Di Shen* and Xiaoquan QiAbstract*CorrespondenceInstitute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, 30 Bai Shi Qiao Road, Beijing 100081, China
Before ultra-dry technology can be adopted in China, the short- and long-term effects of drying seeds to very low water contents must be evaluated. To do this, the viability and vigour of seeds of five vegetable species (Chinese cabbage, Brassica campestris L. ssp. pekinensis; green Chinese onion, Allium fistulosum L. var. Gaganeum makino; chive, Allium tuberosum; cucumber, Cucumis sativus; and pepper, Capsicum frutescens L.) were measured immediately after drying to water contents between 5 and 1%, and after 3 years' storage at constant temperatures of 40, 4 and -20°C and ambient temperatures of 25±10°C (day) and 15±10°C (night). Percentage germination of seeds after 1 day (early count) and 2 weeks (final count), emergence in soil, seedling mass and conductivity of leachate following imbibition were used to evaluate seed performance. There were no differences in initial germination percentages and only minor differences in leakage rates of seeds dried to different levels, indicating that imbibitional damage was not problematic in these seeds. After 3 years' storage, there was no evidence of deterioration in samples stored at ambient temperatures, 4°C or -20°C. Final germination percentage was lower in Chinese cabbage and pepper seeds stored at 40°C and water contents greater than 4%. Reductions in early germination counts, soil emergence and / or seedling mass of Chinese cabbage, cucumber, pepper and Chinese onion seeds stored for 3 years at 40°C and water contents less than about 2% suggested that drying to extremely low water contents had detrimental effects on longevity. In contrast, storage at 40°C had a promoting effect on the germination of chive seeds, suggesting that they were after-ripened at the elevated temperature. Our results suggest that there may be an optimum water content for seed storage and that different germination assays have different sensitivities in detecting deterioration.
Keywords: germplasm, seed longevity, seed storage, temperature, moisture content, ultra-dry, after-ripening, Brassica campestris, Allium fistulosum, Allium tuberosum, Cucumis sativus, Capsicum frutescens.
Introduction
Current genebank standards recommend that seeds be dried to 5% water and stored at -18°C (FAO/IPGRI, 1994). The concept of ultra-dry seed storage suggests that the requirement for refrigerated storage can be diminished if seeds are stored at very low water contents (FAO/IPGRI, 1994; IBPGR, 1985; Ellis et al., 1989, 1990). There is interest in adapting this technology in Chinese germplasm facilities where the cost of refrigeration is prohibitive (e.g. Zheng, 1994; Cheng et al., 1997). Before the technology is used, the water content which gives maximum longevity to seeds stored at ambient temperatures must be determined. Also, the potential risks of drying seeds to low water contents must be addressed, as some reports have indicated that drying to very low water contents damages seeds (Nakamura, 1975; Nutile, 1964). The reduction in vigour with extreme drying may be a result of imbibitional damage (e.g. Ellis et al., 1990), and this can be ameliorated by slow rehydration (Gu and Xu, 1985). Alternatively, the reduction in vigour with extreme drying may be a result of faster deterioration (e.g. Vertucci and Roos, 1990). The purpose of this study was to determine how drying seeds to low water contents affects their viability and vigour and to evaluate the difference in longevities attained when seeds are stored using current and ultra-dry standards.
Materials and methods
Seeds of Chinese cabbage (Brassica campestris L. ssp. pekinensis cv. 87-3), green Chinese onion (Allium fistulosum L. var. Gaganeum makino cv. Zhangquidacong), chive (Allium tuberosum cvs TianjinDaqingmiao and 791), cucumber (Cucumis sativus cv. Zhongnong No. 5), and pepper (Capsicum frutescens cv. Nongfa) were harvested in 1992 (Chinese cabbage, pepper, cucumber) or 1993 (onion, chives). Initial water contents of seeds were about 9% (chive), 7.4% (green Chinese onion) and 4.9% (Chinese cabbage, cucumber and pepper). Seeds were dried to different water contents by a step-wise increase in temperature and the ratio of silica gel weight to seed weight ('method of going up temperature in grade', Qi and Shen, 1994). Seeds were mixed with activated silica gel in a ratio of 4:1 (seed: silica gel) and placed in a ventilated incubator (LRH-250-G illuminating incubator, Guangdong Medical Apparatus, Beijing) at 15°C for 15-30 days to achieve water contents of 5-3% water. To achieve water contents of 2 and 1%, seeds that had been dried to 3% were mixed with silica gel at ratios of 2:1 and 1:1, respectively, and stored for an additional 20-30 days at 25°C then 35°C. Total drying times were 41 days (Chinese cabbage, cucumber, pepper) and 58 days (green Chinese onion, chive). The water content of seeds was determined by comparing fresh and dry weights, dry weights being measured after heating seeds at 103°C for 17 h (ISTA, 1985). Water contents (wc) are expressed on a fresh weight basis.
The immediate effects of extreme drying were evaluated by comparing the percentage germination and the amount of electrolytes leaked during imbibition of seeds that were not dried and seeds that were dried to 1% water (Halmer and Bewley, 1984; Shen and Qi, 1994). To distinguish between damage resulting from extreme drying and damage incurred during imbibition, seeds dried to 1% water were either prehydrated before imbibition or placed directly in the imbibing media (Gu and Xu, 1985; Shen and Qi, 1994). In a three-step prehydration treatment, seeds were hydrated to about 12-16% by placing them at 4°C over a saturated MgCl2 solution (RH=30%) for 72 h (wc » 5-7%), and then over a saturated Mg(NO3)2 solution (RH=50%) for 72 h (wc » 8-10%) and then in a humidity-controlled room (RH=75%) at 4°C for 72 h. In a one-step prehydration treatment, seed water content was elevated to 12-16% by placing seeds at 4°C and 75% RH for 1 week. Seeds were germinated at 28°C (cucumber), 25°C (Chinese cabbage, pepper) and 20°C (green Chinese onion and chive) in boxes containing 0.7% agar as a germination matrix. The percentage germination of three replicates of 100 seeds each was evaluated after 2 weeks' incubation. Leakage of electrolytes during imbibition was measured from seeds that were initially rinsed with deionized water and then soaked at 25°C for 8 h in 50 ml deionized water. The conductivity of the soak water was measured using a DDS-IIA conductivity meter (Xiao Shan Science Apparatus, Beijing). Three replicates of 100 seeds each (all species except cucumber) or 30 seeds each (cucumber) were used in this assay.
To determine the effect of water content and temperature on seed longevity, seeds with water contents adjusted from about 5% to about 1% water were sealed in foil-laminate bags and placed in four temperature regimes: 40, 4 and - 20°C, and ambient conditions of the laboratory in Beijing. The treatment at room temperature (25±10°C in the day and 15±10°C at night) was believed to most closely reflect storage conditions used if ultra-dry technology is adopted. After 3 years' storage, a sample of seeds at each water content/temperature combination was evaluated for germination and other vigour parameters (Shen and Qi, 1994). To avoid imbibitional damage, seeds were prehydrated according to the one-step procedure described above. Three replicates of 100 seeds were germinated on agar as described above and percentage germination was measured after 1 day (early count) and 2 weeks (final count). In a separate assay, the percentage of seeds emerging from soil was measured by planting five replicates of 25 seeds in a vermiculite/sand (w/w 1:1) mix. Seeds were grown in a greenhouse at temperatures of about 25-30°C in the day and 15-20°C at night, and percentage emergence was evaluated after about 2 weeks. After 15-25 days, the fresh weight of the seedlings in each box was measured. The amount of electrolytes leaked from stored seeds during imbibition was also measured following the procedures described above.
Results and discussion
The potential of ultra-dry technology was tested by determining the short- and long-term effects of extreme drying on seed survival. Short-term effects were evaluated by drying seeds to about 1% water and germinating them immediately (Table 1). The initial germination percentages of Chinese cabbage, chive, cucumber and pepper seeds were greater than 90%. Green Chinese onion seeds had a lower initial percentage germination of about 68%. Drying to 1.6±0.3% water had no apparent effect on initial germination percentages irrespective of prehydration treatment (Table 1). These results are consistent with the conclusion that drying seeds to very low water contents has no immediate adverse effect on seed viability (Cheng et al., 1991; Ellis et al., 1989,1990) and does not change the levels of free fatty acids or dehydrogenase activity (Jin et al., 1993). In this study, there was no evidence of imbibitional damage, perhaps because the agar base used to germinate seeds distributed water evenly and slowly, obviating the need for prehydration treatments.
A more rigorous test of imbibitional damage was conducted by soaking seeds in distilled water and measuring the amount of cellular constituents leaked from cells (Table 2) (Halmer and Bewley, 1984). For onion and pepper, there were no significant differences in the conductivity of the soak water in dried and non-dried samples. For Chinese cabbage and cucumber, the conductivity of the soak water was greater in dried samples compared with samples that were not dried, and the prehydration treatment did not ameliorate the greater leakage (Table 2). This suggests that ultra-drying may cause a change in the semi-permeability of membranes in some seeds which is not completely reversed by exposure to 75% RH and 4°C.
Table 1. The effect of drying and rehydration on germination percentage of various vegetable seeds. Seeds were prehydrated by step-wise increase of relative humidity at 4°C from 30 to 50 to 75% (three-step) or by placing directly at 75% RH at 4°C for 7 days (one-step). Values in parentheses represent standard deviation of the mean of three replicates
|
|
Water content (%) |
Germination percentage |
||||
|
Species, cultivar |
Not dried |
Dried |
Before drying |
Dried, not prehydrated |
Dried, three-step prehydration |
Dried, one-step prehydration |
|
Chinese cabbage, 87-3 |
4.8 |
1.6 |
93.5 (1.0) |
93.3 (1.4) |
94.0 (2.3) |
94.3 (2.1) |
|
Cucumber, Zhongnong, No. 5 |
4.9 |
1.3 |
95.0 (3.8) |
91.3 (2.8) |
91.3 (4.8) |
91.3 (3.7) |
|
Onion, Zhangqiudacong |
7.4 |
1.7 |
67.8 (4.0) |
69.6 (5.7) |
69.9 (3.4) |
71.0 (4.3) |
|
Pepper, Nongfa |
5.1 |
1.3 |
98.8 (1.3) |
97.3 (1.5) |
97.9 (1.6) |
96.8 (1.5) |
|
Chive, 791 |
8.9 |
1.6 |
no data |
78.5 |
no data |
no data |
|
Chive, TianjinDaqingmiao |
9.1 |
1.9 |
no data |
91.8 |
no data |
no data |
|
|
|
Conductivity of soak water (mS/cm/g seed) |
||||
|
|
Water content (%) |
Before drying |
Dried, not prehydrated |
Dried, three-step prehydration |
Dried, one-step prehydration |
|
|
Species, cultivar |
Not dried |
Dried |
||||
|
Chinese cabbage, 87-3 |
4.8 |
1.6 |
79.4 (13.7) |
106.4 (12.0) |
120.1 (10.3) |
120.5 (3.3) |
|
Cucumber, Zhongnong, No. 5 |
4.9 |
1.3 |
10.5 (0.6) |
16.5 (0.6) |
15.6 (1.0) |
17.9 (0.9) |
|
Onion, Zhangqiudacong |
7.4 |
1.7 |
130.1 (14.7) |
120.6 (6.1) |
117.9 (8.2) |
118.6 (7.7) |
|
Pepper, Nongfa |
5.1 |
1.3 |
101.8 (5.0) |
51.8 (3.5) |
47.8 (1.9) |
93.8 (6.1) |
|
Chive, 791 |
8.9 |
1.6 |
no data |
36.3 |
no data |
no data |
|
Chive, TianjinDaqingmiao |
9.1 |
1.9 |
no data |
38.0 |
no data |
no data |
Storage of seeds at higher moisture contents is expected to cause more rapid deterioration (e.g. IBPGR, 1985; FAO/IPGRI, 1994). This result was clearly observed in Chinese cabbage and pepper seeds stored at 40°C. In these species, final germination (Fig. 1), emergence from soil (Fig. 2), early germination (Fig. 3) and seedling mass (Fig. 4) were less in seeds stored at about 5% water compared with seeds stored under drier conditions. Chinese cabbage seeds stored at 40°C and about 5% water leaked more solutes during imbibition than seeds stored under drier or colder conditions (Fig. 5). The detrimental effect of high moisture storage was not detected by the conductivity assay in pepper seeds, even though germination was reduced to 0% (Fig. 5). Significant reductions in early germination were observed in cucumber seeds stored at 40°C, and the effect was greatest for seeds stored at about 5% water (Fig. 3). There appeared to be a slight, though not statistically significant, reduction in seedling mass from onion seeds stored at 40°C and 3.8 or 5.7% water (Fig. 4). Deterioration of cucumber and onion seeds was not consistently detected by other growth assays.
There was no evidence of deterioration in seeds of either chive cultivar stored at 40°C. In contrast to other species, early germination rates in chive increased as the water content of storage increased (Fig. 3). The acceleration of germination was more pronounced at higher storage temperatures, suggesting that the phenomenon was related to dry after-ripening (Leopold et al., 1988; Esashi et al., 1993; Foley, 1994).
Data presented for 40°C storage show that the longevity of Chinese cabbage, cucumber, onion and pepper can be improved by drying seeds to water contents less than 5%. It is now necessary to establish whether there is a limit to the beneficial effects of drying. Data collected from the percentage germination, emergence and electrolyte leakage assays do not address this question because deterioration was not detected with these assays in any samples stored at water contents less than 4% (Figs 1, 2 and 5). The early germination (Fig. 3) and seedling mass (Fig. 4) assays provide some evidence of deterioration in seeds stored at 40°C and extremely low water contents. These data suggest optimum water contents for storage at 3% (Chinese cabbage and pepper), 3.5% (cucumber) and 2.2% (onion). The optimum water content for Chinese cabbage agrees well with a critical water content of 2.8% reported previously for other Brassica spp. (Ellis et al., 1989). However, the optimum predicted here for onion is considerably less than the critical value of 3.5% determined earlier for that species (Ellis et al., 1990).
Of the several assays used to detect seed aging, germination percentage had the lowest within-treatment variability, and seedling mass and electrolyte leakage had the greatest (compare size of error bars in Figs 1-5). Measurements of early germination (Fig. 3) and seedling mass (Fig. 4) gave greater indications of change than the other assays, and it remains to be seen whether the other assays eventually give similar information.
Summary
Measurements of germination and growth were made immediately after seeds were dried and after 3 years' storage to determine the short- and long-term effects of ultra-drying. Consistent with some aspects of the ultra-drying concept, life spans of Chinese cabbage, cucumber, onion and pepper seeds stored at 40°C were enhanced by drying to water contents less than 5%. However, drying seeds to water contents less than 2 or 3% had some detrimental effects on longevity, demonstrating that there are potential risks of over-drying seeds. The true potential of ultra-dry technology - how long ultra-dried seeds survive at ambient temperatures compared to refrigerated conditions - cannot be evaluated from this experiment until deterioration at ambient temperature, 4°C or -20°C is detected.
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