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a Syngenta Crop Protection AG, Basel, Switzerland
b Department of Plant and Soil Science, 1405 Veterans Drive, University of Kentucky, Lexington, KY, USA 40546-0312
* Corresponding author (dtekrony{at}uky.edu)
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ABSTRACT |
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 TOPNOTES ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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300 g kg–1 seed moisture, which was slightly after physiological maturity (PM, maximum dry seed weight). The effect of freezing on seed germination and vigor was the same when (i) ears were frozen attached or detached from the plant; (ii) ears were exposed to different freezing rates; or (iii) seeds with sugary and starchy endosperm were frozen. The degree of freezing injury at a given temperature and duration of freezing was similar across four F1 hybrids, but seed from one F2 hybrid was injured slightly less at a given moisture content. Thus, the stage of seed development must be considered by seed companies before making harvesting decisions when facing a potential predicted freezing event. Our results suggest that a seed producer will have higher germination and vigor if they harvest immature seeds (400 g kg–1 SMC) before the freezing event instead of after they are exposed to freezing temperatures.
Abbreviations: AA, accelerated aging • PM, physiological maturity • SMC, seed moisture content • ST, starchy endosperm • SU, sugary endosperm
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INTRODUCTION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Early freezing studies with corn by Kiesselbach and Ratcliff (1920) demonstrated that exposure of seeds that had >300 g kg–1 moisture content to –2°C for 24 h reduced germination. Rossman (1949a) used shorter freezing durations, exposing seeds on unhusked ears to –3.3°C for 16 h, and reported no effect on germination, regardless of the hybrid or SMC. When he exposed ears from F1 hybrids at 500 and 400 g kg–1 SMC for 8 and 16 h at –6.7°C, germination was reduced by as much as 48%, however no reduction occurred at lower SMC (300 g kg–1). When the exposure temperature was lowered to –10°C, 4 h was sufficient to reduce seed germination. Goodsell (1948) concluded that an exposure to –5.6°C was sufficient to induce injury in hybrid corn seed at SMC as low as 285 g kg–1.
Although freezing injury has been shown to reduce germination of immature seeds, few studies have investigated the impact of freeze injury on seed vigor. Fick (1989) reported declines in germination and seed vigor as measured by cold test germination when seeds were frozen at –6°C at various stages of seed development, however the trends between the two tests were similar. Neal (1961) reported cold test germination was reduced for inbred corn seed (SMC >250 g kg–1) when exposed to –6°C for 4 h, however germination was not reported. Judd et al. (1982) used the AA vigor test to determine the effects of freezing injury on soybean seed and found no differences in AA germination between control seed and those exposed to –2°C for up to 32 h. When the seed was frozen at lower temperatures (–7 or –12°C), AA germination was significantly reduced below the unfrozen control, however significant declines also occurred in standard germination.
Since relatively little is known about the effect of freezing temperatures on seed vigor, the objective of these experiments was to determine the effect of freezing on the seed germination and vigor of seed from several corn hybrids frozen at different stages of development and maturation.
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MATERIALS AND METHODS |
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In 1998, after thawing, the ears were husked and placed in a low temperature (
20°C, 25% relative humidity) dryer until seed moisture was <200 g kg–1, after which they were hand shelled and the seed was air dried to <130 g kg–1. In 1999 and 2000 the ears were husked and placed in a forced-air ear dryer (30°C, ambient relative humidity) until seed moisture was <130 g kg–1. All seeds were sealed in polyethylene bags and stored at 10°C until seed quality evaluations were made within 6 mo of harvest. Before testing, a portion of seed from each sample was treated with the fungicides Captan 400 [N-(trichloromethylthio)cyclohex-4-ene-1,2-dicarboximide] and Apron FL (metalaxyl) [methyl-N-(2,6-dimethylphenyl)-N-(2-methoxyacetyl)-DL-alaninate] at the labeled rates of 0.55 and 0.70 g a.i. kg–1 seed, respectively (Bayer Crop Science LP, Research Triangle Park, NC).
Plant vs. Ear Freezing
In 1998, a preliminary experiment was conducted to determine if differences in seed freezing injury occurred when ears were frozen on or detached from the plant. Two harvests were made for F1 hybrid A and three harvests for F2 hybrid B. At each harvest 15 ears with husks and shanks intact were removed from the plants and compared with ears attached to nine plants that were harvested by cutting the plant at the second internode above the soil surface. The harvested ears and plants with ears were preconditioned at 10°C (ears) or 1°C (plants) for 3 h before freezing at –6 ± 1°C. After 3, 6, and 9 h exposure, five detached ears and three plants were removed from the freezing environment and transferred to preconditioning chambers before drying, shelling, and testing for quality.
Freezing Rate
This experiment was conducted to determine if the rate of change in air temperature during freezing altered seed germination and vigor. Seeds from F1 hybrids A (2000) and F (1999) were subjected to two rates of temperature decline: (i) slow freeze, a gradual decrease from 10 to –6°C over a 4 h period (–4°C h–1) in a programmable growth chamber and (ii) fast freeze, directly transferring the ears from the 10°C chamber to –6°C. After the air temperature reached –6°C in both treatments, sets of ears were held at that temperature for 6 h before thawing at 10°C.
Air Temperature and Duration
The effect of freezing temperature (–6 vs. –11°C) and duration (4 vs. 7 h) on seed germination and vigor was examined using seeds from three to five ears of hybrids B and C in 1998 and 1999 with sampling as described in Table 1.
Genotype and Years
Seed germination and vigor was measured in three to five ears of F1 hybrid A in 1998, 1999, and 2000 and F2 hybrid B in 1998 and 1999; following freezing at –6°C for 6 (1998) or 7 h (1999, 2000) with sampling as described in Table 1.
Endosperm Composition
In 1999 and 2000, the effect of endosperm composition on seed germination and vigor was determined using a su sweet corn hybrid ‘Silver Queen’ which was (i) sib-mated to produce F2 seed with sugary endosperm (hybrid D) and (ii) mated with a dent corn pollinator to produce F2 seed with starchy endosperm (hybrid E). One to two ears per hybrid were sampled as shown in Table 1 and frozen at –11°C for 7 h.
Seed Quality Evaluations
Seed moisture content (fresh weight basis) and dry seed weight was determined by removing 20 kernels from the center of each ear from the nonfrozen control and drying at 105°C for 72 h. Seed quality of the control and frozen seed was assessed using the standard germination test (AOSA, 2001), the saturated cold test described by TeKrony and Woltz (1997), the AA test (Hampton and TeKrony, 1995) with conditions of 43°C for 72 h, and the bulk conductivity test (AOSA, 2002). Fungicide-treated seed was used in all tests, except the conductivity test. The number of seeds tested varied between 50 and 200 depending on the availability of seed.
Data Analysis
These experiments utilized a randomized complete block design with two replications at each stage of seed development. Regression analysis using individual observations was used to evaluate the relationship between the stage of seed development (SMC) and seed quality. Models (linear, quadratic, or higher orders) were selected by evaluating the coefficient of determination and the significance of the additional terms in the higher order models. The 95% confidence intervals around the regression lines were used to compare differences among years.
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RESULTS |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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90% at all stages of seed development in all years for both hybrids (Fig. 4, 5). Exposure of seed to –6°C for 6 or 7 h early in seed development reduced germination to very low levels (60%) (exception was hybrid B in 1998). However, germination increased as the seeds developed reaching 80% at PM (hybrid B) or slightly after PM (300 g kg–1 SMC, hybrid A). Following freezing the standard germination of hybrid B was consistently higher than hybrid A. Germination of frozen seed of hybrid A (Fig. 4) was similar in 1998 and 1999, but much lower in 2000, while the germination of seed from hybrid B was always lower in 1999 than in 1998 (Fig. 5).
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300 g kg–1 SMC). When control seeds of hybrids A and B were subjected to the stresses imposed by the cold test, germination was consistently >80% for all years (Fig. 4, 5). The regression slopes for cold test germination following freezing at various stages of seed development were nearly identical to standard germination, but cold test germination was usually higher than AA germination. The cold test germination following freezing exceeded 80% at PM for hybrid B, but slightly after PM for hybrid A.
Conductivity levels of frozen seed were not significantly different than those for the control seed at all harvests of hybrids A and B (data not shown).
There were few differences among years in standard, AA or cold test germination following freezing and germination increased to >80% for nearly all harvests at or after PM, except hybrid A in 2000.
Endosperm Composition
Nearly all unfrozen control seeds from a maternal su-mutant sweet corn that was sib-mated to produce sugary endosperm or mated with a dent pollinator to produce starchy endosperm had high standard germination (>90%) throughout development in 1999 and 2000 (Fig. 6). Germination of seed frozen at –6°C for both endosperm types increased from <60% for very immature seeds to >80% as the seed matured.
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DISCUSSION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Air temperatures approach freezing levels gradually in the field as temperatures decrease in the autumn and during the diurnal temperature cycle. However, in previous freezing studies (Kiesselbach and Ratcliff, 1920; Goodsell, 1948; Rossman, 1949a, 1949b; Neal, 1961; Fick, 1989) the corn plants, ears or seed were placed directly into constant freezing temperatures. We simulated a gradual change in temperature by placing seeds in an environment where temperature changed at a rate of –4°C per hour (10 to –6°C over 4 h) and compared this to placing ears directly into –6°C (Fig. 2). In general, the germination and vigor of seeds frozen slowly were slightly lower than those of seeds on ears frozen quickly, however, significant differences between the two rates occurred only in isolated harvests. In both treatments, the ears were held at –6°C for 6 h, however, seed temperature measurements revealed that some seeds in the slow-freeze treatment reached the freezing point temperature up to 120 min before seeds in the fast-freeze treatment froze (Woltz, 2003). We also found that thawing rate after freezing (fast vs. slow) had no effect on seed quality. These results justify a rapid freezing treatment to study the effects of low temperature in the field on corn seed quality.
When seeds were exposed to –6°C for as little as 4 h, standard germination of seeds harvested before PM was reduced (Fig. 3). As the temperature decreased to –11°C and/or the duration of exposure increased from 4 to 7 h at either –6 or –11°C, germination declined to lower levels over a wider range in moisture contents including PM. The degree of injury decreased as the seeds matured for all maize hybrids studied, however no reductions in germination occurred at low moisture levels (<200 g kg–1) for all freezing treatments. These same general trends in germination response have been observed in previous studies (Kiesselbach and Ratcliff, 1920; Goodsell, 1948; Rossman, 1949a, 1949b). All studies have shown that temperatures that could occur during an early season freezing event, are sufficient to reduce germination. In our studies, in which the freezing point temperature was measured, the corn embryos froze at temperatures warmer than –4°C when the SMC is greater than 300 g kg–1 (Woltz et al., 2005). Thus, as seeds mature the germination percentage after freezing improves, however, it was not until after PM (300 g kg–1 SMC) where no germination differences occurred between exposed and control seeds.
The reductions in standard germination associated with freezing injury were the result of higher levels of dead seed or abnormal seedlings at the conclusion of the test (data not shown). When ears were exposed to –6°C, the proportion of dead seed did not increase significantly, except in the most immature harvests, however, as the temperature was lowered to –11°C after 4 or 7 h of exposure a significantly higher number of dead seeds occurred at all harvests before PM. Immature seed frozen at –6°C had higher levels of abnormal seedlings, however, the abnormality was not as traditionally described in seed testing (AOSA, 2001). Instead, we observed significant increases in seeds which had swollen mesocotyls in all hybrids, which we classified as abnormal seedlings. Tetrazolium chloride staining in seed samples which exhibited large numbers of swollen mesocotyls showed small, isolated areas at the basal end of the embryos where there were discrete areas of cells still capable of respiration. Thus, it is conceivable that these swollen mesocotyls may be an indicator of freeze injury in a standard germination test.
Although the corn seed industry is concerned about seed viability and germination, they are equally concerned about the effects of freezing temperatures on seed vigor. Thus, we evaluated seed vigor using AA germination and the most widely used vigor test for maize— the cold test. Across all years and field corn hybrids, there were few differences between cold test germination and standard germination following freezing (Fig. 1, 2, 4, 5). In seven of eight genotype x year comparisons the cold test germination following freezing reached maximum levels when the SMC had declined to <300 g kg–1, which coincided with maximum levels of standard germination, but was slightly after PM. Only for hybrid A in 2000 (Fig. 4) did maximum cold test levels occur at lower SMC (250 g kg–1). Fick (1989) also found few differences after freezing between standard germination and a sterile cold test germination. The similarities between these two tests supports Rossman's (1949b) conclusion that freezing injury is manifested as an "all or nothing" effect. Examination of the embryos injured by freezing in our studies with tetrazolium chloride showed that embryos either stained normally, indicating no seed injury, or the embryos were unstained or only very slightly stained, indicating that the seeds were dead (data not shown). If exposure to freezing temperatures caused a hidden injury effect, it did not affect germination in the cold test.
The AA test has been used for many years as a rapid measure of seed deterioration which relates to storability and seed vigor (Hampton and TeKrony, 1995). In this study the AA germination of the unfrozen control seed was usually lower than the cold test or standard germination for all hybrids. Freezing the immature seed severely reduced the AA germination to low levels across all years and genotypes. As the seed continued to develop the AA reached maximum germination slightly after PM (SMC <300 g kg–1) and there were no differences between AA and standard germination for the control or freezing treatments (Fig. 4,5). These reductions in AA following freezing were the result of an increase in dead seeds, rather than more abnormal seedlings (swollen mesocotyls) as found in the standard germination test (data not shown). Thus, it appears that germination following aging may be more sensitive than cold test in detecting seed vigor lost due to freezing. Since the AA test for corn seed has been related to seed storability (Delouche and Baskin, 1973) and field performance (TeKrony et al., 1989; Wilson et al., 1992; Woltz and TeKrony, 2001), seed damaged by freezing temperatures also may have reduced carryover and poor stand establishment potential, problems not detected by standard germination or the cold test.
The stage of seed development must be seriously considered when seed companies must make harvest decisions in face of a predicted freezing event. Our results suggest that a seed producer will have higher germination and vigor, if they harvest immature seeds before instead of after they are exposed to freezing temperatures. In all genotype x year comparisons control seeds harvested slightly before PM (400 g kg–1 SMC) had already reached maximum seed germination and vigor levels. This supports previous corn maturation studies in our laboratory, which showed that maximum standard and cold test germination was reached by black layer stage 3 (mid-milk line stage 3) which was slightly before PM which occurred at black layer 4 (TeKrony and Hunter, 1995). Thus, harvesting at this stage slightly before PM should result in acceptable germination and vigor while delaying harvest until after a freeze can potentially lower seed vigor.
Seeds from the F2 hybrid B exhibited less injury from freezing temperatures than seed from the F1 hybrids (Fig. 1
–5). These results support the conclusion of Neal (1961) that seed produced from hybrids was injured less by freezing temperatures than inbred parent seed, but they refute the observations of Rossman (1949a) who found F2 seed to be less tolerant to freezing than their F1 parent. The differential response between F1 and F2 seed in our studies was probably due to ear size and husk thickness. The additional mass of the larger ears on the F2 plant increased the time it took the embryos to reach their freezing point temperature, reducing the number that froze. The husks of the F2 ears from hybrid B were thicker and more tightly attached than the husks on the F1 maternal parents. The husk plays an important role in insulating the seeds (Rossman, 1949b; Fick, 1989). There was little difference in the seed freezing point temperature between F1 and F2 hybrids (Woltz et al., 2005). The combination of husk protection and the larger ear mass probably delayed the onset of freezing of the F2 seeds and produced the results in Fig. 3, 4, and 5. Thus, the variation observed among hybrids may be more closely associated with husk characteristics than seed characteristics.
Rossman (1949b) reported that sweet corn lines were more tolerant to freezing than dent corn lines. In our studies there was also a trend for seeds with sugary endosperm to have slightly higher standard and AA germination than those with starchy endosperm following freezing, however, the effect of endosperm composition (sugary or starchy) was not significant for standard germination, AA or cold test germination (Fig. 6).
Corn seed exposed to freezing temperatures before maturity may suffer large reductions in germination and vigor. The impact of exposure to freezing temperature on germination and vigor of corn seed was influenced by seed maturity, genotype, and the severity of exposure to freezing conditions (temperature x time), but not by freezing rate, attachment to the plant, or endosperm composition (sugary vs. starchy) The most immature seed had the greatest reductions. As the seeds matured, they became more tolerant to freezing and, once the seed reached 300 g kg–1 SMC, which was slightly after PM, they were no longer injured by exposure to freezing temperatures. Thus, if a frost is predicted before this stage of maturity, a seed producer will have higher seed vigor if the seeds are harvested before rather than after the freezing event.
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Received for publication August 31, 2005.
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REFERENCES |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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M. DeVries, A. S. Goggi, and K. J. Moore Determining Seed Performance of Frost-Damaged Maize Seed Lots Crop Sci., September 1, 2007; 47(5): 2089 - 2097. [Abstract] [Full Text] [PDF]
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