Xiang-Yue Zeng, Run-Zheng Chen, Jia-Rui Fu* and Xue-Wu ZhangAbstract*CorrespondenceDepartment of Biology, Zhongshan University, Guangzhou, 510275, China
The effects of water content on seed survival and vigour were studied in cucumber (Cucumis sativus L.) seeds dried to water contents between 2.4 and 7.2% and stored for up to 1 year at ambient temperatures (27±8°C) in Guangzhou, China. Seeds dried to 2.4% water were slightly susceptible to imbibitional damage, but the injury could be avoided by slow imbibition in polyethylene glycol solutions or by pre-humidification at 100% RH. During storage, percentage germination declined steadily and the aging rate increased with increasing water content. Activity of the antioxidant enzymes, catalase, peroxidase and superoxide dismutase declined and leakage of electrolytes increased from the stored seeds, with greater changes observed in seeds stored at progressively higher water contents. These results show that aging results in a general deterioration of seed cells, and indicate that these changes can be slowed by drying seeds to water contents as low as 2.4%.
Keywords: cucumber, Cucumis sativus, seed, antioxidant enzymes, imbibitional injury, seed storage, water content, ultra-dry.
Introduction
Water content and temperature are the main factors influencing seed longevity during storage. Reducing the storage temperature prolongs seed storage life, but the cost of a low-temperature storehouse is prohibitive for a developing country. Lowering the water content of seeds also prolongs storage life. The International Plant Genetic Resources Institute (IPGRI) recommends that seeds be dried to 5±2% water prior to storage at -18°C (FAO/IPGRI, 1994). Several reports have demonstrated that some seeds survive longer if they are stored at water contents less than 5%. Longevity of sesame seeds stored at 50°C was increased 40-fold by lowering the seed water content from about 5% to around 2% (Ellis et al., 1986). Similar results were obtained with other seed species, and it has become generally accepted that if seeds are stored at temperatures of 25°C or greater, drying to water contents less than 5% enhances storage life, especially of seeds containing high amounts of lipid (Ellis et al., 1986, 1988, 1989, 1990; Vertucci and Roos, 1990; Zhou and Bi, 1993). The idea of storing seeds at water contents less than the recommended value of 5% was termed 'ultra-dry storage' or 'ultra-low moisture content storage' (FAO/IPGRI, 1994). Ultra-dry storage at room temperature may permit long-term conservation of germplasm in facilities where refrigeration is not available.
While drying to extremely low water contents did not have harmful effects on the quality of oil-rich seed (Zhou and Bi, 1993), some species such as rice, green gram and soybean were damaged by excessive drying (Zhi and Bi, 1991; Zhang et al., 1994; Jing and Zheng, 1994). Before ultra-dry technology is adopted, it is necessary to determine the species for which this technology is appropriate and whether there are physiological or biochemical effects on seeds that have been dried and stored at extremely low water contents.
Cucumber (Cucumis sativus L.) seed generally has poor longevity compared to other seed species with an average P50 of 4.9 years when stored in unregulated conditions in temperate climates (Priestley, 1986). The potential of prolonging storage life in cucumber by drying seeds to very low water contents has not been reported. This possibility was tested by measuring the germination percentage and enzyme activities of seeds after 12 months' storage at water contents between 2 and 7%.
Materials and methods
Cucumber (Cucumis sativus cv. Xia Qing Si Hao) seeds were harvested in 1994 at the Guangdong Agriculture Research Institute, Guangzhou, Guangdong Province, and placed in a storehouse at 5°C and 70% RH until used in December 1994. The initial moisture content of the seeds was 7.2% and initial germination percentage was 95.8%.
Seeds were dried from 7.2% water to 5.6, 3.4 and 2.4% water by placing in a vacuum desiccator containing CaCL2 (weight of CaCl2:seeds = 5:1) for 4, 8 and 20 days, respectively. The water content of seeds was determined by drying 50 seeds at 105°C for 16 h (ISTA, 1985). Water content measurements are the average of three replicates. Dried seeds were sealed in bottles and stored on the laboratory bench. Laboratory temperatures at the onset of the experiment were low (15-20°C), but increased in the summer months to 30-35°C. The average yearly temperature was 28°C.
To ensure that seeds which had been dried to very low water contents were not damaged during imbibition, dried seeds were imbibed slowly prior to each germination assay. Germination percentage, seedling growth and leakage of electrolytes were compared for seeds dried to 2.4% water and prehydrated using different methods. Immediately after drying, seeds were either placed in 20% PEG 6000 solution for 1 or 3 days, or placed in a sealed box above water at 28°C for 5 days. There were few differences between prehydration treatments, and so seeds were prehydrated using the latter method in all subsequent assays.
The viability and vigour of seeds were evaluated from germination assays in which 50 seeds were sown in rectangular plastic boxes filled with sterilized moist perlite. Each seed was submerged in the moist perlite and the boxes were kept in the dark at a temperature of 28°C for 4 days. Vigour was evaluated by the growth of the seedling and is expressed as the average length of the hypocotyl and radicle (cm) after 4 days incubation (Abdul-Baki and Anderson, 1973). The amount of electrolytes leaked from seeds during imbibition was measured using a DDS-IIA conductivity meter (Xiao Shan Science Apparatus, Beijing). Seeds (1 g per treatment) were prehumidified at 100% RH and 28°C for 5 days and then submerged in 50 ml distilled water. Conductivity of the soak water was assayed in 2 or 4 h intervals for the first 12 h of imbibition, and the rate of electrolyte leakage was calculated from the linear regression of the conductivity versus time relationship. Each germination and leakage assay was replicated three times.
The activity of antioxidant enzymes following storage was compared for seeds stored for 12 months at different water contents. Seeds were prehumidified and planted as described above. For the peroxidase and catalase assays, 0.2 g dw of seeds with testa removed (about 10 seeds) was harvested after 2 days' incubation and ground in 5 ml distilled water according to the method of Luo et al. (1989). The mixture was centrifuged at 4000 r.p.m. for 15 min and the supernatant was used in the enzyme assay. For the peroxidase assay, 50 ml of the supernatant was mixed with 3 ml of 0.05 mM guaiacol and 0.1 M sodium phosphate buffer (pH 7.0) to form the end product tetraguaiacol, which was quantified by measuring absorbance at 470 nm after 5 min. One unit of enzyme activity was defined as a unit increase in A470/min/g (Luo et al., 1989). Catalase activity was assayed by the rate at which H2O2 was consumed. A 10 ml sample of the crude supernatant was mixed with 3 ml of 0.01 mM H2O2 and 50 mM sodium phosphate buffer (pH 7.0). The decrease of absorbance at 240 nm was monitored for 3 min. One unit of enzyme activity was defined as a 0.1 unit decrease in A420/min/g (Luo et al., 1989). Similar methods were used to measure the activity of superoxide dismutase, except that 0.5 g of seed (about 25 seeds) were prepared from each treatment, and seeds were germinated for 1 day only (Huang and Chen, 1990). A 100 ml sample of the crude supernatant was mixed with 30 ml of 50 mM phosphate buffer (pH 7.8, 8.9 g Na2HPO4 and 6.8 g KH2PO4 in 1 L distilled water), 58 mg methionine, 0.87 mg EDTA and 1.375 mg nitric blue tetrazolium. After 15 min, the solution was assayed by measuring absorbance at 560 nm.
Results
Cucumber seeds were dried to very low water contents, and it was important to establish whether this treatment caused damage at the onset of the storage experiment and whether the damage could be ameliorated with slow imbibition. Prehydration had no detectable effect on percentage germination, but gave slightly increased axis growth and reduced levels of electrolyte leakage (Table 1, top portion). Prehydrated seeds gave viability and vigour measurements similar to control seeds that were not dried. These data suggest that there was some sensitivity to imbibitional damage that was completely reversible by slow prehydration, and the extent of the damage was not sufficient to reduce viability. The different prehydration treatments had similar effects, although the 28°C, 100% RH treatment appeared to give slightly more vigorous seeds, with longer axis length and less leakage. Because of this, and because of the greater simplicity of the treatment, prehumidification over water was used in all subsequent assays.
The viability and vigour of cucumber seeds declined during 1 year of storage at room temperature, and the extent of change was greater in seeds stored at higher water contents (Fig. 1). After 1 year, the percentage germination of seeds containing 7.2% water decreased from 95.8% to 17%, while germination of seeds stored at 2.4% water decreased only slightly to 91% (Fig. 1, Table 2). Axis growth declined, the rate of electrolyte leakage increased and the level of antioxidant activity decreased with the decline in germination (Table 2).
Table 1. The effect of different humidification treatments on viability and vigour of cucumber seeds dried to 2.4% water immediately after drying and after 12 months of storage. Values in parentheses represent the standard deviation of the mean of three replicates
|
Treatment |
Germination (%) |
Axis length (cm) |
Conductivity of soak water after 12 h imbibition (mS/cm/g) |
|
Immediately after drying |
|||
|
Not dried |
95.8 (0.5) |
16.3 (0.2) |
18.2 (1.4) |
|
Dried + no prehydration |
93.3 (1.7) |
14.1 (0.3) |
28.8 (0.8) |
|
20% PEG, 1 days |
95.1 (0.8) |
15.7 (0.4) |
17.6 (1.1) |
|
20% PEG, 3 days |
94.6 (0.5) |
16.3 (0.2) |
17.1 (0.6) |
|
100% RH, 5 days |
95.3 (0.5) |
17.2 (0.3) |
16.6 (0.4) |
|
After 12 months' storage at ambient
temperature |
|||
|
Not dried |
17.0 (0.8) |
8.2 (0.6) |
30.1 (0.2) |
|
Dried + no prehydration |
83.3 (0.5) |
12.1 (0.4) |
23.0 (0.7) |
|
20% PEG, 1 days |
89.1 (0.8) |
13.7 (0.3) |
19.6 (0.4) |
|
20% PEG, 3 days |
91.4 (0.5) |
15.4 (0.5) |
18.9 (0.3) |
|
100% RH, 5 days |
92.2 (0.9) |
15.5 (0.3) |
18.6 (0.4) |
Figure 1. Time course for reduction in germination percentage of cucumber seeds stored at indicated water contents at ambient temperatures in Guangzhou, China.
To determine whether seeds became more sensitive to imbibitional injury with aging, we repeated the prehydration study after seeds had been stored for 1 year (Table 1, bottom). There was a greater difference in the percentage germination between prehydrated and non-prehydrated seeds in the stored samples compared to the fresh seeds; however, this difference was not reflected in the leakage assay and only slightly in the axis growth assay.
Discussion
Scientists have known for years that seed longevity improves if seeds are dried to low water contents (e.g. Justice and Bass, 1978) and this certainty forms the basis of the IPGRI recommendation of 5+2% water for safe storage of seeds (FAO/IPGRI, 1994). In most instances, the life span of seeds increases when they are dried to water contents as low as 5%. The question addressed here and in other studies of ultra-dry technology (Ellis et al., 1988; Zhi and Bi, 1991; Zhou and Bi, 1993; Hu and Gong, 1994; Jing and Zheng, 1994) is whether further drying can prolong seed life, especially if seeds are to be stored at ambient temperatures. Drying to water contents less than 5% appears to be beneficial to seeds containing high amounts of lipid (Ellis et al., 1988; Zhou and Bi, 1993; Hu and Gong, 1994), but appears to be detrimental to other seeds (Zhi and Bi, 1991; Jing and Zheng, 1994). The present experiments demonstrate that the shelf life of cucumber seeds stored at ambient temperatures in Guangzhou, China is extended by drying seeds to water contents as low as 2.4% (Fig. 1, Table 1).
Table 2. Physiological characteristics of cucumber seeds stored for 12 months at different water contents and ambient temperatures. Values in parentheses represent the standard deviation of the mean of three replicates. Values for enzyme activities are units (see Methods)
|
Physiological trait |
Water content during storage |
||||
|
|
2.4 |
3.4 |
5.6 |
7.2 |
|
|
Germination (%) |
91 (3) |
88 (3) |
65(2) |
17(2) |
|
|
Axis length (cm) |
15.5 (0.3) |
15.4 (0.3) |
13.4 (0.3) |
8.2 (0.1) |
|
|
Leakage rate (mS/cm/g/h) |
0.56 (0.14) |
0.64 (0.14) |
0.91 (0.20) |
1.05 (0.22) |
|
|
Enzyme activity |
|
|
|
|
|
|
|
Peroxidase |
95.2 (0.6) |
82.7 (0.3) |
71.1 (0.7) |
27.1 (0.5) |
|
Catalase |
5302 (17) |
5304 (14) |
4203 (83) |
2602 (15) |
|
|
Superoxide dismutase |
110.3 (6.1) |
107.2 (2.2) |
102.5 (0.1) |
87.3 (0.4) |
|
In summary, we have shown that cucumber seeds will deteriorate almost completely within 1 year if stored at ambient temperatures in Guangzhou, China (27±8°C). Deterioration is significantly slower when seeds are dried to progressively lower water contents. Associated with the decline in germination was a decrease in axis growth, antioxidant enzyme activity and an increase in electrolyte leakage. Collectively, these data show a general deterioration of cells in aging cucumber seeds.
Acknowledgements
The authors wish to thank Christina Walters (USDA-ARS National Seed Storage Laboratory) for her helpful comments.
References
Abdul-Baki, A.A. and Anderson, J.D. (1973) Vigor determination in soybean seed by multiple criteria. Crop Science 13, 630-633.
Ellis, R.H., Hong, T.D. and Roberts, E.H. (1986) Logarithmic relationship between moisture content and longevity in sesame seeds. Annals of Botany 57, 499-503.
Ellis, R.H., Hong, T.D. and Roberts, E.H. (1988) A low-moisture-content limit to logarithmic relations between seed moisture content and longevity. Annals of Botany 61, 405-408.
Ellis, R.H., Hong, T.D. and Roberts, E.H. (1989) A comparison of the low-moisture-content limit to the logarithmic relation between seed moisture and longevity in twelve species. Annals of Botany 63, 601-611.
Ellis, R.H., Hong, T.D., Roberts, E.H. and Tao K.-L. (1990) Low moisture content limits to relations between seed longevity and moisture. Annals of Botany 65, 493-504.
FAO/IPGRI (1994) Genebank Standards. Rome, Food and Agriculture Organization of the United Nations/International Plant Genetic Resources Institute.
Huang, X.-L. and Chen, R.-Z. (1990) Seed Physiology Experimental Handbook. Beijing, Chinese Agricultural Press.
Hu, J. and Gong, L.-Q. (1994) Effect of ultra-dry process and storage on tomato and pepper seeds. Seeds (China) 5, 27-30.
ISTA (1985) International rules for seed testing. Seed Science and Technology 13, 299-355.
Jing, X.M. and Zheng, G.H. (1994) Discussion of the sensitivity of 'Heihe 5' and 'Heihe 3' soybean seeds to desiccation. Seeds (China) 5, 27-30.
Justice, O.L. and Bass, L.N. (1978) Principles and Practices of Seed Storage. Agriculture Handbook No. 506. Washington, D.C., US Government Printing Office.
Luo, G.-H., Wang, A.-G., and Shao, C.-B. (1989) Injury of high concentrations of oxygen to seedlings of rice and the activity of antioxidant enzymes. Acta Botanica Austrio Sinica [South China Institute of Botany Academica Sinica, Guangzhou] 4, 139-142.
Priestley, D.A. (1986) Seed Aging. Ithaca and London, Cornell University Press.
Simon, E.W. and Raja Harun, R.M. (1972) Leakage during seed imbibition. Journal of Experimental Botany 23, 1076-1085.
van Bilsen, D.G.J.L., Hoekstra, F.A., Crowe, L.M. and Crowe, J.H. (1994) Altered phase behavior in membranes of aging dry pollen may cause imbibitional leakage. Plant Physiology 104, 1193-1199.
Vertucci, C.W. and Roos, E.E. (1990) Theoretical basis of protocols for seed storage. Plant Physiology 94, 1019-1023.
Zhang, Y.-N., Tao, M. and Gou, X.-R. (1994) Research of grain and green gram seeds. Seeds (China) 4, 29-31.
Zhi, J.-Z. and Bi, X.-H. (1991) The effect of ultra-low moisture content storage on viability and physiology of rice seeds. Seeds (China) 4, 19-23.
Zhou, X.-S. and Bi, X.-H. (1993) Effect of ultra-low moisture content storage on physiology of several high oil seeds. Seeds (China) 5,12-15.
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