The effects of storing seeds under extremely dry conditions - Walters, C. & Engels, J.

Christina Walters¹* and Jan Engels²

¹ United States Department of Agriculture, Agricultural Research Service, National Seed Storage Laboratory, 1111 South Mason Street, Fort Collins, CO, USA

²International Plant Genetic Resources Institute, Via delle Sette Chiese 142, 00145 Rome, Italy

*Correspondence

From traditional wisdom, we know that dry seeds survive longer than moist seeds. This axiom was extended to mean 'drier is better' and formed the basis of longevity equations introduced in the 1950s and 1960s (Roberts, 1972; Justice and Bass, 1978). The 'drier-the-better' concept conjures visions of seed life spans extending into infinity as the water content of seeds approaches 0%. Tests of this assumption began in earnest in the late 1980s when it was clearly shown that seed life spans could not be prolonged indefinitely by progressively drying seeds (Ellis et al., 1986, 1988, 1989, 1990a,b). Rather, seed life spans were finite, and the water content below which longevity could not be improved was considered a critical water content (Ellis et al, 1986, 1988, 1989, 1990a,b; Vertucci and Leopold, 1987b; Vertucci and Roos, 1990).

The existence of a critical water content is the most important point in the seed storage debate. Groups at both the University of Reading, UK and the National Seed Storage Laboratory, USA have shown that drying below a certain water content will not improve seed longevity (Ellis et al, 1988,1989, 1990a,b; Vertucci and Roos, 1990; Vertucci et al, 1994a). There is also undisputed evidence that the value of the critical water content varies among species in an inverse relationship with the lipid content of the seed (Ellis et al., 1989, 1990; Vertucci and Roos, 1990). One of the more powerful findings is that the water activity that corresponds to the critical water content appears to be constant among species (Roberts and Ellis, 1989; Ellis et al., 1989, 1990a,b; Vertucci and Roos, 1990). The Reading group discovered this using an empirical approach to determine the low-moisture-content limit to the logarithmic relationship between water content and longevity (e.g. Roberts and Ellis, 1989; Ellis et al, 1989, 1990a,b). Alternatively, Walters and colleagues used a thermodynamic approach to study the role of water in biological systems and critical water contents for physiological processes (e.g. Vertucci and Leopold, 1986, 1987a,b; Leopold and Vertucci, 1989; Vertucci and Roos, 1990). It is remarkable that the laboratories at Reading and the NSSL arrived at the conclusion of critical water activities independently using completely different approaches and species.

The issues of the current debate are elaborations of the initial discovery of a critical water activity for seed longevity. These issues are important, but secondary to the understanding that there are limits to the beneficial effects of drying seeds: (i) how is longevity affected by drying to water contents less than the critical value?, (ii) what is (are) the value(s) of the critical water activity(ies)?, and (iii) how does the critical water activity or critical water content change with temperature? Answers to these questions will provide the basis for seed storage protocols for germplasm conservation purposes.

How is longevity affected by drying to water contents less than the critical value?

Intuitively, we know that removing every last bit of water from seeds is detrimental. Thus early reports of low germination in seeds stored under extremely dry conditions (Kosar and Thompson, 1957; Nutile, 1964; Nakamura, 1975; Woodstock et al., 1976; Nishiyama, 1977) were not unexpected. However, the mechanism of the damage was unclear: there were insufficient data on the initial effects of drying seeds to extremely low water contents and/or precautions against imbibitional stress to convincingly rule out desiccation or imbibitional damage. New reports using numerous species (Vertucci and Roos, 1990; Carpenter and Boucher, 1992; Vertucci et al., 1994a; Dickie and Smith, 1995; Ellis et al., 1995; Buitink et al., 1998; Chai et al., 1998; Hu et al., 1998a,b; Kong and Zhang, 1998; Shen and Qi, 1998) have demonstrated that detrimental effects were not initially evident, but became more apparent with time. In other words, the seeds aged more rapidly under extremely dry conditions. Hence we can now conclude with confidence that drying to extremely low water contents may shorten seed longevity. This conclusion is consistent with the finding that over-drying foods causes more rapid deterioration (Rockland, 1969; Labuza, 1980).

The reason why detrimental effects of over-drying were detected in some experiments and not in others remains unresolved. Data sets that address the question of seed deterioration at low water contents are rare since the experiments are logistically difficult. Because aging rates are generally slow under dry conditions, researchers have sought more sensitive detectors of deterioration than percentage germination assays. Assays measuring changes in radicle growth or time to germination often give the appearance of more rapid deterioration. For example, significant deterioration was detected within 8 weeks in lettuce seeds stored at 7% water and 35°C when root growth or germination time was monitored, and after 16 weeks when percentage germination was monitored (Ellis et al., 1995). Even using similar assays and storage conditions, there can be major differences in deterioration rates among samples from the same species, and these are attributed to differences in seed quality among cultivars and lots (e.g. Hay et al., 1997; Chai et al., 1998; reviewed by Walters, 1998a). This variability in aging kinetics confounds predictions of longevity for specific water content and temperature combinations.

The experimental design is critical in determining whether a particular data set addresses the controversy about extremely dry storage conditions. Experiments where a single water content or a narrow range of water contents is considered may give important information about potential longevity, but give no information about the role of water. Data sets containing two moisture treatments may give information about an overall effect of water content, but they cannot be used to show a break in the trend. At least three water contents are required, and one of the water contents must be sufficiently low to rule out the possibility of overlooking any detrimental effects of drying. The best data sets have many water content treatments over a large range of water contents beginning at close to 0% water, and deterioration detected in most, if not all the moisture treatments. There are few data sets that meet these criteria (Kosar and Thompson, 1957; Ellis et al., 1988, 1989, 1990a,b, 1992, 1995; Vertucci and Roos, 1990; Carpenter and Boucher, 1992; Vertucci et al., 1994a; Dickie and Smith, 1995; Buitink et al., 1998; Chai et al., 1998; Hu et al., 1998a; Kong and Zhang, 1998; Shen and Qi, 1998). The global project studying deterioration of lettuce seeds was initiated to evaluate a 'model' design for this type of research (Walters et al., 1998).

Among experiments that are appropriate to evaluate the effect of extremely low water contents on seed longevity, there is evidence showing no effect of water content below a critical value (Ellis et al., 1988, 1989, 1990a,b, 1992, 1995; Kong and Zhang, 1998); detrimental effects (Kosar and Thompson, 1957; Ellis et al., 1989, 1990a,b; Vertucci and Roos, 1990; Carpenter and Boucher, 1992; Vertucci et al, 1994a; Dickie and Smith, 1995; Buitink et al., 1998; Chai et al., 1998; Hu et al., 1998a; Kong and Zhang, 1998; Shen and Qi, 1998); and in a few cases, a progressively beneficial effect of drying to extremely low water contents (1% for chive at 40°C, Kong and Zhang, 1998; 1.7% for yew, Walters-Vertucci et al., 1996; and 2% for sesame at 50°C, Ellis et al., 1986). Even when experiments were conducted using the same species at the same temperature, conflicting results have been reported. For example, detrimental effects of storing Brassica pekinensis at 40°C and <3% (Shen and Qi, 1998), as opposed to no effect of water content for the same species stored at 40°C and water contents between 0.5 and 3.0% (Kong and Zhang, 1998). For the majority of species tested there were no detrimental effects of extreme drying when seeds were stored at or above 50°C, but the longevities for all treatments were relatively short. However, for many species a definite reduction in longevity was observed when seeds were dried to water contents less than 2-3% and subsequently stored at or below 45°C. This introduces the possibility that some of the conflicting results may be attributed to the different temperatures used in the experiments.

The observation that seeds stored at extremely low water contents have reduced longevity, even if that observation is inconsistent, points out the potential risks of over-drying. Thus for many seeds there is an optimum moisture level for storage at which longevity is maximized. Drying below the optimum is counterproductive. This finding introduces another critical water content - the water content below which seeds are damaged.

What are the values of the critical /optimum water contents and water activities?

The presence of critical water contents for longevity have been demonstrated in numerous seeds from diverse phylogenetic backgrounds. Two critical water contents, the water content that gives maximum longevity (wc₁) and the water content below which aging rates increase (wc₂) have been identified. The range of water contents between wc₁ and wc₂ represents the optimum water contents for seed storage. From seed storage experiments published thus far, it is unclear whether wc₁ and wc₂ are the same value (i.e. there is an optimum water content), or whether the values are different (there is an optimum range of water contents) and this is because it is difficult to detect significant differences in aging rates among similar water contents within the 4-year time span of most of the experiments. The range of water contents that give maximum longevity define the 'margin of error' between drying seeds to prolong seed life spans and over-drying seeds at the expense of seed life spans. Experiments on the effect of water content and temperature on aging rates of pea seeds (Vertucci et al., 1994a) and pollen grains (Buitink et al., 1998) show that the margin of error narrows as storage temperature increases.

The mean and standard deviation of critical water content values for storage at different temperatures are given in Table 1. These data are compiled from experiments conducted in incubators at 65, 45, 40 or 25°C or under ambient room conditions where the temperatures fluctuated from about 18-26°C. For the 65°C and 40-5°C treatments, the optimum water contents for storage range between 3 and 4%, and at ambient conditions they range between 4 and 6%. These values are within the range of the FAO/IPGRI recommendation of 5±2% water (FAO/IPGRI, 1994). The major source of concern is the variation among species that is represented by the '±2%' in the IPGRI standards. The standards provide few criteria to evaluate whether a seed should be stored at 3 or 7% water. Usually, genebank operators make a conservative decision and adjust seed water contents to about 5%. This decision often results in storing seeds wetter than the optimum [e.g. the optimum water content for flax at ambient temperatures is about 3.2% (Chai et al., 1998)], or drier than the optimum [e.g. the optimum water content for rice is about 7% (Hu et al., 1998a)]. The seed storage debate originated from efforts to determine the appropriate water content for storage for any species without doing the long-term experiment to determine the value directly.

It is clear that a certain amount of the variation in critical water contents is related to the lipid content of the seed. Lipid composition determined through table values (Spector, 1956; Earle and Jones, 1962; Jones and Earle, 1966) for the species considered in Table 1 range from 1 to 45% of the mass of the seed. When regressed against lipid content, regression coefficients (r²) ranged from about 0.68 (both 65°C and 40-45°C) to about 0.84 (18-26°C). The relationship between lipid content and critical water content is not surprising, as one expects that much of the water in the seed will be in the aqueous regions of the seed and not in the lipid bodies. Water content can therefore be corrected to reflect the non-lipid component of the seed by

wc_corr = wc/(1 - %lipid)

where wc_corr is the water content assuming water is not associated with the lipids in seeds, and %lipid is the fraction of the seed mass containing lipids (measured directly or determined from tables). This calculation increases the value of critical water content and slightly reduces the variability among species (Table 1).

Much of the variation in critical water content could be eliminated if water activity were used as the measure of water levels. Water activity describes the water concentration in thermodynamic terms - i.e. its availability for reactions. In a package containing seeds and air, the water activity of the seeds can be determined by measuring the RH of the air (water activity=RH/100). Water sorption isotherms describe the equilibrium relationship between temperature, water content and RH for a particular seed. Isotherms used by Ellis and colleagues were constructed using an instrument that measures the equilibrium RH above seeds adjusted to different water contents (Ellis et al., 1989, 1990a,b, 1995). Isotherms used by Walters and colleagues were constructed by measuring the equilibrium water content of seeds incubated at known relative humidities. Isotherms from the two groups have identical properties at RH>25%, but vary considerably in shape at lower relative humidities. Isotherms by Ellis and colleagues bend sharply at about 10% RH (termed the inflection point) and water content-RH data for RH£9% are not available (Ellis et al., 1989, 1990a,b, 1995). The concavity of isotherms constructed by Walters and colleagues varies with temperature, and water contents are >0 as RH approaches zero (Vertucci and Leopold, 1986, 1987a,b; Vertucci and Roos, 1990, 1993a,b; Vertucci et al., 1994b; Walters-Vertucci et al., 1996; Buitink et al., 1998; reviewed by Walters, 1998a). For a justification of her approach, the interested reader is referred to the NSSL perspective statement in this supplement of Seed Science Research (Walters, 1998b).

Table 1. Values determined for the critical water content for seed longevity at different storage temperatures. Critical water contents are taken from the seed storage experiments cited in this paper

Temperature	No. of species	Average lipid content	Not corrected for lipid content		Corrected for lipid content
			wc1	wc2	wc1	wc2
65	25	15	3.9±1.2	(3.0±0.6)*	4.6±0.8	(3.8±1.0)
40-45	19	25	3.4±1.2	2.9±1.4	4.5±0.9	3:8+1.2
22±4	15	27	5.6±3.3	3.9±1.8	6.9±2.5	5.0+1.4

* Detected for only two species: beet and mungbean.

How does the critical water activity or critical water content change with temperature?

From the above discussion it is clear that there is a reasonable level of consensus on the optimum RH for seed storage: either the water content corresponding to 10% RH at 20°C according to Ellis and colleagues (Ellis et al., 1989, 1990a), or the water content corresponding to 20% RH at the storage temperature according to Walters and colleagues. Further, both groups predict that the optimum RH should be almost constant among storage temperatures (Ellis et al., 1989, 1990a; Vertucci and Roos, 1993a; Vertucci et al., 1994a). This is significant progress towards a consensus on how best to store seeds at ambient or refrigerated temperatures, and an average at 15% RH would seem like the most reasonable way to resolve the entire controversy. Unfortunately, this simple resolution does not address one of the most important issues of the seed storage debate: how the optimum water contents change with storage temperature.

Ellis and colleagues (Ellis et al., 1989, 1990a,b, 1991) contend that the value of critical water content is not likely to vary greatly among different storage temperatures. Walters and colleagues, on the other hand (Vertucci and Roos, 1993a,b; Vertucci et al., 1994a; Walters-Vertucci et al., 1996; Buitink et al., 1998; reviewed by Walters, 1998a) contend that the water contents giving maximum longevity increase with decreasing temperature. The data compiled in Table 1 do not resolve the question: the critical water content values are identical for temperatures of 65 and 40-45°C, as Ellis and colleagues predict, but increase as the storage temperature decreases from 40-45°C to 18-26°C, as Walters and colleagues predict.

As with the controversy over the RH values corresponding to wc₁ and wc₂, the differing shapes of isotherms used by Ellis and Walters at low RH explain the differing predictions. The isotherms used by Ellis and colleagues (e.g. Mazza et al., 1990) have similar water content-RH relationships at temperatures ³25°C (i.e. the isotherms overlap at low RH); hence, given the same low RH of 11%, water contents will be very similar for temperatures ³25°C. The same conclusion cannot be drawn at temperatures £25°C. The isotherms constructed by Walters and colleagues have unique water content-RH combinations for each temperature (i.e. the isotherms do not overlap at low RH); hence given the same low RH, optimum water contents must be different for different temperatures (Vertucci and Roos, 1993a,b; reviewed by Walters, 1998a,b).

Implications for ultra-dry technology

The concept of ultra-dry technology was first introduced as a means to reduce or avoid the requirement for refrigeration in germplasm facilities with economic constraints (IBPGR, 1985; Ellis et al., 1986). Research was initiated to determine the potential for ultra-dry storage in obtaining the longest seed life spans possible for seeds stored at ambient temperatures, and perhaps even achieving longevities comparable to those obtained by cold-temperature storage (FAO/IPGRI, 1994; Zheng et al., 1998). Originally, ultra-dry meant water contents less than 5% and was intended for species with high lipid contents. However in 1994, ultra-dry was redefined as drying seeds to 10-12% RH at 20°C (FAO/IPGRI, 1994). The former definition reflected the ability to enhance seed longevity of oily seeds by drying them below the IPGRI standard of 5% water. The latter definition reflected the belief that the water content achieved by equilibrating seeds at 10% RH and 20°C gave maximum longevity (i.e. wc₁). For many seeds, these two definitions mean very different things, and it is not always clear which definition is used in a particular research project.

Based on the data available, can we say whether ultra-dry storage will place seeds at optimum moisture levels for storage? When ultra-dry is defined as water contents less than 5%, the answer to this question is, probably if seeds are stored at 65°C or 40-45°C (for most seeds studied, wc₁ <5%; Table 1). If seeds are stored at ambient conditions of 18-26°C, the answer is maybe, depending on the lipid content of the seed and the risk of over-drying (Table 1). When ultra-dry is defined as the water content in equilibrium with 10% RH at 20°C, both Ellis and Walters agree that seeds will be at or very near the optimum water content if they are stored at temperatures between 25 and 35°C. However, these predictions must not be extrapolated when storing seeds at temperatures £20°C; based on her research at lower temperatures, Walters believes that this excessive drying would be counter-productive to seed longevity for most species.

Unfortunately, the early assumption of ultra-dry technology - that comparable longevities of seeds can be achieved under non-refrigerated conditions if seeds are dried sufficiently (e.g. Zheng et al., 1998) - cannot be fully realized. The longevity of seeds, even at the optimum water content, decreases as storage temperature increases. In other words, there is no substitute for refrigeration when trying to prolong seed life spans. Refrigerated facilities are not always possible; under these circumstances, seed water contents should be optimized by adjusting them to a specific RH. In tropical areas where temperatures range from 25-35°C, the optimum water content can be achieved by drying to 15% RH (ambient temperatures) or at 20°C and 10% RH. From a practical point of view, applying these conditions should allow numerous national programmes to conserve their seed collections with high viability for increased durations.

References

Buitink, J., Walters, C, Hoekstra, F.A. and Crane, J. (1998) Storage behavior of Typha latifolia pollen at low water contents: interpretation on the basis of water activity and glass concepts. Physiologia Plantarum 102 (in press).

Carpenter, W.J. and Boucher, J.F. (1992) Temperature requirements for the storage and germination of Delphinum × cultorum seed. HortScience 27,989-992.

Chai, J., Ma, R., Li, L. and Du, Y. (1998) Optimum moisture contents of seeds stored at ambient temperatures. Seed Science Research 8, Supplement No. 1, 23-28.

Dickie, J.B. and Smith, R.S. (1995) Observations on the survival of seeds of Agathis spp. stored at low moisture contents and temperatures. Seed Science Research 5, 5-14.

Earle, F.R. and Jones, Q. (1962) Analyses of seed samples from 113 plant families. Economic Botany 26, 221-250.

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. (1990a) Low-moisture-content limits to relations between seed longevity and moisture. Annals of Botany 65, 493-504.

Ellis, R.H., Hong, T.D. and Roberts, E.H. (1990b) Moisture content and the longevity of seeds of Phaseolus vulgaris. Annals of Botany 66, 341-348.

Ellis, R.H., Hong, T.D. and Roberts, E.H. (1991) Seed moisture content, storage, viability and vigour (correspondence). Seed Science Research 1, 275-277.

Ellis, R.H., Hong, T.D. and Roberts, E.H. (1992) The low-moisture-content limit to the negative logarithmic relation between seed longevity and moisture content in three subspecies of rice. Annals of Botany 69, 53-58

Ellis, R.H., Hong, T.D. and Roberts, E.H. (1995) Survival and vigour of lettuce (Lactuca sativa L.) and sunflower (Helianthus annuus L.) seeds stored at low and very low moisture contents. Annals of Botany 76, 521-534.

FAO/IPGRI (1994) Genebank Standards. Rome, Food and Agricultural Organization of the United Nations/ International Plant Genetic Resources Institute.

Hay, F.R., Probert, R.J. and Smith, R.D. (1997) The effect of maturity on the moisture relations of seed longevity in foxglove (Digitalis purpurea L.). Seed Science Research 7, 341- 349.

Hu, C., Zhang, Y, Tao, M., Hu, X. and Jiang, C. (1998a) The effect of low water contents on seed longevity. Seed Science Research 8, Supplement No. 1, 35-39.

Hu, X., Zhang, Y., Hu, C., Tao, M. and Chen, S. (1998b) A comparison of methods for drying seeds: vacuum freeze drier versus silica gel. Seed Science Research 8, Supplement No. 1, 29-33.

IBPGR (1985) Cost-effective Long-term Seed Stores. Rome, International board for Plant Genetic Resources.

Jones, Q. and Earle, F.R. (1966) Chemical analyses of seeds. II. Oil and protein content of 759 species. Economic Botany 20, 127-155.

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.

Kong, X.-H. and Zhang, H.-Y. (1998) The effect of ultra-dry methods and storage on vegetable seeds. Seed Science Research 8, Supplement No. 1, 41-45.

Kosar, W.F. and Thompson, R.C. (1957) Influence of storage humidity on dormancy and longevity of lettuce seed. Proceedings of the American Society of Horticultural Science 70, 273- 276.

Labuza, T.P. (1980) The effect of water activity on reaction kinetics of food deterioration. Food Technology 34, 36-59.

Leopold, A.C. and Vertucci, C.W. (1989) Moisture as a regulator of physiological reaction in seeds, pp 51-68 in Stanwood, P.C. and McDonald, M.B. (Eds) Seed Moisture (Special Publication No. 14). Madison, Wisconsin, Crop Science Society of America.

Leopold, A.C., Sun, W.Q. and Bernal-Lugo, I. (1994) The glassy state in seeds: analysis and function. Seed Science Research 4, 267-274.

Mazza, G., Jayas, D.S. and White, N.D.G. (1990) Moisture sorption isotherms of flax seed. Transactions of the American Society of Agricultural Engineers 33, 1313-1318.

Nakamura, S. (1975) The most appropriate moisture content of seeds for their long life span. Seed Science and Technology 3, 747-759.

Nishiyama, I. (1977) Decrease in germination activity of rice seeds due to excessive desiccation in storage. Japanese Journal of Crop Science 46, 1111-1118.

Nutile, G.E. (1964) Effect of desiccation on viability of seeds. Crop Science 4, 325-328.

Roberts, E.H. (1972) Storage environment and the control of viability, pp 14-58 in Roberts, E.H. (Ed.) Viability of Seeds. London, Chapman & Hall.

Roberts, E.H. and Ellis, R.H. (1989) Water and seed survival. Annals of Botany 63, 39-52.

Rockland, L.B. (1969) Water activity and storage stability. Food Technology 23, 1241-1251.

Shen, D. and Qi, X. (1998) Short- and long-term effects of ultra-drying on germination and growth of vegetable seeds. Seed Science Research 8, Supplement No. 1, 47-53.

Smith, R.D. (1992) Seed storage, temperature and relative humidity. Seed Science Research 2, 113-116.

Spector, W.S. (1956) Handbook of Biological Data. Philadelphia/London, W.B. Saunders.

Vertucci, C.W. and Leopold, A.C. (1986) Physiological activities associated with hydration level in seeds, pp 35-49 in Leopold, A.C. (Ed.) Membranes, Metabolism, and Dry Organisms. Ithaca, NY/London, Comstock Publishing.

Vertucci, C.W. and Leopold, A.C. (1987a) Water binding in legume seeds. Plant Physiology 85, 224-231.

Vertucci, C.W. and Leopold, A.C. (1987b) Relationship between water binding and desiccation tolerance in tissues. Plant Physiology 85, 232-238.

Vertucci, C.W. and Roos, E.E. (1990) Theoretical basis of protocols for seed storage. Plant Physiology 94, 1019-1023.

Vertucci, C.W. and Roos, E.E. (1991) Seed moisture content, storage, viability and vigour: response (correspondence). Seed Science Research 1, 277-279.

Vertucci, C.W. and Roos, E.E. (1993a) Theoretical basis of protocols for seed storage. II. The influence of temperature on optimal moisture levels. Seed Science Research 3, 201-213.

Vertucci, C.W. and Roos, E.E. (1993b) Seed storage, temperature and relative humidity: response (correspondence). Seed Science Research 3, 215-216.

Vertucci, C.W., Roos, E.E. and Crane, J. (1994a) Theoretical basis of protocols for seed storage. III. Optimum moisture contents for pea seeds stored at different temperatures. Annals of Botany 74, 531-540.

Vertucci, C.W., Crane, J., Porter, R.A. and Oelke, E.A. (1994b) Physical properties of water in Zizania embryos in relation to maturity status, water content and temperature. Seed Science Research 4, 211-224.

Walters, C. (1998a) Understanding the mechanisms and kinetics of seed aging. Seed Science Research 8, 223-244.

Walters, C. (1998b) Ultra-dry technology: perspective from the national Seed Storage Laboratory, USA. Seed Science Research 8, Supplement No. 1 11-14.

Walters, C., Kameswara Rao, N. and Hu, X. (1998) Optimizing seed water content to improve longevity in ex situ genebanks. Seed Science Research 8, Supplement No. 1, 15-22.

Walters-Vertucci, C., Crane, J. and Vance, N.C. (1996) Physiological aspects of Taxus brevifolia seeds in relation to seed storage characteristics. Physiologia Plantarum 98, 1-12.

Williams, R.J., Hirsh, A.G., Takahashi, T.A. and Meryman, H.T. (1993) What is vitrification and how can it extend life? Japanese Journal of Freezing and Drying 39, 1-10.

Woodstock, L.W., Simkin, J. and Schroeder, E. (1976) Freeze drying to improve seed storability. Seed Science and Technology 4, 301- 311.

Zheng, G.H., Jing, X.M. and Tao, K.L. (1998) Ultradry seed storage cuts costs in gene bank. Nature 393, 223-224.

US Department of Agriculture, 1998