TY - JOUR
AU - Steadman, Kathryn J.
AB - Abstract The influence of temperature, light environment, and seed hydration on the rate of dormancy release in Lolium rigidum (annual ryegrass) seeds during hydrated storage (stratification) was investigated. In a series of experiments, seeds were subjected to a range of temperatures (nine between 5 °C and 37 °C), light (white, red, far‐red, and dark), and hydration (4–70 g H2O 100 g–1 FW) during stratification for up to 80 d. Samples were germinated periodically at 25/15 °C or constant 15, 20, or 25 °C with a 12 h photoperiod to determine dormancy status. Dark‐stratification was an alternative, but not equivalent dormancy release mechanism to dry after‐ripening in annual ryegrass seeds. Dormancy release during dark‐stratification caused a gradual increase in sensitivity to light, but germination in darkness remained negligible. Germination, but not dormancy release, was greater under fluctuating diurnal temperatures than the respective mean temperatures delivered constantly. Dormancy release rate was a positive linear function of dark‐stratification temperature above a base temperature for dormancy release of 6.9 °C. Dormancy release at temperatures up to 30 °C could be described in terms of thermal dark‐stratification time, but the rate of dormancy release was slower at ≤15 °C (244 °Cd/probit increase in germination) than ≥20 °C (208 °Cd/probit). Stratification in red or white, but not far‐red light, inhibited dormancy release, as did insufficient (<40 g H2O 100 g–1 FW) seed hydration. The influence of dark‐stratification on dormancy status in annual ryegrass seeds is discussed in terms of a hypothetical increase in available membrane‐bound phytochrome receptors. After‐ripening, annual ryegrass, germination modelling, phytochrome, stratification, thermal time, weed, winter annual. Received 20 August 2003; Accepted 22 December 2003 Introduction Seeds of winter annuals are often dormant at maturity. A period of after‐ripening, involving exposure of dry seeds to high summer temperatures, is usually the requirement for dormancy release in winter annuals (Baskin and Baskin, 1998), and annual ryegrass (Lolium rigidum Gaud.) is no different (Steadman et al., 2003b). Recent research has revealed that otherwise strongly dormant annual ryegrass seeds can be stimulated to germinate using an alternative mechanism, circumventing the need for dry after‐ripening to release primary dormancy. Dormant seeds do not germinate when hydrated in light or dark conditions, but fully hydrated storage (hereafter termed ‘stratification’) in darkness for 2–3 weeks sensitizes seeds to light, renders them conditionally dormant, and germination occurs upon subsequent prolonged exposure to light (Steadman, 2002). Seeds of Veronica arvensis L., Papaver rhoeas L., and Viola arvensis Murr. may respond similarly, as more germinate from the soil surface following removal of a black polyethylene film cover after 7 weeks than if seeds are covered with transparent polyethylene film (Froud‐Williams et al., 1984). Dark‐stratification as a means to release primary dormancy by increasing light‐sensitivity has been applied to Matricaria inodora L. (Hartmann et al., 1997), but while short periods of dark‐stratification initially release dormancy in Arabidopsis thaliana (L.) Heynh. and Sisymbrium officinale (L.) Scop., extended treatment induces skotodormancy (Derkx and Karssen, 1993; Derkx et al., 1993). Skotodormancy is a secondary dormancy that involves the reduction in sensitivity to light in light‐requiring seeds brought about by dark‐stratification, which occurs in a number of species such as Lactuca sativa L. (Thanos and Georghiou, 1988), Sorghum halipense (L.) Pers. (Hsiao and Huang, 1988) and Hygrophila auriculara (Schumach.) Haines (Amritphale et al., 1993). Temperature is the environmental factor that has been shown to influence the level of dormancy in seeds, as it alters the range of conditions in which germination will occur, and sensitivity to germination stimulants such as light, nitrate, ethylene, and smoke (Vleeshouwers et al., 1995). While water does not appear itself to alter seed dormancy, it can modulate the effect of temperature on dormancy release (Benech‐Arnold et al., 2000; Steadman et al., 2003b). There is some evidence that other environmental factors may also modify the influence of temperature on the degree of dormancy. Low oxygen suppresses the ability of high temperatures to induce secondary dormancy in Sisymbrium officinale (Karssen, 1980/81). Light can inhibit dormancy release that occurs during conditioning (i.e. warm dark‐stratification) of Orobanche and Striga seeds, suppressing the increase in sensitivity to host‐derived germination stimulants such as strigol (Parker and Riches, 1993; Joel et al., 1995; Takeuchi et al., 1995). Predictive models for the emergence of major agricultural weeds, such as annual ryegrass, must account for dormancy release, as it exerts important control over emergence timing (Forcella et al., 2000). Thermal time theory has proved to be useful in describing the role of temperature in dormancy release through after‐ripening for annual ryegrass, based on the linear increase in rate of dormancy release above a minimum (base) temperature for dormancy release rate (Steadman et al., 2003a, b). Thermal after‐ripening time has also been used to predict dormancy release under warm, dry conditions in Bromus tectorum L. and Elymus elymoides (Raf.) Swezey (Bauer et al., 1998; Meyer et al., 2000). Similarly, dormancy release under cool, moist conditions has been described in terms of thermal stratification time in Aesculus hippocastanum L. and Polygonum aviculare L. (Pritchard et al., 1996; Batlla and Benech‐Arnold, 2003; Steadman and Pritchard, 2004) due to the linear increase in dormancy release rate as temperature reduces below a maximum (ceiling) temperature. Dormancy release during dark‐stratification is faster as temperature increases in Arabidopsis thaliana, Sisymbrium officinale, Matricaria inodora, and Orobanche spp. (Derkx and Karssen, 1993; Derkx et al., 1993; Hartmann et al., 1997; Kebreab and Murdoch, 1999). Thus, thermal time may also be useful in describing this form of dormancy release. With this in mind, the effect of dark‐stratification on dormancy status of annual ryegrass was explored. The effect of temperature on the efficacy of dark‐stratification was investigated in terms of thermal time, and the role that the light environment and seed water content played in modulating temperature‐regulation of dormancy release were examined. Materials and methods Four experiments were performed. (1) The effect of dark‐stratification on germination at a range of temperatures: seeds were stratified in darkness at 20 °C for 0–41 d and then germinated at 15, 20, 25, or 25/15 °C with a 12 h photoperiod. (2) The role of temperature during stratification on dormancy release: seeds were stratified in darkness or in a 12 h photoperiod at nine temperatures (5–37 °C) for 0–80 d and then germinated at 25/15 °C with a 12 h photoperiod. (3) The impact of light environment during stratification on dormancy release: seeds were stratified in continuous red, far‐red, darkness, or light at 25/15 °C for 0–21 d and then germinated at 25/15 °C with a 12 h photoperiod. (4) The impact of seed water content during dark‐stratification on dormancy release: seeds containing 5–70 g H2O 100 g–1 FW were stored in darkness at 20, 25, or 25/15 °C for 14 d and then germinated at 25/15 °C with a 12 h photoperiod. Plant material Mature annual ryegrass (Lolium rigidum Gaud.) spikes were collected from within a wheat crop in long‐term cropped fields from the Department of Agriculture research station at Wongan Hills, Western Australia (30°50′ S, 116°43′ E) on 24 December 1999 (seed water content 7.7±0.1 g H2O 100 g–1 FW) or 6 November 2000 (13.6±0.3 g H2O 100 g–1 FW). Spikes were threshed and florets (hereafter called seeds) separated from chaff by sieving and forced‐air separation. Seeds were equilibrated over 100% glycerol for 2 weeks and sealed inside aluminium foil bags. The 1999 collection (7.0±0.1 g H2O 100 g–1 FW) was stored at 20 °C for 5 months, and the 2000 collection (7.8±0.1 g H2O 100 g–1 FW) was stored at –20 °C until use. Measurement of seed water content Seed water content (WC) was measured in triplicate by weighing seeds, drying at 103 °C for 17 h, and reweighing (International Seed Testing Association, 1999). Results are expressed on a fresh weight (FW) basis. Dormancy release treatments Experiments 1 and 2: Seeds from the 2000 collection were stratified at full hydration (seed water content >60 g H2O 100 g–1 FW) by placing them on 6 mm deep solidified agar‐water (1% w/v) in 9 cm diameter circular Petri dishes. One dish containing 50 seeds was stratified per replicate. Dishes were placed in constant temperature incubators or rooms set at 9, 15, 20, 25, 30, or 37 °C, 12‐hourly alternating 25/15 °C or 12‐hourly alternating 30/10 °C for stratification times of up to 80 d. Light (12 h d–1) from Osram 40 W fluorescent white light tubes provided a photosynthetic photon flux of 30–45 µmol m–2 s–1 at shelf level; dishes were wrapped in aluminium foil when darkness was required. Samples were taken periodically for germination testing and measurement of seed water content. Experiment 3: Seeds from the 1999 collection were stratified on solidified agar‐water as above. Dishes were placed in a controlled environment room set at 25/15 °C in which four light environments (white, red, far‐red, and darkness) were compared. Kodak Wratten gelatin filters were used to filter light from Sylvania Clean‐ace bulbs, resulting in 20 µmol m–2 s–1 at 660 nm using filter no. 29 for the red environment (R:FR=9) and 2 µmol m–2 s–1 at 730 nm using filter no. 87 for the far‐red environment (R:FR=0.06), measured using a SKR 100 red/far‐red meter (Skye Instruments, Llandrindod Wells, UK). Dishes were removed after 4, 14, and 21 d for germination testing and measurement of seed water content. Experiment 4: Dark‐stratification was also performed for 14 d at 20, 25, or 25/15 °C using seeds (2000 collection) at water contents below 60 g H2O 100 g–1 FW. For stratification at 25 °C, seed water content was set by controlled hydration (Taylor et al., 1998). Seeds were placed into capped 5 ml scintillation vials with a specific amount of water and attached to a rotating drum for the first 24 h to allow an even distribution of water amongst the seeds. For stratification at 20 °C and 25/15 °C, seeds were placed in atmospheres of controlled relative humidity (RH), based on the moisture isotherm for annual ryegrass (Steadman et al., 2003b), using silica crystals, or saturated solutions of Mg(NO3)2 (55% RH), KCl (89% RH), BaCl2 (91% RH), and KNO3 (93% RH), or solutions of glycerol in water of 20% w/w (96% RH), 12% (98% RH), 6% (99% RH), and 0% (100% RH) (Sun, 2002). Vials or chambers containing seeds were wrapped in foil to provide darkness, and placed at the relevant temperature for 14 d. At the end of this period, seeds were removed for germination and water content measurement. Germination and viability tests Once seeds had dark‐stratified for the required amount of time, any aluminium foil wrapping was removed and dishes were placed inside clear plastic bags in constant temperature rooms set at 15, 20, or 25 °C, or in an incubator set at 12‐hourly alternating 25 °C and 15 °C. Osram 40 W fluorescent white light tubes provided 30–45 µmol m–2 s–1 at shelf level for 12 h d–1. The criterion for germination was visible radicle protrusion. The number of germinated seeds was counted regularly throughout the test up to a total stratification/germination time of 94 d. Inviability of soft, ungerminated seeds was confirmed by cutting through the seed. Samples of firm, ungerminated seeds were assessed for viability by slicing longitudinally to expose the endosperm and incubating in 1% 2,3,5‐triphenyltetrazolium chloride solution for 24 h in the dark at 30 °C (International Seed Testing Association, 1999). The extent of pink staining was observed through a microscope, and complete staining of the embryo and aleurone was required to score a seed as viable. Germination is expressed as a percentage of the total seeds in the test. Statistical analysis Thermal dark‐stratification time (θ °Cd) above the base temperature for dormancy release (Tb) accumulated by seeds was calculated by multiplying the thermal units above Tb accumulated per day (TDS–Tb) by the time to the sampling date (t) according to equation (1), where the dark‐stratification temperature (TDS) was the average temperature experienced by the seeds measured periodically by a TTec datalogger (Temperature Technology, South Australia). θ(TDS–Tb)t(1) The package Genstat version 6 was used for statistical analysis (Genstat 5 Committee, 1993). For comparison of dormancy release rate (DRR) between treatments, linear regression lines (equation (2)) through germination percentage (G) on a probit scale versus thermal dark‐stratification time (θ), where G0 is the proportion of seeds that germinate at the start of the experiment, were compared using generalized linear modelling with a probit link function (Steadman et al., 2003b). probit(G)=(θ×DRR) + probit (G0)(2) Data were excluded where dormancy release reached a maximum and no further increase in germination was measured. Multiple regression lines were constrained to have the same slope and/or intercept and analysed for a statistically significant increase in deviance using the F‐distribution. Results The seedlot used for most of the experiments (2000 collection) was highly dormant at the start of the experiments; germination was low and dependent on light and temperature. When a 12 h photoperiod was provided to unstratified seeds, a small proportion (20%) of the population germinated at 25/15 °C (Fig. 1A), but <5% germinated at constant 15, 20, or 25 °C (Fig. 1B, C, D). Less than 2% of seeds germinated during hydrated storage in the dark (dark‐stratification), but following a short period of dark‐stratification at 20 °C a greater proportion of the seeds germinated when subsequently exposed to a 12 h photoperiod. The germinable fraction increased as dark‐stratification time extended, with ≥30 d dark‐stratification at 20 °C eliciting over 80% germination when tested at 25/15 °C (Fig. 1A). This period of dark‐stratification also improved germination at constant temperatures (Fig. 1B, C, D), but these remained sub‐optimal for germination compared with alternating 25/15 °C (Fig. 1A). While germination was clearly better when fluctuating temperatures were provided (Fig. 1), fluctuations in temperature per se during the dark‐stratification phase did not increase dormancy release. Germination progress curves following dark‐stratification at 20 °C (Fig. 2D) and 25/15 °C (Fig. 2I) were similar, and dark‐stratification at 30/10 °C, with the same average temperature, was less effective (Fig. 2H). Dark‐stratification was least effective when performed at cold temperatures, with seeds dark‐stratified at 5 °C (Fig. 2A) showing similar germinability to seeds that had no dark‐stratification treatment (Fig. 1A). As the temperature of dark‐stratification was raised, dormancy release increased. Seeds lost dormancy fastest during dark‐stratification at 30 °C and 4 weeks enabled almost every seed to germinate when subsequently placed in a 12 h photoperiod at 25/15 °C (Fig. 2F). The higher temperature (37 °C) was supra‐optimal for dark‐stratification, with some improvement in germination following dark‐stratification for up to 11 d, but germination reduced thereafter (Fig. 2G). Seed viability was high following dark‐stratification in all treatments except those at 37 °C for 6, 11, 16, and 22 d during which 3, 7, 23, and 61% of seeds died, respectively. Stratification of seeds in light was much less effective than stratification in darkness. A small improvement in germination was observed following light‐stratification at some of the warmer temperatures prior to germination at 25/15 °C (Fig. 3). However, the maximum improvement in germination was 20%, elicited by 27 d light‐stratification at 30 °C (Fig. 3F); the equivalent treatment involving dark‐stratification produced 98% germination (Fig. 2F). Only in seeds light‐stratified at 37 °C for 11 d and 22 d was the lack of germination caused by viability loss, for which 12% and 51%, respectively, died during the treatment. In a separate experiment using seeds collected from the same site but in a different year (1999), darkness was also substantially better than white light for promoting dormancy release during stratification (Fig. 4A, B). Moreover, far‐red light during stratification promoted dormancy release to a similar extent as darkness, but red was similar to white light in inhibiting dormancy release (Fig. 4). The major change in germination response as dark‐stratification time extended was the increase in the proportion of the population responding, rather than any alteration in the rate of germination (Fig. 2). The rate of dormancy release, in terms of the increase in the proportion of the population responding, was faster when dark‐stratification took place at warmer rather than cooler temperatures (Fig. 5). Dark‐stratification at 20 °C produced the same rate of dormancy release as 25/15 °C, but seeds dark‐stratified at 30/10 °C behaved differently even though they experienced the same average temperature, initially losing dormancy at a similar rate but reaching a maximum final germination of 55%. Likewise, 59 d dark‐stratification had the same effect as 41 d in seeds at 10 °C (Fig. 5). Linear regression lines were fitted to the final percentage germination measured 94 d after the start of imbibition on a probit scale, excluding data for which no further reduction in dormancy was observed following dark‐stratification at 10 °C or 30/10 °C. The slope of the linear regression lines increased linearly with dark‐stratification temperature up to 30 °C (Fig. 6), and the point at which the line intersected with the temperature axis gave the base temperature (Tb) for dormancy release as 6.9 °C (Fig. 6). Using Tb, the thermal time accumulated during the dark‐stratification phase of each treatment was calculated. Dormancy release in response to thermal dark‐stratification time was dependent on treatment temperature. The increase in germinable fraction on a thermal dark‐stratification time‐scale separated into two distinct groups according to treatment temperature (Fig. 7). Seeds stratified at the cooler temperatures of 10, 15, and 30/10 °C lost dormancy at the same rate as each other (Fig. 7A; P=0.056), and seeds stratified at the warmer temperatures of 20, 25, 30, and 25/15 °C lost dormancy at the same rate as each other (Fig. 7B; P=0.071), but dormancy release at cooler temperatures was significantly slower than at warmer temperatures (P <0.001). Seeds stratified at 5 °C did not change in their dormancy status because this temperature was below Tb and so no thermal time was accumulated. The relationship between dormancy release and thermal time for seeds stratified at 37 °C was confounded by accelerated seed ageing and death, and did not fit with the other treatments (Fig. 7A), even when germination was calculated as a percentage of viable seeds (data not shown). Annual ryegrass seeds must contain enough water for a change in dormancy status to take place during storage in darkness. As seed water content reduced from 45% FW to 35% FW, the proportion of the population able to respond to dark‐stratification reduced dramatically, and seeds containing less than 30% water showed no change in germinability following 14 d in darkness compared with unstratified seeds (Fig. 8). Discussion The reduction in dormancy that occurs in annual ryegrass seeds during dark‐stratification was temperature dependent. There was a linear increase in rate of dormancy release as temperature increased above a base temperature, which in the 2000 cohort was 6.9 °C. This simple relationship between rate of dormancy release and temperature allows storage time and temperature to be combined to form a single variable, thermal time. Each seed in the population will have its own specific thermal dark‐stratification time requirement in order to become responsive to light. Membranes are believed to be the primary site of temperature perception and ‘memory’, and thus the site at which temperature acts to control dormancy and germination in seeds. Changes are hypothesized to occur through the alteration of phospholipid headgroup spacing, which influences binding of peripheral proteins and thereby controls signal transduction pathways (Hilhorst, 1998; Hallett and Bewley, 2002). Temperature can alter phospholipid headgroup spacing directly, with a rise in temperature resulting in an increase in membrane fluidity and cross‐sectional area, with phase transitions occurring at key temperatures. This change in membrane fluidity, along with inherent temperature–activity relationships of enzymes, also allows temperature to control adjustments to membrane lipid composition. A gradual alteration in the level of fatty acid desaturation occurs during temperature acclimation in many organisms (Nishida and Murata, 1996), and may explain the ability of seeds to ‘remember’ their thermal history and accumulate thermal units towards dormancy release or induction (Hilhorst, 1998; Hallett and Bewley, 2002). Differences in membrane fluidity may be responsible for different dormancy release rates in annual ryegrass seeds dark‐stratified at warm (≥20 °C) and cool (≤15 °C) temperatures (Fig. 7). This range (15–20 °C) appears to be critical to dormancy in a range of species under hydrated conditions as, for example, over 17 °C is required for the induction of skotodormancy in Lactucasativa (Thanos and Georghiou, 1988) and dormancy release only takes place below approximately 15–17 °C in Rumex spp. (Totterdell and Roberts, 1979), Aesculushippocastanum (Steadman and Pritchard, 2004), Polygonum aviculare (Batlla and Benech‐Arnold, 2003) and a variety of deciduous fruit tree species (Seeley and Damavandy, 1985). In the specific case of seeds that require light for germination, such as Arabidopsis thaliana and Sisymbrium officinale, an hypothesis has been developed to describe dormancy relief and induction in terms of temperature‐dependent changes in membrane fluidity controlling the availability of an unknown membrane‐bound phytochrome receptor (X) (Hilhorst, 1998). According to the hypothesis, the temperature and time‐dependent synthesis of X is the relief of dormancy, and subsequent temperature‐dependent changes in the receptor between available (Xa) and unavailable/inactive (Xi) forms are responsible for the temperature range over which germination is possible. Available receptors (Xa) are able to bind phytochrome (Pfr), formed on exposure to red light, resulting in an active phytochrome–receptor complex (Xp) which, when enough is present, can trigger germination (Hilhorst, 1998; Vleeshouwers and Bouwmeester, 2001). The data presented here for annual ryegrass are consistent with this hypothesis, as the proportion of seeds responding to germination stimulation by light increases in a temperature and time‐dependent manner (Fig. 7). Furthermore, it allows the extension of the hypothesis, because dormancy release in annual ryegrass (i.e. formation of Xa) proceeds at a substantially higher rate in darkness than in light (Figs 2, 4). Far‐red is similar to darkness for promoting dormancy release during stratification, and red light is equivalent to white light in retarding dormancy release (Fig. 4), suggesting inhibition by phytochrome. During dark‐stratification, when any Pfr reverts to the inactive Pr and no new Pfr is produced, levels of Xa build up to a threshold level that enables germination to be stimulated when light is eventually provided. During stratification in light, Xa is formed at the same temperature‐dependent rate as in darkness, but the threshold level is not reached because of inhibition by phytochrome. In this case Pr is converted to Pfr, which binds to any Xa as it is synthesized to form Xp, and Xp has a limited lifetime before it degrades and is no longer capable of stimulating germination. Accordingly, the fact that dormancy release occurred in 20% of seeds light‐stratified at 30 °C (Fig. 3F) may be explained by the rate of formation of Xa at this warm temperature exceeding the rate of degradation of Xp, allowing the threshold level of Xp to be reached in 20% of the population. Dark‐stratification promotes a fast alternative, but not equivalent, dormancy release mechanism to dry after‐ripening in annual ryegrass seeds. Dormancy release during dark‐stratification involves an increase in sensitivity to light, resulting in conditionally dormant seeds that can be stimulated to germinate by subsequent exposure to light. However, seeds remain unable to germinate in darkness, so dark‐stratification does not result in complete removal of dormancy. By contrast, seeds that undergo dry after‐ripening for dormancy release slowly become able to germinate both with and without a light stimulus (Steadman et al., 2003b). While dark‐stratification and dry after‐ripening are similar in the way that temperature controls dormancy release rate, with both mechanisms being dependent on thermal time, seed water content ultimately controls whether darkness will promote rapid dormancy release. Dark‐stratification requires a high level of hydration for dormancy release to occur, the critical level of approximately 40 g H2O 100 g–1 FW corresponding to around –3 MPa. Thus, this mechanism can only occur in seeds that contain water at hydration levels 4 and 5 (Vertucci and Farrant, 1995), in line with the theory that integrated enzymic processes and protein synthesis are required to form a hypothetical phytochrome receptor (Xa) for dormancy release during dark‐stratification. Dormancy release of seeds stratified in darkness at 10 °C and 30/10 °C reached a maximum after 40 d and 20 d, respectively, and no further improvement in germination was gained by extending stratification time (Fig. 5). This indicates that while c. 50% of the population slowly accumulated thermal time towards dormancy release under these conditions, the rest of the population were not able to. A possible explanation for this may be that Tb for dormancy release was not identical for each seed, but a distribution in Tb existed amongst the population. If for some seeds Tb was ≥10 °C, thermal time would not accumulate during stratification at 10 °C. Alternatively, as a winter annual, seeds of annual ryegrass may be expected to enter secondary dormancy in response to hydrated storage at cold temperatures (Baskin and Baskin, 1998). Many species exhibit cycles in dormancy status, with dormancy relief occurring during the season preceding that most suitable for sustaining plant growth, and dormancy induction occurring just prior to the period during which conditions are likely to prohibit survival. Thus the limited dormancy release in treatments involving stratification at 10 °C may result from the interaction between dormancy relief and induction. In conclusion, it has been established that dormancy in annual ryegrass seeds can be relieved in hydrated seeds by stratification in darkness as well as after‐ripening in dry seeds. Both mechanisms show similar relationships between rate of dormancy release and temperature that allow the use of thermal time theory to simplify their measurement and prediction. Dormancy release during after‐ripening of annual ryegrass under field temperatures can be predicted using thermal time (Steadman et al., 2003a,b). Dormancy release during dark‐stratification is more complex, as the dependence of dormancy release on storage temperature leads to different responses in seeds at warm (≥20 °C) and cool (≤15 °C) temperatures in terms of thermal time. Furthermore, stratification must be completed under conditions that exclude red light, such as burial or under a complete canopy. Clearly these constraints require further investigation before dormancy status in annual ryegrass seeds under field conditions can be accurately modelled. Acknowledgements This research was funded by the Grains Research and Development Corporation of Australia. Thanks to the Department of Agriculture, Western Australia for access to the seed collection site and Julie A Plummer (University of Western Australia) for helpful discussions regarding the manuscript. View largeDownload slide Fig. 1. Role of germination temperature on the response of dormant annual ryegrass seeds to dark‐stratification (DS). Seeds were stratified on dishes containing solidified agar‐water in complete darkness at 20 °C for 0, 10, 20, 30, and 41 d. Dishes were then moved to 25/15 °C (A), 25 °C (B), 20 °C (C), or 15 °C (D) and exposed to a 12 h photoperiod until the end of the experiment at 94 d. Less than 2% of seeds germinated during dark‐stratification; the first symbol on the x‐axis for each curve indicates the point at which exposure to light began. Germination of seeds that received no period of dark‐stratification is indicated by the open symbols. Bars are ±1 se of the mean of three replicates. View largeDownload slide Fig. 1. Role of germination temperature on the response of dormant annual ryegrass seeds to dark‐stratification (DS). Seeds were stratified on dishes containing solidified agar‐water in complete darkness at 20 °C for 0, 10, 20, 30, and 41 d. Dishes were then moved to 25/15 °C (A), 25 °C (B), 20 °C (C), or 15 °C (D) and exposed to a 12 h photoperiod until the end of the experiment at 94 d. Less than 2% of seeds germinated during dark‐stratification; the first symbol on the x‐axis for each curve indicates the point at which exposure to light began. Germination of seeds that received no period of dark‐stratification is indicated by the open symbols. Bars are ±1 se of the mean of three replicates. View largeDownload slide Fig. 2. Role of dark‐stratification (DS) temperature and duration on dormancy release in annual ryegrass seeds. Seeds were stratified on agar‐water in complete darkness at constant 5 °C (A), 10 °C (B), 15 °C (C), 20 °C (D), 25 °C (E), 30 °C (F), 37 °C (G), 12 hourly alternating 30/10 °C (H), or 25/15 °C (I). Following incubation for between 6 d and 41 d, dishes were moved to a 12 h photoperiod at 25/15 °C until the end of the experiment at 94 d. Less than 2% of seeds germinated during dark‐stratification; the first symbol on the x‐axis for each curve indicates the point at which exposure to light began. Bars are ±1 se of the mean. For comparison, germination of seeds that received no dark‐stratification period is indicated by the open symbols in Fig. 1A. View largeDownload slide Fig. 2. Role of dark‐stratification (DS) temperature and duration on dormancy release in annual ryegrass seeds. Seeds were stratified on agar‐water in complete darkness at constant 5 °C (A), 10 °C (B), 15 °C (C), 20 °C (D), 25 °C (E), 30 °C (F), 37 °C (G), 12 hourly alternating 30/10 °C (H), or 25/15 °C (I). Following incubation for between 6 d and 41 d, dishes were moved to a 12 h photoperiod at 25/15 °C until the end of the experiment at 94 d. Less than 2% of seeds germinated during dark‐stratification; the first symbol on the x‐axis for each curve indicates the point at which exposure to light began. Bars are ±1 se of the mean. For comparison, germination of seeds that received no dark‐stratification period is indicated by the open symbols in Fig. 1A. View largeDownload slide Fig. 3. Germination progress curves for imbibed annual ryegrass seeds during light‐stratification (LS) in nine different temperature regimes. Seeds were placed on agar‐water at constant 5 °C (A), 10 °C (B), 15 °C (C), 20 °C (D), 25 °C (E), 30 °C (F), 37 °C (G), 12 hourly alternating 30/10 °C (H), or 25/15 °C (I). On two occasions between 11 d and 80 d, dishes were moved to 25/15 °C, where they remained until the end of the experiment at 94 d. Seeds were exposed to a 12 h photoperiod throughout stratification and subsequent germination testing. Bars are ±1 se of the mean. View largeDownload slide Fig. 3. Germination progress curves for imbibed annual ryegrass seeds during light‐stratification (LS) in nine different temperature regimes. Seeds were placed on agar‐water at constant 5 °C (A), 10 °C (B), 15 °C (C), 20 °C (D), 25 °C (E), 30 °C (F), 37 °C (G), 12 hourly alternating 30/10 °C (H), or 25/15 °C (I). On two occasions between 11 d and 80 d, dishes were moved to 25/15 °C, where they remained until the end of the experiment at 94 d. Seeds were exposed to a 12 h photoperiod throughout stratification and subsequent germination testing. Bars are ±1 se of the mean. View largeDownload slide Fig. 4. Role of light quality during stratification on dormancy release in annual ryegrass seeds. Seeds were stratified on agar‐water in continuous darkness (A), white (B), far‐red (C), or red (D) light at 25/15 °C. After 4, 14, or 21 d dishes were moved to a germination test at 25/15 °C with a 12 h photoperiod of white light. Up to 5% of seeds germinated during stratification in darkness or far‐red, and up to 25% in white or red light; the first symbol on the x‐axis for each curve indicates the point at which the germination test began. Germination of seeds that received no period of stratification is indicated by the open symbols. Bars are ±1 se of the mean. View largeDownload slide Fig. 4. Role of light quality during stratification on dormancy release in annual ryegrass seeds. Seeds were stratified on agar‐water in continuous darkness (A), white (B), far‐red (C), or red (D) light at 25/15 °C. After 4, 14, or 21 d dishes were moved to a germination test at 25/15 °C with a 12 h photoperiod of white light. Up to 5% of seeds germinated during stratification in darkness or far‐red, and up to 25% in white or red light; the first symbol on the x‐axis for each curve indicates the point at which the germination test began. Germination of seeds that received no period of stratification is indicated by the open symbols. Bars are ±1 se of the mean. View largeDownload slide Fig. 5. Relationship between dark‐stratification (DS) temperature and the proportion of seeds subsequently germinating at 25/15 °C. Seeds were stratified on agar‐water in complete darkness for up to 80 d at constant 5, 10, 15, 20, 25, 30, 37 °C, or 12 h alternating 30/10 °C or 25/15 °C. Dishes were then moved to 25/15 °C with a 12 h photoperiod until final germination was measured at 94 d. The data are shown with logistic curves fitted to all except DS at 5 °C and 37 °C. Bars are ±1 se of the mean. View largeDownload slide Fig. 5. Relationship between dark‐stratification (DS) temperature and the proportion of seeds subsequently germinating at 25/15 °C. Seeds were stratified on agar‐water in complete darkness for up to 80 d at constant 5, 10, 15, 20, 25, 30, 37 °C, or 12 h alternating 30/10 °C or 25/15 °C. Dishes were then moved to 25/15 °C with a 12 h photoperiod until final germination was measured at 94 d. The data are shown with logistic curves fitted to all except DS at 5 °C and 37 °C. Bars are ±1 se of the mean. View largeDownload slide Fig. 6. Relationship between rate of dormancy release (probits per day) and temperature of dark‐stratification. The linear regression line, plotted through the temperatures between 5 °C and 30 °C (filled symbols), intercepts the x‐axis at the base temperature for dormancy release (Tb) of 6.9 °C. Dormancy release at 37 °C (open symbol) was excluded from regression analysis. Bars are ±1 se of the mean. View largeDownload slide Fig. 6. Relationship between rate of dormancy release (probits per day) and temperature of dark‐stratification. The linear regression line, plotted through the temperatures between 5 °C and 30 °C (filled symbols), intercepts the x‐axis at the base temperature for dormancy release (Tb) of 6.9 °C. Dormancy release at 37 °C (open symbol) was excluded from regression analysis. Bars are ±1 se of the mean. View largeDownload slide Fig. 7. Influence of thermal dark‐stratification time (°Cd) on final germination (on a probability % or probit scale) attained at 25/15 °C. Seeds were stratified on agar‐water in complete darkness (DS) for up to 80 d at 10 °C, 15 °C, 30/10 °C, and 37 °C (A) or 20 °C, 25 °C, 30 °C, and 25/15 °C (B) before being exposed to 25/15 °C with a 12 h photoperiod. Final germination (G) was counted at the end of the experiment at 94 d. Regression lines were not significantly different for seeds that were dark‐stratified at 10 °C, 15 °C, and 30/10 °C (P=0.056) and 20 °C, 25 °C, 30 °C, and 25/15 °C (P=0.071), but the two groups were significantly different from each other (P<0.001). Thus two regression lines of the form probit(G)=DRR–0.7465 are shown, for which DRR=0.41 and 0.48 probits per 100 °Cd for the solid lines in (A) and (B), respectively. The dashed line in (A) shows the regression line for the data in (B), and the dashed line in (B) shows the regression line for the data in (A). Data for dormancy release at 5 °C (data not shown) and 37 °C, and where no further increase in germination was observed (10 °C and 30/10 °C, open symbols), were excluded from linear regression analysis. Bars are ±1 se of the mean. View largeDownload slide Fig. 7. Influence of thermal dark‐stratification time (°Cd) on final germination (on a probability % or probit scale) attained at 25/15 °C. Seeds were stratified on agar‐water in complete darkness (DS) for up to 80 d at 10 °C, 15 °C, 30/10 °C, and 37 °C (A) or 20 °C, 25 °C, 30 °C, and 25/15 °C (B) before being exposed to 25/15 °C with a 12 h photoperiod. Final germination (G) was counted at the end of the experiment at 94 d. Regression lines were not significantly different for seeds that were dark‐stratified at 10 °C, 15 °C, and 30/10 °C (P=0.056) and 20 °C, 25 °C, 30 °C, and 25/15 °C (P=0.071), but the two groups were significantly different from each other (P<0.001). Thus two regression lines of the form probit(G)=DRR–0.7465 are shown, for which DRR=0.41 and 0.48 probits per 100 °Cd for the solid lines in (A) and (B), respectively. The dashed line in (A) shows the regression line for the data in (B), and the dashed line in (B) shows the regression line for the data in (A). Data for dormancy release at 5 °C (data not shown) and 37 °C, and where no further increase in germination was observed (10 °C and 30/10 °C, open symbols), were excluded from linear regression analysis. Bars are ±1 se of the mean. View largeDownload slide Fig. 8. Influence of water content on the response of annual ryegrass seeds to dark‐stratification. Seeds at various levels of hydration were stored in darkness at 20 °C, 25 °C, or 25/15 °C for 14 d. Seeds were then placed on solidified 1% agar‐water at 25/15 °C with a 12 h photoperiod for 14 d to germinate. To allow results for dark‐stratification at three different temperatures to be plotted together, the % germination scale is normalized with respect to % germination of unstratified seeds at the lower end of the scale, and % germination of 14 d dark‐stratified fully hydrated seeds at the upper end of the scale. A logistic curve is fitted to the data: y=100/(1+e–(x–38.5)/4.9), r2=0.945. View largeDownload slide Fig. 8. Influence of water content on the response of annual ryegrass seeds to dark‐stratification. Seeds at various levels of hydration were stored in darkness at 20 °C, 25 °C, or 25/15 °C for 14 d. Seeds were then placed on solidified 1% agar‐water at 25/15 °C with a 12 h photoperiod for 14 d to germinate. 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TI - Dormancy release during hydrated storage in Lolium rigidum seeds is dependent on temperature, light quality, and hydration status
JF - Journal of Experimental Botany
DO - 10.1093/jxb/erh099
DA - 2004-04-01
UR - https://www.deepdyve.com/lp/oxford-university-press/dormancy-release-during-hydrated-storage-in-lolium-rigidum-seeds-is-qP9doQn1Oh
SP - 929
EP - 937
VL - 55
IS - 398
DP - DeepDyve
ER -