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a Dep. of Agronomy, Iowa State Univ., Ames, IA 50011
b Dep. of Statistics, Iowa State Univ., Ames, IA 50011
* Corresponding author (adknapp{at}iastate.edu)
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ABSTRACT |
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 TOPNOTES ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Abbreviations: TZ, 2,3,5-triphenyl tetrazolium chloride • GA, gibberellic acid
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INTRODUCTION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Ahring and Frank (1968) investigated the effect of prechill on seed germination of two eastern gamagrass seed lots. Germination increased with up to 6 wk of prechill at 5 to 10°C and then appeared to decline after longer prechill periods. Prechill has been recommended and used with certain success to reduce dormancy in this species (USDA, 1991); however, to be effective, the seed must have imbibed water near its maximum capacity and must be kept moist throughout the prechill period. Further, a long prechill treatment (6–8 wk) is required, after which the prechilled seeds should be transferred to conditions favorable for germination without being allowed to dry (Gamagrass Seed Company, 1999). Nevertheless, the results are inconsistent and unpredictable (Kindiger, 1994).
In previous work, Tian et al. (2002) tested the roles of the cupule and pericarp on seed dormancy of eastern gamagrass. Cupule removal followed by pericarp scarification resulted in germination of all viable seeds. It was concluded that cupule removal and pericarp scarification overcame the seed dormancy in eastern gamagrass. However, establishing a mechanical system to remove the cupule and scarify the pericarp without damaging the caryopses may be difficult. The difficulty may arise because the cupule shape and size of eastern gamagrass is not uniform, the embryo extends the length of the caryopsis, and the embryo is not recessed relative to the surface of the caryopsis.
A stimulatory effect of GA3 on germination of dormant seed has been reported for many plant species, such as lettuce (Lactuca sativa L.) (Lona, 1956), most cereals (Mott, 1978; Agrawal, 1981; Prasad et al., 1983), and many other grasses (Mott, 1974; Hagon, 1976; Fulbright et al., 1983). Anderson (1985) applied GA3 to eastern gamagrass seed of one seed lot collected near Carbondale, IL. Both nonhulled and dehulled seeds were soaked in 1.0 g L-1 (0.003 M) of GA3 solution for 24 h. Gibberellic acid was somewhat effective at breaking dormancy of dehulled seeds, increasing germination percentage from 40 to 65% after 30 d of germination. The effect of GA3 on germination of seeds with their cupule intact was slight, with germination percentage increasing 5 to 8% when compared with untreated seed.
Nikolaeva (1977) stated that hormonal treatment had little effect if the seeds were in coat-imposed dormancy. Seed germination of wild oats (Avena fatua L.) (Hsiao, 1979a) and wild buckwheat {Polygonum convolvulus L. [= Fallopia convolvulus (L.) A. Love]} (Hsiao, 1979b) was not influenced by GA3 until the seed coat was made more permeable by NaOCl. In a study of the effect of GA3 on the germination of yellow rocket (Barbarea vulgaris R. Br.) seed, Taylorson (1976) demonstrated that seed scarification or buffering the substrate pH at 3.0 increased responsiveness of the seeds to GA3. He suggested that GA3 uptake is a limiting factor in the stimulation of germination in intact seeds.
Toole and Cathey (1961) studied the response of light-requiring seeds lettuce and Virginia peppergrass (Lepidium virginicum L.) to GA3, and observed that a buffered (citrate phosphate, pH 3.2) solution of GA3 was more effective in stimulating seed germination in the dark than unbuffered solutions. Palevitch and Thomas (1976) showed that the stimulation of celery (Apium graveolens L.) seed germination by GA3 was enhanced by decreasing the pH of the incubation solution below the pKa of gibberellin. This was accomplished by adding low-pH compounds such as buffers, weak acids (e.g., citric acid), or by titration with strong acids, such as HCl. Collectively, these previous studies suggest that buffering GA3 below its pKa may enhance its uptake and stimulatory effect of GA3 on germination of eastern gamagrass seeds.
The enhancement of GA3 activity by reducing its pH was explained in two ways (Palevitch and Thomas, 1976). First, lowering the pH of the GA3 solution increased the proportion of the undissociated form of GA3 in the solution, thus facilitating GA3 movement through the lipid membranes of seeds (Toole and Cathey, 1961). Second, under low pH conditions, hydrogen ions might affect the acidic bonds of the cell walls or stimulate the activity of certain cell-wall-degrading enzymes which react more efficiently in an acidic environment, a phenomenon known as the acid effect (Evans, 1974).
The studies reported herein were conducted to assess the influence of GA3 solution buffered below its pKa (3.8) on the germination of decupulated and intact caryopses of eastern gamagrass. The two major named cultivars from several production years were used to assess the variability in response across different seed lots.
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MATERIALS AND METHODS |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Seed lots of ‘Iuka’ and ‘Pete’ eastern gamagrass used in these studies were produced in 1995, 1996, and 1997 for Experiment 1 and in 1996, 1997, and 1998 for Experiment 2. Seeds were received in November of their year of production and stored at 4.4°C and 46% relative humidity until needed. The first study was initiated in March 1998 and the second study in March 1999. Viability of seeds was determined in January 1998 for 1995, 1996, and 1997 seed used in both experiments and in January 1999 for 1998 seed used in Experiment 2. Tetrazolium (TZ; 2,3,5-triphenyl tetrazolium chloride) tests indicated that viability was between 87 and 97% across the different cultivars and production years of the seed lots (Table 1).
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Eastern gamagrass seeds were randomly sampled from each seed lot. In Experiment 1, the cupule (including the lemma and palea) was removed by cutting through it at the juncture of rachis and glume with a razor blade. Care was taken to ensure that the caryopsis was not scarified during this procedure. Twenty-five caryopses were soaked for 24 h at 25 ± 1°C in a 100- by 15-mm plastic Petri dish with two sheets of Whatman No. 1 filter paper (Whatman Chemical Separation, Inc., Clifton, NJ) moistened with 5 mL of the appropriate test solution. A similar procedure was used for Experiment 2 except that 50 seeds were sampled from each lot, the cupules were left intact, 9 mL of treatment solution were used, and soaking was performed for either 24 or 48 h. After the proper exposure time, caryopses or cupules were washed for 60 s under distilled water to remove any remnant treatment solution. The pH of the test solution in treatment dishes was checked using a micro-combination pH electrode (Orion Research Inc., Beverly, MA) to determine that buffering capacity during incubation was sufficient.
Immediately after washing, seeds for each treatment were placed in 13- by 13- by 3.5-cm covered containers with two layers of Anchor Steel blue seed germination paper (Anchor Paper Co., St. Paul, MN) moistened with distilled water. One box, containing 25 randomly sampled seeds for Experiment 1 and 50 seeds for Experiment 2, was considered as an experimental unit. All germination tests were performed at 20/30°C alternating night/day temperature (16-h night/8-h day; Ahring and Frank, 1968). Light was supplied in conjunction with the day temperature period.
Seed from Experiment 1 was germinated in a Percival (Perry, IA) model 1-35 incubator with two 40-W cool-white fluorescent lights set vertically on each the left and right side. Replication for Experiment 1 was done in time. Seed from Experiment 2 was germinated simultaneously with each replication in a different incubator. Seed for two replications were placed in Hoffman (Albany, OR) model SG30 incubators with three 40-W cool-white fluorescent lights oriented vertically in each the front and rear. Seed for the third replication was germinated in a Conviron (Pembina, ND) model G30 germinator with four 40-W cool-white fluorescent lights vertically oriented on each the left and right sides. Temperature of each chamber was measured with a thermograph and was generally within 1°C of the set temperatures. Photon flux density (400–700 nm) was measured in all chambers using a Licor (Lincoln, NE) LI-190SA Quantum Sensor and was 20 ± 5 µmol m2 s-1 when the sensor was held vertically (facing the lights) in the center of the incubators.
Germination counts were made every 7 d for 28 d. Seeds were considered germinated if the coleoptile exceeded the seed in length and the seedling was normal according to the seedling evaluation criteria of the Association of Official Seed Analysts for comparable grasses (Association of Official Seed Analysts, 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, ungerminated seeds were examined by TZ tests and classified as dormant or dead.
The experimental design for both studies was a randomized complete block and each study was analyzed independently. Treatments in Experiment 1 were factorial combinations of six seed lots by five test solutions replicated four times. Experiment 2 contained factorial combinations of six seed lots by four test solutions by two exposure times with three replications. Germination percentages were collected from each dish and adjusted based on the viability of each seed lot. Germination data from each week were then subjected to analysis of variance according to General Linear Model (GLM) procedure of the Statistical Analysis System (SAS Inst., Cary, NC). Analysis of germination across time, from 7 to 28 d was accomplished using Proc Mixed of SAS with the auto regressive [ar(1)] covariance structure and the Satterthwaite approximation of degrees of freedom. Mean comparisons were performed using LSD or Duncan's Multiple Range tests at P < 0.05. The coefficients of variation for germination was 18 and 102% in Experiments 1 and 2, respectively.
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RESULTS |
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Experiment 2
A test of pH during soaking of intact cupules found that the pH of the buffer solution and buffered GA3 solution was at or below 3.8 at 24 and 48 h. Thus, the buffered GA3 solution was below the pKa of GA3 and kept protonated during the seed treatment period. The pH of the unbuffered GA3 solution, at 4.3 ± 0.07 and 6.1 ± 0.03 at 24 and 48 h, respectively, rose above the pKa of GA3.
There were no significant differences in germination response to exposure of intact cupules to treatment for 24 and 48 h, therefore data for the two periods were combined (Fig. 2). Treatment with 0.001 M GA3 solution increased germination from 9 to 16% when compared with soaking in water alone. Germination response from treatment with buffered GA3 solution was similar to soaking in water. Treatment with buffer solution alone had a negative impact on germination, decreasing it from 9 to 5% when compared with soaking in water. Tetrazolium tests performed at the end of the 28 d germination period indicated that soaking in the buffer solution did not increase the proportion of dead seeds (data not shown).
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DISCUSSION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Gibberellic acid is important in the promotion and maintenance of germination (Bewley, 1997), while embryos are considered to be the source of GA3, the GA3 requirement for germination in eastern gamagrass could imply inadequate endogenous levels or inadequate GA3 synthesis in the embryos. Mechanisms by which GA3 stimulates seed germination have been suggested. Simpson (1990) proposed that GA3 could promote formation of low molecular weight mono- and disaccharides, which assist the intracellular generation of negative water potentials that aid radicle emergence. However, Welbaum and Bradford (1990) found no increase in turgor pressure before radicle protrusion in muskmelon (Cucumis melo L.) seeds.
Endosperm weakening during tomato (Lycopersicon esculentum Mill.) seed germination has been extensively studied (Groot and Karssen, 1987; Haigh and Barlow, 1987; Groot et al., 1988). Seed germination of GA3–deficient tomato mutants was absolutely dependent on application of GA3 unless the endosperm and testa layers opposing the radicle were removed (Groot and Karssen, 1987). Groot et al. (1988) found that GA3 application increased the activity of galactomannan hydrolyzing enzymes in the mutant seeds. They concluded that the key role of GA3 in seed germination is to induce the synthesis of endosperm cell wall-degrading enzymes to hydrolyze the layers surrounding the radicle until they no longer act as physical barriers to radicle emergence.
Our previous study (Tian et al., 2002) indicated that the outer layers of the embryo restricted germination of eastern gamagrass. Seeds germinated readily when the cupule was removed and the pericarp was scarified. The stimulatory effect of GA3 on seed germination of eastern gamagrass could involve weakening those structures through enzymatic degradation. Also, accumulating osmotic solutes could increase the hydrostatic pressure of embryo tissues and help expanding radicles burst through barrier tissues. In addition, a loosened cell wall would promote water and oxygen uptake needed for expansive growth of the emerging radicle.
It should be noted that GA3 did not break down the dormancy of all caryopses when applied to seeds with cupules removed. Eastern gamagrass has an indeterminate fruiting pattern in that different spikes on a single plant develop during an extended time period. At harvest, seeds are not uniform in age and the dormancy levels could vary due to differences in physiological stage and environmental conditions experienced during seed development.
The stimulatory effect of GA3 is restricted to periods when the seeds are capable of responding to the hormone because of receptor availability or activity. The majority of eastern gamagrass seeds used in this study responded to GA3. The failure of a small portion of the seeds to respond to GA3 could be due to insensitivity or the presence of other factors limiting the processes leading to germination. For example, GA3 application did not improve the seed germination of genetically pure lines of wild oats with very deep seed dormancy unless the seeds were afterripened before GA3 treatment (Upadhyaya et al., 1982). The authors hypothesized that several blocks exist in dormant seeds. Increased sensitivity of seeds to GA3 by afterripening or slow drying has been reported (Evans and Young, 1975; Nicholls, 1979, 1986). This sensitivity of seeds to dormancy-breaking agents is proposed to be related to membrane-bound or membrane-associated receptor proteins within the embryonic cells (Taylorson, 1988; Hooley et al., 1991; Vleeshouwers et al., 1995). It has been hypothesized that receptor proteins move to the membrane surface and become exposed during afterripening.
Our results generally agree with those of Anderson (1985) in that GA3 resulted in significant enhancements in eastern gamagrass germination when cupules were removed from seeds, but made only a slight difference in seeds with intact cupules. However, germination levels obtained from application of GA3 to decupulated seed were higher in the current study than the 65% reported by Anderson. This could be due to the use of buffered GA3 solutions or differences in genotype, production environments, handling and processing, age, and incubation temperature.
Buffering GA3 below its pKa was not effective at breaking dormancy in eastern gamagrass when the cupule remained on the seed. Significant but small increases in germination occurred when cupules were soaked in GA3 dissolved in water. The response to GA3 in water varied considerably among the lots tested. These results suggest that there are one or more cupule (including lemma and palea)-mediated dormancy mechanisms in eastern gamagrass. These mechanisms appear very strong in some lots and prevented the action of GA3. In other lots, the cupule-mediated dormancy was weaker and the GA3 stimulated germination.
Previous research has shown that a cold-moist (4.4°C) stratification period of 40 to 60 d followed by exposure to >25°C was necessary for many eastern gamagrass seeds to germinate when their cupules were intact (Ahring and Frank, 1968; Anderson, 1985). Dormancy mechanisms affected by these prechilling conditions are unknown for this species. Germination of seeds in contact with their removed cupules had greater germination than cold-moist stratified seed or dry seed with the cupule removed, suggesting that there is no chemical inhibitor in the cupule (Anderson, 1985). Soaking cupule-enclosed seed in sodium hypochlorite solution did not stimulate germination, adding further weight to this argument (Anderson, 1985; Ahring and Frank, 1968). Germination was not enhanced by subjecting seeds to ethylene chlorohydrin vapors and solutions or using salt solutions of 0.1 to 0.8% KNO3 as moistening agents (Ahring and Frank, 1968). Lack of evidence for chemical germination inhibitors suggests that the cupule dormancy mechanism is physical. This hypothesis is supported, in some ways, by recent work that suggested that the integrity of the cupule must be reduced before germination will proceed normally (Springer et al., 2001).
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ACKNOWLEDGMENTS |
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NOTES |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Received for publication April 3, 2002.
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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This article has been cited by other articles:
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  L. R. Gibson, E. Z. Aberle, A. D. Knapp, K. J. Moore, and R. Hintz Release of Seed Dormancy in Field Plantings of Eastern Gamagrass Crop Sci., January 31, 2005; 45(2): 494 - 502. [Abstract] [Full Text] [PDF]
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C. Rogis, L. R. Gibson, A. D. Knapp, and R. Horton Enhancing Germination of Eastern Gamagrass Seed with Stratification and Gibberellic Acid Crop Sci., March 1, 2004; 44(2): 549 - 552. [Abstract] [Full Text] [PDF]
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