1

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:

iromagosa@pvcf.udl.es

Keywords: Germination, seed dormancy, mutagenesis, barley, abscisic acid,

Short title

Differential germination of a barley mutant.

2

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.

3

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

4

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

5

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.

6

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

7

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

8

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.

9

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

10

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.

11

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

12

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

13

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.

14

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.

15

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

16

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

17

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.

18

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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)