Maturation temperature and rainfall influence seed dormancy ...

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Australian Journal of Agricultural Research, 2004, 55, 1047–1057

Maturation temperature and rainfall influence seed dormancy characteristics of annual ryegrass (Lolium rigidum) Kathryn J. SteadmanA,D , Amanda J. ElleryB , Ross ChapmanB , Andrew MooreC , and Neil C. TurnerB A Western

Australian Herbicide Resistance Initiative, School of Plant Biology, Faculty of Natural and Agricultural Sciences, University of Western Australia, Crawley, WA 6009, Australia. B CSIRO Plant Industry, Private Bag No. 5, Wembley, WA 6913, Australia. C CSIRO Plant Industry, GPO Box 1600, Canberra, ACT 2601, Australia. D Corresponding author; email: [email protected]

Abstract. The role of temperature and rainfall during seed development in modulating subsequent seed dormancy status was studied for Lolium rigidum Gaud. (annual ryegrass). Climatic parameters relating to geographic origin were compared with annual ryegrass seed dormancy characteristics for seeds collected from 12 sites across the southern Western Australian cropping region. Seed germination was tested soon after collection and periodically during subsequent after-ripening. Temperature in the year of seed development and long-term rainfall patterns showed correlations with aspects of seed dormancy, particularly the proportion of seeds remaining dormant following 5 months of after-ripening. Consequently, for one population the temperature (warm/cool) and water supply (adequate/reduced) during seed development were manipulated to investigate the role of maternal environment in the quantity and dormancy characteristics of seeds produced. Seeds from plants grown at warm temperatures were fewer in number, weighed less, and were less dormant than those from plants grown at cool temperature. Seeds that developed under both cool temperature and reduced moisture conditions lost dormancy faster than seeds from well-watered plants. Seed maturation environment, particularly temperature, can have a significant effect on annual ryegrass seed numbers and seed dormancy characteristics. Additional keywords: climate, drought, germination, plant growth, seed production, water stress, weed.

Introduction In annual ryegrass (Lolium rigidum Gaud.), the most important weed of southern Australian cropping systems, seed dormancy is well timed to ensure emergence during the winter growing season. Flowering and seed development occur during spring, and seed fall occurs in early summer; seeds will generally spend the summer months on or near the soil surface. Seed dormancy prevents potentially fatal germination during infrequent and sporadic rainfall events that occur during the summer in the southern Australian Mediterranean-type climate, but dormancy is gradually lost in response to the high summer temperatures so that emergence of most of the population is permitted during the subsequent autumn–winter growing season (Steadman et al. 2003a, 2003b). Dormancy release in annual ryegrass seeds is controlled by temperature, and can be modelled using thermal time concepts. However, different populations collected from the Western Australian wheatbelt, and seedlots collected in different years from the same site, exhibit large variation in dormancy characteristics, in both the initial level © CSIRO 2004

of dormancy at the beginning of the summer, and the rate at which dormancy is lost in response to summer temperature (Steadman et al. 2003a, 2003b). Accurate prediction of the extent and timing of weed emergence can provide important information to assist annual ryegrass management. If predictive models for emergence are to be improved, seed dormancy status must be incorporated (Forcella et al. 2000; Grundy 2003). Consequently, potential cause(s) of inter-population and inter-year variation in dormancy release parameters must be identified. The environment in which seeds develop on the parent plant often plays a pivotal role in determining dormancy status. Temperature, water supply, shading, daylength, and nutrient supply are the main factors that have been found to modify the proportion of mature seeds exhibiting dormancy in studies with a wide range of species (Fenner 1991; Wulff 1995; Baskin and Baskin 1998; Gutterman 2000). Temperature has a particularly consistent correlation with seed dormancy among species. Seeds that develop at warm temperatures are generally less dormant at maturity than 10.1071/AR04083

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those that develop at cooler temperatures (Fenner 1991; Baskin and Baskin 1998). For example, Lolium multiflorum Lam. seeds become larger and more dormant as maturation temperature is reduced from 27◦ C to 21◦ C to 15◦ C (Wiesner and Grabe 1972). Other grasses, such as Festuca arundinacea Schreb. and Avena fatua L., respond similarly (Boyce et al. 1976; Peters 1982b). The effect of drought during seed development on dormancy is less consistent and largely species-dependent (Baskin and Baskin 1998), although in studies with grass species, drought-stressed plants of Avena fatua, Bromus tectorum L., and Sorghum halepense (L.) Pers. produced fewer dormant seeds than plants with adequate water (Peters 1982a; Benech-Arnold et al. 1992; Meyer and Allen 1999). Climatic variables can form a selection pressure during long-term exposure, with adaptation to the local environment leading to genotypic differentiation (Linhart and Grant 1996). For example, Avena fatua L. and A. barbata Pott ex Link plants in northern areas of the cropping region in Western Australia produce seeds that are less dormant than those in the south (Paterson et al. 1976). It is possible that similar generalisations may apply for annual ryegrass; indeed, it has been mentioned that ‘in northern agricultural districts the dormancy cycle is complete by the opening of the following growing season’, but not necessarily in other areas of the Western Australian cropping region (Pearce and Quinlivan 1971). Of the environmental factors with the potential to modify plant characteristics, it is relatively easy to acquire historical temperature and rainfall data, which are important when considering the long-term aim of developing a predictive emergence model. Additionally, as with seed dormancy status, temperature and rainfall exhibit large variation among localities and years across the agricultural regions of Western Australia (Department of Agriculture Western Australia 2004), so it is these parameters that are considered in the present study. The role of temperature and rainfall in modulating annual ryegrass seed dormancy status was studied in two ways. Firstly, long-term climate parameters relating to geographic origin, and climate parameters during the year of seed set, were related to dormancy release characteristics for 12 populations collected from the cropping region of southern Western Australia. Subsequently, we experimentally manipulated the maternal environment for one population and measured the effect on quantity of seeds produced, their dormancy level at maturity, and dormancy release characteristics. Materials and methods Expt 1: effect of collection site on dormancy Mature annual ryegrass (Lolium rigidum Gaud.) tillers were collected from cropping fields at Esperance (33◦ 37 S, 121◦ 48 E), Geraldton (28◦ 78 S, 114◦ 61 E), Hyden (32◦ 45 S, 118◦ 86 E), Jennacubbine (31◦ 27 S, 116◦ 43 E), Mullewa (28◦ 33 S, 115◦ 29 E), Moora (30◦ 65 S, 116◦ 01 E), Merredin (31◦ 31 S, 118◦ 10 E), Ravensthorpe (33◦ 26 S,

K. J. Steadman et al.

119◦ 24 E), and Wongan Hills (30◦ 50 S, 116◦ 43 E) in Western Australia. A total of 12 collections were made at the end of the growing season around the time of crop harvest in one or more of 1998, 1999, and 2000. Tillers were threshed soon after collection and florets (hereafter called seeds) separated from chaff by sieving and forced-air separation. During this process, seeds were stored in paper bags at 22◦ C for approximately 3 weeks until germination and dormancy release tests were commenced. Seeds of all populations were placed in constant temperature incubators set at 20, 40, and 60◦ C for after-ripening. In addition, the seeds collected from Jennacubbine in 1998 and 1999 were also afterripened at 30 and 50◦ C. Four replicate samples of 50 seeds were removed from the after-ripening treatments at 2-week intervals for germination testing. After-ripening continued for up to 170 days. Seeds were germinated in 9-cm-diameter circular Petri dishes containing filter paper (MN 615, Macherey-Nagel) soaked with water. One dish containing 50 seeds was germinated per replicate. Dishes were sealed with Parafilm and placed in a controlled-environment growth cabinet set at 12-h alternating 25 and 17◦ C (25/17◦ C), optimal for germination (Steadman et al. 2003a). Light (12 h per day during the warm phase) was provided by five 1000-W Powerstar HQI-T E40 lamps and eight 100-W incandescent globes (Osram Australia, Subiaco, WA), resulting in a photosynthetic photon flux of 600–700 µmol/m2 .s and a red : far-red ratio of 2.0 at shelf level. The number of germinated seeds was counted 14 days after the start of the test; the criterion for germination was visible radicle protrusion. Ungerminated seeds were assessed for viability by slicing transversely to expose the endosperm, and incubating in 1% (w/v) 2,3,5-triphenyltetrazolium chloride solution for 24 h in the dark at 22◦ C. Evidence of pink staining, required to score a seed as viable, was observed through a microscope. Logistic regression was used to examine the significance of 3 factors (population, after-ripening temperature, and after-ripening time) on the proportion of seeds germinating. The sum of all proportions germinating at the start of the experiment (Time 0) was compared with that at the first sample point (Time 1) for the 5 after-ripening temperatures using a 1-sided chi-square test. For those temperatures that exhibited an initial reduction in germinability at the start of after-ripening, the logistic regression was plotted with Time 1 as the starting point instead of Time 0. Separate logistic growth curve models were fitted for each combination of population and after-ripening temperature. Due to large variation within some replicates, which prevented convergence during curve fitting, the total proportion germinating over the 4 replicates was used as the dependent variable with an appropriate weighting based on the reciprocal of the standard error of this amount. This lead to a total of 40 logistic models of the form: Gi =

Gmax 1 + exp[b + DRR(ti − t0 )]

(1)

where Gi is the fraction germinating at time i (ti ) and ti − t0 is the number of days of after-ripening since the start of the experiment (t0 ) experienced by the sample taken at ti . The maximum fraction of seeds losing dormancy is represented by parameter Gmax (the asymptote), and the slope parameter DRR describes the dormancy release rate. Parameter estimates for Gmax , DRR, and the initial germination at t0 , G0 , were obtained for each of the models and a new dataset was created containing these values to carry out subsequent modelling. Weighted ANOVA and regression techniques were used in the following manner. ANOVA was carried out with dependent variables G0 , Gmax , and DRR to investigate relationships of these variables with population and storage temperature. Least square (LS) means were calculated where appropriate. Stepwise regression techniques were used on the variables G0 , Gmax , and DRR to establish whether relationships existed between these parameters and the climate data for the particular site and year combination (population). The climate data used for

Annual ryegrass seed dormancy

(1) long-term average annual rainfall; (2) long-term average early season rainfall (February–April); (3) long-term early season rainfall as a fraction of long-term annual rainfall; (4) total growing season rainfall (May–October) in year of seed production; (5) total late season rainfall (September–November) during period of seed development; (6) long-term average autumn temperature (May–July); (7) average temperature during September in year of seed development; (8) average temperature during October in year of seed development; (9) average temperature during November in year of seed development. Statistical analysis was carried out using SAS (SAS Institute Inc., Cary, NC, USA).

Expt 2: effect of maternal environment on dormancy Seeds collected from Jennacubbine in 2000 and stored at 22◦ C were germinated in seedling trays containing potting mix in a glasshouse at CSIRO, Floreat, WA, on 10 April 2002. Seedlings at the 2-leaf stage of development were transplanted into 120 free-draining pots (10 cm diam. and 48 cm deep) at a density of 3 plants per pot on 29 April 2002. Pots were previously filled with 10 kg of sandy loam soil (5 parts sand + 1 part loam) with a slow-release fertiliser containing 15% N, 3.9% P, 12.5% K, 7% S, 3.4% Ca, 2% Mg, 0.3% Fe, 0.01% B, 0.007% Zn, and 0.004% Mo added at 1 g/kg soil. The weight of the pots containing the dry soil mixture was measured. Pots were watered, allowed to drain for 6 h, and re-weighed to determine the field (pot) capacity. Subsequently, the plants were watered 3 times per week to maintain the soil near field capacity. At the 6-leaf stage, plants were thinned to 1 plant/pot. Regular watering continued until the start of anthesis. To achieve maximum uniformity in flowering time, the first 10 plants to flower were discarded. Thereafter, as each plant flowered, the first 2 spikes to flower on each plant were tagged and dated, and the plant was placed into one of 2 controlled-environment growth cabinets. Flowering date was defined as the date on which the first anthers were fully emerged and ranged from 23 August to 9 October 2002 amongst the plants. Pots were randomly allocated to 4 treatments: (1) cool temperature, well-watered; (2) warm temperature, well-watered; (3) cool temperature, droughted; (4) warm temperature, droughted. One controlledenvironment growth cabinet was set to alternate between day/night temperatures 20/15◦ C (cool), and the second cabinet was set at 30/25◦ C (warm), on 12-h cycles. Light (12 h/day during the warm phase) was provided by five 1000-W Powerstar HQI-T E40 lamps and eight 100-W incandescent globes, resulting in a photosynthetic photon flux of 800–1000 µmol/m2 .s and a red : far-red ratio of 2.0 at plant level. Plants allocated to the drought treatment were weighed 3 times per week, and where the plant-available soil water fell below 20%, the pots were watered to return the soil to field capacity. Plants allocated to the control (‘well-watered’) treatments received 2 L water each, 3 times per week. Plants that became heavily infested with aphids or exhibited extremely slow maturation were discarded to exclude outliers. The final number of plants remaining in each treatment from which seeds were harvested was 16 (warm, control), 16 (warm, drought), 10 (cool, control), and 18 (cool, drought).

1049

At harvest maturity (23 October–20 November 2003), determined as the point at which there was no green colour remaining in the stems, the 2 tagged spikes were harvested from each plant, the seeds threshed out by hand, and placed into a single paper envelope. The seeds were then frozen until used in the after-ripening and germination tests, which, in our experience, retains both viability and dormancy status for many years. The remaining spikes were harvested as a bulk sample, weighed, and stored in paper bags at room temperature (20◦ C). For each bulk sample, the number of spikes was counted and 10 spikes chosen randomly for more detailed counts. The number of spikelets on each of the 10 spikes was counted, the seeds were then threshed out by hand, and the total number of seeds from the 10 spikes counted. The total number of seeds produced by the plant was estimated by multiplying the average number of seeds per spike by the number of spikes. For 8 representative plants, 2 from each treatment, the seeds from the rest of the spikes were threshed out and counted. This allowed the total number of seeds estimated from the 10 spikes to be compared with the actual number of seeds determined by direct counting. Seed dry weight was measured according to International Seed Testing Association (1999) guidelines; 100 seeds were dried at 103◦ C for 24 h and then weighed and the average weight per seed calculated. The seeds from the first 2 spikes to flower from each plant, previously placed in paper envelopes and stored in the freezer, were placed in an incubator at constant 30◦ C for after-ripening on 14 January 2003. Samples were taken immediately after removal from the freezer and then every 4 weeks for germination testing to measure dormancy release. For each parent plant, one dish containing 25 seeds was germinated as described for Expt 1, at each of 2 temperature regimes, 25/17◦ C and 20/12◦ C. The number of germinated seeds was counted 21 days after the start of the test and ungerminated seeds were assessed for viability using triphenyltetrazolium chloride. Using 8 representative plants (2 from each treatment), we established that the estimated total seed number per plant using the seed count from 10 randomly sampled spikes multiplied by the number of spikes, correlated well (r = 0.969) with the actual seed number arrived at by counting every seed produced per plant (Fig. 1). However, the regression line in Fig. 1 was significantly different (P = 0.012) from the 1 : 1 line, 8000

Actual number of seeds per plant

comparison were calculated from historical records of the daily rainfall and maximum and minimum temperatures measured by automatic weather stations (Department of Agriculture Western Australia 2004) except Geraldton, Hyden, and Moora for which Australian Bureau of Meteorology records were used. Nine parameters were calculated from the data, either specific to the season in which maternal plant growth and seed development occurred, or long-term averages across the years 1986–2000:

Australian Journal of Agricultural Research

y = 0.9656x + 699

r 2 = 0.939 6000

4000

2000

0 0

2000

4000

6000

8000

Estimated number of seeds per plant Fig. 1. Relationship between actual and estimated number of annual ryegrass seeds produced per plant. The numbers were either directly counted (all seeds produced by the plants were removed from the spikes and counted) or estimated (seeds produced by 10 spikes were removed and counted, and the average number of seeds per spike multiplied by the number of spikes).

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indicating that the total seed number was underestimated. The time involved in extracting and counting every seed was prohibitive, so for the other 52 plants the total seed number was calculated using the equation in Fig. 1 relating the estimated to actual seed numbers (actual number = (0.9656 × estimated number) + 699). This approach produced more conservative results during statistical comparison of treatment effects than using the estimated total seed number per plant. Statistical comparisons were made using GenStat version 6.2 (Lawes Agricultural Trust, UK). Generalised linear modelling was used to determine whether the relationship between the estimated total seed number (based on the seed count from 10 randomly sampled spikes) and the counted total seed number (seed count from every spike) was significantly different from unity. To investigate the effect of maturation temperature and water availability on seed production, data were logtransformed prior to analysis to improve normality, and a 2-way ANOVA for unbalanced treatment structure applied. The effects of maturation

and germination environment on seed dormancy characteristics were assessed using ANOVA of angular-transformed germination data (as % of viable seeds) with generalised treatment structure in randomised blocks.

Results Expt 1: effect of collection site on dormancy The change in dormancy status during after-ripening at constant temperatures between 20◦ C and 60◦ C was plotted and logistic regressions fitted (Fig. 2). Seeds after-ripened at constant 50◦ C and 60◦ C exhibited an early reduction in germination, visible as a difference between the first and second germination tests (P < 0.01). The reduction in

100 80 60 40 20 Esperance 2000

Geraldton 2000

Geraldton 1999

0 Jennacubbine 1998

Jennacubbine 1999

Jennacubbine 2000

80

Germination (% at 14 days)

60 40 20 0 80 60 40 20 Hyden 2000

Merredin 2000

Moora 2000

0 Wongan Hills 1998

80 60 20°C 30°C 40°C 50°C 60°C

40 20 Mullewa 2000

Ravensthorpe 2000

0 0

50

100

150

0

50

100

150 0

50

100

150

After-ripening time (days)

Fig. 2. Change in germinability (as % of viable seeds) with increased after-ripening time at 20, 30, 40, 50, or 60◦ C for 12 populations of annual ryegrass collected from the Western Australian wheatbelt at the end of the growing season in 1998, 1999, or 2000. Germination was tested at 25/17◦ C with a 12-h photoperiod for 14 days.

Annual ryegrass seed dormancy

Australian Journal of Agricultural Research

Table 1. Summary of analyses of variance for the effect of afterripening temperature and seed population on the dependent variables initial germination (G0 ), maximum germination (Gmax ), and dormancy release rate (DRR) from logistic regressions through dormancy release curves in Fig. 2 Source

d.f.

MS

F

G0 Population After-ripening temperature

11 4

0.14 0.05

49.53*** 16.36***

Gmax Population After-ripening temperature

11 4

1.42 0.83

37.67*** 22.08***

DRR Including Jennacubbine Population After-ripening temperature

11 4

0.01 0.01

2.24* 1.27

Excluding Jennacubbine Population After-ripening temperature

8 2

0.01 0.01

4.16** 3.66*

*P < 0.05; **P < 0.01; ***P < 0.001.

germination was greater for seeds after-ripened at 60◦ C than at 50◦ C and was not apparently related to loss of viability according to tetrazolium staining. This reduction in germination prevented the initial germination value from being included in the logistic curve-fitting process for seeds at these temperatures. Parameter estimates for the initial germination (G0 ), the maximum germination obtained (Gmax ), and the dormancy release rate (DRR) were obtained from each of the 40 logistic models and a new dataset was created containing these values to carry out subsequent modelling. Graphical comparison

of these 3 parameters across populations (data not shown) indicated that the collections from Jennacubbine provided the most extreme values. Therefore, subsequent comparisons were made with and without these populations to establish the rigour of the relationship between model parameters and climate variables. Parameter G0 was affected by both after-ripening temperature and the source of the seed population (Table 1). The effect of after-ripening temperature was due only to the reduction in germination between the first and second germination tests caused by incubation at 50 and 60◦ C (Fig. 2); G0 at 20, 30, and 40◦ C were not significantly different from each other. Stepwise regression with the climate data as predictors and G0 as the response variable identified 3 potential climate predictors. A higher long-term average February–April rainfall as a fraction of long-term average annual rainfall, a lower rainfall during the growing season in which seed development occurred, and a higher September temperature in the year of seed set were predictors for a higher G0 (R2 = 85%; Table 2). However, removing Jennacubbine from the dataset altered the climate predictors. In this case, lower growing season rainfall and higher September temperature remained as predictors, but a lower October temperature became significantly correlated with a higher G0 (lower dormancy). Parameter Gmax was significantly affected by population and after-ripening temperature (Table 1), with seeds from all populations reaching the highest Gmax when they were afterripened at 40◦ C (Fig. 2). Two climate variables were selected as potential predictors for Gmax by stepwise regression. A higher long-term average February–April rainfall as a fraction of long-term average annual rainfall and a

Table 2. Results of stepwise regression procedure for parameter estimates of G0 , Gmax , or DRR against climate variables and after-ripening temperature Results for parameter estimates of DRR were performed with collections from Jennacubbine excluded Source

d.f.

MS

F

Direction

G0 Long-term Feb.–Apr. rainfall as a fraction of long-term annual rainfall Mean Sept. temperature in year of seed development Growing-season rainfall (May–Oct.) in year of seed development After-ripening temperature R2 = 0.8522

1 1 1 4

3.61 1.90 0.51 0.52

82.19*** 43.21*** 11.63** 6.55***

+ + –

Gmax Long-term Feb.–Apr. rainfall as a fraction of long-term annual rainfall Mean Sept. temperature in year of seed development After-ripening temperature R2 = 0.8596

1 1 4

6.10 3.08 1.97

72.17*** 36.49*** 23.35***

+ +

DRR Mean Oct. temperature in year of seed development Growing-season rainfall (May–Oct.) in year of seed development After-ripening temperature R2 = 0.6871

1 1 2

0.04 0.04 0.02

16.33*** 14.17** 8.91**

– +

**P < 0.01; ***P < 0.001.

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50

(a)

Plant dry weight (g)

40 30 20 10 0

7000

2.5

(b)

(c)

Seed dry weight (mg)

5000 4000 3000 2000 1000 0

2.0 1.5 1.0 0.5 0.0

100

100

(e)

(d )

Seeds per spike

60 40

80 60 40

20

20

0

0

2500

4

(g)

2000

Seeds per spikelet

1500 1000 500

3

2

1

0

ou dr p,

m

W ar

m

te

m te m ar

gh

ed er w at

p,

p,

dr

at w p,

oo

em lt oo

t

t gh ou

ed er

gh ou dr

p, te m

C

W

ar

m

te m m ar

W

t

ed at er w

p,

p, em lt

C oo

oo

lt

em p,

w

dr

at

ou

er

gh

t

ed

0

C

Spikelets per plant

(f )

W

Spikes per plant

80

lt em

Seeds per plant

6000

C

1052

Fig. 3. Effect of seed maturation environment on various yield parameters. During flowering and seed development, annual ryegrass plants were subjected to warm (30/25◦ C) or cool (20/15◦ C) temperatures, with or without a drought treatment. Bars are ± s.e. of the mean (n = 4).

Annual ryegrass seed dormancy

higher mean temperature in September of the year of seed development were predictors for a higher Gmax (R2 = 86%; Table 2). Removing Jennacubbine from the populations assessed did not change the identity or direction of the climate predictors. With all 12 populations included in the analysis, parameter DRR showed borderline (P = 0.048) influence by population but not by after-ripening temperature (Table 1). However, after removal of the 3 collections from Jennacubbine, which gave particularly variable estimates of DRR, the remaining 9 populations were significantly affected by both population and after-ripening temperature. Using the stepwise regression procedure with the climate data as predictors and DRR as the response, 2 climate variables appeared to be predictors of the DRR parameter for these 9 populations. Higher growingseason rainfall and lower October temperature during the year of seed development correlated with a higher rate of dormancy release (R2 = 69%; Table 2). However, inspection of a graph of DRR v. each of these climate predictors revealed that the above-average growing-season rainfall experienced in Geraldton during 2000 (568 mm; long-term average 395 mm) had a major effect on the relationship. Indeed, if this population was removed from the analysis, only the mean temperature in the October of seed set remained a significant climate predictor (R2 = 60%). Expt 2: effect of maternal environment on dormancy The time between anthesis and harvest maturity was shorter in plants subjected to the warm temperature during flowering and seed development (mean: watered, 42 days; drought, 39 days) than those at the cool temperature (mean: watered, 52 days; drought, 51 days). Whereas temperature had no effect on the total dry matter per plant (Fig. 3a, Table 3), it had a significant effect on seed number (Fig. 3b, Table 3). Plants subjected to the cool temperature treatments during flowering and seed development produced over 5000 seeds, whereas plants grown at the warm temperature produced around 3000 (Fig. 3b). This was due to more seeds developing in each spikelet rather than more spikes or spikelets being produced by the plants (Fig. 3d–g; Table 3). Furthermore, the seeds produced by plants at the cool temperature were significantly heavier than seeds that developed at warm temperature (Table 3, Fig. 3c). The effect of drought treatment was less pronounced than temperature, but did have an effect on seed production. Plants that were water-stressed produced fewer seeds than plants grown with unlimited soil moisture (Table 3, Fig. 3b). The germination test performed on seeds that were stored in the freezer since harvest gave a measure of dormancy at maturity. Germination at maturity was affected by both the temperature used for the germination test and the temperature at which the seeds matured on the parent plant (Table 4, Time 0 in Fig. 4). More seeds germinated at 25/17◦ C (Fig. 4b, d) than at 20/12◦ C (Fig. 4a, c), and more

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Table 3. Summary of analyses of variance for the effect of maturation temperature (T-mat) and/or water availability (W-mat) applied during flowering and seed development on seed production Log-transformed values were analysed for all yield parameters Source

d.f.

MS

T-mat W-mat T-mat × W-mat Error

Plant weight 1 1 1 53

0.002 0.046 0.023 0.015

T-mat W-mat T-mat × W-mat Error

No. of seeds 1 1 1 51

0.748 0.122 0.000 0.025

T-mat W-mat T-mat × W-mat Error

Seed dry weight 1 0.226 1 0.009 1 0.021 50 0.007

T-mat W-mat T-mat × W-mat Error

No. of spikes 1 1 1 51

T-mat W-mat T-mat × W-mat Error

No. of spikelets 1 0.025 1 0.018 1 0.001 51 0.024

T-mat W-mat T-mat × W-mat Error

Seeds per spike 1 0.445 1 0.076 1 0.003 51 0.023

19.34*** 3.29 0.13

T-mat W-mat T-mat × W-mat Error

Seeds per spikelet 1 0.462 1 0.056 1 0.003 50 0.021

22.12*** 2.67 0.13

0.038 0.006 0.001 0.021

F 0.12 3.03 1.48

30.13*** 4.90* 0.01

32.53*** 1.29 3.00

1.84 0.28 0.06

1.04 0.74 0.03

*P < 0.05; ***P < 0.001.

seeds germinated from plants that were exposed to warm (Fig. 4c, d) than cool temperature (Fig. 4a, b) during seed development. The drought treatment had no effect on dormancy status at maturity for either of the temperature treatments (Table 4). Dormancy was reduced as after-ripening time progressed for seeds from all of the treatments (Fig. 4, Table 5). The pattern of dormancy loss over time differed between germination temperatures used to test for dormancy, with different initial but similar final germination values (Fig. 4) leading to a significant interaction between after-ripening time and germination temperature (Table 5). For seeds that matured at cool temperatures, germination increased from the initial level of 20% (measured at 20/12◦ C, Fig. 4a) or 40% (measured at 25/17◦ C, Fig. 4b) to an average maximum

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Table 4. Summary of analyses of variance for the effect of maturation temperature (T-mat) and/or water availability (W-mat) applied during flowering and seed development, and germination temperature (T-germ) on the level of dormancy measured at harvest maturity or after-ripened for 5 months at 30◦ C Germination percentages were subjected to angular transformation prior to analysis Source

Harvest maturity MS

d.f. T-mat W-mat T-germ

1 1 1

1947.4 0.5 1337.7

Interactions T-mat × W-mat T-mat × T-germ W-mat × T-germ T-mat × W-mat × T-germ

1 1 1 1

18.3 11.4 4.4 0.6

21

90.1

Error

After-ripened MS

F

d.f.

21.61*** 0.01 14.85***

1 1 1

372.7 42.2 0.2

5.98* 0.68 0.00

1 1 1 1

229.7 8.7 93.2 0.1

3.68 0.14 1.50 0.00

21

62.4

0.20 0.13 0.05 0.01

F

*P < 0.05; ***P < 0.001. 100

(a) Maturation at 20/15°C; germination at 20/12°C

(b) Maturation at 20/15°C; germination at 25/17°C

(c) Maturation at 30/25°C; germination at 20/12°C

(d ) Maturation at 30/25°C; germination at 25/17°C

80

60

Germination (% at 21 days)

40

20

0

80

60

40

control drought

20

0

0

28

56

84

112

140

0

28

56

84

112

140

After-ripening time (days)

Fig. 4. Change in germinability (as % of viable seeds) during after-ripening at 30◦ C for annual ryegrass seeds. Seeds were collected from plants subjected to cool (20/15◦ C; a, b) or warm (30/25◦ C; c, d) temperatures during flowering and seed development. Plants were also either well-watered (control) or subjected to a drought treatment during flowering and seed development. Every 4 weeks, seeds were removed from the after-ripening treatment and germinated for 21 days at 2 different temperature regimes: 20/12◦ C (a, c) and 25/17◦ C (b, d). Bars are ± s.e. of the mean (n = 4).

of 74% during the 5 months of after-ripening. For seeds that matured at warm temperatures, germination increased from 50% (measured at 20/12◦ C, Fig. 4c) or 65% (measured at 25/17◦ C, Fig. 4d) to over 80%. Seeds from drought-stressed

plants lost dormancy significantly faster than seeds from plants grown with ample water, but only when drought was combined with cool maturation temperatures (Table 5, Fig. 4).

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Table 5. Summary of analyses of variance for the effect of water availability (W-mat) applied during flowering and seed development, after-ripening time (Time), and germination temperature (T-germ) on dormancy release of seeds produced from plants at two different maturation temperatures (cool/warm) Germination percentages were subjected to angular transformation prior to analysis Source

d.f.

MS

F

W-mat Time T-germ W-mat × T-germ W-mat × time T-germ × time W-mat × T-germ × time Error

Cool 1 5 1 1 5 5 5 69

786.8 1743.7 1833.3 34.2 76.4 226.3 27.0 53.7

14.82*** 32.85*** 34.54*** 0.64 1.44 4.26** 0.51

W-mat Time T-germ W-mat × T-germ W-mat × time T-germ × time W-mat × T-germ × time Error

Warm 1 5 1 1 5 5 5 69

9.6 639.6 1264.7 78.7 153.8 222.0 15.7 77.6

0.12 8.24*** 16.29*** 1.01 1.98 2.86* 0.20

*P < 0.05; **P < 0.01; ***P < 0.001.

At the end of the 5-month after-ripening period, the only factor that influenced germination was maturation temperature (Table 4). Fewer seeds germinated when they originated from plants grown at the cool temperature (74%) than at the warm temperature (83%). The germination temperature 25/17◦ C no longer produced greater germination than 20/12◦ C, and the presence of drought during seed maturation had no influence on dormancy status following 5 months of after-ripening at 30◦ C (Table 4, Fig. 4). Discussion Temperature during the period of seed development and maturation played a major role in determining dormancy characteristics of the resulting seeds. A higher average September temperature during seed development correlated with higher initial (G0 ) and final (Gmax ) germination (i.e. lower dormancy) for the 12 populations collected from the Western Australian wheatbelt (Table 2). Furthermore, subjecting laboratory-grown plants to warm temperatures during seed development produced seeds that had higher initial and final germination than seeds that developed at cool temperature (Table 4). Thus, although previously no significant relationship between maternal temperature and germination of after-ripened annual ryegrass seeds was observed (Gramshaw 1976), the two approaches used here determined that higher temperatures during seed development correlated with greater germinability (less dormancy) when measured at harvest and following 5 months

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of after-ripening. Maturation temperature also had a profound effect on other aspects of seed production, with warm temperature reducing the number and size of mature seeds produced. These effects of temperature on seed production and dormancy in ryegrass are similar to those previously observed in other species (Fenner 1991; Baskin and Baskin 1998), including grass species (Wiesner and Grabe 1972; Boyce et al. 1976; Peters 1982b). Water availability during seed development had a less pronounced effect on dormancy in annual ryegrass. Although the drought treatment was enough to influence seed production in terms of reducing mature seed numbers (Table 3), there was no significant effect on seed dormancy characteristics at maturity (Table 4). This is in contrast to other grasses, Avena fatua, Sorghum halepense, and Bromus tectorum, in which seeds from droughtstressed plants exhibited reduced dormancy at harvest (Peters 1982a; Benech-Arnold et al. 1992; Meyer and Allen 1999). The intermittent drought treatment methodology used by Benech-Arnold et al. (1992) involved withholding watering for 5 days, resulting in plants showing signs of wilting and the seeds produced showing a strong effect of drought treatment. There were no signs of wilting in response to treatments imposed in the present study; it is possible that a harsher moisture-stress treatment may have had a greater effect. Indeed, there was an indication that lower rainfall during the growing season may be a predictor for reduced dormancy at harvest maturity (G0 ) when 12 populations were compared (Table 2). Although an effect on dormancy of newly matured annual ryegrass seeds was not evident in the initial germination tests using seeds from experimentally droughted plants, an effect on dormancy became apparent during after-ripening. Plants that were grown at cool temperature and drought conditions produced seeds that lost dormancy faster than watered plants, although there was no effect when plants were grown at warm temperature (Fig. 4). However, there was no correlation between rainfall during the period of seed development and dormancy release rate when compared amongst the 12 populations. Growing-season rainfall initially appeared to correlate with dormancy release rate, with higher rainfall being linked to slower dormancy release, but this correlation was affected by a single outlier and no correlation was evident when the outlier was removed. Thus, although less consistent than that for maturation temperature, there was some indication of a link between lower rainfall during seed development and reduced dormancy for annual ryegrass from the two different experimental approaches. The use of long-term climate variables in this exercise was to allow for the potential for local adaptation to have occurred, as annual ryegrass may be expected to have adapted to particularly risky environments by increasing the proportion of the population being dormant initially (G0 ) and remaining dormant into future seasons (Gmax ). For example, high levels

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of seed dormancy at dispersal are associated with habitats with a high probability of summer storms that could trigger premature germination into a lethal environment (Meyer et al. 1997). Annual rainfall during February to April was initially included as a measure of the likelihood of summer storms. However, a greater amount of annual rainfall falling during February to April as a proportion of the whole year correlated with a higher Gmax (Table 2). Thus, annual ryegrass growing in areas that have a higher proportion of rainfall falling early in the year, prior to the start of the growing season, appears to retain fewer seeds in the seedbank for future years. Therefore, rather than being considered as risky, summer rainfall may reduce the need for annual ryegrass to conserve seeds for future years, possibly through restocking soil water in advance of the growing season. Indeed, in the Mediterranean climatic regions of Western Australia the presence of stored soil moisture resulting from summer rainfall significantly improves early growth and yield of wheat, particularly in drier regions with heavy soils (Asseng et al. 2001). More detailed research, involving the separation of genetic and maternal environment effects, is needed to further investigate this hypothesis. Dormancy release rate is known to be dependent on after-ripening temperature, with an increase in temperature resulting in faster dormancy release (Steadman et al. 2003a, 2003b). In the present study, after-ripening temperature also played a role in influencing the proportion of seeds remaining dormant after 5 months of after-ripening (Gmax ). Seeds stored at constant 50◦ C and 60◦ C exhibited an early reduction in germination, which was maintained throughout afterripening (Fig. 2) and not caused by viability loss. Similarly, Gramshaw (1972) also noticed that temperatures greater than 50◦ C retarded dormancy release. In reality, soil temperatures reach this range for only a few hours per day during summer and so these treatments were extreme. Storage at 40◦ C was optimal for dormancy release for all 12 populations, resulting in more seeds ultimately losing dormancy than in cooler and warmer temperatures. Previously, predictive models have used after-ripening temperature as a regulator of dormancy release rate, but not the proportion of dormant seeds actually losing dormancy (Steadman et al. 2003a, 2003b). In fact, at least 2 of 4 populations that were each after-ripened on the soil surface at the extreme northern and southern ends of the Western Australian wheatbelt had greater germination (Gmax was higher) in the north, where average daily temperature was 7◦ C higher, than in the south (Steadman et al. 2003a). Future predictive modelling efforts will have to account for the influence of temperature on the overall proportion of the population losing dormancy as well as the rate at which dormancy release occurs. Germination temperature had a major effect on the number of seeds that germinated, irrespective of maturation treatment. Early in the after-ripening period, more seeds germinated at 25/17◦ C than at 20/12◦ C. Thus, some seeds

K. J. Steadman et al.

in the population have a narrower temperature window over which they will germinate than others. For example, for seeds matured at 20/15◦ C, 20% of the population could germinate at either temperature, a further 20% could germinate at 25/17◦ C but not 20/12◦ C, and the remainder did not germinate at either temperature (Fig. 3a, b). As seeds lose dormancy during after-ripening, this window gradually widens, with more of the seeds becoming able to germinate at both temperatures. After 5 months of after-ripening, there was no longer a differentiation between test temperatures (Table 4). Similarly, annual ryegrass seeds used in the study by Turner et al. (2001) had after-ripened for over 1 year, and germinated well over a wide range of alternating temperature regimes. Cocks and Donald (1973) and Gramshaw (1976) reported an influence of germination temperature for seeds that had been after-ripened; this may be expected to be due to some residual dormancy within the populations used. In summary, using 2 different approaches, we have shown that environmental conditions experienced by the parent plant during seed development, particularly temperature and to a lesser extent moisture, can significantly alter subsequent dormancy characteristics of seeds produced. Additionally, fewer seeds appear to remain dormant for future years when they are produced in areas that tend to have a greater proportion of rainfall falling prior to the growing season, suggesting that annual ryegrass may have adapted to local environmental pressures. Further investigation is indicated, particularly into the role of genetic adaptation to local environmental conditions, to enable these factors to be taken into account in future predictive emergence models. Acknowledgments We thank Sandy Nedelkos, Sheena Pollock, and Christiane Ludwig for invaluable technical support. Thanks to Kevin Murray (Statistical Consulting Group, UWA) for the logistic regression analysis. This research was funded by the Grains Research and Development Corporation (CSP270). References Asseng S, Turner NC, Keating BA (2001) Analysis of water- and nitrogen-use efficiency of wheat in a Mediterranean climate. Plant and Soil 233, 127–143. doi: 10.1023/A:1010381602223 Baskin CC, Baskin JM (1998) ‘Seeds, ecology, biogeography, and evolution of dormancy and germination.’ (Academic Press: San Diego, CA) Benech-Arnold RL, Fenner M, Edwards PJ (1992) Changes in dormancy level in Sorghum halepense seeds induced by water stress during seed development. Functional Ecology 6, 596–605. Boyce KG, Cole DF, Chilcote DO (1976) Effect of temperature and dormancy on germination of tall fescue. Crop Science 16, 15–18. Cocks PS, Donald CM (1973) The germination and establishment of two annual pasture grasses (Hordeum leporinum Link and Lolium rigidum Gaud.). Australian Journal of Agricultural Research 24, 1–10.

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Department of Agriculture Western Australia (2004) ‘Climate impacts and weather information.’ Available online at http://www. agric.wa.gov.au/climate/USDA Fenner M (1991) The effects of the parent environment on seed germinability. Seed Science Research 1, 75–84. Forcella F, Benech-Arnold RL, Sanchez R, Ghersa CM (2000) Modelling seedling emergence. Field Crops Research 67, 123–139. doi: 10.1016/S0378-4290(00)00088-5 Gramshaw D (1972) Germination of annual ryegrass seeds (Lolium rigidum Gaud.) as influenced by temperature, light, storage environment, and age. Australian Journal of Agricultural Research 23, 779–787. Gramshaw D (1976) Temperature/light interactions and the effect of seed source on germination of annual ryegrass seeds (Lolium rigidum Gaud.). Australian Journal of Agricultural Research 27, 779–786. Grundy AC (2003) Predicting weed emergence: a review of approaches and future challenges. Weed Research 43, 1–11. doi: 10.1046/J.13653180.2003.00317.X Gutterman Y (2000) Maternal effects on seeds during development. In ‘The ecology of regeneration in plant communities’. (Ed. M Fenner) pp. 59–84. (CAB International: Wallingford, UK) International Seed Testing Association (1999) International rules for seed testing. Seed Science and Technology 27(Suppl.). Linhart YB, Grant MC (1996) Evolutionary significance of local genetic differentiation in plants. Annual Review of Ecology and Systematics 27, 237–277. doi: 10.1146/ANNUREV.ECOLSYS.27. 1.237 Meyer SE, Allen PS (1999) Ecological genetics of seed germination regulation in Bromus tectorum L. Reaction norms in response to a water stress gradient imposed during seed maturation. Oecologia 120, 35–43. doi: 10.1007/S004420050830 Meyer SE, Allen PS, Beckstead J (1997) Seed germination regulation in Bromus tectorum (Poaceae) and its ecological significance. Oikos 78, 475–485.

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Paterson JG, Goodchild NA, Boyd WJR (1976) Effect of storage temperature, storage duration and germination temperature on the dormancy of seed of Avena fatua L. and Avena barbata Pott ex Link. Australian Journal of Agricultural Research 27, 373–379. Pearce GA, Quinlivan BJ (1971) The control of annual (‘‘Wimmera’’) ryegrass in cereal crops. Journal of Agriculture Western Australia 12, 58–62. Peters NCB (1982a) Production and dormancy of wild oat (Avena fatua) seed from plants grown under soil water stress. Annals of Applied Biology 100, 189–196. Peters NCB (1982b) The dormancy of wild oat seed (Avena fatua L.) from plants grown under various temperature and soil water conditions. Weed Research 22, 205–212. Steadman KJ, Bignell GP, Ellery AJ (2003a) Field assessment of thermal after-ripening time for dormancy release prediction in annual ryegrass (Lolium rigidum) seeds. Weed Research 43, 458–465. doi: 10.1046/J.0043-1737.2003.00363.X Steadman KJ, Crawford AD, Gallagher RS (2003b) Dormancy release in Lolium rigidum seeds is a function of thermal after-ripening time and seed water content. Functional Plant Biology 30, 345–352. doi: 10.1071/FP02175 Turner NC, Thompson CJ, Rawson HM (2001) Effect of temperature on germination and early growth of subterranean clover, capeweed and Wimmera ryegrass. Grass and Forage Sciences 56, 97–104. doi: 10.1046/J.1365-2494.2001.00253.X Wiesner LE, Grabe DF (1972) Effect of temperature preconditioning and cultivar on ryegrass (Lolium sp.) seed dormancy. Crop Science 12, 760–764. Wulff RD (1995) Environmental maternal effects on seed quality and germination. In ‘Seed development and germination’. (Eds J Kigel, G Galili) pp. 491–505. (Marcel Dekker: New York)

Manuscript received 6 April 2004, accepted 12 August 2004

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