Published in Crop Sci. 44:549-552 (2004).
© 2004 Crop Science Society of America
677 S. Segoe Rd., Madison, WI 53711 USA
SEED PHYSIOLOGY, PRODUCTION & TECHNOLOGY
Enhancing Germination of Eastern Gamagrass Seed with Stratification and Gibberellic Acid
Carla Rogis,
Lance R. Gibson*,
Allen D. Knapp and
Robert Horton
Dep. of Agronomy, Iowa State Univ., Ames, IA 50011
* Corresponding author (lgibson{at}iastate.edu).
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ABSTRACT
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Eastern gamagrass (Tripsacum dactyloides, L.), a warm season, perennial grass with great potential for forage and conservation uses, has a high level of seed dormancy, making establishment difficult. Stratification at 4°C for 6 wk is a standard method for providing germinable gamagrass seed. Earlier research showed that gibberellic acid (GA3) increased germinability of gamagrass caryopses removed from the cupule, but was less effective when the caryopses remained in the cupule. We hypothesized that gibberellic acid together with stratification may increase germination of seed above levels obtained by stratification or gibberellic acid alone. This study assessed the germination of three seed lots of eastern gamagrass to 1 mM GA3 and exposure to 4°C for 0 to 7 wk. Seed soaked in GA3 solution averaged 43% germination during the first 3 wk of stratification and was significantly higher than the germination of water soaked seed averaging 35% germination. Seed reached peak germinability after 4 wk of stratification and remained at this level during the final weeks. After 4 wk of stratification, germination levels of GA3 and water treated seed were similar at 64 to 70%. The most pronounced effect of GA3 was more rapid germination of seed in all of the stratification durations tested.
Abbreviations: GA, gibberellic acid
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INTRODUCTION
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EASTERN GAMAGRAS is a warm-season, perennial grass species found from the northeastern and north central USA and south into Mexico, Central America, the Caribbean, and into Bolivia and Paraguay in South America (Newell and De Wet, 1974). High productivity (Fine et al., 1990) and quality (Horner et al., 1985; Burns et al., 1996) relative to other warm-season grasses make it attractive as a forage crop. Plant characteristics of high leaf proportion and low dead tissue ratio can lead to high daily gains for animals grazing eastern gamagrass (Burns et al., 1992). Gamagrass also has high potential for use in conservation practices. Its natural drought and wetness tolerance, perennial nature, and growth from a strong crown, make it a good species for grass hedges used to control water runoff and soil erosion from row-crop fields (Dewald et al., 1996). It can tolerate herbicides used on the adjacent crops, shading from cultivated crops, and climatic extremes.
Despite its potential, problems with stand establishment have limited the use of eastern gamagrass. Seed dormancy is very strong in this species and less than 10% of newly harvested seed typically germinate (Ahring and Frank, 1968; Tian et al., 2002). Ahring and Frank (1968) reported that cold, wet stratification of seed for 6 wk increased germination to 58 to 66%. Anderson (1985) also noted that a cold, moist period of 60 d in 4°C enhanced eastern gamagrass seed germination. It is unclear from these reports if dormant seed remained at the end of the stratification period.
Gibberellin plant hormones have also been shown to overcome seed dormancy in eastern gamagrass. The seed dispersal unit for eastern gamagrass is a caryopsis surrounded by the lemma, palea, and a hardened fruit case made from the glumes and rachis (Hitchcock and Chase, 1950). This hardened fruit case is referred to as a cupule (Galinat and Craighead, 1964; Anderson, 1985). Low molar solutions of gibberellic acid increased the germination of gamagrass caryopses removed from the cupules, but had only small effects on dormancy when caryopses remained in the cupules (Anderson, 1985; Tian et al., 2003). Removal of the cupule and application of GA3 increased germination to within a few percentage points of the total amount of viable seed (Tian et al., 2003). Germination was 34 percentage points more with cupule removal and GA3 application, than with cupule removal and no GA3 application (Tian et al., 2003).
Because cupule removal is labor intensive and risks the integrity of the caryopsis, it is necessary to find methods of increasing germination of intact seed. In the following study, we tested whether combinations of known germination promoters, stratification and GA3, could more completely break seed dormancy and promote seed germination in eastern gamagrass than the individual application of these promoters.
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MATERIALS AND METHODS
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Two seed lots of ‘Iuka’ eastern gamagrass produced in 1998 and 2000 and one lot of ‘Pete’ produced in 2000 were used for this study. Seed lots were received from Gamagrass Seed Company, Falls City, NE, in November of their year of production and stored at 4°C and 40% relative humidity until needed. Seed lots were immediately returned to cold storage after being sampled for experimental use.
Seed were imbibed for 24 h with either 1 mM gibberellic acid (GA3) solution or deionized water. Two sheets of blue blotter paper (Anchor Paper Co., St. Paul, MN) were placed in 12.07- by 12.07-cm plastic boxes and saturated with 27 mL of GA3 solution or deionized water. After the liquid had been applied to the blotter paper, 9 g of seed were placed in the boxes and the lids were sealed. The boxes were maintained at 25°C for 24 h before being placed in cold temperatures.
After the 24-h imbibition period, the seed were placed in cold temperatures for a 1- to 7-wk stratification period. The plastic boxes with blotter paper, liquid, and seed were placed in the cold temperature room at 4°C and 40% relative humidity. One set of seed was randomly selected for a 0-wk stratification treatment and germinated immediately after the imbibition period.
A box for each imbibition treatment was removed from the cold room at weekly periods to test the effect of stratification period length on germination. New plastic boxes were prepared with two sheets of blue blotter paper and saturated with 24 mL of deionized water. Fifty seed with intact cupules were randomly selected from each imbibition treatment and placed in a box for germination testing. Fifty seed of dry (unimbibed) seed were randomly sampled from the original lots for germination testing as well.
The germination tests were performed at 30/20°C alternating temperature (Ahring and Frank, 1968) with light (four 40W cool-white fluorescent lights vertically oriented on each the left and right sides of the germinator) and 30°C for 8 h daily. Darkness and 20°C temperature were combined for the other 16 h of a daily cycle. Germination counts were made every 7 d for 28 d. Seed were considered germinated if the coleoptile exceeded the seed in length and the seedling was normal according to the seedling evaluation criteria of AOSA for comparable grasses (AOSA, 1992). Normal seedlings were removed as they were counted. Water was added to each germination box as needed to maintain optimum moisture levels. After 28 d of incubation, abnormal seedlings were counted and ungerminated seed were examined by tetrazolim (TZ, 2,3,5-triphenyl tetrazolium chloride) tests (AOSA, 1998) and classified as dormant or dead. Not all cupules contained a caryopsis, so germination was calculated as the percentage of normal seedlings produced from the total number of caryopsis containing cupules in each germination box.
Statistical Analysis
The study was conducted as a randomized factorial design with three replications. The experimental unit was 9 g of seed that were imbibed with gibberellic acid or deionized water and placed in cold temperature from 0 to 7 wk. Dry seed was germinated each week and used as a control. The Proc GLM procedure of SAS (SAS Institute, Cary, NC) was used to analyze final germination (Day 28 of the germination period), abnormal seedlings, dormant seed, and dead seed. The Proc Mixed procedure of SAS was used with an autoregressive covariance structure to analyze the repeated measurements of germination over the 28-d germination period. Significance of main effects and interactions of GA treatment, seed lot, duration of stratification, and germination time were determined by an F test. Tukey's test (Steel and Torrie, 1980a) was used to make mean comparisons of main effects. Statistically significant interactions were subjected to further ANOVA by the slice command of Proc GLM. Germination for each week of the stratification period was compared to germination at 7 wk of stratification with Dunnett's procedure (Steel and Torrie, 1980b) to determine the amount of stratification required for peak germination. The significance level for all comparisons was P < 0.05.
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RESULTS
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Total Seed Germination
The main effects of GA treatment, seed lot, and duration of stratification influenced the amount of seed germination at the conclusion of the 28-d germination period. There was a significant interaction between GA treatment and duration of stratification, which was caused by the inclusion of the dry control, which had no change in final germination percentages depending on weeks of exposure (Fig. 1)
. Because the comparison of interest was between the GA3 and water treatments, germination data of the dry seed were removed and the data set was reanalyzed. The week x treatment interaction was no longer present in this analysis and the main effects of GA treatment and duration of stratification could be more clearly interpreted. The Iuka 2000 seed lot had 3 percentage points less germination than the Iuka 1998 and Pete 2000 seed lots when analyzed with and without the dry seed. However, this difference was only significant when the dry seed were included in the analysis.
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Fig. 1. Germination of eastern gamagrass seed after treatment with water or GA3 and stratification for 0 to 7 wk. Average of three seed lots—Iuka produced in 1998 and 2000 and Pete produced in 2000. Counts were taken on Days 7, 14, 21, and 28 of the germination-testing period.
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Germination of both GA3 and water treated seed peaked at 4 wk of exposure to stratification and was maintained at these levels through the remaining 4 wk of stratification. Total germinations increased for both GA3 and water treated seed with each week of additional stratification from Week 0 until Week 4. The final germination ranged from 64 to 70% with 4 to 7 wk of stratification, compared to 8 to 16% for dry seed.
Analysis of 0 through 3 wk of exposure to stratification revealed significant differences in final germination due to GA treatment, lot, and duration of stratification. GA3 treated seed averaged 8 percentage points higher final germination than water-treated seed during this period (Fig. 1). The differences among lots occurred because the final germination counts of Iuka seed produced in 2000 were 7 percentage points less than the germination of Iuka produced in 1998 and Pete seed produced in 2000. Differences in GA treatment, lot and duration of stratification did not occur for Weeks 4 through 7 of the stratification treatments.
Germination Rate
Analysis of variance was performed on the data set that included the normal seedling counts made every 7 d of the germination period to determine if there were differences in rate at which seed germinated. GA treatment x germination time and duration of stratification x germination time interactions occurred when this analysis was performed both with and without dry seed. In addition there were three-way interactions between GA treatment, duration of stratification, and germination time and seed lot, duration of stratification, and germination time. The interactions that included GA treatment resulted from more rapid germination in GA3 treated seed than the water treated seed (Fig. 1). During the first 3 wk of stratification, the GA3 stimulated greater amounts of germinating seed at Days 7, 14, 21, and 28 of the germination period. There was little difference in germination between GA3 and water treated seed at 14, 21, and 28 d of the germination period for 4 to 7 wk of stratification. But, the number of seedlings at Day 7 of the germination period was 43% for GA3 treated seed compared with 25% in water treated seed indicating more rapid germination with GA3 treated seed.
The three seed lots responded differentially to combinations of duration of stratification and germination time. They had similar germination at Day 7 of the germination period during the first 4 wk of stratification, but Iuka 2000 had 6% greater germination than the other two lots for 5 to 7 wk of stratification. At Days 14, 21, and 28 of the germination period Iuka 1998 had 5 to 6% less germination than the other two seed lots for 0 to 3 wk of stratification. There were no differences in germination among lots with at least 4 wk of stratification and 14 d of germination time.
Abnormal Seedlings, Dormant Seed, and Dead Seed
Abnormal seedlings were counted at the conclusion of the 28-d germination period and the remaining seed were determined to be dormant or dead by a tetrazolium test. There were no interactions between main effects when only the GA3 and water stratification treatments were included in the analysis. The number of abnormal seedlings was influenced by both GA treatment and duration of stratification. GA3 treated seed averaged 3% abnormal seedlings compared with two for untreated and water treated seed (Fig 2 .) This small difference suggested that stratification with GA3 did not adversely affect the normal development of the seedlings. No stratification and 1 wk of stratification produced between 4 and 5% abnormal seedlings compared to an average of 2% abnormal seedlings for 2 to 7 wk of stratification.
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Fig. 2. Abnormal seedlings, dormant seed, and dead seed of eastern gamagrass after treatment with water or GA3 and stratification for 0 to 7 wk. Average of three seed lots—Iuka produced in 1998 and 2000 and Pete produced in 2000. Counts were determined after a 28-d germination-testing period.
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The amount of dormant seed decreased with each week of stratification and after 7 wk there was only 3 and 7% dormant seed remaining in the GA3 and water treated seed, respectively, compared with 73% dormancy in seed that did not receive stratification treatment (Fig. 2). Iuka produced in 2000 averaged 6 percentage points more dormant seed over the course of the 7 wk of stratification than Iuka 1998 or Pete 2000.
The two stratification treatments (with and without GA3) produced similar amounts of dead seed, which were greater than dead seed recorded at the completion of the germination for the unstratafied seed (Fig. 2). The amount of dead seed in dry, water treated, and GA3 treated seed was 13 to 16% for 0 to 4 wk of stratification. With 5 to 7 wk of stratification, the amount of dead seed in the water treated and GA3 treated seed increased to 20 to 26%. Since there was no increase in germination for 5 to 7 wk of stratification, the reduction in seed dormancy over this period was caused by increasing amounts of seed death. Seed of Iuka from 1998 and Pete from 2000 averaged approximately 17% dead compared with 14% for Iuka harvested in 2000.
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DISCUSSION
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The results of this study provide several previously unreported insights into the dormancy and germination of eastern gamagrass seed. Combinations of GA3 and stratification for 4 wk or more did not increase final germination of eastern gamagrass seed above levels attained by stratification without GA3. However, GA3 did stimulate more rapid and greater germination when seed was stratified for 3 wk or less. Once the dormancy was completely broken with 4 wk of stratification, the GA3 was effective at increasing the rate at which the seed germinated. The increased rate of germination could have an impact on the success of eastern gamagrass establishment. More rapid germination could give seedlings an increased ability to compete with weeds, insect pests, and soil pathogens. Mueller et al. (2000) and Aberle et al. (2003) found that eastern gamagrass stand establishment was sensitive to dry soil conditions in the weeks following planting. More rapid germination could increase the chance of generating an acceptable stand if eastern gamagrass seed was planted into moist, soil that dried over the course of the establishment period. Field studies are required to determine if combinations of GA3 and stratification increase field establishment success.
The duration of stratification required for breaking the seed dormancy in eastern gamagrass was less in our study than in previous reports. Stratification for 4 wk broke nearly all of the seed dormancy and there was no difference among three seed lots tested. The level of germination remained stable with additional stratification for up to 7 wk. Ahring and Frank (1968) stated "6 to 8 wk on moist substrate at 5 to 10°C was sufficient to promote germination to within 60 to 80% of all live seed," which has become an industry standard. A closer examination of their data indicated that one lot required 6 wk for maximum germination; however, another required only 2 wk. Our levels of final germination were similar to those reported by Ahring and Frank (1968). Anderson (1985) reported that 30 d of stratification had very little influence on eastern gamagrass seed germination, while 60 and 90 d of stratification increased germination to about 40% of the live seed compared to 8% in unstratified seed. Seed were soaked on moistened substrates in germination boxes in our study and the two previous studies. However, in our studies, we soaked the seed at room temperature for 24 h before exposing the seed to cold temperatures. Reducing the stratification duration for eastern gamagrass seed from 6 to 4 wk could reduce seed costs and stand establishment expenses.
The reports of Ahring and Frank (1968) and Anderson (1985) did not address the fate of the eastern gamagrass seed that remained ungerminated after peak germination was reached with stratification. A decrease in germination with more than 6 wk of stratification as reported by Ahring and Frank (1968) could result from seed death or cycling back into dormancy. Final germination did not increase after 4 wk of stratification in our study. But, the number of dead seed increased with each week of stratification past 5 wk until very few dormant seed remained with 7 wk of stratification. These trends suggest that the remaining ungerminated seed were not capable of producing viable seedlings and would die with additional weeks of stratification.
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NOTES
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This research was supported by the Iowa Agriculture and Home Economics Experiment Station and the Leopold Center for Sustainable Agriculture.
Received for publication December 20, 2002.
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REFERENCES
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- Aberle, E.Z., L.R. Gibson, A.D. Knapp, P.M. Dixon, K.J. Moore, E.C. Brummer, and Roger Hintz. 2003. Optimum planting procedures for eastern gamagrass. Agron. J. 95: 1054–1062.[Abstract/Free Full Text]
- Ahring, R.M., and H. Frank. 1968. Establishment of eastern gamagrass from seed and vegetative propagation. J. Range Manage. 21:27–30.
- Anderson, R.C. 1985. Aspects of the germination ecology and biomass production of eastern gamagrass (Tripsacum dactyloides, L.). Bot. Gaz. (Chicago) 46:353–364.
- AOSA. 1992. Seedling evaluation handbook. Contrib. No. 35. p. 84–87. Association of Official Seed Analysts. Las Cruces, NM.
- AOSA. 1998. Rules for testing seeds. Association of Official Seed Analysts. Las Cruces, NM.
- Burns, J.C., D.S. Fisher, and K.R. Pond. 1996. Quality of eastern gamagrass compared with switchgrass and flaccidgrass when preserved as hay. Postharvest Biol. Tech. 7:261–269.
- Burns, J.C., D.S. Fisher, K.R. Pond, and D.H. Timothy. 1992. Diet characteristics, digesta kinetics, and dry matter intake of steers grazing eastern gamagrass. J. Anim. Sci. 70:1251–1261.[Abstract]
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- Tian, X., A.D. Knapp, K.J. Moore, E.C. Brummer, and T.B. Bailey. 2002. Cupule removal and caryopsis scarification improves germination of eastern gamagrass seed. Crop Sci. 42:185–189.[Abstract/Free Full Text]
- Tian, X., A.D. Knapp, L.R. Gibson, R. Struthers, K.J. Moore, E.C. Brummer, and T.B. Bailey. 2003. Response of eastern gamagrass seed to gibberellic acid buffered below it pKa. Crop Sci. 43:927–933.[Abstract/Free Full Text]
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ABSTRACT
INTRODUCTION
