In vitro techniques are very useful for conserving plant biodiversity, including (a) genetic resources of recalcitrant seed and vegetatively propagated species, (b) rare and endangered plant species and (c) biotechnology products such as elite genotypes and genetically engineered material. Explants from recalcitrant seed and vegetatively propagated species can be efficiently collected under field conditions using in vitro techniques. In vitro culture techniques ensure the production and rapid multiplication of disease-free material. Medium-term conservation is achieved by reducing growth of plant material, thus increasing intervals between subcultures. For long-term conservation, cryopreservation (liquid nitrogen, −196°C) allows storing plant material without modification or alteration for extended periods, protected from contaminations and with limited maintenance. Slow growth storage protocols are routinely employed for a large number of species, including numerous endangered plants, from temperate and tropical origin. Cryopreservation is well advanced for vegetatively propagated species, and techniques are ready for large-scale experimentation in an increasing number of cases. Research is much less advanced for recalcitrant species due to their seed characteristics, viz., very high sensitivity to desiccation, structural complexity and heterogeneity in terms of developmental stage and water content at maturity. However, various technical approaches should be explored to develop cryopreservation techniques for a larger number of recalcitrant seed species. A range of analytical techniques are available, which allow understanding physical and biological processes taking place in explants during cryopreservation. These techniques are extremely useful to assist in the development of cryopreservation protocols. In comparison with crop species, only limited research has been performed on cryopreservation of rare and endangered species. Even though routine use of cryopreservation is still limited, an increasing number of examples where cryopreservation is used on a large scale can be found both in genebanks for crops and in botanical gardens for endangered species.

Use of biotechnologies for the conservation of plant biodiversity

Contents Preface Contributors Chapter 1 Long-term ex situ conservation of Biological Resources and the role of Biological Resource Centres Glyn N. Stacey and John G. Day Chapter 2 The Process of Freeze-drying Gerald Adams Chapter 3 Principles of Cryopreservation David E. Pegg Chapter 4 Lyophilization of Proteins Paul Matejschuk Chapter 5 Vacuum-drying and Cryopreservation of Prokaryotes Brian J. Tindall Chapter 6 Freeze-drying of Yeast Cultures Chris Bond Chapter 7 Cryopreservation of Yeast Cultures Chris Bond Chapter 8 Freeze-drying Fungi using a Shelf-freeze-drier C. Shu-hui Tan, Cor W. van Ingen and Joost A. Stalpers Chapter 9 Cryopreservation and Freeze-drying of Fungi employing Centrifugal and Shelf Freeze-drying Matthew J. Ryan and David Smith Chapter 10 Cryopreservation of Microalgae and Cyanobacteria John G. Day Chapter 11 Cryopreservation of Plant Cell Suspensions Brian W. W. Grout Chapter 12 Cryopreservation of Shoot-tips and Meristems Erica E. Benson, Keith Harding and Jason W. Johnston Chapter 13 Cryopreservation of Desiccation Tolerant Seeds Hugh W. Pritchard Chapter 14 Cryopreservation of Fish Sperm Eugeny Kopeika, Julia Kopeika and Tiantian Zhang Chapter 15 Cryopreservation of Avian Spermatozoa Graham Wishart Chapter 16 Cryopreservation of Animal and Human Cell Lines Christopher B. Morris Chapter 17 Cryopreservation of Haematopoietic Stem/Progenitor Cells for Therapeutic Use Suzanne M. Watt, Eric Austin and Sue Armitage Chapter 18 Cryopreservation of Human Embryonic Stem Cell Lines Charles J. Hunt and Paula M. Timmons Chapter 19 Cryopreservation of Primary Animal Cell Cultures Glyn N. Stacey and Stuart Dowall Chapter 20 Cryopreservation of Red Blood Cells and Platelets Andreas Sputtek Chapter 21 Cryopreservation of Mammalian Semen Mark R. Curry Chapter 22 Cryopreservation of Mammalian Oocytes Sharon J. Paynter and Barry J. Fuller Chapter 23 Cryopreservation of Mammalian Embryos Barry J. Fuller and Sharon J. Paynter Glossary

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Cryopreservation and freeze-drying Protocols

The premise of this paper is that the chemical potential of water strongly influences aging reactions in seeds, and that there will be an optimal chemical potential of water for seed longevity. If this is true, the optimum moisture content for storage and the optimal drying protocols should vary with the storage temperature, but should be predictable from water sorption isotherms. Isotherms for pea, soybean and peanut are given for temperatures between 65° and −150°C. Relative humidity/moisture content relationships were determined directly for temperatures between 5° and 50°C using saturated salt solutions. At extreme temperatures, isotherms were calculated from heat capacity measurements using differential scanning calorimetry or extrapolations of van't Hoff analyses. The family of isotherms was used to predict optimum moisture contents at storage temperatures between 65°C and −150°C. The optimal moisture contents for these three species are consistently shown to increase as the storage temperature is lowered.

https://www.cambridge.org/core/services/aop-cambridge-core/content/view/S0960258500001793

Theoretical basis of protocols for seed storage. II. The influence of temperature on optimal moisture levels.

Discrete categories of seed physiology can be explained through a unified concept of the structural and molecular mobility responses within cells to drying.
 Tolerance of desiccation is typically described by a threshold or low water content limit to survival. This convention provides fairly good distinction between orthodox and recalcitrant seeds, which show thresholds of less than about 0.07 and greater than about 0.2 g H2O g DW−1, respectively. Threshold water contents, however, are not direct measures of the intensity of water stress tolerated by seeds, nor are they measures of cell response to water stress. More direct criteria, that accommodate both spatial and temporal effects of water loss, are required to explain variation of desiccation tolerance and longevity in seeds from diverse genetic backgrounds and growth conditions. This essay presents the argument that changes in cellular volume directly quantify primary responses to desiccating stress in a context that also links damage, as cellular constituents compress, and protection, as compressed molecules form stabilizing structure. During desiccation, fluid cytoplasm solidifies, and the newly formed spatial relationships among molecules determine whether and how long viability is maintained. The diversity of seed behaviors suggests complexity and opportunity to discover molecules and mechanisms that regulate survival and perception of time in cells that lack metabolic function.

Orthodoxy, recalcitrance and in-between: describing variation in seed storage characteristics using threshold responses to water loss.

The effect of rate of dehydration was assessed for embryonic axes from mature seeds of Camellia sinensis and the desiccation sensitivity of axes of different developmental stages was estimated using electrolyte leakage. Rapidly (flash) dried excised axes suffered desiccation damage at lower water contents (0.4 g H2O (g DW)−1) than axes dried more slowly in the whole seed (0.9 g H2O (g DW)−1). It is possible that flash drying of isolated axes imposes a stasis on deteriorative reactions that does not occur during slower dehydration. Differential scanning calorimetry (DSC) of the axes indicated that the enthalpy of the melting and the amount of non-freezable water were similar, irrespective of the drying rate.Very immature axes that had completed morphogenesis and histodifferentiation only were more sensitive to desiccation (damage at 0.7 g H2O (g DW)−1) than mature axes or axes that were in the growth and reserve accumulation phase (damage at 0.4 g H2O (g DW)−1). As axes developed from maturity to germination, their threshold desiccation sensitivity increased to a higher level (1.3−1.4 g H2O (g DW)−1). For the very immature axes, enthalpy of the melting of tissue water was much lower, and the level of non-freezable water considerably higher, than for any other developmental stage studied.There were no marked correlations between desiccation sensitivity and thermal properties of water. Desiccation sensitivity appears to be related more to the degree of metabolic activity evidenced by ultrastructural characteristics than to the physical properties of water.

Effects of developmental status and dehydration rate on characteristics of water and desiccation-sensitivity in recalcitrant seeds of Camellia sinensis

Seed viability was maintained in four cultivars of oil palm (Elaeis guineensis Jacq.) during 12 months of hermetic storage at 15°C with 10–12% moisture content (embryo moisture contents of 19–21%). The viability of both these and drier seeds was reduced greatly during this period at cooler storage temperatures of 0°C and −20°C, however. For example, intact seeds at 6.1–7.4% moisture content (embryo moisture contents of 9.1–12.0%, at which freezing damage would not be expected) lost viability more rapidly at 0°C and −20°C than at 15°C. Moreover, desiccation to 4–5% moisture content (4–6% embryo moisture content) reduced seed lot viability in some but not all cultivars. The results confirm earlier reports that oil palm is not recalcitrant, but neither is it orthodox. Thus, seed storage behaviour in oil palm appears to be intermediate between these categories. Additional results are presented which suggest that the seeds of the royal palm (Oreodoxa regia HBK) may also show intermediate seed storage behaviour.

https://www.cambridge.org/core/services/aop-cambridge-core/content/view/S0960258500000726

U. Soetisna

Papers

Seed storage behaviour in Elaeis guineensis