II. Use of cryopreservation and reproductive technologies for conservation of genetic resources

5. The potential of cryopreservation and reproductive technologies for animal genetic resources convervation strategies

Sipke Joost Hiemstra, Tette van der Lende and Henri Woelders

5.1 SUMMARY

Ex situ conservation of genetic material from livestock and fish through cryopreservation is an important strategy to conserve genetic diversity in these species. Conservation strategies benefit from advances in cryopreservation and reproductive technologies. Choice of type of genetic material to be preserved for different species depends highly on objectives, technical feasibility (e.g. collection, cyropreservation), costs and practical circumstances.

5.2 INTRODUCTION

Global diversity in domestic animals is considered to be under threat. A large number of domestic animal breeds are endangered worldwide, in a critical status or already extinct. Of the 6 379 domestic animal breed populations, 9 percent are in critical condition and 39 percent are endangered (FAO, 2000). There is worldwide consensus on the global decline in domestic animal diversity and the need to conserve genetic diversity. The vast majority of aquatic genetic resources are found in wild populations of fish, invertebrates and aquatic plants. Domestication of aquatic species has not proceeded to the same level as it has in crop and livestock sectors. According to FAO, there are more than a thousand common aquatic species harvested by humans in major fisheries and thousands more species harvested in small-scale fisheries. The number of species in aquaculture is growing and several important species rely on the collection of brood stock or seed from natural populations.

In farm animals, trends in within-breed diversity are as important as between-breed diversity to be able to cope with changing requirements and future demands in breeding and selection. A small effective population size in rare or endangered breeds requires monitoring of within-breed diversity and conservation programmes to maintain within-breed diversity. Several authors also emphasized the reduction in effective population sizes of widely used domestic animal breeds (e.g. Weigel, 2001). Although introgression of genes for specific traits or characteristics from local breeds to commercial breeds has been very limited so far, Notter (1999) suggested that, as in plants, useful genes may exist in lowly productive types and recommended systematic programmes for genetic resources conservation, evaluation and use.

There are several options to conserve genetic diversity. In general, in situ conservation or conservation by utilization is preferred as a mechanism to conserve breeds. A breed has to evolve and adapt to changing environments and efforts should be promoted to create a need for products or functions of the breed. Conservation without further development of the breed or without expected future use is not a desirable strategy. However, in addition to in situ conservation, methods or techniques to maintain live animals outside their production or natural environment (ex situ live) or through cryopreservation of germplasm (ex situ) are set up to preserve (germplasm of) rare breeds as well as the more widely used commercial breeds. Moreover, cryopreservation of germplasm is a very good ex situ strategy to conserve existing allelic diversity for future use.

There is a growing interest in ex situ conservation strategies, serving a variety of objectives (ERFP, 2003). In many countries, ex situ conservation represents an integral component of conservation strategies (Blackburn, 2004). Some strategies focus primarily on preservation of germplasm of rare breeds, but in general there is consensus that ex situ collections should be established for all breeds with the aim to capture as much allelic or genetic diversity in conservation programmes as possible. Whereas in situ conservation or use of animal genetic resources is not necessarily dependent on high-tech approaches or facilities, the efficiency and efficacy of ex situ conservation strategies will certainly benefit from advances in cryopreservation and reproductive technology. Since ex situ conservation activities are in general rather costly, debate is ongoing on priorities, different methodologies and future use and benefits of cryopreservation and reproductive technology.

This chapter focuses on ex situ conservation. An overview of the state of the art in cryopreservation and reproductive technology for farm animals and fish is followed by a discussion on the implications for ex situ conservation strategies. This chapter is restricted to the main agricultural species; with regard to aquatic species, it deals with fish only and focuses on aquaculture rather than fisheries.

5.3 STATE OF THE ART IN CRYOPRESERVATION TECHNOLOGY

5.3.1 Cryobiologic principles

Cryopreservation allows virtually indefinite storage of biological material without deterioration over a time scale of at least several thousands of years (Mazur, 1985), but probably much longer. Important progress in cryobiology was achieved in the second half of the previous century. Much progress resulted from empirical studies. In later years, progress was also strongly stimulated by the development of fundamental theoretical cryobiology.

In so-called “slow cooling” methods, the biological material is cooled at a range of cooling rates that are fast enough to prevent “slow cooling damage” but slow enough to allow sufficient dehydration of the cells to prevent intracellular ice formation (IIF) (Mazur, Leibo and Chu, 1972). The dehydrated cells in the “unfrozen fraction” that remains between the masses of ice will ultimately reach a stable glassy state, or “vitrify”. In so-called “vitrification methods”, the water content is lowered before cooling by adding high concentrations of cryoprotective agents (CPA). Thus, no ice is formed at all, and the entire sample will vitrify. This allows fast cooling rates without risk of IIF. The CPA concentration of vitrification solutions can be minimized by using very high cooling and thawing rates. By using extremely high cooling rates, vitrification is possible even in complete absence of CPAs (Isachenko et al., 2004).

5.3.2 Semen

Semen of most livestock species can be frozen adequately. In addition, dedicated freezing media and equipment for collecting, packing, freezing and inseminating semen have been developed and are available commercially for a large number of bird and mammal livestock species. In the cattle artificial insemination (AI) industry, in which bulls are selected for “freezability” of their semen, the post-thaw semen quality is quite good, featuring 50 to 70 percent motile spermatozoa. Pregnancy or calving rate is the same as that of fresh semen, provided that higher sperm dosages are used for frozen sperm. For other mammalian species, the percentage of post-thaw motile sperm or membrane-intact sperm is generally somewhat lower, but a fair post-thaw viability can be expected for most species. For many species, the fertility of frozen semen is found to be lower than that of fresh semen. This may depend on the site of semen deposition, the morphology of the female genital tract, and the ability to detect heat or ovulation. For instance in sheep, very poor results are obtained with cervical semen deposition when using frozen rather than fresh ram semen (Molinia, Evans and Maxwell, 1996). There may be considerable differences between breeds and between males in the “freezability” of the semen. As a consequence, frozen semen of some genetically interesting breeds or males may not be suitable as a gene bank resource, or can be used only with a poor efficiency.

As regards avian livestock species, semen-freezing techniques for fowl, turkey, goose, and duck render a fair post-thaw sperm survival of up to 60 percent live spermatozoa. Reasonable insemination results with frozen-thawed semen have been reported for the major avian livestock species (Blesbois and Labbé, 2003; for more references, see Hammerstedt and Graham, 1992). However, there is a striking variation between studies in the reported percentages of fertilized eggs, as listed in Hammerstedt and Graham (1992), ranging from 9 to 91 percent. Moreover, the number of spermatozoa that gives maximal fertilization levels in chickens is much higher for frozen-thawed semen compared with fresh semen (Wishart, 1985).

More than 200 fish species with external fertilization have been tested for sperm cryopreservation (Blesbois and Labbé, 2003). The present state of the art for many species of fish seems to be adequate for the purpose of gene banking. The insemination ratios used may vary according to species and procedure between 104 and 107 spermatozoa per egg. Even in fish species like the African catfish, in which semen can only be obtained by testis destruction or death of the male, enough semen can be obtained from one single male to produce close to 106 larvae (Viveiros, So and Komen, 2000). Thus, for gene bank purposes, storage of only one single vial or straw would be sufficient to generate plenty of progeny of that male.

The freezing media vary widely between the classes (mammals, birds, fish), but also between species within a class. Most media feature a saline or saccharide bulk osmotic support, a suitable CPA at concentrations varying from 0.2–1.5 M, and various protective macromolecular additives, mostly milk and egg yolk components, or lipid components from vegetal origin (Bousseau et al., 1998). Milk or egg yolk is often used in media for mammalian semen. In mammalian semen, the egg yolk and milk components protect the spermatozoa during cooling, freezing and thawing (Watson, 1976). These additives are generally not used in freezing media for avian and fish species, although in a few studies with fish semen, egg yolk was found to confer protection against cryodamage (e.g. Cabrita et al., 1998).

Glycerol is widely used as a suitable CPA in mammalian, bird, and fish species. However, in poultry it is found that glycerol is contraceptive, i.e. the semen must be washed free of glycerol after thawing (Hammerstedt and Graham, 1992). The type of CPA used varies widely between species, and occasionally within one species, a CPA has been successfully used in one study and was found to be unsuitable in another study with the same species (Viveiros, So and Komen, 2000). Glycerol is used in most mammalian species. In avian species, dimethyl sulphoxide (DMSO), Ethylene glycol (EG), dimethylacetamide (DMA) and dimethylformamide (DMF) are also frequently used. In fish species, glycerol, DMA, DMF, DMSO and methanol are often used.

Semen is generally cryopreserved with “slow cooling” methods. Optimal cooling rates for freezing semen are mostly found between 10 and 100 °C/min. To some extent, the reported differences may be related to the use of different types of CPA and different CPA concentrations. An extreme example is that fowl semen can be effectively frozen at a cooling rate of approximately 600 °C/min when using dimethylacetamide as CPA, but not using glycerol (Woelders et al., in preparation). CPAs may differ widely in the cell membrane permeability, and may also affect the membrane permeability to water. These parameters greatly affect the velocity of dehydration, and therewith the optimal range of cooling rates.

5.3.3 Oocytes

In the last ten years, considerable progress has been made with cryopreservation of oocytes. Viable oocytes have been recovered after freezing and thawing in a great number of species (see references in Woelders, Zuidberg and Hiemstra, 2003). Successes have been reported in post-thaw oocyte maturation, fertilization, and embryo development in a number of species. Live-born young from embryos produced from cryopreserved oocytes have been reported in cattle (Otoi et al. 1996; Abe et al.,2005), mice (Frydman et al., 1997), (Stachecki et al., 2002), rats (Nakagata, 1992), horses (Maclellan et al., 2002) and humans (Stachecki and Cohen, 2004). The present efficiency and reliability of using frozen thawed oocytes for generating offspring is still much lower than with cryopreserved embryos. Freezing oocytes of avian and fish species is not successful (Blesbois and Labbé, 2003), however, largely because of the large size, the high lipid content, and the polar organization (vegetal and animal pole) of bird and fish ova.

5.3.4 Embryos or embryonic cells

In cattle, cryopreservation of embryos is highly successful. Both slow freezing and vitrification protocols are effective. The success of cryopreservation depends on the stage of the embryo, that is, especially good results are obtained with blastocysts. Cryopreservation of embryos resulting in live offspring has been reported for most of the important (mammalian) livestock species (reviewed by Paynter et al., 1997; see also Cognie et al., 2003; Squires et al., 2003). Cryopreservation of pig embryos has long been problematic due to extreme chilling sensitivity and high lipid content of the pig embryos. However, recent studies have focused on overcoming these problems and produced successful vitrification methods for cryopreservation of pig embryos (e.g. Vajta, 2000; Nagashima et al., 1999; Dobrinsky et al., 2000).

Embryo cryopreservation is not viable in birds (Blesbois and Labbé, 2003) and fish (Hagedorn et al., 2004) species, largely because of the same limitations as in the case of avian and fish oocytes, i.e. the large size, the high lipid content, and the polar organization of the ova and the early embryos of fish and birds. However, in birds and fish species, cryopreservation of isolated embryonic cells is an option. Post-thaw survival of blastomeres was demonstrated in rainbow trout, carp and medaka (see references in Blesbois and Labbé, 2003). Embryonic cells and recipient embryos can be used to produce chimeric embryos. Provided that the gonads become populated with primordial germ cells from the donor embryo, such chimeric embryos can be used to produce future progeny of the donor genotype (e.g. Nakagawa, Kobayashi and Ueno, 2002). In chicken, the primordial germ cells (PGC) can be specifically harvested. Recently, improvement of the efficiency of producing chimerae with donor genotype germinal cells was achieved by depleting PGC from the recipient embryos using busulfan (Song et al., 2005).

5.3.5 Somatic cells

Cryopreservation of somatic cells proved to be possible for a number of cell types. In early studies, the methods came down to adding 5 to 10 percent of a suitable cryoprotectant, such as glycerol or DMSO to the suspension of cells in culture medium, and placing tubes with a few millilitres of that suspension at -80 °C in a mechanical freezer. In fact, this simple procedure is still effectively used today. Obviously, with this simple procedure the rate of cooling cannot be controlled; in fact in many publications the cooling rate is unknown. There are only a few studies in which controlled rate freezers were used, e.g. with skin fibroblast.

5.3.6 Further progress

More attention to fundamental aspects of cryobiology should enable further progress in cryopreservation methods. A fundamental approach has been taken in many studies concerning mammalian semen and embryos (Gilmore et al., 1996, Liu et al., 2000; Chaveiro et al., 2004), but fewer concerning avian and fish semen (e.g. Viveiros et al., 2001). Recently, a theoretical model was presented to predict the optimal cooling programme for “slow cooling” freezing methods (Woelders and Chaveiro, 2004). The model indicated that a non-linear cooling profile could give better results than linear freezing programmes. This and other models (cf. Liu et al., 2000) also demonstrate that the optimal cooling rate can be expected to be inversely related to the CPA concentration, and in fact, this is found in empirical studies. Therefore, it is important to address both factors in empirical optimization studies. It can also mean that a lower concentration of CPA would become feasible provided that a higher cooling rate is used. Further improvement could result from preventing delayed ice formation or “supercooling”, e.g. by using so-called “directional solidification” methods (Woelders et al., 2005). Improving the freezing methods can raise the general level so that even the semen of “bad freezers” would have an adequate post-thaw sperm survival (ibid.).

Attempts to vitrify spermatozoa have not been successful to date. It has recently been shown that vitrification of human spermatozoa is possible in the absence of CPA by using an extremely high cooling rate of 720 000 °C/min (Isachenko et al., 2004). In this way, damage due to the presence of the CPA, chilling injury and ice formation may be avoided. Further improvement of vitrification techniques is especially important for freezing cells that are sensitive to chilling, e.g. to “outrun” spindle microtubule depolymerization in metaphase II oocytes. Very high cooling rates can be applied in the open-pulled straw (OPS) technique (Vajta et al., 1998) or by using the cryoloop (Lane, Schoolcraft and Gardner, 1999; Isachenko et al., 2004). However, interrupted slow cooling methods can also be highly effective, as a fully normal and functional spindle can reform after thawing (Stachecki and Cohen, 2004).

5.4 STATE OF THE ART IN REPRODUCTIVE TECHNOLOGY

5.4.1 Artificial insemination (AI)

In several species, AI techniques and strategies have been improved and knowledge on the fate of sperm in the female genital tract (e.g. phagocytosis) improved during the last decades. However, there are large differences between species in insemination techniques and pregnancy rates using fresh or frozen semen. In cattle and pigs, existing AI infrastructure allows easy collection and future use of semen, but only in cattle has the use of frozen semen replaced the use of fresh semen. In pig production, disadvantages of using frozen semen (reduced fertility, high freezing, storage and transport costs) are still greater than the advantages.

In sheep, surgical (laparoscopic) AI gives much better pregnancy rates than cervical AI. However, laparoscopic AI is more laborious and also more invasive than cervical AI. Molinia et al. (1996) showed that the difference in pregnancy rates between surgical and non-surgical AI was even larger with frozen semen than with fresh semen: 70 percent versus 20 percent pregnancy with non-surgical AI (with 180 x 106 sperm) vs. surgical AI (with 10 x 106 sperm). It is believed that frozen-thawed sperm are less motile and lack stamina to transverse the highly viscous cervical mucus, but phagocytosis of the sperm by leukocytes is also considered a cause of the reduced fertility. Development of a non-surgical technique to reach the oviductal end of the uterine horns as closely as possible would enhance the efficiency and ease of use of cryopreserved semen in sheep. Such deep intrauterine insemination techniques have been developed in pigs (reviewed by Vazguez et al., 2005) and may in general contribute to the more efficient use of semen (less sperm per insemination).

AI can be used successfully in poultry, but is not used extensively in any domestic avian species except turkeys where it is used almost exclusively for commercial flock production (reviewed by Donoghue and Wishart, 2000).

5.4.2 Embryo transfer

Surgical embryo transfer could be possible in principle in all mammalian livestock species. In contrast, non-surgical embryo transfer is only possible in cattle (routinely performed), horses and pigs, although still not as efficient as in cattle and horses. For embryo transfer purposes, embryos can either be flushed from donors or can be produced in vitro. Surgical embryo collection is in principle possible in all mammalian livestock species. In contrast, non-surgical embryo collection is only possible in cattle and horses. After surgically shortening the long uterine horns of the pig, non-surgical recovery of embryos has also been proven possible in pigs (Hazeleger et al., 1989). Although ethical issues have prevented the further use of this method, it may be used in specific situations, e.g. to collect large numbers of embryos in very rare pig breeds in a relatively short time. The efficiency of non-surgical embryo collection in cattle, and to a lesser extent in horses, can be improved by hormonal induction of superovulation.

In vitro production of embryos by in vitro maturation and fertilization of oocytes is possible for major livestock breeds but the efficiency varies between species. Oocytes for this purpose can either be collected by aspiration of immature oocytes from ovaries from slaughtered (or deceased) animals or by the use of ovum pick-up techniques in live animals. The latter techniques are presently mainly in use in cattle and horses but could also be used in other livestock species.

5.4.3 Reproductive cloning

Reproductive cloning involves the collection of oocytes, culture and in vitro maturation of oocytes, enucleation of oocytes, transfer of (somatic) nuclei to, or fusion of the somatic cells with, enucleated oocytes, culture of the resulting embryos, and finally, embryo transfer into recipients of the same or a highly related species (reviewed by Galli, Lagutina and Lazzari, 2003). The use of nuclear transfer means that the original mitochondrial genotype of the nucleus donor is lost.

In mammals, live offspring have been obtained from embryos generated from somatic cells in a number of species, i.e. sheep, cattle, mice, pigs, goats, horses, rabbits and cats. Until now, cloning has failed in rats, rhesus monkeys and dogs. Remarkably, some success (embryo development but no live offspring) was even obtained when bovine oocytes were used as recipients for somatic nuclei from other mammalian species (e.g. Dominko et al., 1999). It must be emphasized, however, that current techniques are inadequate to be used safely and efficiently for procreation. In all published research, only a small proportion of embryos produced by using somatic cells developed into live offspring, i.e. typically less than 4 percent (Paterson et al., 2003). The low overall success rate is the cumulative result of inefficiencies at each stage of the cloning process. Many pregnancies are terminated by abortion and full-term pregnancies frequently result in abnormal offspring. It therefore seems that current cloning techniques introduce errors that affect prenatal development. Even apparently healthy live-born offspring could have anomalies that only become apparent later in life, or in the next generation of animals. On a long time horizon it is very likely that cloning methodology will become both reliable and efficient.

In fish, successful cloning has been reported by Lee et al. (2002). In their experiments with zebrafish, an overall success rate of 2 percent was achieved. To the best knowledge available, no successful cloning has been reported in poultry.

5.4.4 Miscellaneous emerging reproductive technologies

Transplantation of ovarian tissue and germ cells (e.g. PGC or spermatogonial stem cells [SSC]) are emerging technologies with potential for future use in conservation programmes. Autotransplantation of ovarian tissue has been developed to restore fertility in women after aggressive chemotherapy resulting in ovarian failure. Successful transplantation of ovarian tissue has been reported in rodents, sheep, marmoset monkeys and humans (references in Donnez et al., 2004). Successful whole sheep ovary cryopreservation and autotransplantation has recently been reported (Revel et al., 2004). The potential of ovary transplantation as a tool in genetic conservation is underlined by the work of Dorsch et al. (2004). Their experiments demonstrate that transplantation of rat ovaries can be used as a tool for the rescue of rat strains where females are unable to reproduce despite having normal ovarian cycles.

Germ cell transplantation research has been developed as a unique approach for the study of gametogenesis and germ line manipulation. To date, successful germ cell transplantations have been reported in several livestock species, e.g. transplantation of SSC in cattle (Izadyar et al., 2003) and goats (Honaramooz et al., 2003), but also in poultry (e.g. Park et al., 2003) and fish (e.g. Takeuchi, Yoshizaki and Takeuchi, 2003). As far as application of this technology in fish is concerned, fascinating results of allogenic transplantation in rainbow trout have been reported (ibid.). By transplanting PGC of donors into the peritoneal cavity of hatching recipient embryos, live fry with donor-derived phenotype were produced from gametes of PGC-recipients.

Although many hurdles have to be overcome, these emerging technologies may enable production of gametes or offspring of rare or extinct breeds in the long term by abundantly available individuals of related common breeds. The first steps to overcome limitations for homologous transfer of ovarian tissue and germ cells are now underway; the development of an effective recipient preparation protocol in mice (Brinster et al., 2003) is an example.

5.5 IMPLICATIONS FOR EX SITU CONSERVATION STRATEGIES

The choice of type of material to be preserved and sampling strategies depends, inter alia, on the objectives of cryopreservation programmes (ERFP, 2003). Decisions will be different between species because of variation in technical feasibility, costs and practical circumstances for cryopreservation of different types of material. In general, cryopreservation and associated reproductive technologies are costly; the main limitations for extensive development of ex situ collections are high costs of collection and limited use of preserved material (FAO, 2004). Costs of sampling, collection, freezing, storage and use of genetic material differ between species, and optimum strategies depend on local circumstances, availability of technology and costs of labour and facilities. Gandini and Pizzi (2003) reviewed the literature on conservation costs (in situ and ex situ) and concluded that published information on ex situ conservation costs was very limited and not very timely. Labroue et al. (2001) calculated total costs for creating pig semen storage among four European countries at about 30 000 euros per breed and 15 euro per dose. Costs of cryopreservation of pig semen in the Netherlands (1999–2001) were estimated at approximately 10 euro per dose, based on costs for labour, laboratory materials and infrastructure, assuming a freezing capacity of six ejaculates per day. Transport costs of the semen from the AI centre to the freezing facility and costs for semen collection are not included in this figure.

The Centre for Genetic Resources (CGN) observed that costs of collecting and freezing of semen of different species vary from less than 1 euro per dose (cattle) to more than 20 euro per dose (sheep and poultry). The higher costs in sheep and poultry are due to the much higher handling, training, collection and freezing costs per dose of semen and the lack of AI infrastructure in these species. As an alternative for semen collection and freezing of ejaculated semen, CGN concluded that collection and freezing of epididymal semen of culled rams is a cost-effective method to conserve genetic diversity in sheep breeds (Woelders et al., in preparation).

Differences in generation interval and reproductive rates between species may also influence decision-making in conservation programmes. In some species it is possible to regenerate a breed very quickly with inexpensive, sometimes less sophisticated methods, compared to other species. For example, in fish the regeneration of an extinct species with stored semen is feasible through backcrossing since fish species have a low generation interval and a high annual turnover. In contrast, such a strategy in horse or cattle would be time-consuming and extremely expensive. In these species, cryo-banking of embryos rather than sperm is highly preferable.

Costs of embryo collection and freezing are much higher than those for semen collection and freezing. However, regeneration costs using embryos are much lower than those for semen (repeated backcrossing). Many conservation programmes focus on freezing of semen only. If the aim is to conserve breeds and taking into account the loss of mitochondrial DNA and the time lag to re-establish a breed by backcrossing, collection and cryopreservation of embryos is underrated. In this context, cryopreservation of somatic cells does not seem to be a good alternative for cryopreservation of embryos, even if the efficiency of cloning is largely improved. Upfront costs of freezing somatic cells may be very low, but mitochondrial DNA is not conserved and the efficiency of subsequent steps in reproductive cloning can never beat the efficiency of cryo-conservation and implantation of embryos. Storage of both oocytes and semen may also be efficient in terms of sampling and freezing costs. However, high costs are associated with in vitro fertilization and ovum pick-up (OPU). Costs are expected not to be lower overall than when using embryos instead of semen plus oocytes.

When survival of material after freezing/thawing improves and the chance of pregnancy increases, costs of sampling and freezing of gene bank material will drop because less genetic material is needed in the gene bank to generate a sufficient number of live offspring. Furthermore, if freezability of semen of genetically important males can be improved substantially (especially in the case of “bad freezers”), sampling costs will drop even more.

Cryopreservation technology strongly affected reproduction in livestock. Ironically, unsustainable use of cryopreservation can cause a decline in genetic diversity, but at the same time its use is beneficial when applied to conservation programmes. For example, in dairy cattle the combined use of genetic evaluation, AI and frozen semen, and more recently, several other reproductive technologies, has resulted in high genetic gain in the Holstein Friesian (HF) breed and thus stimulated their worldwide, large-scale use at the cost of local dairy breeds and a decline in the effective population size of the HF breed. As a side effect, however, know-how on cryopreservation, AI and other reproductive technologies developed for use in cattle has turned out to be of major importance for the conservation of breeds of various species.

5.6 CONCLUSIONS

• In ex situ conservation programmes with the aim to conserve breeds, collection and cryopreservation of embryos is underrated, and should be given more attention.

• Advances in cryopreservation and reproductive technology have contributed and will continue to contribute to the efficiency and effectiveness of conservation programmes.

• Species in which cryopreservation and reproductive technology are less developed can benefit from advances in other species.

• Decision-makers in conservation programmes should regularly reconsider the balance between objectives, costs, technical feasibility and practical feasibility.

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6. Status of cryopreservation technologies in plants (crops and forest trees)

Bart Panis and Maurizio Lambardi

6.1 SUMMARY

Over the past decades, plant cryopreservation technologies have been evolving rapidly, opening the door to the possibility of long-term storage of valuable genetic resources of many crop and forest species. From the original slow-cooling approach, research has moved to easier and more reproducible techniques, which allow the complete vitrification of extra- and intra-cellular liquids. This chapter describes concisely the procedures that have been proposed in time for the cryopreservation of a wide range of tissues and organs, such as cell suspensions, embryogenic callus, pollen, meristematic tissues, seeds and embryo axes. In addition, the most important achievements in the cryopreservation of herbaceous, hardwood and softwood species are discussed.

6.2 INTRODUCTION

It is estimated that up to 100 000 plants, representing more than one-third of the world's plant species, are currently threatened or face extinction in the wild (BGCI, 2005). Preservation of the plant biodiversity is essential for classical and modern (genetic engineering) plant breeding programmes. Moreover, this biodiversity provides a source of compounds to the pharmaceutical, food and crop protection industries. Since the 1970s, large numbers of landraces and wild relatives of cultivated crops have been sampled and stored in ex situ gene banks. It is estimated that six million samples of plant genetic resources are held in national, regional, international and private gene bank collections around the world (IPGRI, 2004). Storage of desiccated seeds at low temperature, the most convenient method to preserve plant germplasm, is not applicable to crops that do not produce seed, such as bananas, nor to those with recalcitrant seed - non-orthodox seed that cannot be dried to moisture contents that are low enough for storage, for instance, many tropical trees, nor to plant species that are propagated vegetatively to preserve the unique genomic constitution of cultivars, such as fruit and several timber and ornamental trees. For these species, although clonal orchards play a pre-eminent role in assisting conservation programmes, their maintenance requires large areas of land and high running costs, mainly for pruning operations, weed and pest management and irrigation. Further, they are prone to environmental stresses such as heavy frosts and flooding and to hazards such as pests, diseases and genetic alterations; hence, valuable germplasm can be easily lost (genetic erosion). In vitro collections, established for some vegetatively propagated species and maintained by means of traditional micropropagation, is labour-intensive as well, and there is always the risk of losing accessions due to contamination, human error or somaclonal variation (i.e. mutations that occur spontaneously in tissue culture, with a frequency that increases with repeated subculturing).

Today, tissue culture technology offers two major options to ex situ conservation of valuable plant germplasm - slow growth storage (i.e. the medium-term conservation of stock cultures at few degrees above zero) and cryopreservation (Benson, 1999). Cryopreservation, or freeze-preservation at ultra-low temperature (-196 °C, i.e., the temperature of liquid nitrogen), is a sound alternative for the long-term conservation of plant genetic resources since biochemical and most physical processes are completely arrested under these conditions. As such, plant material can be stored for unlimited periods. Moreover, in addition to its use for the conservation of genetic resources, cryopreservation proved to be extremely useful for the safe long-term storage of plant tissues with specific characteristics, such as medicinal- and alkaloid-producing cell lines, hairy root cultures, and genetically transformed (Elleuch et al., 1998) and transformation-competent culture lines (Gordon-Kamm et al., 1990). Recently, it was also proven that cryotherapy can be successfully applied to eradicate viruses from plum, banana and grape (Brison et al., 1997; Helliot et al., 2002; Wang et al., 2003). However, despite the fact that cryogenic procedures are now being developed for an increasing number of recalcitrant seeds and in vitro tissues/organs, the routine utilization of cryopreservation for the preservation of plant biodiversity is still limited.

6.3 THE THEORETICAL BASIS OF PLANT CRYOPRESERVATION

Cryopreservation of biological tissues can be successful only if intra-cellular ice crystal formation is avoided since this causes irreversible damage to cell membranes, thus destroying their semi-permeability. In nature, some plant species adopted systems where ice crystal formation at sub-zero temperatures can be avoided through the synthesis of specific substances (such as sugars, proline and proteins) that lower the freezing-point in the living plant cells, resulting in “supercooling”. Such “avoidance” of crystallization, while still maintaining a minimal moisture level needed to maintain viability, is not possible when dealing with ultra-low temperatures of cryopreservation. Crystal formation, without an extreme reduction of cellular water, can only be prevented through vitrification. Vitrification refers to the physical transition process from an aqueous solution to an amorphous and glassy (i.e. non-crystalline) (Sakai, 2000). Two requirements must be met for a cell to vitrify: rapid freezing rates and a concentrated cellular solution. Rapid freezing rates (6 °C/sec) are normally obtained by plunging explants enclosed in a cryovial into liquid nitrogen. Higher cooling rates can be obtained by enclosing the meristems in semen straws, resulting in cooling rates of about 60 °C/sec, or using a “droplet freezing protocol” where the material is placed on aluminium foil strips plunged directly into liquid nitrogen, giving rise to cooling rates of 130 °C/sec (Panis, Piette and Swennen, 2005; Schäfer-Menuhr, Schumacher and Mix-Wagner, 1997).

The cell cytosol can be concentrated through air drying, freeze dehydration, application of penetrating or non-penetrating substances (cryoprotectants), or adaptive metabolism (hardening). For a solution to be vitrified at high cooling rates, a reduction in water content to at least 20 to 30 percent is required. For dehydration, the following techniques are applied:

6.3.1 Air drying

Samples are usually dried by the sterile airflow of a laminar airflow cabinet. However, with this method there is no control of temperature and air humidity, both strongly influencing evaporation rates. More reproducible is the air-drying method that uses closed vials containing a fixed amount of silica gel (Uragami, Sakai and Nagai, 1990).

6.3.2 Freeze dehydration

Since plant cells rarely contain ice-nucleating agents, during a slow cooling process, crystallization is initiated first in the extra-cellular spaces. Since only a proportion of the water that contributes to the extra-cellular solution undergoes a transition into ice, the remaining solution becomes increasingly concentrated and thus hypertonic to the cell. To restore the osmotic equilibrium, cellular water will leave the protoplast, resulting in cell dehydration. Generally, freezing rates of 0.5 to 2 °C/min, depending on the type and physiological state of the plant material, are applied. These slow-cooling rates can be obtained using computer-driven cooling devices, stirred methanol baths, and propanol containers held at -70 °C.

6.3.3 Non-penetrating cryoprotective substances

Osmotic dehydration can be obtained through the application of non-penetrating cryoprotective substances, such as sugars, sugar alcohols and high molecular weight additives such as polyethylene glycol (PEG).

6.3.4 Penetrating cryoprotective substances

Commonly used penetrating cryoprotective agents are dimethyl sulphoxide (DMSO) and glycerol. DMSO is preferred for many applications due to its extreme rapid penetration into the cells. Where DMSO toxicity is a problem, glycerol or amino acids (e.g. proline) are often applied.

6.3.5 Adaptive metabolism (hardening)

Hardening is a process that increases the plant's ability to survive the impact of unfavourable environmental stress. In nature this is triggered by environmental parameters, such as reduction in temperature and shortening of day length. Osmotic changes and abscisic acid (ABA) treatments can also have similar effects. Hardening can result in a considerable increase of proteins, sugars, glycerol, proline and glycine betaine, which all contribute to increasing the osmotic value of the cell solutes.

Most hydrated tissues, however, do not withstand dehydration to moisture contents needed for vitrification (20 to 30 percent) due to solution and mechanical effects. Exceptions are pollen, seeds and somatic embryos of most orthodox seed species. The key for successful cryopreservation thus shifts from freezing tolerance to dehydration tolerance. This tolerance can be induced by chemical cryoprotection with substances like sugars, amino acids, DMSO, glycerol. The mode of action of most of these substances is, however, still far from being understood. Alternatively, tolerance to dehydration can also be induced by adaptive metabolism. For example, it has been observed that cold acclimation in nature often leads to the accumulation of specific proteins, sugars, polyamines and other compounds that can protect cell components during drying. Sugar treatments also result in alterations in protein (Carpentier et al., 2005) and membrane composition (Ramon et al., 2002), the latter influencing both its flexibility and permeability.

6.4 AVAILABLE PLANT CRYOPRESERVATION PROTOCOLS

All cryopreservation protocols described in literature use the above-mentioned techniques or combinations. The most commonly applied protocols are:

6.4.1 Air drying (flash drying, normal drying)

This method is directly applicable to orthodox seed, zygotic embryos and pollen of many common agricultural and horticultural species. Some of these orthodox seeds can even withstand drying below 3 percent moisture content, without any damage and reduction of viability. Flash (or ultra-rapid) drying proved to be beneficial for recalcitrant zygotic embryos of some plant species (Berjak et al., 2000).

6.4.2 Classical slow-cooling (or slow-freezing) protocol

This was the first “standard” protocol developed for hydrated plant tissues (Withers and King, 1980). It is based on slow cooling of specimens (at a rate of 0.5 to 2 °C/min) in the presence of a cryoprotectant solution, generally containing DMSO at a 5 to 15 percent concentration. When during the slow-cooling process a temperature of about -40 °C is reached, the intra-cellular solution is considered to be concentrated enough to vitrify on a subsequent liquid nitrogen plunge. This method is mainly used today for cryopreservation of non-organized tissues, such as cell suspensions and calli.

6.4.3 Encapsulation/dehydration

In this method, developed by Fabre and Dereuddre (1990), explants (usually meristems or embryos) are firstly encapsulated in alginate beads (which can also contain mineral salts and organic substances), thus forming “synthetic seeds” (artificial seeds or synseeds). These synseeds are then treated with a high sucrose concentration, dried down to a moisture content of 20 to 30 percent (under airflow or using silica gel) and subsequently rapidly frozen in liquid nitrogen. Although the procedure can be considered rather lengthy and labour-intensive, it is observed that the presence of a nutritive matrix (the bead) surrounding the explant can promote its regrowth after thawing.

6.4.4 Vitrification

First reports on the use of a vitrification solution with plant tissues appeared in 1989 (Langis et al., 1989; Uragami et al., 1989). The technique relies on treatment of the explants with a concentrated vitrification solution for variable periods of time, from 15 minutes up to two hours, followed by a direct plunge into liquid nitrogen (“vitrification/one-step freezing”). This results in both intra- and extra-cellular vitrification. The vitrification solution consists of a concentrated mixture of penetrating and non-penetrating cryoprotective substances. The most commonly applied solution, called “Plant Vitrification Solution no. 2” (PVS2), consists of 30 percent glycerol, 15 percent ethylene glycol, 15 percent DMSO (all percentage v/v) and 0.4 M sucrose (Sakai, Kobayashi and Oiyama, 1990). Fifteen years after its first report, vitrification is today by far the most widely used cryopreservation protocol. The success of the procedure can be attributed to its ease, high reproducibility and to the fact that it can successfully be applied to a wide range of tissues and plant species.

6.4.5 Other protocols

Other available methods are the “droplet freezing” (Schäfer-Menuhr, Schumacher and Mix-Wagner, 1997), the “preculture method” (Panis et al., 1996) and the “preculture/dehydration” (Dumet et al., 1993). Up to now these techniques have been applied to only a limited number of plant species and are not described in detail in this review. A recent and promising technique, “encapsulation/vitrification” (Sakai, 2000), is mentioned below in greater detail.

6.5 APPLICATION OF CRYOPRESERVATION TO HERBACEOUS SPECIES

6.5.1 Cell suspensions and callus cultures

Cell suspension and callus cultures are often cryopreserved using the classical slow-cooling protocol. The main aim of cryopreserving these non-organized tissues is not the long-term storage of the genetic diversity, but the conservation of specific features of these tissues that can be lost during normal in vitro maintenance. It has been repeatedly reported that the morphogenetic potential of embryogenic callus lines is not affected by their storage in liquid nitrogen. Moreover, it was proven that cryopreservation did not affect the expression of a foreign sam gene in transgenic Papaver somniferum cells (Elleuch et al., 1998), and was beneficial for pyrethrin biosynthesis by Chrysanthemum cinerariaefolium cell cultures (Hitmi, Sallanon and Barthomeuf, 1997). Also, the production of regenerable protoplasts is not influenced by cryopreservation, as shown for rice (Jain, Jain and Wu, 1996; Meijer et al., 1991), Festuca and Lolium species (Wang et al., 1994). Moreover, cryopreserved rice (Cornejo, Wong and Blechl, 1995) and maize calli (Gordon-Kamm et al., 1990) proved to be a constant source of regenerable cell cultures for the production of transgenic plants. Moukadiri, Lopes and Cornejo (1999) showed that rice calli, which were stored in liquid nitrogen, had a higher competence for transformation, as indicated by transient gene expression levels. In banana and grape, comparable levels of transient expression, as well as stable transformation, were obtained from cryopreserved and non-cryopreserved suspension cells (Panis et al., 2004; Wang et al. 1994). Large-scale cryopreservation of non-organized cultures is reported for coffee (Florin, Brulard and Lepage, 1999), oil palm (Dumet et al., 1994) and banana (Panis et al. , 2004).

6.5.2 Cryopreservation of pollen

Pollen is stored for facilitating crosses in breeding programmes, distributing and exchanging germplasm among locations, and preserving nuclear genes of germplasm, as well as for studies in basic physiology, biochemistry, fertility and biotechnology involving gene expression, transformation and in vitro fertilization (Towill and Waters, 2000). For example, preservation of pollen can be very useful for cross-pollination of cultivars differing in flowering period. There are methods for the cryopreservation of pollen from many crops (Towill, 1985.), but its application is still rather low and limited to a few research centres (Engelmann, 2004).

6.5.3 Cryopreservation of meristematic tissues

Shoot-meristematic tissues are the most commonly used explants for the cryopreservation of vegetatively propagated species, such as fruit trees and many root and tuber crops. Also, in view of the lower chance for somaclonal variation, organized tissues such as meristems are often preferred over non-organized tissues, such as calli and cell suspensions. Most recent reports deal with encapsulation/dehydration and vitrification methods. It is presumed that in view of its rather severe freeze-dehydration, the classical slow-cooling method is effective in retaining the integrity of individual cells, but less efficient in retaining the tissue integrity necessary for meristem survival. There are many scientific publications on the cryopreservation of meristems (“shoot tips”), but its large-scale application is mainly found in fruit crop germplasm collections (see 6.6.2. “cryopreservation of hardwood trees”) in case of herbaceous species, the number of accessions stored in liquid nitrogen is significantly smaller, but continuously growing.

At the German Collection of Micro-Organisms and Cell Cultures (DSMZ, Braunschweig, Germany), meristems of 519 old potato varieties are cryopreserved using the droplet freezing method (Mix-Wagner, Schumacher and Cross, 2003), while at the International Potato Centre (CIP, Lima, Peru), 345 potato accessions are preserved using the vitrification protocol (Panta, personal communication). At K.U. Leuven, Belgium, 306 banana accessions are currently safely stored using the droplet vitrification method (Panis, Piette and Swennen, 2005), representing more than one-quarter of the world's banana collection (INIBAP, 2005). Significant efforts are also made for cassava (Roca et al., 2000), garlic (Kim et al., 2004; Volk, Maness and Rotindo, 2004), mint (Hirai and Sakai, 1999; Towill and Bonnart, 2003) and Australian endangered species (Turner et al., 2001).

6.5.4 Cryopreservation of seeds

Seeds of most common agricultural and horticultural species are tolerant to desiccation and exposure to liquid nitrogen (Stanwood, 1985). Cryogenic storage of orthodox seeds can be considered an alternative to the traditional storage at -20 °C. For some plants species, seed longevity at -20 °C is only a few years and can thus be increased considerably through storage in the vapour phase of liquid nitrogen. In most cases, simple drying to lower the moisture content to 5 to 10 percent is sufficient to resist ultra-low temperatures. Celery is an example of orthodox seed cryopreservation (Gonzalés-Benito et al., 1995).

6.6 APPLICATION OF CRYOPRESERVATION TO WOODY SPECIES

Up to now seed and field collections have been the only reliable option for the long-term germplasm preservation of woody species. However, numerous forest Angiosperms (e.g. Acer spp., Quercus spp., chestnut, horsechestnut and many tropical species) have non-orthodox seeds with a very limited storage period. Fruit trees, which are mainly vegetatively propagated, require the conservation of huge numbers of accessions in clonal orchards (including old and newly selected cultivars, local varieties and wild material) where a periodic and careful monitoring of the preserved trees is essential. The preservation of the Prunus European germplasm, for instance, requires the maintenance of over 30 000 accessions in field repositories spread over 21 countries (Gass, Tobutt and Zanetto, 1996). Hence, cryopreservation should be regarded as a strategically important support to traditional in-field banks. Indeed, the combination of traditional conservation approaches with the potential of cryogenic technology represents an important step forward in minimizing the risks of accidental loss of woody plant genetic resources.

6.6.1 Organs and tissues for woody plant cryopreservation

Recent advances in plant tissue culture have greatly increased the variety of organs and tissues that can be used for storage in liquid nitrogen. Among the explants described above for herbaceous species, three categories are mainly used for woody plant cryopreservation:

1. the shoot tips, differentiated organs which are used for the preservation of vegetatively propagated plants, such as many fruit and timber tree cultivars for which the maintenance of genetic fidelity is fundamental. Shoot tips (1–2 mm, on average) are obtained from apical or axillary buds, excised from in vitro-grown shoot cultures. Under sterile conditions, the bud is handled in order to obtain the apical meristem, surrounded by some of the original leaf primordia and leaflets (Niino et al., 1992);

2. the seeds or the isolated embryo axes for the preservation of species that are mainly reproduced by seeds. For those species characterized by non-or sub-orthodox seeds, which cannot be stored in traditional seed-banks, cryopreservation should be regarded as an important option for the long-term conservation of genetic resources (Pence, 1995);

3. lines of embryogenic callus, which permit the germplasm conservation of plants with non-orthodox seeds, as well as the maintenance of valuable embryogenic lines utilized in bioengineering, e.g. avoiding the loss of embryogenic potential due to repeated subculturing or allowing the storage of transgenic material while field trials are ongoing.

6.6.2 Cryopreservation of hardwood trees

In recent years, the “vitrification/one-step freezing” technique has been continuously improved and applied to an increasing number of hardwood species. The application of this cryogenic technique to woody species became very popular after the PVS2 solution was introduced for the cryopreservation of Citrus sinensis nucellar cells (Sakai, Kobayashi and Oiyama, 1990). To date, the PVS2 solution has been successfully used for the cryopreservation of shoot tips from several economically-important hardwood genera, such as Malus (Niino et al., 1992), Pyrus (ibid.; Tahtamouni and Shibli, 1999), Prunus (Chunnuntapipat et al., 2000; De Carlo, Benelli and Lambardi, 2000; Niino et al., 1997), Castanea (Vidal et al., 2005), Populus (Lambardi, 2002) and Vitis (Matsumoto and Sakai, 2000), easily achieving survival rates higher than 50 percent (Lambardi and De Carlo, 2003). Highest percentages of shoot-tip survival (over 90 percent) are reported for Prunus jamasakura(Niino et al., 1997) and Populus alba (Lambardi, Fabbri and Caccavale, 2000). It is important to note that when loading explants with the PVS2 solution, post-thaw survival is to a great extent influenced by the duration of treatment, which must be long enough to ensure sufficient cell dehydration without cytotoxic effects. When the solution is applied at 25 °C, exposure time of woody-plant shoot tips ranges from 20 (Matsumoto et al., 2001) to 120 min (Niino et al., 1997; Vidal et al., 2005). Alternatively, to reduce the risk of toxicity, a chilled solution (0 °C) can be used (Caccavale, Lambardi and Fabbri, 1998; Valladares et al., 2004), or a gradient of concentration is applied (e.g. 50 percent for 30 min followed by 100 percent for 50 min) (Matsumoto and Sakai, 2000). Following the storage in liquid nitrogen, rapid warming in a waterbath is required. This will avoid recrystallization and ensures a proper recovery of the vitrified material. Thawing temperatures ranging from 20 °C to 45 °C have been proposed for woody species, 40 °C (corresponding approximately to a warming rate of 180 °C/min) being by far the most commonly applied (Lambardi and De Carlo, 2003).

In addition, the “encapsulation/dehydration” technique has repeatedly shown itself to be very effective for the long-term conservation of hardwood species. Effective protocols have already been developed for several important species, such as Malus, Pyrus and Prunus spp., with post-thaw shoot-tip survival and regrowth rates often exceeding 80 percent (Lambardi and De Carlo, 2003).

“Encapsulation/vitrification” is a new technique (Sakai, 2000), combining the encapsulation of explants with the application of a vitrification mixture. It has already been successfully applied to apple (Paul, Daigny and Sangwan-Norreel, 2000) and plum (De Carlo, Benelli and Lambardi, 2000) shoot tips. On the other hand, it showed to be less effective than a classic “vitrification/one-step freezing” approach for the long-term preservation of juvenile shoot tips of common ash (Schoenweiss, Meier-Dinkel and Rüdiger, 2005) showing that further improvement of protocols is necessary.

Recently, the cryopreservation method for dormant-vegetative buds (winter buds), based on the original procedure described by Sakai (1960), was applied to 1 915 apple accessions (Towill et al., 2004). Accordingly, winter-collected scions were desiccated in a cold room at -5 °C to 30 percent moisture and cooled down slowly (1 °C per hour) to -30 °C, where they remained for 24 hours prior to being transferred to the vapour phase of liquid nitrogen (-160 °C). After retrieval from storage and grafting onto rootstocks, over 90 percent of the accessions showed a survival higher than 30 percent. Similarly, a successful multi-step pre-freezing procedure was developed for mulberry and pear winter buds. Here, the hardening treatment consisted in a 5 °C daily reduction of bud temperature to bring it down from 0 °C to -30 °C, followed by direct immersion in liquid nitrogen (Niino, 2000). A more classic “vitrification/one-step freezing” approach has been proposed for the cryopreservation of persimmon (Diospyros kaki) winter-dormant axillary buds, directly collected from the field (Matsumoto et al., 2001).

Compared to shoot tips, reports dealing with the cryopreservation of embryogenic calli and somatic embryos are limited for hardwood trees. The number of temperate hardwood species for which a “one-step freezing” procedure has been developed has increased in recent years include Castanea sativa (Correidoira et al., 2004), Fraxinus angustifolia (Tonon et al., 2001), Quercus suber (Valladares et al., 2004), Olea europaea (Lambardi et al., 2002) and Aesculus hippocastanum (Lambardi, De Carlo and Capuana, 2005.). In addition, embryogenic calli of some tropical forest and fruit species have been successfully cryopreserved (Sudarmonowati, 2000; Wu et al., 2003). In the above-mentioned reports, isolated somatic embryos or pieces of embryogenic callus are used as explants and treated with PVS2 or, alternatively, encapsulated and dehydrated prior to a direct plunge into liquid nitrogen.

For hardwood species, in addition to the direct immersion of specimens in liquid nitrogen, the use of the slow-cooling technique is still sporadically reported. With the slow cooling approach, shoot-tip survival is variable, ranging from a minimum of 34 percent (Juglans regia [de Boucaud and Brison, 1995]) up to a maximum of 92 percent (Malus spp. [Zhao et al., 1999]). Also, a combination of “encapsulation/dehydration” and slow cooling has been proposed for the cryopreservation of walnut somatic embryos (de Boucaud, Brison and Negrier, 1994).

6.6.3 Cryopreservation of softwood trees

Cryopreservation plays an important role in the clonal selection programmes of conifers, based on somatic embryogenesis. Effective procedures based upon the slow-cooling approach have been successfully developed for embryogenic cultures of numerous species of Picea, Pinus, Larix, Abies and Pseudotsuga. An important programme for the cryopreservation of conifer germplasm has been developed in British Columbia, where over 5 000 embryogenic lines are presently stored in liquid nitrogen (Cyr, 2000) At present, no reports are available dealing with cryopreservation of conifer shoot tips.

6.6.4 Seed and embryonic axis cryopreservation

As concerns the long-term storage of seeds, effective cryopreservation procedures have been developed for both whole seeds and excised embryonic axes from many temperate (Pence, 1995) and tropical (Marzalina and Krishnapillay, 1999) woody species. Pre-dessiccation to a moisture content below 20 percent, followed by slow cooling or by direct immersion in liquid nitrogen, is the common methodology applied to orthodox seeds. However, very promising results have also been obtained with woody species characterized by sub-orthodox or non-orthodox (recalcitrant) seeds. In polyembryonic Citrus and related species, for instance, various procedures have been developed for the cryostorage of their seeds (Cho et al., 2001; Cho et al, 2002; Rhadamani and Chandel, 1992), and the regrowth of both zygotic and nucellar embryos has been reported (Lambardi et al., 2004). Both kinds of embryos are very important for evolution, breeding, and propagation of Citrus species.

6.6.5 Cryostored woody plant germplasm

Today, promising examples of cryogenic repositories for woody species are available. In addition to the aforesaid collection of conifer embryogenic lines in British Columbia, the following cryo-banks can be mentioned (Reed, 2001):

• the National Seed Storage Laboratory (NSSL) of Fort Collins (USA), with around 2 100 accessions of apple (dormant buds);

• the National Clonal Germplasm Repository (NCGR) of Corvallis (USA), with over 100 accessions of pear (shoot tips);

• Association Forêt-Cellulose (AFOCEL) of France, with over 100 accessions of elm (dormant buds);

• the National Institute of Agrobiological Resources (NIAR) of Japan, with around 50 accessions of mulberry.

Some tropical and sub-tropical woody species are also now cryopreserved, e.g. at Institut de Recherche pour le développement (IRD) of France (80 accessions of oil palm [Engelmann, 2004]), and at the National Bureau of Plant Genetic Resources (NBPGR), India (numerous accessions of citrus, jackfruit, almond, litchi and tea [Reed, 2001]).

6.7 GENETIC INTEGRITY OF PLANTS FROM CRYOPRESERVATION

The occurrence of somaclonal variation has been reported several times as a consequence of the introduction, manipulation and regeneration of plants under in vitro conditions. Since cryopreservation involves many in vitro steps, careful attention must be given to avoid the incidence of somaclonal variation before and after the conservation of germplasm in liquid nitrogen. On the other hand, during storage the main peculiarities of the cryopreservation technology (e.g. the blocked metabolism of cells and the absence of subcultures) reduce the risks of genetic and epigenetic alterations to nil. Some threats to genetic stability arise from particular reactions (free radical formation, molecular damage due to ionizing radiation) that might still occur at the temperature of -196 °C (Grout, 1990) as well as from the common practice of using DMSO as cryoprotectant at concentrations up to 10 percent. Although the number of reports studying these aspects in detail are still limited, the fact that up to now no clear evidence of morphological, cytological or genetic alterations due to cryopreservation has been produced is promising. (For a thorough review, see Harding, 2004.)

6.8 CONCLUSIONS

Although the slow-cooling approach was already introduced in the 1970s, for a long time cryopreservation of plant tissues was not studied on a wide scale. This was mainly due to the complexity of procedures and the high cost of computer-driven cryo-freezers. With the development in the early 1990s of “new” and more simplified cryopreservation protocols, based on the prevention of intra- and extra-cellular ice crystals by means of cell dehydration followed by direct immersion of explants in liquid nitrogen, the cryostorage of genetic resources has become a realistic target for many plant species. Nowadays, although the vitrification protocol could be considered a “standardized” protocol, a large amount of the work is still performed in the framework of academic studies and involves only one or a few accessions per plant species. This is mainly due to the complex and time-consuming optimization of procedures that is always required before an efficient cryopreservation protocol is established for a new species or accession. The two most important parameters that need to be optimized for each species and tissue are the preparation phase of tissues for the dehydration phase (most important are sugar and/or cold treatments) and the length of exposure to the vitrification solution (or, in the case of encapsulated explants, to air flow or silica gel). Research should move in the direction of simplifying and standardizing the procedures as much as possible in order to make the technology available to a wide range of public institutions and private companies. Moreover, to facilitate the development of even more efficient cryopreservation protocols, a better knowledge of the physico-chemical background of cryopreservation is needed. This can only be obtained through fundamental studies that involve both thermal analysis and a thorough examination of the different parameters that can influence the cryo-behaviour, such as endogenous sugars, membrane composition, oxidative stress and cryoprotective proteins. These parameters are now investigated for different plant species in the framework of the European FP5 project, Establishing Cryopreservation Methods for Conserving European Plant Germplasm Collections (CRYMCEPT). (See www.agr.kuleuven.ac.be/dtp/tro/CRYMCEPT.)

6.9 ACKNOWLEDGEMENTS

B. Panis gratefully acknowledges the financial support of the Directorate General of International Collaboration (DGIC), Belgium, the International Network for the Improvement of Banana and Plantain (INIBAP) within the framework of the Genetic Improvement Group of the Global Programme for Musa Improvement (PROMUSA). Part of this study has also been carried out with financial support from the Commission of the European Communities' specific cooperative research programme, Quality of Life and Management of Living Resources, QLK5-2002–1279, CRYMCEPT. This study does not necessarily reflect the Commission's views and in no way anticipates its future policy in this area. M. Lambardi's research activity is financially supported by the National Research Council (CNR) of Italy, Agri-food Department Project, Risorse biologiche e tutela dell'agroecosistema. Special thanks are due to Carla Benelli and Anna De Carlo for their valuable and competent participation in the Cryopreservation Working Group of the Istituto per la valorizzazione del legno e delle specie arboree (IVALSA).

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