Dormancy, ABA content and sensitivity to exogenous ABA...
Transcript of Dormancy, ABA content and sensitivity to exogenous ABA...
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Dormancy, ABA content and sensitivity to exogenous ABA application
of a barley mutant during seed development and after ripening
I. Romagosa1*, D. Prada1, M.A. Moralejo1, A. Sopena1, P. Muñoz1, A.M. Casas2,
J.S. Swanston 3 and J.L. Molina-Cano1
1 Centre UdL-IRTA, Av. Alcalde Rovira Roure 177, 25178 Lleida, Spain.
2 Estación Experimental de Aula Dei, CSIC, P.O.Box 202, 50080 Zaragoza, Spain
3 Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK
* To whom correspondence should be addressed: Fax: +34 973 702502. E-mail:
Keywords: Germination, seed dormancy, mutagenesis, barley, abscisic acid,
Short title
Differential germination of a barley mutant.
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Abstract
Assessment of dormancy inception, maintenance and release was studied for
artificially dried immature seeds harvested in Triumph and its mutant line TL43
throughout seed development, under different environmental conditions in Spain and
Scotland (low and high dormancy inducing conditions, respectively). TL43 showed
strongly reduced dormancy compared with Triumph. Both TL43 and Triumph
seemed to follow a similar pattern of release of dormancy across the seasons, with
relative differences in the stage of development when the seed was capable to start to
germinate. Immature seed viability through grain filling of TL43 and Triumph was
more closely related with the dry matter deposition pattern than to genotypic
differences. Abscisic acid (ABA) content was also studied through the seed
development period. No genotypic differences in ABA pattern in the course of grain
development could be detected, whilst significant differences were observed for
germination percentage (GP). The total amount of ABA in Triumph and TL43 was
much higher in Scotland than in Spain for each developmental stage. These values
suggest that endogenous ABA content alone through grain development could not
explain differences in GP between genotypes within environments. The initial
germination (GP0) played a key role in the sensitivity to ABA. For the same
developmental stage TL43 is more insensitive to exogenous ABA than Triumph, but
seeds from TL43 and Triumph with the same initial germination showed the same
ABA response. Endogenous ABA content seems not to affect the later response to
different exogenous ABA concentrations. Therefore differences in ABA sensitivity
on post-harvest mature grain seem to be mainly determined by the initial germination
percentage.
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Introduction
Dormancy is defined as the inability of a viable seed to germinate under conditions
otherwise perfectly adequate for germination. Expression of dormancy is affected by
genetic and environmental factors, particularly the conditions prevailing during seed
development and storage after harvest (Corbineau and Côme, 1996). Low
temperature and high relative humidity during grain development seem to be the
main environmental factors inducing dormancy in barley (Zoppolo et al., 1982;
Buraas and Skinnes, 1984; Strand,1989). Peripheral tissues of the kernel rather than
the embryo itself may induce or maintain dormancy in barley (Van Beckum et al.,
1993), and in most cereals (Black et al., 1987). The coat is considered to exert its
control through its influence on the embryonic activity and emergence by limiting
oxygen supply to the embryo (Simpson, 1990; Benech-Arnold et al., 1999).
Inception of dormancy seems to occur at early stages of seed development. In some
species, artificially dried seeds were capable of precocious germination during the
second week after anthesis and then dormancy gradually set in during the third week
(Bianco et al., 1994, Romagosa et al., 1999). A desiccation period seems to be
needed in order to proceed from seed development to germination (Kermode, 1995).
The mechanisms that relieve dormancy during after-ripening are unknown, but may
involve non-enzymatic oxidative reactions (Leopold et al., 1988; Esashi et al., 1993;)
or may result from turnover of products inhibiting germination (Dyer, 1993).
The genetics and biochemistry of dormancy are not yet fully understood. Mutants
were developed in the model plant Arabidopsis thaliana that, besides changes in
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ABA content and response, showed either absence of, or alteration in dormancy
(Koornneef et al., 1984; reviewed in Karssen, 1995). However, despite the economic
importance of germination in malting barley, dormancy is less understood in this
species. Early investigations by Freistedt (1935) and Moorman (1942), were
continued by Buraas and Skinnes (1984) who indicated that dormancy was highly
heritable, controlled by several recessive genes and not affected by cytoplasmic
factors. Recent attempts to study the genetics of dormancy in barley have utilised
molecular and mapping techniques for quantitative trait loci (QTL), based on a
doubled-haploid line (DHL) population derived from the Steptoe (high dormancy) x
Morex (low dormancy) cross. Four regions of the barley genome were associated
with most of the differential genotypic expression of dormancy (Oberthur et al.,
1995; Han et al., 1996; Larson et al., 1996; Romagosa et al., 1999).
The phytohormone abscisic acid plays a major role in development and germination
of plant seeds. During early seed development, ABA is involved in embryogenesis,
whereas at a later stage of seed development it seems to prevent precocious seed
germination. However, the actual mechanisms of dormancy are quite unknown
(Bewley, 1997). Some of the ABA responses are rapid and involve the modification
of ion fluxes, whereas others are long term and involve changes in gene expression
(Kermode, 1995). A major action of ABA in seeds is the regulation of gene
expression; particularly the induction of several different kinds of polypeptides
(Skriver and Mundy, 1990) and the inhibition of genes for certain reserve mobilising
enzymes (Jacobsen and Chandler, 1987). Not only ABA content, but also ABA
sensitivity may influence seed dormancy and germination. The free ABA content is
highest in developing seeds and is generally relatively low or even undetectable in
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mature seeds, though in several species considerable amounts of ABA are detected
(Black, 1991). The appearance of ABA peaks during development could be
manipulated by different growth conditions (Radley, 1976; Weidenhoeft et al., 1988;
Walker-Simmons and Sessing, 1990). In barley, ABA peaked earlier in high-
temperature-grown barley than in low-temperature-grown grains, and cultivars with
a lower level of dormancy have less ABA during development (Goldbach and
Michael, 1976; Wang et al., 1995). Quarrie et al., (1988), found that ABA levels in
barley embryos were up to 10-times greater than those in the endosperm.
The role of ABA in seed dormancy has been studied in Arabidopsis by using mutants
defective in either ABA biosynthesis or mode of action of this hormone (Koornneef
et al., 1998). ABA-insensitive (abi1-abi5) mutants were selected by germinating at
ABA concentrations that normally inhibit germination (Koornneef et al., 1984).
These mutants affect many ABA responses (abi1 and abi2) or primarily seed
germination (abi3, abi4 and abi5).
The absence of barley mutants with reduced dormancy and sensitivity to ABA in
germination indicated the need for a mutation experiment with the specific objective
of developing such genotypes. Recently, Molina-Cano et al. (1999) induced mutants
in the barley cultivar Triumph. One of them, TL43, exhibited reduced dormancy and
seemed to tolerate a ten-fold increase in ABA, before germination was inhibited,
compared to its original cultivar. In the present study we present a detailed
characterisation of this mutant and its original genotype on dormancy and ABA
sensitivity during seed development and after ripening.
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Material and Methods
Plant material
The mutant line TL43 was obtained from cv. Triumph following the mutagenic
treatment and selection procedure described in detail by Molina-Cano et al. (1999).
The near-isogenic nature of these two lines was confirmed by means of molecular
markers (Molina-Cano et al., 1999). Both genotypes were field grown under low and
high dormancy inducing conditions in 1997, 1998 and 1999 at Lleida (Spain), and in
1998 and 1999 at Dundee (Scotland). There were very contrasting growing regimes
at both sites: fall-sown in Spain and spring-sown in Scotland, i.e. long- vs. short-day
conditions during grain-filling.
Inception and release from dormancy during grain development
In order to provide the same developmental stage for all the samples, approximately
250 spikes were tagged in the field plots at ear emergence. Starting two weeks after
tagging, and twice a week through the grain filling process, 10 spikes per entry were
harvested and hand threshed. Two kernels per spike were previously bulked for
determination of dry weight and moisture content. The remaining seeds were placed
in a forced-ventilation oven at 37ºC for 24 hours at which time they reached a
constant weight. These artificially dried seeds were kept at -20ºC until the last
sample was taken at the end of grain filling. After thawing, germination tests for all
samples conserved at –20ºC were immediately conducted in the dark at 19ºC in petri
dishes lined with 3 sheets of Whatman no. 1 filter paper, saturated with 3 ml distilled
water. Three replications of 40 seeds were carried out per genotype x sampling date.
After 3 days, germinated seeds (those where the coleoptile had emerged through the
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hull) were counted and expressed as a germination percentage of the total (GP). The
remaining seeds were kept at room temperature and germination tests were then
repeated, after 66 days post harvesting (DPH) in 1997 and 144 DPH in 1998.
Comparing these two assays, the initial test and the repeated one after the storage
period, it was possible to distinguish dormancy from lack of viability in artificially
dried immature seed.
Quantification of ABA during grain development
Samples of ten spikes, each distributed across all the grain development period, were
harvested from each genotype. They were immediately preserved in liquid nitrogen
at –80ºC up to the moment to determine the ABA content. In order to analyse the
endogenous ABA content, the samples were thawed and 2 grains from the central
part of each spike were lyophilised. The analyses were carried out on the embryo-
half of each grain, to avoid the dilution effect on ABA caused by the endosperm.
Samples were milled to obtain the 50 mg of dry flour that were extracted with water
(20:1). ABA determination was done with the method of Phytodetek-ABA (IDEXX
Laboratories, Inc. One Idexx Drive, Westbrook, ME 04092, USA), based on Mertens
et al. (1983) and Weiler (1984), and results were expressed as ng ABA g-1 DW.
Three, five and six sampling dates across the grain filling process were analyzed in
1997, 1998 and 1999 respectively. Furthermore, ABA level was also measured in
1999 in mature dry grains grown in Spain and Scotland one month after harvest.
Sensitivity to ABA during germination
Germination tests on post-harvest seed stored at room temperature were conducted
by adding exogenous ABA into the incubation medium according to the same
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germination protocol as described previously. The assays were performed 75 days
post harvest (DPH) in 1998, and 30 DPH in 1999. The experiment layout was as
follows: (1) 3 replications of 40 seeds of width between 2.5 and 2.8 mm, and (2)
ABA concentrations in the germination medium of 0, 10-8, 10-7, 10-6, 10-5, 10-4 and
10-3 M [()-cis,trans-abscisic acid, Sigma Chemical Corporation].
Statistical analysis
Data from grain filling and germination test to encompass the onset, maintenance
and release of dormancy during grain development were fitted to a logistic model,
using SAS/STAT (1991) procedures. The following non-linear equation was fitted:
GP(%)=A/(1+exp(B·time-C)); where A represents the maximum GP; B is the time
needed to reach GP=50%, i.e. LD50; and C is a coefficient without a clear and direct
biological meaning, that together with A and B determines the inflexion point of the
curve.
The results from the studies on ABA sensitivity were also fitted using SAS
procedures to the following logistic model: GP(%)=A/{1+[log (ABA)/B] C}. These
parameters have a direct biological meaning: A represents the germination of the
untreated seeds (GP0); B the ABA concentration that reduces germination of the
untreated control to half, LD50; and C together with A and B determines the inflexion
point of the curve. The comparison of these parameters by means of the homogeneity
test (Kimura, 1980) allows us to analyse the differences in the response to exogenous
ABA.
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Results
Dormancy during seed development
Assessment of dormancy inception and release was based on germination studies for
artificially dried seeds taken in Triumph and its mutant line TL43 throughout seed
development (Figure 1). TL43 showed strongly reduced dormancy compared with
Triumph in 1997 and 1998, in Spain (Figures 1A-1B, 1C-1D). Both TL43 and
Triumph seemed to follow a similar pattern of release of dormancy across the
seasons, with relative differences in the stage of development when the seed was
capable to start to germinate. No precocious germination prior to dormancy inception
as shown in some barley genotypes (Romagosa et al., 1999) was seen as both lines
were fully dormant at the beginning of the germination test two weeks after anthesis.
The environmental influence on dormancy inception could be clearly shown
comparing the results from Spain 1997 vs. Spain 1998 and Scotland 1998. In Spain
97, TL43 started to loose dormancy progressively from over 500 degree days after
anthesis (DDAA) while Triumph remained dormant until about 700 DDAA. The
mutant showed higher germination percentage (GP) than Triumph at the end of the
grain filling process (87 % against 73 %, respectively). In Spain 98, TL43 showed a
lower level of precocious germination starting at 500 DAA and reaching a 25 %
germination at seed maturity, while Triumph was fully dormant through the grain
filling period and at seed maturity. TL43 and Triumph did not germinate throughout
seed development in the 1998 Scottish conditions (Figures 1E-1F).
The germination tests were repeated after dry storage at room temperature to break
dormancy and check the viability of the not fully developed seeds. The storage
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period required for dormancy release varied among the years. In 1997 when the
seeds developed under environmental conditions not imposing high levels of
dormancy, dormancy disappeared after 66 days postharvest (DPH), when the most
fully developed samples almost reached 100 % GP. In 1998 the required period for
dormancy release was longer than 144 days, when the fully developed grains reached
around 80 % GP. For every genotype x season combination and by comparing both
curves 1 DPH and 66 or 144 DPH it was possible to assess and distinguish between
dormancy and viability effects. Germination percentage after storage at room
temperature, which basically reflects seed viability, was less affected by genetic and
environmental effects. Genotypic differences in the germination profiles, once
dormancy was released, were not as large as in the case of seeds germinated at 1
DPH. Therefore, seed viability through seed development of TL43 and Triumph was
very similar within a given environment (Figure 1). In fact, the viability curve seems
to be closely related with the dry matter deposition pattern, rather than to genotypic
differences. In Spain in the two studied years, both genotypes had the highest
germination rate when they reached about 80% of their constant end dry weight at
seed maturity. A similar trend was observed in Scotland 98 (Figures 1E-1F),
however under these conditions the dormancy of these grains was not fully released
after 144 DPH and, therefore, these samples did not reached 100% GP. Contrasting
differences in barley growing conditions exist between the two sites, particularly in
relation to the phenology and length of the barley lifecycle as determined mainly by
daylength during the early phases of plant development and temperature and
moisture conditions at later stages.
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ABA content during seed development
The ABA content was also studied during the seed development period (Figure 2).
The amount of endogenous ABA decreased along the grain filling period at all
experiments. This decrease seems necessary to trigger the germination process since
ABA content was lowest at seed maturity, when the GP was highest. However,
within a given growing environment, no genotypic differences in ABA pattern along
grain development period could be detected. For both years in Spain, there were no
differences in ABA content between Triumph and TL43, whereas significant
differences were observed for GP (Figures 2A-2B and 2C-2D). Genotypic
differences for GP at seed maturity were large (TL43: 87 %, Triumph 73 %, in Spain
97 vs. TL43: 25 %, Triumph: 0 % in Spain 98), but the corresponding differences in
ABA concentration were small and not significant (110 and 121 vs. 738 and 788 ng/g
DW for TL43 and Triumph respectively, in the two studied years). Likewise, in
Scotland 98 there were no genotypic differences in ABA pattern, but the total
amounts of ABA in Triumph and TL43 were much higher than in Spain for each
developmental stage (Figures 2E-2F). There were no genotypic differences in the
pattern of endogenous ABA content under Scottish conditions, since both genotypes
reached an ABA peak at the same developmental stage around 600 DDAA.
These values suggest that endogenous ABA content alone could not explain
differences in GP throughout grain development and at the end of the grain filling
period between genotypes and within environments.
Endogenous ABA content and response to exogenous ABA in mature dry seed
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Response curves to different concentrations of ABA in the germination medium are
shown in Figure 3. Germination was carried out with mature dry grain stored at room
temperature one and three months after ripening, in 1998 and 1999. Data were fitted
to a dose-response logistic model and very good levels of fit were obtained (r2
ranging from 0.94 to 0.99). These curves were compared by means of the
homogeneity test (Kimura, 1980).
Three different curves were compared in this figure: seeds grown in Spain in 1998
and 1999 (Spain 98 and 99) and in Scotland in 1999 (Scotland 99). In Spain 98,
response to exogenous ABA in mature dry seed was determined in three seed lots:
TL43 at 75 DPH, Triumph at 75 DPH and TL43 at 7 DPH. The latter had
approximately the same initial germination percentage as Triumph (75 DPH) at a
different after ripening developmental stage.
The initial germination level played a key role in the sensitiveness to ABA. TL43 (7
DPH) had a statistically significantly different ABA response profile (GP0 = 48.7 %,
LD50 = 1.6 10-5 M ABA) than TL43 (75 DPH) (GP0 = 92.8 %, LD50 = 1.1 10-4 M
ABA). However, TL43 (7 DPH) was not significantly different than Triumph (75
DPH) (GP0= 44.4 %, LD50 = 3.1 10-5 M ABA) (Figure 3). That is, for the same
developmental stage TL43 is relatively more insensitive to exogenous ABA than
Triumph, but they do have the same ABA response at any given common GP0.
Additional experiments were performed to identify the influence of the endogenous
ABA content on the sensitivity of barley grains to exogenous application of ABA.
The endogenous ABA content was analysed in barley seed grown in Spain and
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Scotland in 1999 one month after-harvest, using the same seed lot that was used to
study the response to exogenous ABA application in germination (Table 1). At every
site, the endogenous ABA content was not statistically significant between the two
genotypes (2101 and 2092 ng/g DW for Triumph and TL43 in Spain; 2255 and 1942
ng/g DW for Triumph and TL43 in Scotland). However, as is shown in Figure 3,
significant differences in sensitivity to exogenous ABA were found (LD50 Triumph =
1.5 10-5 vs. LD50 TL43 = 8.6 10-5 M ABA in Spain and LD50 Triumph = 1.7 10-5 vs. LD50
TL43 = 4.8 10-5 M ABA in Scotland). Genotypes with lower initial germination (GP0
Triumph = 66.3 vs. GP0 TL43 = 76.6 % in Spain and GP0 Triumph = 35.3 vs. GP0 TL43 = 77.6
% in Scotland) were more sensitive to exogenous ABA application in the
germinating media. Therefore, endogenous ABA content seems not to affect the later
response to different exogenous ABA concentrations. On the contrary, differences in
ABA sensitivity on post-harvest mature grain seem to be mainly determined by the
initial germination percentage.
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Discussion
In a previous paper, the phenotypic characterisation of TL43 and Triumph was
presented (Molina-Cano et al., 1999). The mutant TL43 appeared to be slightly later
in heading, with shorter stems, smaller and lighter kernels, and lower yield than its
parental genotype Triumph, when grown in Spain. TL43 showed, however, a fairly
normal phenotype under Scottish growing conditions. TL43 consistently showed
reduced dormancy levels under a series of environmental conditions across a number
of growing years in Spain and Scotland. The objectives of this work were: (1) to
assess the pattern of inception, maintenance and release of dormancy during seed
development and after ripening; (2) to study the role of endogenous ABA in
determining these processes; and (3) to characterise the genotypic sensitivity of seed
to increasing exogenous ABA concentrations.
Although dormancy was highly variable across years and sites, the two genotypes
seemed to follow a relatively similar pattern of inception of dormancy. Both TL43
and Triumph were fully dormant at the first sampling date, two weeks after anthesis.
The viability and dormancy profiles for TL43 and Triumph across seasons did not
reveal differences in the time that dormancy inception took place. Release of primary
dormancy through seed development took place in TL43 at an earlier point of the
grain filling process. Dormancy of TL43 was reduced earlier during seed
development, compared to Triumph under Spanish growing conditions. TL43 was
able to undergo precocious germination at an earlier developmental age and reached
higher GP at seed maturity. Viability of the developing seeds was much less affected
by genotypic and environmental differences than dormancy.
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There is considerable circumstancial evidence that ABA is involved in regulating the
onset of dormancy and in maintaining the dormant state (Black, 1991; Bewley,
1997). The role of ABA content in imposing dormancy was not unmistakably shown
in this study. No clear correlation between ABA content of the grains and dormancy
has been shown in some studies carried out in barley (Boivin et al., 1995) or in other
cereals (Berrie et al., 1979; Dunwell, 1981; Walker-Simmons, 1987; Walker-
Simmons and Sessing, 1990). In our study, when differences in germination
percentage of developing grains between genotypes and between environments were
large, the corresponding differences in endogenous ABA were small. But on the
other hand, and for both lines, ABA concentrations under Scottish conditions, which
impose high levels of dormancy in the grain, were always higher. The initiation of
dormancy may also depend partially or wholly on seed sensitivity to ABA whereas
the amount of hormone would be less relevant (Walker- Simmons, 1987). Recently,
White et al. (2000) showed that although ABA is required for seed maturation, active
gibberellins (GA) are also present in developing maize embryos. The relative
amounts of ABA and GA, rather than the concentration of ABA alone, determine
whether maize developing embryos undergo precocious germination or quiescence
and maturation (White and Rivin, 2000).
TL43 was neither similar to the ABA-deficiens (aba) mutants reported in
Arabidopsis thaliana (reviewed in Karssen, 1995, and Koornneef et al., 1998), nor
to the rdo mutants which showed reduced-dormancy and similar contents and
sensitivity to ABA, also in Arabidopsis (Léon-Kloosteerziel et al., 1996). TL43 is
not an ABA-deficient mutant and showed enhanced insensitiveness to exogenous
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application of ABA in germination. TL43 behaviour shows some similarities to the
abi (ABA-insensitive mutants), defined as those able to maintain germination at
ABA concentrations ten-times higher than the wild type, with similar level of
endogenous ABA concentration (Koornneef et al., 1984). In this study, no clear
differences in endogenous ABA concentrations were found between the two
genotypes across grain filling and after ripening. Therefore, differences in dormancy
release were not associated to this character. Distinct sensitivity to exogenous ABA
application to germination was confirmed using mature grain grown under several
environmental conditions. TL43 maintained the ability to germinate at exogenous
ABA concentrations five to ten times higher than the wild type, across changing
environmental conditions, as measured by the LD50. However, sensibility to ABA
seemed to be driven by the initial germination percentage of the seed lot. Both traits,
decreased dormancy and insensibility to ABA, in this 'abi-like' mutant could be
expression of a common phenomenon rather than independent phenotypical events.
The phenotype of TL43 resembles that of abi3, one of the Arabidopsis ABA
insensitive mutants. Extreme abi3 mutants are characterised by severely disturbed
seed maturation as shown by a strong reduction in storage proteins and many seed-
maturation specific transcripts, desiccation sensitivity of mature seeds, lack of seed
dormancy, and a very reduced sensitivity to the inhibiting effect of ABA in seed
germination (Parcy et al., 1994). The aminoacid sequence of ABI3 (Giraudat et al.,
1992) revealed that this gene is homologous to the maize Viviparous-1 (Vp1) gene
(McCarty et al., 1991). Both genes encode transcription factors with seed specific
expression. Unsuccessful attempts have been made to relate the seed dormancy (SD)
QTL identified in barley with the maize and rice Vp1 sequences (F. Han, personal
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communication). Current research is underway by crossing TL43 and Triumph with
both Steptoe and Morex to try to relate the mutation present in TL43 with the SD1 to
SD4 quantitative trait loci identified in the Steptoe (highly dormant) x Morex (not
dormant) cross. Recently, the wheat Vp1 homoeologues (vp1A, vp1B, vp1D) have
been located in the long arm of chromosome 3 (Bailey et al., 1999). However, none
of the barley SD QTL (Han et al., 1996) are in this genomic region. Some clues may
come from cloning the Vp1 homologous gene from Triumph and TL43, which is
currently being done.
Acknowledgements
The financial support of the INIA (Instituto Nacional de Investigación y Tecnología
Agraria y Alimentaria) and CICYT (Comisión Interministerial de Ciencia y
Tecnología), which funded the Spanish teams and of the Scottish Office, Agriculture,
Environment and Fisheries Department (SOAEFD) to JS Swanston are gratefully
acknowledged.
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References
Bailey PC, McKibbin RS, Lenton JR, Holdsworth MJ, Flintham JE, Gale MD.
1999. Genetic map locations for orthologous Vp1 genes in wheat and rice.
Theoretical and Applied Genetics 98, 281-284.
Benech-Arnold RL, Giallorenzi MC, Frank J, Rodriguez V. 1999. Termination of
hull-imposed dormancy in barley is correlated with changes in embryonic
ABA content and sensitivity. Seed Science Research 9, 39-47.
Berrie AMM, Buller D, Don R, Parker W. 1979. Possible role of volatile fatty acid
and abscisic acid in the dormancy of oats. Plant Physiology 563, 758-764.
Bewley JD. 1997. Seed Germination and Dormancy. The Plant Cell 9, 1055-1066.
Bianco J, Garello G, Le Pege-Degivry MT. 1994. Release of dormancy in
sunflower embryos by dry storage: involvement of gibberellins and abscisic
acid. Seed Science Research 4, 57-62.
Black M. 1991. Involvement of ABA in the physiology of developing and maturing
seeds. In: Davies WJ, Jones HG, eds. Abscisic Acid: Physiology and
Biochemistry. Oxford: BIOS Scientific, 99-124.
Black M, Butler J, Hughes M. 1987. Control and development of dormancy in
cereals. In: Mares D, ed. Fourth Internacional Symposium on Preharvest
Sprouting in Cereals. Boulder: Westview Press, 379-392.
Boivin P, Kohl S, Clamagirand V. 1995. Barley endogenous phytohormones and
malting performance. Bios 255, 119-124.
Buraas T, Skinnes H. 1984. Genetic investigations on seed dormancy in barley.
Hereditas 101, 235-244.
Corbineau F, Côme D. 1996. Barley seed dormancy. Bios 261, 113-119.
19
Dunwell JM. 1981. Dormancy and germination in embryo of Hordeum vulgare L.
Effect of dissection, incubation temperature and hormone application. Annals
of Botany 48, 203-213.
Dyer WE. 1993. Dormancy associated embryonic mRNAs and proteins in imbidding
Avenua fatua (L.) caryopses. Physiologia Plantarum 88, 201-211.
Esashi Y, Ogasawara M, Gorecki R, Leopold AC. 1993. Possible mechanisms of
afterripening in Xanthium seeds. Physiologia Plantarum 87, 359-364.
Freistedt P. 1935. Neue Zielsetzungen in der Gerstenzüchtung. Zeitschrift für
Züchtung, Reihe A, Planzenzüchtung 20, 169-209.
Goldbach H, Michael G. 1976. Abscisic acid content of barley grains during
ripening as affected by temperature and variety. Crop Science 16, 797-799.
Giraudat J, Hauge BM, Valon C, Smalle J, Parcy F, Goodman HM. 1992.
Isolation of the Arabidopsis ABI3 gene by positional cloning. The Plant Cell 4,
1251-1261.
Han F, Ullrich SE, Clancy JA, Jitkov V, Kilian A, Romagosa I. 1996.
Verification of barley seed dormancy loci via linked molecular markers.
Theoretical and Applied Genetics 92, 87-91.
Jacobsen JV, Chandler PM. 1987. Gibberellin and abscisic acid in germinating
cereals. In: Davies PJ, ed. Plant Hormones and Their Role in Plant Growth
and Development. Boston: Martinus Nijhoff, 164-193.
Karssen CM. 1995. Hormonal regulation of seed development, dormancy and
germination studied by genetic control. In: Kigel J, Galli G, eds. Seed
development and germination. New York: Marcel Dekker, Inc., 164-193.
Kermode AR. 1995. Regulatory mechanisms in the transition from development to
germination: interactions between the embryo and the seed environment. In:
20
Kigel J, Galli G, eds. Seed development and germination. New York: Marcel
Dekker, Inc, 273-332.
Kimura DK. 1980. Likelihood methods for the Von Bertalanffy Growth Curve.
United States Fishery Bulletin 77, 765-776.
Koornneef M, Reuling G, Karssen CM. 1984. The isolation and characterization of
abscisic acid-insensitive mutants of Arabidopsis thaliana. Physiologia
Plantarum 61, 377-383.
Koornneef M, Léon-Kloosterziel KM, Schwartz SH, Zeevart JAD. 1998. The
genetic and molecular dissection of abscisic acid biosynthesis and signal
transduction in Arabidopsis. Plant Physiologie et Biochemistrie 36, 83-89.
Larson S, Bryan G, Dyer W, Blake T. 1996. Evaluating gene effects of a major
barley seed dormancy QTL in reciprocal backcross populations. Journal of
Quantitative Trait Loci [online], Volume 2, Article 4. In:
http://probe.nalusda.gov:8000/otherdocs/jqtl/Larson et al. 1996
Léon-Kloosterziel KM, van de Bunt GA, Zeevaart J, Koornneef M. 1996.
Arabidopsis mutants with a reduced seed dormancy. Plant Physiology 110,
233-240.
Leopold AC, Glenister R, Cohn MA. 1988. Relationship between water content
and afterripening in red rice. Physiologia Plantarum 74, 659-772.
Mertens R, Deus-Neumann B, Weiler EW. 1983. Monoclonal antibodies for the
detection and quantitation of the endogenous plant growth regulator, Abscisic
acid. FEBS Letters 160, 269-272.
McCarty DR, Hattori T, Carson CB, Vasil V, Vasil IK. 1991. The viviparous 1
developmental gene of maize encodes a novel transcriptional activator. Cell 66,
895-905.
21
Molina-Cano JL, Sopena A, Swanson JS, Moralejo MA, Casas AM, Ubieto A,
Pérez-Vendrell AM, Lara I, Romagosa I. 1999. A mutant induced in malting
barley cv. Triumph with reduced dormancy and ABA response. Theoretical
and Applied Genetics 98, 347-355.
Moorman B. 1942. Untersuchungen über Keimruhe bei Hafer und Gerste. Kuhn
Archiv 56, 41-79.
Oberthur L, Blake TK, Dyer WE, Ullrich SE. 1995. Genetic analysis of seed
dormancy QTL in reciprocal backcross populations. Journal of Quantitative
Trait Loci [online], Volume 1, Article 5. In:
http://probe.nalusda.gov:8000/otherdoc/jqtl/jqtl1995-05/dormancy.html
Parcy F, Valon C, Raynal M, Gaubier-Comella P, Delseny M, Giraudat J. 1994.
Regulation of gene expression programs during Arabidopsis seed development:
roles of the ABI3 locus and of endogenous abscisic acid. The Plant Cell 6,
1567-1582.
Quarrie SA, Tuberosa R, Lister P. 1988. Abscisic acid in developing grains of
wheat and barley genotypes differing in grain weight. Plant Growth Regulation
7, 3-17.
Radley M. 1976. The development of wheat grain in relation to endogenous growth
substances. Journal of Experimental Botany 27, 1009-1021.
Romagosa I, Han F, Clancy JA, Ullrich SE. 1999. Individual locus effects on
dormancy during seed development and after-ripening in barley. Crop Science
39, 74-79.
SAS Institute Inc. 1991. SAS user´s Guide. Statistics. Version 6.11. Cary: SAS
Institute.
22
Simpson GM. 1990. Seed dormancy in grasses. Cambridge: Cambridge University
Press.
Skriver K, Mundy J. 1990. Gene expression in response to abscisic acid and
osmotic stress. The Plant Cell 2, 503-512.
Strand E. 1989. Studies on seed dormancy in small grain species. I. Barley.
Norwegian Journal of Agricultural Science 3, 85-99.
Van Beckum J, Libbenga K, Wang M. 1993. Abscisic acid and gibberellic acid-
regulated responses of embryos and aleurone layers isolated from dormant and
non dormant barley grains. Physiologia Plantarum 89, 483-489.
Walker-Simmons M. 1987. ABA levels and sensitivity in developing wheat
embryos of sprouting resistant and susceptible cultivars. Plant Physiology 84,
61-66.
Walker-Simmons M, Sessing J. 1990 Temperature effects on embryonic abscisic
acid levels during development of wheat grain dormancy. Journal of Plant
Growth Regulation 9, 51-56.
Wang M, Heimovaara-Diijkstra S, Van Duijn B. 1995. Modulation of germination
of embryos isolated from dormant and nondormant barley grains by
manipulation of endogenous abscisic acid. Planta 195, 586-592.
Weidenhoeft T, Hagemann M, Chevalier P, Walker-Simmons M, Ciha AJ. 1988.
Field studies on abscisic acid and embryonic germinability in winter wheat.
Field Crops Research 18, 271-278.
Weiler EW. 1984. Immunoassay of plant growth regulators. Annual Review of Plant
Physiology 35, 85-95.
23
White CN, Rivin CJ. 2000. Gibberellins and seed development in maize. II.
Gibberellin synthesis inhibition enhances abscisic acid signaling in cultured
embryos. Plant Physiology 122, 1089-1097.
White CN, Proebsting WM, Hedden P, Rivin CJ. 2000. Gibberellins and seed
development in maize. I. Evidence that gibberellin/abscisic acid balance
governs germination versus maturation pathways. Plant Physiology 122, 1081-
1088.
Zoppolo J, Lafon M, Chaussat R. 1982. Obtention de semences d´orge dormantes
en milieu climatique artificiel. Comptes Rendus de L’Academie de Agriculture
de France 68, 124-133.
24
Table 1. ABA content, measured as ng ABA/ g DW, one month after-harvest of
Triumph and TL43 mature grain. Same letter indicates no significant differences among
genotypes within a given location in 1999, according to a Duncan test (p 0.05).
Genotype Spain Scotland
Triumph
2101 a
2255 a
TL43
2092 a
1942 a
25
Figure 1. Germination (%) of artificially-dried seed at 1 day post-harvest (dormancy
profile) and 66 and 144 days post-harvest, in 1997 and 1998, respectively (viability
profile) during seed development expressed as degree days after-anthesis (DDAA). (A)
TL43 Spain 1997; (B) Triumph Spain 1997; (C) TL43 Spain 1998; (D) Triumph Spain
1998; (E) TL43 Scotland 1998; (F) Triumph Scotland 1998. The dotted lines show the
grain filling process, expressed as percentage of the constant end dry weight at seed
maturity.
Figure 2. ABA content of artificially-dried seed at 1 day post-harvest during seed
development expressed as degree days after-anthesis (DDAA). (A) TL43 Spain 1997;
(B) Triumph Spain 1997; (C) TL43 Spain 1998; (D) Triumph Spain 1998; (E) TL43
Scotland 1998; (F) Triumph Scotland 1998. The lines and points show the dormancy
profile. Vertical bars represent the standard error of the mean of ABA content.
Figure 3. Genotypic sensitivities of Triumph and TL43 mature dry seeds to increasing
ABA concentrations (logarithmic scale on the X axis) in the germination medium. (A)
Spain 1998: seven and 75 days post harvest (DPH); (B) Spain 1999 at 30 DPH; (C)
Scotland 1999 at 30 DPH.
26
Fig.1
27
Fig 2
0
20
40
60
80
100
0
20
40
60
80
100
0
20
40
60
80
100
0
20
40
60
80
100
0
20
40
60
80
100DRY WEIGHT (%)
0
20
40
60
80
100GERMINATION (%)
r2=0.985r2=0.994r2=0.992
r2=0.976
r2=0.939
r2=0.894
r2=0.965
r2=0.994 r2=0.992r2=0.985 r2=0.972
r2=0.958
r2=0.982
r2=0.904r2=0.980
A B
C D
E F
Dry weightViability prof ileDormancy prof ile
TL43 Scotland 98
TRIUMPHScotland 98
TL43 Spain 98
TRIUMPHSpain 98
TL43 Spain 97
TRIUMPHSpain 97
200 400 600 800
THERMAL TIME (DDAA)
200 400 600 800
THERMAL TIME (DDAA)
28
Fig 3
0
2000
4000
6000
8000
100000
2000
4000
6000
8000
100000
20
40
60
80
100GERMINATION (%)
0
2000
4000
6000
8000
10000ABA CONTENT (ng/ g DW)
0
20
40
60
80
100
0
20
40
60
80
100
A B
C D
E F
ABA content Dormancy profile
200 400 600 800
THERMAL TIME (DDAA)
200 400 600 800
THERMAL TIME (DDAA)
TL43Spain 97
TRIUMPHSpain 97
TL43Spain 98
TRIUMPHSpain 98
TL43Scotland 98
TRIUMPHScotland 98
100
80
60
40
20
0
r2=0.975 r2=0.916
10-8 10-7 10-6 10-5 10-4 10-3
Scotland 99
r2=0.995r2=0.986
r2=0.941
0
20
40
60
80
100GERMINATION (%)
TL43 Triumph TL43 (7 DPH)
0
20
40
60
80
100
r2=0.979 r2=0.988
Spain 99
Spain 98
ABA (M)