Stratification in Switchgrass Seeds Is Reversed and Hastened by Drying
 
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© 2001 Crop Science Society of America
SEED PHYSIOLOGY, PRODUCTION & TECHNOLOGY
Stratification in Switchgrass Seeds Is Reversed and Hastened by Drying
a Dep. of Horticulture, Virginia Polytechnic Inst. and State Univ., Blacksburg, VA 24061
b Dep. of Crop and Soil Environmental Sciences, Virginia Polytechnic Inst. and State Univ., Blacksburg, VA 24061
* Corresponding author (dparrish@vt.edu )
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Switchgrass (Panicum virgatum L.) seed dormancy is a major obstacle to successful establishment of this multi-purpose species. We have investigated the influences of prolonged stratification, poststratification drying, restratification, and afterripening on germinability of ‘Cave-in-Rock’ switchgrass seeds. Germination can be increased many-fold to
80% with 14 d of stratification, if the seeds are moved directly to germination without drying. However, we have found germinability (but not viability) may decrease by half or more if the stratified seeds are first dried and then rehydrated for germination testing. The reappearance of dormancy (secondary dormancy) during poststratification drying is herein called reversion. During poststratification drying, dormancy reversion increased as the degree of desiccation increased. Extended stratification (for 42 d) prevented reversion. Afterripening also reduced the potential for reversion. Stratification and afterripening appeared to work additively to remove revertibility. Restratifying dried seeds showed that, while a drying interruption caused reversion, it also decreased the dormancy variability within a seedlot and shortened the total stratification time needed to obtain maximum germination compared with continuous stratification. Switchgrass seeds can be moved toward greater germinability by stratification, but drying following insufficient stratification can lead to dormancy reversion.
Abbreviations: MC, moisture content
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INTRODUCTION |
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| TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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SWITCHGRASS IS A TALL-GROWING, widely adapted, warm-season perennial with uses that include forage, soil conservation, wildlife habitat, and biofuel feedstock (Wolf and Fiske, 1995; Sanderson et al., 1996). Establishment of switchgrass, however, can be a major problem, partly because of seed dormancy. Some cultivars may have up to 95% of their seeds dormant at the time of harvest, and those seeds may need as long as 2 yr of afterripening to become germinable.
The efficacy of stratification (periods of wet, cool exposure) in breaking seed dormancy is well documented in many species (Lewak and Rudnicki, 1977; Nikolaeva, 1977; Bewley and Black, 1982; Mayer and Poljakoff-Mayber, 1989). Dormancy of switchgrass seedlots can largely be overcome by stratification (Zarnstorff et al., 1994; Wolf and Fiske, 1995). However, the stratified seeds must be dried before mechanical planting, and we have observed that drying frequently reduces germinability. This decrease in germinability is not a result of lost viability; the seeds that revert to dormant status will again become germinable if restratified. Secondary dormancy induced by drying after stratification has been reported in seeds of Malus (Kaminski, 1974), Prunus (Haut, 1932), Polygonum spp. (Justice, 1944; Staniforth and Cavers, 1979), Pyrus spp. (Westwood and Bjornstad, 1968), and Pinus taeda L. (Barnett, 1972). By contrast, partial drying of dormant (freshly harvested) or partly dormant (stratified for 1 mo) seeds of Zizania palustris L. increased subsequent germination rate and percentage (Aldridge and Probert, 1992).
The secondary dormancy induced by drying seeds following stratification is herein termed reversion to distinguish it from other types or causes of secondary dormancy. The object of this research is to study the reversion phenomenology in switchgrass to better understand survival and propagation of this important species under both natural and agricultural conditions.
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MATERIALS AND METHODS |
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Seedlots
‘Cave-in-Rock’ switchgrass seeds were used throughout this study. Seedlots 19-92 and 26-92 were purchased in February 1993, seedlot 12-93 in November 1993, and seedlot 13-94 in January 1995 from Osenbaugh Grass Seeds (Lucas, IA). Seedlots 1-94 and 1-98 were hand-collected from plots in Blacksburg, VA, in September of 1994 and 1998, respectively. Seedlot 1-92, which was highly germinable, was a composite of 1992-harvested seedlots that had been naturally after-ripened during storage for nearly 2 yr at ambient (laboratory) temperatures. All seedlots were cleaned with a seed blower (Ames Power Count Co., Brookings, SD) so that average seed weight was >1.85 mg seed-1 (540,000 seeds kg-1). All seedlots (except 1-92, which was held at ambient temperature) were stored at 5°C and
110 g kg-1 moisture content (MC) until experiments were conducted a few months after purchasing or harvesting.
Dormancy Reversion as Influenced by Extent of Poststratification Drying
The germination of seeds immediately following stratification was compared with the germination of seeds that were stratified and then partially or fully air-dried to determine if dormancy reversion was influenced by the degree of desiccation. Twenty-five grams of 13-94 seeds were placed in a cloth bag, immersed in water for 24 h, drained, sealed in a plastic bag, and stratified at 5°C for 14 d. Seeds were then spread in an open, shallow container and dried for 3 d with forced air (except during the first 6 h). The ambient drying air was 22 to 25°C with a relative humidity of 30 to 70%. Under these conditions, the seed MC decreased to 150 g kg-1 after 12 h of drying, and was around 90 g kg-1 after 72 h. Seeds were frequently stirred to ensure uniform moisture loss. Subsamples were periodically tested for MC, which was expressed on a dry-weight basis, that is, (Fresh Wt - Dry Wt)/Dry Wt, after drying at 95°C until constant weight. At each sampling time, subsamples were also tested for germination by scattering seeds on hydrated 38 by 25.5 cm germination paper towels (Anchor Paper Co., St. Paul, MN) that were rolled and placed in plastic bags. The standard germination test for switchgrass seeds (Association of Official Seed Analysts, 1993) includes 15/30°C (16-/8-h) cycles for 28 d. To eliminate the effect of stratification during the 15°C period, the seeds were germinated at a constant 30°C for 10 d in a dark incubator (Model 6, Precision Scientific, Chicago, IL). Most seeds germinated in the first 3 d, and there was very little if any additional germination increase after 10 d. Seedlings were counted as germinants when either the coleoptile or radicle was at least 5 mm long. Percentage dormancy reversion of stratified seeds was calculated as: (germination without drying – germination with drying)/germination without drying.
Dormancy Reversion as Influenced by Duration of Stratification
Eighty-gram samples of seedlots 1-92, 19-92, and 26-92 were stratified at 5°C. After 14, 28, 42, or 56 d of stratification, subsamples were removed and placed on paper towels as described above and germinated at a continuous 30°C in the dark. Other subsamples, removed after 14, 28, 42, or 56 d, were forced-air dried at ambient temperature for 3 d. The dried samples were then placed on paper towels, rehydrated, and either germinated at 30°C for 10 d or further stratified at 10°C for 14 d before germination at 30°C for 10 d.
The Effect of Interrupting Stratification with Drying
Seeds of seedlot 1-98 placed on paper towels were forced-air dried for 3 d after stratification at 5°C for 7 or 14 d. The dried seeds were then rehydrated and further stratified for up to 42 d (thus, their total stratification time was 7 or 14 d longer), and then germinated at 30°C for 10 d. The germination of seeds whose stratification was interrupted by drying at 7 or 14 d was compared with seeds that were continuously stratified.
Dormancy Reversion as Influenced by Afterripening
Subsamples of 40 g from seedlots 1-92, 12-93, 1-94, and 13-94 were equilibrated to an initial MC of 50, 75, 100, or 135 g kg-1 by either adding water or vacuum drying. These MC-adjusted seeds were then stored in sealed jars at 5, 21, or 30°C for 12 mo. Subsamples of seedlot 13-94 at each MC were also held at 45°C and sampled periodically across 12 mo. Seed MC was determined as above for each treatment at the beginning, middle, and end of storage. The MCs reported for each storage temperature were averaged across seedlots and MC sampling times.
Germination testing procedures using rolled paper towels were the same as above for seeds sampled at all times, MC, and temperature. Germination of nonstratified seeds was obtained by placing seeds in towels at 30°C for 10 d. Other seeds were stratified in towels at 5°C for 14 d, and germination recorded 10 d after transfer to 30°C. Still other seeds were stratified in towels at 5°C for 14 d, forced-air dried at ambient temperature for 3 d, and rehydrated for germination at 30°C for 10 d.
Data Analyses
Germination tests were conducted on three replicates of 100 seeds each and analyzed as a
completely randomized design. Data were not included in this paper for seeds stored at 30°C and 135 g kg-1 MC because most of them were dead after 12 mo of storage. Analyses of variance for all germination variables were performed with PROC GLM of SAS (SAS Institute, 1988). Means were separated using Tukey's Studentized Range (HSD) test (P 
0.05). The square root of germination percentage was arcsin transformed to normalize germination data. Back-transformed data (sin transformation squared) are reported. Sigmoidal curves were fitted to the cumulative germination time course for the stratification interruption study using Microcal Origin (Microcal Software, Inc., Northampton, MA). Daily germination change by stratification, that is, rate of dormancy breaking, was derived from the sigmoidal curves.
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RESULTS |
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Dormancy Reversion as Influenced by Extent of Poststratification Drying
Stratification at 5°C for 14 d raised germination of seedlot 13-94 from 3 to 65%, but a steady decrease in germinability or return of dormancy (i.e., reversion) was observed as MC of the seeds decreased during drying following stratification (Fig. 1). Full air drying to 80 g kg-1 MC decreased subsequent germination values to about half of those of nondried seeds.
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Dormancy Reversion as Influenced by Duration of Stratification
Stratification at 5°C for 14 d increased the germination of seedlots 19-92 and 26-92 from 5 to 81% and from 9 to 70%, respectively (Table 1). Drying reduced subsequent germination of stratified seeds to 45 and 33% for 19-92 and 26-92, respectively. Restratification (14 d at 10°C) increased germination to 83 and 93% for 19-92 and 26-92, respectively, indicating that drying had not decreased seed viability. Stratification for 28 d at 5°C greatly reduced or eliminated the reversion caused by poststratification drying of seedlot 19-92. Reversion was still observed for seedlot 26-92 after 28 d of stratification. Stratification for 42 or 56 d increased germination to more than 90% for both seedlots and eliminated reversion.
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Germination of seedlot 1-92, which had after-ripened for 2 yr at ambient temperature, was high even without stratification, and was increased 17% by 14 d of stratification. No reversion was observed in this seedlot.
The Effect of Interrupting Stratification with Drying
Drying decreased the germination of 1-98 seeds stratified for 7 or 14 d from 26 to 4% or from 44 to 13%, respectively (Fig. 2). However, interrupting stratification by drying increased the efficacy of subsequent stratification as seen by the more rapid change in germination percentage. By 11 or 17 d of total stratification, germination of seeds dried temporarily after a 7- or 14-d stratification, respectively, exceeded those of continuously stratified seeds. The drying treatment appeared to synchronize or homogenize the seedlot's dormancy; the standard deviations for daily changes in germination percentage were 17.3, 5.6, or 5.3 d for seeds that were continuously stratified, dried at Day 7, or dried at Day 14, respectively (Fig. 2, inset). Drying interruption decreased both the amount of and variation in stratification time required to break switchgrass seed dormancy.
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2 of 13.8 (df = 17, P = 0.99), 105 (df = 9, P = 0.31), and (df = 11, P = 0.20) for seeds continuously stratified, dried at Day 7, and dried at Day 14, respectively. The inset shows the daily germination percentage changes (derived from sigmoidal curves and of Gaussian distribution) caused by stratification.
Dormancy Reversion as Influenced by Afterripening
At the outset of this year-long experiment, stratification for 14 d increased germination of all four seedlots tested (Table 2). All seedlots except 1-92 reverted to some degree with the drying treatment. More than two-thirds of the seeds of newly harvested 1-94 and 13-94 seedlots reverted to secondary dormancy when they were stratified for 14 d and dried.
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After 12 mo of storage at 5°C and any MC, most seeds of 1-94 and 13-94 were still dormant. Stratification increased the germination of 1-94 and 13-94, but many seeds reverted to dormancy upon drying. For 1-92 and 12-93, stratification increased the germination after 12 mo of storage, and there was no reversion when seeds were dried. Reversion disappeared more slowly for the newly harvested, more-dormant 1-94 and 13-94 than for the older, less-dormant 1-92 and 12-93 seedlots. After 12 mo at 21°C, some dormancy still remained for 1-94 and 13-94, because stratification increased germination of these two seedlots. There was no reversion except for 1-94 and 13-94 stored at 50 g kg-1 MC.
No reversion occurred in seeds stored at 30°C for 12 mo (Table 2). The tendency toward reversion had disappeared even in the 1994 seedlots, which were neoteric at the outset of this experiment. Stratification still increased germination in several cases, that is, a stratifiable dormancy was still present in some seeds. Afterripening for 12 mo at 30°C did not release all seeds from primary dormancy, especially at the lowest MC.
Storage at 45°C caused much more rapid reduction in primary dormancy than did lower temperatures. In Fig. 3, the difference between curve S and 100% represents the seeds that were either dead or still dormant after 14 d at 5°C. The difference between curves S and SD represents those seeds that reverted into dormancy when stratified seeds were dried. The germination difference between curves SD and N represents those seeds that became germinable when stratified for 14 d and remained germinable after poststratification drying. The germination difference between N and 0% represents the seeds that were nondormant from the outset or that afterripened sufficiently to germinate. The time needed to lose revertibility decreased as MC increased from 48 to 104 g kg-1 (Fig. 3). The degree of reversion was also greatest at 48 g kg-1 as seen in the difference between curves S and SD after 1 mo of storage. It can further be seen, especially at 48 g kg-1 MC, that the revertibility disappeared before seeds were fully afterripened.
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DISCUSSION |
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Biologists tend to regard germination as an on/off process; accordingly, a germinable seed is by definition "on", and a dormant seed is "off", and a seed population is separated into two cohorts: dormant and germinable (nonviable seeds aside). Our data indicated that switchgrass seeds within a population have varying degrees or depths of dormancy, or "offness", because they need varying stratification and/or afterripening times to be released from dormancy. We infer, therefore, that a population of switchgrass seeds exists on a continuum of dormancy, with less dormant ones being released from dormancy more readily.
The reversion phenomenon further suggests that individual seeds in a population can be of varying degrees of germinability, because some of the stratified seeds moved back to dormancy upon drying, while others did not. This suggests that germinable seeds may have a residual dormancy, or a tendency toward secondary dormancy. The residual dormancy depends on how completely primary dormancy has been removed. Our results suggest 42 d of stratification can prevent dormancy reversion even in neoteric switchgrass seedlots. When seeds change from dormancy to nondormancy, they pass through a state known as conditional dormancy, during which they germinate only under a limited range of environmental conditions (Baskin and Baskin, 1985). The dormancy-reversible switchgrass seeds seemed to be in a conditional germinable stage in that they would become dormant again when dried. Baskin and Baskin (1985), Cohn (1996), and Hilhorst (1997) discussed a continuum between dormancy and germination. Welbaum and Bradford (1991) suggested that dormancy, germinability, and vigor in muskmelon (Cucumis melo L.) seeds are linked in a physiological continuum. The reversion phenomenon observed in our study supports the existence of a dormant-to-nondormant transitional zone between dormancy-breaking and germination processes (Cohn, 1996).
Afterripening influenced the reversion of stratified seeds, that is, the effect of afterripening and stratification was additive. Afterripening appeared first to reduce the degree or depth of primary dormancy (decreasing reversion) and then to fully overcome dormancy (germinate without stratification). Revertibility is lost more quickly under conditions that accelerate the afterripening process (Fig. 3) (Shen, 1997).
The mechanism by which dormancy reversion occurs during dehydration is not known. The degree of dormancy reversion clearly increased as seed moisture was reduced. It is perhaps significant that no definite MC threshold was observed for the germinability decrease, that is, reversion began to be evident with very little dehydration. Perhaps there was a threshold at which individual seeds reverted to dormancy, but differences in threshold values and maybe differences in drying status allowed some seeds to revert to dormancy when the MC of seeds in toto had just started to decrease. Kaminski (1974) reported that secondary dormancy in Malus can be prevented by drying stratified seeds at lower than 10°C. This approach did not work for switchgrass seeds; they reverted when desiccated at 5°C (data not shown). No loss of germinability was observed when fully afterripened switchgrass seeds were dried after imbibing at 22 or 30°C for 24 h and then germinated (data not shown). Our previous research indicated that 22°C was the upper temperature limit for switchgrass seed stratification. Reversion apparently is associated with the initial breaking of primary dormancy by stratification, and does not occur in fully stratified or well afterripened seeds.
The complex nature of dormancy in switchgrass seeds improves survivability in the wild. Switchgrass is native to the Great Plains region of the U.S., where sudden temperature and moisture fluctuations occur in the spring. The reimposition of dormancy upon drying of inadequately or marginally stratified seeds might prevent untimely germination in the early spring before temperatures are sufficiently warm. With adequate restratification, dormancy would be lost rapidly, allowing seeds to fully exploit the warm, wet conditions that favor switchgrass establishment.
Although dormancy aids survival in the wild, it hinders successful use of switchgrass as a crop. The stratification-drying-stratification cycle could be used by commercial seed companies to more rapidly break dormancy of newly harvested seeds. More research is needed to determine the most effective stratification-drying-stratification cycle for commercial use.
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ACKNOWLEDGMENTS |
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This publication is part of Zheng-Xing Shen's Ph.D. dissertation, finished in the Department of Crop and Soil Environmental Sciences, Virginia Tech. The research was supported in part by funds from the Bioenergy Feedstock Development Program of the U.S. Department of Energy under contract DE-AC05-00OR22725 with University of Tennessee-Battelle LLC.
Received for publication June 26, 2000.
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Crop Science 2001 41: 1379-1380.[Full Text]
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