ± 0.0d 6.7 ± 3.3cd 0.0 ± 0.0d 63.3 ± 8.8ab 26.7 ± 21.9abcd 33.3 ± 17.6abcd 21.7 ± 6.9ab
7 46.7 ± 24.0abcd 16.7 ± 3.3bcd 13.3 ± 3.3bcd 20.0 ± 0.0abcd 13.3 ± 8.8bcd 13.3 ± 3.3bcd 20.6 ± 4.7ab
Mean 14.3 ± 4.9ab
14.3 ± 2.5ab
6.7 ± 2.3b
27.1 ± 4.7a
28.1 ± 6.1a
19.5 ± 3.8ab
Embryo
stage
1 1.5 2 3 4 5
(Pro-
embryonary
stage)
(Early cleavage poly-
embryonary stage)
(Cleavage poly-
embryonary
stage)
(Early bullet
stage)
(Bullet stage) (Late bullet
stage)
In the last row, the embryo stage is shown. Means followed by the same letter (a, b, c or d) are not significantly different at p\0.05 Tukey’s post
hoc test
1344 Trees (2013) 27:1339–1351
1 3
-
8/18/2019 34 Somatic Embryogenesis in Pinus Halepensis Mill an Important Ecological Species From the Mediterranean Forest
7/13
Experiment 2
In this experiment twelve media were tested. These media
differed in the nitrogen source, the plant growth regulator
combinations or the gellan gum concentration. TheANOVA showed that only the nitrogen source used had a
significant effect on ET initiation and no interaction was
observed (Table 5). Thus, the media containing ED amino
acid mixture presented an initiation percentage of 36.4 %,
whereas the media supplemented with 1 g L-1 casein
hydrolysate and 500 mg L-1 L-glutamine had an initiation
percentage of 15.4 % (data not shown).
When analysing the proliferation stage, it was observed
that this was only significantly ( p\ 0.05) influenced by the
growth regulators (Table 5). At this stage, we also
observed no interactions between the factors studied.
Namely, the combinations of growth regulators DKI andDNB gave significantly ( p\ 0.05) better results (12 and
11.5 %, respectively) than DBA (3.3 %) (Fig. 3).
Maturation and germination
Experiment 1
From the sixteen ECLs tested, thirteen ECLs produced
mature somatic embryos. The ANOVA for these thirteen
ECLs showed that the ECL ( p\0.001) and the interaction
among ECL and AC ( p\ 0.05) had a significant effect onthe maturation process (Table 6). Notably, H49 and H150
only produced somatic embryos when the suspension used
for maturation had AC whereas H58 developed somatic
embryos solely when the suspension lacked AC (Fig. 4).
The ECL H29 was more productive (716 somatic
embryos per gram of ET) than the rest of the ECLs
assayed. Six ECLs produced between 108 and 350 somatic
embryos per gram of ET, whereas six ECLs produced less
than 68 somatic embryos per gram of ET (Fig. 4).
When considering the interaction between the ECL and
presence or absence of AC in the suspension used for
maturation, in five of the thirteen ECLs tested, the presenceof AC was beneficial (Fig. 4). On the contrary, in eight
ECLs the absence of AC in the suspension led to a higher
production of somatic embryos (Fig. 4).
The number of germinated somatic embryos was sig-
nificantly ( p\ 0.001) affected by the ECL tested (Table 6)
and there was no interaction among the ECL and the
presence or absence of AC in the suspension used for
maturation. Three of the thirteen ECLs tested yielded more
than 100 germinated somatic embryos per gram of ET.
Only two of the lines tested produced less than 10 embryos
per gram of ET (Table 7).
Considering the conversion rate, the overall germination
percentage in this experiment was 60 % (data not shown).
The plants were successfully acclimatized in plastic
0
5
10
15
20
25
1 2 3 4 5 6 7
Seed family
E T P r o l i f e r a t i o n ( % )
b b
a
ab
b
ab
ab
Fig. 2 Embryogenic tissue (ET) proliferation (%) in megagameto-
phytes from seven seed families of Pinus halepensis cultured on DCR
medium (Gupta and Durzan 1985) (M ± SE). Different letters show
significant differences at p \ 0.05 by Tukey’s post hoc test
Table 5 ANOVA for embryogenic tissue (ET) initiation and prolif-
eration (%) in Pinus halepensis megagametophytes cultured on DCR
medium (Gupta and Durzan 1985) supplemented with different amino
acid mixtures [(A), ED or 1 g L-1
casein hydrolysate and
500 mg L-1 L-glutamine], plant growth regulator combinations
[(H), DBA, DKI and DNB] or gellan gum concentrations [(G),
2 g L-1
or 3 g L-1
]
Source df ET initiation (%) ET proliferation (%)
Mean square F test p value Mean square F test p value
Amino acid mixture (A) 1 8,294.81 18.22 \0.001 443.41 3.12 n.s.
Growth regulators (H) 2 132.90 0.29 n.s. 594.14 4.19 \0.05
Gellan gum (G) 1 1.56 0.003 n.s. 208.10 1.47 n.s.
A 9 H 2 182.13 0.40 n.s. 133.98 0.94 n.s.
A 9 G 1 380.52 0.83 n.s. 0.54 0.004 n.s.
H 9 G 2 143.81 0.31 n.s. 206.37 1.45 n.s.
A 9 H 9 G 2 15.29 0.03 n.s. 119.45 0.84 n.s.
Error 64 457.73 141.96
n.s. non-significant
Trees (2013) 27:1339–1351 1345
1 3
-
8/18/2019 34 Somatic Embryogenesis in Pinus Halepensis Mill an Important Ecological Species From the Mediterranean Forest
8/13
propagation trays, and their growth is currently being
monitored in the greenhouse.
Experiment 2
The ECL, the maturation medium (A, B, C or D) and the
interaction between them had a significant ( p\ 0.001)
effect on the number of mature somatic embryos obtained
per gram of ET (Table 8). The ECL h42 yielded a signif-
icantly ( p\ 0.05) higher number of somatic embryos (195
somatic embryos per gram of ET) than h19 and h41 (57 and
114 somatic embryos per gram of ET, respectively)
(Table 9). The maturation medium C led to a significantly
( p B 0.05) higher production of somatic embryos (243
somatic embryos per gram of ET) than the other media
tested (from 70.5 to 97.2 somatic embryos per gram of ET)
(Table 9).
The interaction among ECL and maturation medium
showed that ET from h41 and h42 performed better when
cultured on maturation medium C (247 and 435 somatic
embryos per gram of ET), whereas h19 produced its higher
number of somatic embryos (81 somatic embryos per gram
of ET) on maturation medium B (a medium with lower
concentration of sucrose and a higher concentration of
glutamine) (Table 9).
The results regarding germination of the somatic
embryos followed the same trend as for the maturation
process (Table 8). Thus the ECL, the maturation medium
and the interaction between them had a significant( p\ 0.001) effect on the number of germinated somatic
embryos obtained per gram of ET (Table 8). Again, h42
produced a significantly ( p\ 0.05) higher number of ger-
minated somatic embryos (172) per gram of ET than the
other ECLs tested (Table 10). In this sense ET on matu-
ration medium C also yielded a significantly ( p\ 0.05)
higher number of germinated somatic embryos (207) per
gram of ET than the other maturation media tested
(Table 10).
The interaction among ECL and maturation medium
also showed that the highest number of germinated somatic
embryos was obtained on medium C for h41 and h42,whereas for h19 the best results were found on medium B
(Table 10). The overall conversion percentage in this
experiment was 80 % (data not shown). The plantlets were
successfully acclimatized in plastic propagation trays, and
their growth is currently being monitored in the
greenhouse.
Discussion
In a first experiment the effect of culture medium, collec-
tion date and seed family on ET initiation and proliferation
in Pinus halepensis were analysed. Our results show that
these SE stages were strongly affected by culture medium
with the best results obtained after culture on DCR (Gupta
and Durzan 1985) medium. This medium was also found to
be the best for organogenesis in this species (Lambardi
et al. 1993) and for organogenesis and SE in other Pinus
0
5
10
15
20
25
DKI DBA DNB
Plant growth regulator combinations
E T P r o l i f e r a t i o n ( % )
aa
b
Fig. 3 Embryogenic tissue (ET) proliferation (%) in Pinus halepensis
megagametophytes cultured on DCR medium (Gupta and Durzan
1985) supplemented with plant growth regulators [(9 lM 2,4-D and
2.7 lM Kinetin (DKI); 9 lM 2,4-D and 2.7 lM BA (DBA); or
4.5 lM NAA and 4.5 lM 2,4-D and 2.7 lM BA (DNB)] (M ± SE).
Different letters show significant differences at p \ 0.05 by Tukey’s
post hoc test
Table 6 ANOVA for the number of mature somatic embryos and germinated somatic embryos per gram (FW) of embryogenic tissue (ET) in
Pinus halepensis
Source df Somatic embryos per gram of ET Germinated somatic embryos per gram of ET
Mean square F test p value Mean square F test p value
ECL 12 263,388.29 23.36 \0.001 61,330.15 13.49 \0.001
AC 1 10,520.27 0.93 n.s. 3,617.36 0.80 n.s.
ECL 9 AC 12 21,929.27 1.94 \0.05 3,226.48 0.71 n.s.
Error 100 11,274.42 4,547.41
The embryogenic tissue from thirteen embryogenic cell lines (ECLs) was suspended in liquid EDM medium, supplemented with 2 g L -1 of
activated charcoal (AC) or without AC. Somatic embryos were germinated on half strength LP (Quoirin and Lepoivre 1977, modified by Aitken-
Christie et al. 1988) supplemented with 2 g L-1
AC
n.s. non-significant
1346 Trees (2013) 27:1339–1351
1 3
-
8/18/2019 34 Somatic Embryogenesis in Pinus Halepensis Mill an Important Ecological Species From the Mediterranean Forest
9/13
spp. (Miguel et al. 2004; De Diego et al. 2008, 2010;
Aronen et al. 2009). On DCR medium (Gupta and Durzan
1985), ET initiation rates could be significantly affected by
the collection date (Table 4), in agreement with reports on
several Pinus species (Klimaszewska et al. 2001; Har-
greaves et al. 2009; Montalbán et al. 2012a).
SE in Pinus halepensis was initiated in all of the seed
families tested and only differences among two seed fam-ilies were observed, with initiation percentages ranging
from 8.3 to 25.6 % (Table 4). These ET initiation rates are
consistent with reported results in P. taeda (Pullman et al.
2003). Proliferation of ET ranged depending on the family,
from 0.6 to 7.2 % (Fig. 2), in accordance with results in
other Pinus species (Maruyama et al. 2007). At the pro-
liferation stage no significant effect of the collection date
was observed (Table 3) which accords with Hargreaves
et al. (2011); this is contrary to the results reported by
Yildirim et al. (2006) in P. brutia or Montalbán et al.
(2012a) in P. radiata SE. In a second ET initiation and
proliferation experiment, the effect of different nitrogensources, growth regulator combinations and gellan gum
concentrations on the culture medium was assessed. Our
results showed that ET initiation was significantly affected
by the nitrogen source, whereas proliferation was signifi-
cantly affected by the combination of growth regulators
supplemented in the culture medium (Table 5). Initiation
was better when the culture medium was supplemented
with ED amino acid mixture. Although in previous studies
in P. radiata SE we reported that the amino acid mixture
1 g L-1 casein hydrolysate plus 500 mg L-1 L-glutamine
yielded better proliferation rates, we also found that ET
cultured in the presence of ED amino acid mixture per-
formed better in the long term (Montalbán et al. 2012a).
The rates of ET proliferation in this second experiment
were significantly better when the culture medium was
supplemented with DKI or DNB plant growth regulator
combinations. The three plant growth regulator combina-
tions used had 2.7 lM cytokinin and 9 lM auxin. In some
cases, the addition of cytokinins as the sole source of plant
growth regulators is sufficient to induce and maintain SE
Table 8 ANOVA for the number of mature somatic embryos and the number of germinated somatic embryos per gram (FW) of embryogenic
tissue (ET) in Pinus halepensisSource df Somatic embryos per gram of ET Germinated somatic embryos per gram of ET
Mean square F test p value Mean square F test p value
Embryogenic cell line (ECL) 2 58,053.39 15.71 \0.001 50,344.79 17.17 \0.001
Maturation medium (M) 3 60,029.64 19.25 \0.001 46,334.89 15.80 \0.001
ECL 9 M 6 22,196.51 6.01 \0.001 19,909.98 6.79 \0.001
Error 24 3,694.79 2,932.41
The ET was cultured on different maturation media (A, B, C and D). Somatic embryos were germinated on half strength LP (Quoirin and
Lepoivre 1977, modified by Aitken-Christie et al. 1988) supplemented with 2 gL-1
of activated charcoal
0
200
400
600
800
1000
H 4 9
H 5 8
H 1 5 0
H 6 0
H 4 5
H 3 2
H 4 7
H 1 9 5
H 3 0
H 2 3 9
H 2 1 7
H 1 5 3
H 2 9
N º o f m a t u r e s o m a t i c e m b r y o s p e r g r a m o f
e m b r y o g e n i c t i s s u e ( F W )
Embryogenic cell lines
Fig. 4 Mature somatic embryos per gram (FW) of embryogenic
tissue (ET) from thirteen embryogenic cell lines (ECLs) suspended in
liquid EDM medium, with 2 g L-1
of activated charcoal (AC) (black
squares linked by a dotted line), or without AC (white squares linked
by a solid line) (M ± SE)
Table 7 Germinated somatic embryos per gram (FW) of embryo-
genic tissue (ET) in Pinus halepensis (M ± SE)
ECL Germinated somatic embryosper gram (FW) of ET
H29 270.0 ± 52.8a
H30 30.0 ± 10.9c
H32 32.3 ± 11.7c
H45 19.5 ± 9.0c
H47 30.2 ± 15.5c
H49 4.0 ± 4.0c
H58 10.0 ± 8.0d
H60 11.0 ± 6.2c
H150 0.0 ± 0.0
c
H153 101.8 ± 20.6bc
H195 58.2 ± 15.8c
H217 171.0 ± 32.9ab
H239 42.6 ± 11.0c
The somatic embryos were germinated on half strength LP (Quoirinand Lepoivre 1977, modified by Aitken-Christie et al. 1988) sup-plemented with 2 g L-1 of activated charcoal
Means followed by the same letter (a, b, c or d) are not significantlydifferent at p \ 0.05 Tukey’s post hoc test
Trees (2013) 27:1339–1351 1347
1 3
-
8/18/2019 34 Somatic Embryogenesis in Pinus Halepensis Mill an Important Ecological Species From the Mediterranean Forest
10/13
(Krajnaková et al. 2008), but most of the protocols used
auxins (alone or in combination with cytokinins) for SE
induction (Gaj 2004; Klimaszewska et al. 2007). Although
many authors use for proliferation the same or slightly
modified initiation medium, the results obtained for initi-
ation and proliferation suggest that a multi-step protocol
would be better. Montalbán e t a l . (2012b) described a
protocol to induce organogenesis in P. radiata buds fromadult trees and containing two steps, one in presence of
high cytokinin concentration and another in absence of
plant growth regulators. Furthermore, Fernández-Guijarro
et al. (1995) in Quercus suber SE reduced the high con-
centrations of BAP and NAA present in a first step of the
process to lower levels in a second one.
The ECLs obtained in the aforementioned experiments
were subjected to maturation. In maturation experiment
1, where the effect of AC in the ET suspension was
assessed, over 80 % of the lines tested produced somatic
embryos. In this case, our results showed that the ECL
and the interaction among ECL and AC had a significant
effect on the number of somatic embryos produced
(Table 6). The mean numbers of somatic embryos per
gram of ET ranged from 7 to 716 (Fig. 4); these results
are consistent with others obtained in P. radiata
(Montalbán et al. 2010) o r in P. pinaster (Lelu-Walteret al. 2006). When the interaction between ECLs and AC
was considered, a clear trend was not found (Fig. 4).
Similar results were obtained in P. sylvestris SE (Lelu-
Walter et al. 2008) where the effect of AC depended on
the line tested. AC plays an essential role in micro-
propagation of conifers for its capacity of adsorbing
phenolics and residual plant growth regulators (Thomas
2008; De Diego et al. 2010), but its effect on maturation
depends also on the species tested, being beneficial in
Table 10 Germinated somatic embryos per gram (FW) of embryogenic tissue (ET) in Pinus halepensis (M ± SE)
ECL Germinated somatic embryos per gram of ETMaturation media
Mean
Low sucrose High sucrose
Low glutamine High glutamine Low glutamine High glutamine
A B C D
h19 28.6 ± 14.3c
66.7 ± 26.5bc
33.3 ± 33.3bc
52.4 ± 17.2bc
45.2 ± 11.2b
h41 72.7 ± 8.6bc
64.1 ± 19.6bc
188.0 ± 40.8b
21.4 ± 11.3c
86.5 ± 21.2b
h42 97.8 ± 19.4bc 115.6 ± 49.5bc 400.0 ± 61.1a 75.6 ± 24.8bc 172.2 ± 43.8a
Mean 66.3 ± 12.5b
82.1 ± 19.1b
207.1 ± 58.0a
49.8 ± 12.2b
The ET was cultured on different maturation media (A, B, C and D). Somatic embryos were germinated on half strength LP (Quoirin and
Lepoivre 1977, modified by Aitken-Christie et al. 1988) supplemented with 2 g L-1
of activated charcoal (AC). Different letters show significant
differences at p\ 0.001 by Tukey’s post hoc test. In the last row and the last column appear the mean for the number of somatic embryos (%) onthe ECLs and maturation media tested, respectively (M ± SE)
Means followed by the same letter (a, b, c or d) are not significantly different at p \0.05 Tukey’s post hoc test
Table 9 Mature somatic embryos per gram (FW) of embryogenic tissue (ET) in Pinus halepensis (M ± SE)
ECL Somatic embryos per gram of ET
Maturation media
Mean
Low sucrose High sucrose
Low glutamine High glutamine Low glutamine High glutamine
A B C D
h19 28.6 ± 14.3c 81.0 ± 26.5bc 47.6 ± 47.6c 71.4 ± 16.5bc 57.1 ± 14.0b
h41 89.7 ± 7.4bc 68.4 ± 23.8c 247.9 ± 52.5b 51.3 ± 31.2c 114.3 ± 27.9b
h42 115.6 ± 27bc 142.2 ± 49.5bc 435.6 ± 54.1a 88.9 ± 17.8bc 195.6 ± 45.5a
Mean 78.0 ± 15.8b
97.2 ± 21.0b
243.7 ± 61.6a
70.5 ± 14.4b
The ET was cultured on different maturation media (A, B, C and D). Different letters show significant differences at p \ 0.001 by Tukey’s post
hoc test. In the last row and the last column appear the mean for the number of somatic embryos (%) on the ECLs and maturation media tested,
respectively (M ± SE)
Means followed by the same letter (a, b, c or d) are not significantly different at p \0.05 Tukey’s post hoc test
1348 Trees (2013) 27:1339–1351
1 3
-
8/18/2019 34 Somatic Embryogenesis in Pinus Halepensis Mill an Important Ecological Species From the Mediterranean Forest
11/13
some of them and detrimental in others (Montalbán et al.
2010).
In a second maturation experiment, the effect of sucrose
concentrations and different nitrogen sources (with high or
standard glutamine concentration) on the maturation
medium was assessed. The ANOVA for the number of
somatic embryos per gram of ET showed a significant
effect of both factors analysed (ECL and maturationmedium) and a significant effect of the interaction between
factors (Table 8). Previous research with Tamarillo has
shown that the manipulation of sucrose concentration in the
development medium increased the number of morpho-
logically normal somatic embryos (Correia et al. 2012).
Furthermore, several authors (Kumar and Thomas 2012;
Rode et al. 2012) demonstrated that the addition of higher
concentrations of sucrose with and without higher ABA
concentrations significantly increased the embryogenic
response. The effect of sucrose concentration has been also
widely studied in different Pinus species and, as discussed
by Yildirim et al. (2006), the suitability of a higher or alower sucrose concentration for maturation depends on the
species studied. Previous work carried out with radiata pine
in our laboratory showed a significant increase of the
number of somatic embryos per gram of ET by increasing
the sucrose concentration (from 30 to 60 g L-1) (Mont-
albán et al. 2010). However, as Montalbán et al. (2010)
demonstrated and Percy et al. (2001) discussed, variation
among ECLs for maturation yields is an attribute common
to several conifer species. Some studies have shown the
importance of nitrogen source compounds for the prolif-
eration and maturation of somatic embryos in different
species (Pérez-Rodrı́guez et al. 2006). Gerdakanenh et al.
(2011) postulated that stimulation of embryogenesis and
embryo development was strictly dependent on the type
and concentration of amino acid in the medium. Mihaljevic
et al. (2011) have shown in Cucurbita pepo L. that later
stage embryos developed only after a re-supply of nitrogen
in the form of nitrate or L-glutamine. In pine SE, it has been
suggested that the carbohydrate to nitrogen ratio of the
culture medium may represent a key factor responsible for
the expression of certain glutamine synthetase-related and
photosynthesis-related genes (Pérez-Rodrı́guez et al. 2006).
In our experiment, in the most productive lines, the mat-
uration medium with a low concentration of glutamine and
a high concentration of sucrose led to the highest number
of somatic embryos per gram of ET, whereas in the less
productive line, the best results were obtained after culture
of ET on a maturation medium with a high concentration of
glutamine and a low concentration of sucrose (Table 9).
Ramarosandratana et al. (2001) also pointed out that
somatic embryo maturation in maritime pine was highly
variable and depended both on the ECL and the maturation
medium. Moreover, Divakaran and Nair (2011) observed
the different effect of increasing L-glutamine concentration
in the number of germinated embryos in Musa acuminata.
The somatic embryos obtained in both maturation
experiments were successfully germinated on half strength
LP (Quoirin and Lepoivre 1977, modified by Aitken-
Christie et al. (1988)), with conversion percentages ranging
from 60 to 80 %, which is in accordance with the results
reported by Choudhury et al. (2008) in P. kesiya and Percyet al. (2000) in P. monticola. Although germination has
been accomplished in other Pinus species with a post-
maturation treatment (Maruyama and Hosoi 2012) or on
media without AC (Klimaszewska et al. 2001), this ger-
mination procedure and medium has been proven to be
suitable for micropropagation in species such as P. radiata
(Montalbán et al. 2010; Montalbán et al. 2011b).
To the best of our knowledge, this is the first report on
P. halepensis SE for which we have developed a simple
protocol that can be an efficient procedure for large-scale
somatic embryo production. The authors consider that this
protocol will be a very useful tool for the propagation of selected families/crosses and for genetic engineering of this
species.
Acknowledgments We thank Charlie B. Low for his statistical
advice. This research was funded by Ministerio de Ciencia y Tec-
nologı́a—Spain (AGL2005-08214-CO2-02) and Departamento de
Agricultura y Pesca-Gobierno Vasco (VEC2004021 and
VED2007014), who granted I. A. M. with a PhD scholarship.
References
Abelló MA (1998) Historia y Evolución de las Repoblaciones
Forestales en España. Universidad Complutense de Madrid,
Madrid, p 749
Aitken-Christie J, Singh AP, Davies H (1988) Multiplication of
meristematic tissue: a new tissue culture system for radiata pine.
In: Hanover JW, Keathley DE (eds) Genetic manipulation of
woody plants. Plenum Publishing Corp, New York, pp 413–432
Aronen T, Pehkonen T, Ryyananen L (2009) Enhancement of somatic
embryogenesis from immature zygotic embryos of Pinus
sylvestris. Scan J For Res 24:372–383
Botella L, Santamarı́a O, Dı́ez JJ (2010) Fungi associated with the
decline of Pinus halepensis in Spain. Fungal Divers 40:1–11
Breton D, Harvengt L, Trontin JF, Bouvet A, Favre JM (2005) High
subculture frequency, maltose-based and hormone-free medium
sustained early development of somatic embryos in maritime
pine. In Vitro Cell Dev Biol-Plant 41:494–504
Choudhury H, Kumaria S, Tandon P (2008) Induction and maturation
of somatic embryos from intact megagametophyte explants in
Khasi pine (Pinus kesiya Royle ex. Gord.). Curr Sci 95:
1433–1438
Correia S, Cunha AE, Salgueiro L, Canhoto JM (2012) Somatic
embryogenesis in Tamarillo (Cyphomandra betacea):
approaches to increase efficiency of embryo formation and plant
development. Plant Cell Tiss Org Cult 109(1):143–152
Davis JM, Becwar MR (2007) Developments in tree cloning.
Developments in fibres and Fibre treatment series. PIRA
International Ltd, Surrey, p 69
Trees (2013) 27:1339–1351 1349
1 3
-
8/18/2019 34 Somatic Embryogenesis in Pinus Halepensis Mill an Important Ecological Species From the Mediterranean Forest
12/13
De Diego N, Montalbán IA, Fernández de Larrinoa E, Moncaleán P
(2008) In vitro regeneration of Pinus pinaster adult trees. Can J
For Res 38:2607–2615
De Diego N, Montalbán IA, Moncaleán P (2010) In vitro regeneration
of adult Pinus sylvestris L. trees. S Afr J Bot 76:158–162
Divakaran SP, Nair AS (2011) Somatic embryogenesis from bract
cultures in diploid Musa acuminate cultivars from South India.
Sci Hortic 131:99–102
Fernández-Guijarro B, Celestino C, Toribio M (1995) Influence of
external factors on secondary embryogenesis and germination in
somatic embryos from leaves of Quercus suber L. Plant Cell Tiss
Org Cult 41:99–106
Gaj MD (2004) Factors influencing somatic embryogenesis induction
and plant regeneration with particular reference to Arabidopsis
thaliana (L.) Heynh. Plant Grow Reg 43:27–47
Gerdakanenh M, Mozafari AA, Sioseh-mardah A, Sarabi B (2011)
Effects of different aminoacids on somatic embryogenesis of
strawberry (Fragaria x Ananassa Duch.). Acta Physiol Plant
33(5):1847–1852
Gupta PK, Durzan DJ (1985) Shoot multiplication from mature trees
of Douglas fir and sugar pine. Plant Cell Rep 4:177–179
Hargreaves CL, Reeves CB, Find JI, Gough K, Josekutty P, Skudder
DB, Van der Maas SA, Sigley MR, Menzies MI, Low CB,
Mullin TJ (2009) Improving initiation, genotype capture, and
family representation in somatic embryogenesis of Pinus radiata
by a combination of zygotic embryo maturity, media, and
explant preparation. Can J For Res 39:1566–1574
Hargreaves CL, Reeves CB, Find JI, Gough K, Menzies MI, Low CB,
Mullin TJ (2011) Overcoming the challenges of family and
genotype representation and early cell line proliferation in
somatic embryogenesis from control-pollinated seeds of Pinus
radiata. New Zeal J For Sci 41:97–114
Klein T, Cohen S, Yakir D (2011) Hydraulic adjustment underlying
drought resistance of Pinus halepensis. Tree Physiol 31:637–648
Klimaszewska K, Park YS, Overton C, MacEacheron I, Bonga JM
(2001) Optimized somatic embryogenesis in Pinus strobus L.
In Vitro Cell Dev Biol-Plant 37:392–399
Klimaszewska K, Trontin JF, Becwar M, Devillard C, Park YS, Lelu-
Walter MA (2007) Recent progress on somatic embryogenesis of
four Pinus spp. Tree For Sci Biotechnol 1:11–25
Krajnaková J, Gömöry D, Häggman H (2008) Somatic embryogenesis
in Greek fir. Can J For Res 38:760–769
Kumar GK, Thomas TD (2012) High frequency somatic embryogen-
esis and synthetic seed production in Clitoria Ternatea Linn.
Plant Cell Tiss Org Cult 110(1):141–151
Lambardi M, Sharma KK, Thorpe TA (1993) Optimization of in vitro
bud induction and plantlet formation from ma-ture embryos of
Aleppo pine (Pinus halepensis Mill.). In Vitro Cell Dev Biol-
Plant 29:189–199
Lelu-Walter MA, Bernier-Cardou M, Klimaszewska K (2006)
Simplified and improved somatic embryogenesis for clonal
propagation of Pinus pinaster (Ait.). Plant Cell Rep
25(8):767–776
Lelu-Walter MA, Bernier-Cardou M, Klimaszewska K (2008) Clonalplant production from self- and cross-pollinated seed families of
Pinus sylvestris (L.) through somatic embryogenesis. Plant Cell
Tiss Org Cult 92(1):31-45
MacKay JJ, Becwar MR, Park YS, Corderro JP, Pullman GS (2006)
Genetic control of somatic embryogenesis initiation in loblolly
pine and implications for breeding. Tree Genet Genomes 2:1–9
Maestre FT, Cortina J (2004) Are Pinus halepensis plantations useful
as a restoration tool in semiarid Mediterranean areas? For Ecol
Manage 198:303–317
Maruyama TE, Hosoi Y (2012) Post-maturation treatment improves
and synchronizes somatic embryo germination of three species
of Japanese pines. Plant Cell Tiss Org Cult 110:45–52
Maruyama E, Hosoi Y, Ishii K (2007) Somatic embryogenesis and
plant regeneration in yakutanegoyou, Pinus armandii Franch.
var. amamiana (Koidz.) Hatusima, an endemic and endangered
species in Japan. In Vitro Cell Dev Biol Plant 43:28–34
Miguel C, Gonçalves S, Tereso S, Marum L, Maroco J, Oliveira M
(2004) Somatic embryogenesis from 20 open-pollinated families
of Portuguese plus trees of maritime pine. Plant Cell Tiss Org
Cult 76:121–130
Mihaljevic S, Radic S, Bauer N, Garic R, Mihaljevic B, Horvat G,
Leljak-Levanic D, Jelaska S (2011) Ammonium-related meta-
bolic changes affect somatic embryogenesis in pumpkin (Cu-
curbita pepo L.). J Plant Physiol 168(16):1943–1951
Moncaleán P, Alonso P, Centeno ML, Cortizo M, Rodrı́guez A,
Fernández B, Ordás RJ (2005) Organogenic responses of Pinus
pinea cotyledons to hormonal treatments: BA metabolism and
cytokinin content. Tree Physiol 25:1–9
Montalbán IA, De Diego N, Moncaleán P (2010) Bottlenecks in Pinus
radiata somatic embryogenesis: improving maturation and
germination. Trees Struct Funct 24:1061–1071
Montalbán IA, De Diego N, Aguirre-Igartua E, Setién A, Moncaleán
P (2011a) A combined pathway of somatic embryogenesis and
organogenesis to regenerate radiata pine plants. Plant Biotechnol
Rep 5(2):177–186
Montalbán IA, De Diego N, Moncaleán P (2011b) Testing novel
cytokinins for improved in vitro adventitious shoots formation
and subsequent ex vitro performance in Pinus radiata. For
84:363–373
Montalbán IA, De Diego N, Moncaleán P (2012a) Enhancing
initiation and proliferation in radiata pine (Pinus radiata D.
Don) somatic embryogenesis through seed family screening,
zygotic embryo staging and media adjustments. Acta Physiol
Plant 34:451–460
Montalbán IA, Novak O, Rolcik J, Strnad M, Moncaleán P (2012b)
Endogenous cytokinin and auxin profiles during in vitro organ-
ogenesis from vegetative buds of Pinus radiata adult trees.
Physiol Plant. doi:10.1111/j.1399-3054.2012.017019.x
Oliveras I, Martı́nez-Vilalta J, Jimenez-Ortiz T, Lledó MJ, Escarré A,
Piñol J (2003) Hydraulic properties of Pinus halepensis, Pinus
pinea and Tetraclinis articulata in a dune ecosystem of Eastern
Spain. Plant Ecol 169:131–141
Park YS (2002) Implementation of conifer somatic embryogenesis in
clonal forestry: technical requirements and deployment consid-
erations. Ann For Sci 59:651–656
Park YS, Barrett JD, Bonga JM (1998) Application of somatic
embryogenesis in high-value clonal forestry: deployment,
genetic control, and stability of cryopreserved clones. In Vitro
Cell Dev Biol- Plant 34:231–239
Percy RE, Klimaszewska K, Cyr DR (2000) Evaluation of somatic
embryogenesis for clone propagation of western white pine. Can
J For Res 30:1867–1876
Percy REL, Livingston NJ, Moran JA, Von Aderkas P (2001)
Desiccation, cryopreservation and water relations parameters of
white spruce (Picea glauca) and interior spruce (Picea glauca x
engelmannii complex) somatic embryos. Tree Physiol21(18):1303–1310
Pérez-Rodrı́guez MJ, Suárez MF, Heredia R, Ávila C, Breton D,
Trontin JF, Filonova L, Bozhkov P, Von Arnold S, Harvengt L,
Cánovas FM (2006) Expression patterns of two glutamine
synthetase genes in zygotic and somatic pine embryos support
specific roles in nitrogen metabolism during embryogenesis.
New Phytol 169:35–44
Quoirin M, Lepoivre P (1977) Études des milieu adaptés aux cultures
in vitro de Prunus. Acta Hortic 78:437–442
Ramarosandratana A, Harvengt L, Bouvet A, Calvayrac R, Pâques M
(2001) Effects of carbohydrate source, polyethylene glycol and
gellam gum concentration on embryonal-suspensor mass (ESM)
1350 Trees (2013) 27:1339–1351
1 3
http://dx.doi.org/10.1111/j.1399-3054.2012.017019.xhttp://dx.doi.org/10.1111/j.1399-3054.2012.017019.x
-
8/18/2019 34 Somatic Embryogenesis in Pinus Halepensis Mill an Important Ecological Species From the Mediterranean Forest
13/13
proliferation and maturation of maritime pine somatic embryos.
In Vitro Cell Dev Biol-Plant 37:29–34
Rode C, Lindhorst K, Braun HP, Winkelmann T (2012) From callus
to embryo: a proteomic view on the development and maturation
of somatic embryos in Cyclamen persicum. Planta
235(5):995–1011
Thomas TD (2008) The role of activated charcoal in plant tissue
culture. Biotechnol Adv 26(6):618–631
Walter C, Find JI, Grace LJ (1998) Somatic embryogenesis and
genetic transformation in Pinus radiata. In: Jain SM, Gupta PK,
Newton RJ (eds) Somatic embryogenesis in woody plants, vol 4.,
Kluwer Academic PubDordrecht, The Netherlands, pp 491–504
Yildirim T, Kaya Z, Isik K (2006) Induction of embryogenic tissue
and maturation of somatic embryos in Pinus brutia TEN. Plant
Cell Tiss Org Cult 87:67–76
Trees (2013) 27:1339–1351 1351
1 3