PLANT CRYOPRESERVATION: PROGRESS AND PROSPECTS FLORENT ENGELMANN*
Cirad, Station de Roujol, 97170 Petit-Bourg, Guadeloupe, French West Indies
(Received 12 August 2003; accepted 4 February 2004; editor J. Aitken)
Summary
Cryopreservation (liquid nitrogen,21968C) represents the only safe and cost-effective option for long-term conservation of germplasm of non-orthodox seed species, vegetatively propagated species, and of biotechnology products. Classical cryopreservation techniques, which are based on freeze-induced dehydration, are mainly employed for freezing undifferentiated cultures and apices of cold-tolerant species. New cryopreservation techniques, which are based on vitrification of internal solutes, are successfully employed with all explant types, including cell suspensions and calluses, apices, and somatic and zygotic embryos of temperate and tropical species. The development of cryopreservation protocols is significantly more advanced for vegetatively propagated species than for recalcitrant seed species. Even though its routine use is still limited, there are a growing number of examples where cryopreservation is employed on a large scale for different types of materials, including seeds with orthodox and intermediate storage behaviour, dormant buds, pollen, biotechnology products, and apices sampled fromin vitroplantlets of vegetatively propagated species. Cryopreservation can also be employed for uses other than germplasm conservation, such as cryoselection, i.e., the selection through freezing of samples with special properties, or cryotherapy, i.e., the elimination of viruses from infected plants through apex cryopreservation. Because of its high potential, it is expected that cryopreservation will become more frequently employed for long-term conservation of plant genetic resources.
Key words: cryopreservation techniques; genetic resources; large-scale application; long-term storage; genebank; non-orthodox seed species; vegetatively propagated species.
Introduction
Most agricultural crops have orthodox seeds, i.e., seeds that can be dehydrated down to low moisture contents and can thus be stored at low temperature for extended periods (Roberts, 1973). However, conservation in seed form is problematic for three main categories of plant species. The first category includes crops such as banana and plantain, which do not produce seeds and are thus vegetatively propagated. The second category comprises species such as potato or sugarcane, which have both sterile genotypes and genotypes which produce orthodox seeds. However, the seeds are generally highly heterozygous and are thus of limited interest for conserving particular genotypes. These species are thus mainly maintained by means of vegetative propagation. The third category consists of fruit and forest tree species, especially from tropical origin, which produce recalcitrant seeds, i.e., seeds that cannot be dried to sufficiently low moisture level to permit their storage at low temperature (Roberts, 1973). Conservation in seed form is also still problematic for a large number of species, termed intermediate (Ellis et al., 1990). Genetic resources of all these species are traditionally conservedex situas whole plants in field collections. Field conservation presents major drawbacks which limit its
efficacy and threaten the safety of plant genetic resources conserved in this way. Indeed, collections remain exposed to natural disasters, attacks by pests and pathogens, and moreover, labor costs and the requirement for technical personnel are very high. In addition, distribution and exchange from field genebanks is difficult because of the vegetative nature of the material and the greater risks of disease transfer from country to country (Engelmann, 1997b).
The development of biotechnology has led to the production of a new category of germplasm including clones obtained from elite genotypes, cell lines with special attributes, and genetically transformed material (Engelmann, 1992). This new germplasm is often of high added-value and very difficult to produce. The development of efficient techniques to ensure its safe conservation is therefore of paramount importance.
Cryopreservation, i.e., the storage of biological material at ultra-low temperature, usually that of liquid nitrogen (21968C), is the only technique currently available to ensure the safe and cost-efficient long-term conservation of these different types of germplasm. At this temperature, all cellular divisions and metabolic processes are stopped. The plant material can thus be stored without alteration or modification for a theoretically unlimited period of time. Moreover, cultures are stored in a small volume, protected from contamination, and require a very limited maintenance.
In this paper, we present a brief overview of the various cryopreservation techniques available, of the achievements made *Author to whom correspondence should be addressed: Email florent.
engelmann@cirad.fr 1054-5476/04 $18.00+0.00
and problems faced with cryopreservation of vegetatively propagated and recalcitrant species, and of the current utilization of cryopreservation for long-term storage of plant germplasm.
Cryopreservation Techniques
Some materials, such as orthodox seeds or dormant buds, display natural dehydration processes and can be cryopreserved without any pretreatment. However, most of the experimental systems employed in cryopreservation (cell suspensions, calluses, shoot tips, embryos, etc.) contain high amounts of cellular free water and are thus extremely sensitive to freezing injury since most of them are not inherently freezing-tolerant. Cells have thus to be dehydrated artificially to protect them from damage caused by crystallization of intracellular water into ice (Mazur, 1984). The techniques employed and the physical mechanisms upon which they are based are different in classical and new cryopreservation techniques (Withers and Engelmann, 1998). Classical techniques involve freeze-induced dehydration, whereas new techniques are based on vitrification. Vitrification can be defined as the transition of water directly from the liquid phase into an amorphous phase or glass, while avoiding the formation of crystalline ice (Fahy et al., 1984).
Classical Cryopreservation Techniques
Classical cryopreservation techniques involve slow cooling down to a defined prefreezing temperature, followed by rapid immersion in liquid nitrogen. With temperature reduction during slow cooling, cells and the external medium initially supercool, followed by ice formation in the medium (Mazur, 1984). The cell membrane acts as a physical barrier and prevents the ice from seeding the cell interior and the cells remain unfrozen but supercooled. As the temperature is further decreased, an increasing amount of the extracellular solution is converted into ice, thus resulting in the concentration of intracellular solutes. Since cells remain supercooled and their aqueous vapour pressure exceeds that of the frozen external compartment, cells equilibrate by loss of water to external ice. Depending upon the rate of cooling and the prefreezing temperature, different amounts of water will leave the cell before the intracellular contents solidify. In optimal conditions, most or all intracellular freezable water is removed, thus reducing or avoiding detrimental intracellular ice formation upon subsequent immersion of the specimen in liquid nitrogen. However, too intense freeze-induced dehydration can incur different damaging events due to concentration of intracellular salts and changes in the cell membrane (Meryman et al., 1977). Thawing should be as rapid as possible to avoid the phenomenon of recrystallization in which ice melts and reforms at a thermodynamically favorable, larger and more damaging crystal size (Mazur, 1984).
Classical freezing procedures include the following successive steps: pregrowth of samples, cryoprotection, slow cooling (0.5 – 2.08C min21) to a determined prefreezing temperature (usually around 2408C), rapid immersion of samples in liquid nitrogen, storage, rapid thawing, and recovery. Classical techniques are generally operationally complex since they require the use of sophisticated and expensive programmable freezers. In some cases, their use can be avoided by performing the slow freezing step with a domestic or laboratory freezer (Kartha and Engelmann, 1994; Engelmann, 1997b).
Classical cryopreservation techniques have been successfully applied to undifferentiated culture systems such as cell suspensions and calluses (Kartha and Engelmann, 1994; Withers and Engelmann, 1998). In the case of differentiated structures, these techniques can be employed for freezing apices of cold-tolerant species (Reed and Chang, 1997).
New Cryopreservation Techniques
In vitrification-based procedures, cell dehydration is performed prior to freezing by exposure of samples to concentrated cryoprotective media and/or air desiccation. This is followed by rapid cooling. As a result, all factors that affect intracellular ice formation are avoided. Glass transitions (changes in the structural conformation of the glass) during cooling and rewarming have been recorded using thermal analysis (Sakai et al., 1990). Vitrification-based procedures offer practical advantages in comparison to classical freezing techniques. Like ultra-rapid freezing (above), they are more appropriate for complex organs (shoot tips, embryos) which contain a variety of cell types, each with unique requirements under conditions of freeze-induced dehydration. By precluding ice formation in the system, vitrification-based procedures are operationally less complex than classical ones (e.g., they do not require the use of controlled freezers) and have greater potential for broad applicability, requiring only minor modifications for different cell types (Engelmann, 1997b).
A common feature to all these new protocols is that the critical step to achieve survival is the dehydration step, and not the freezing step, as in classical protocols. Therefore, if samples to be frozen are amenable to desiccation down to sufficiently low water contents (which vary depending on the procedure employed and the type and characteristics of the propagule to be frozen) with no or little decrease in survival in comparison to non-dehydrated controls, no or limited further drop in survival is generally observed after cryopreservation (Engelmann, 1997b).
Seven different vitrification-based procedures can be identified: (1) encapsulation –dehydration; (2) vitrification; (3) encapsulation – vitrification; (4) dehydration; (5) pregrowth; (6) pregrowth – dehydration; and (7) droplet freezing.
Encapsulation – dehydration. The encapsulation – dehydration procedure is based on the technology developed for the production of artificial seeds. Explants are encapsulated in alginate beads, pregrown in liquid medium enriched with sucrose for 1– 7 d, partially desiccated in the air current of a laminar airflow cabinet or with silica gel to a water content around 20% (fresh weight basis), then frozen rapidly (Dereuddre et al., 1991). Survival is high and growth recovery of cryopreserved samples is generally rapid and direct, without callus formation. A modified protocol has been proposed by Sakai et al. (2000) in which encapsulation and pregrowth in medium with sucrose and glycerol are performed simultaneously. This technique has been applied to apices of numerous species from temperate and tropical origin as well as to cell suspensions and somatic embryos of several species (Engelmann, 1997b; Engelmann and Takagi, 2000).
as the glycerol-based PVS2 solution (Sakai et al., 1990) which has a molarity of 7.8M, rapid freezing and thawing, removal of cryoprotectants and recovery. In order to reduce the toxicity of the vitrification solution, a modified vitrification protocol has been developed recently in which apices are first treated with half-strength vitrification solution, then with a full-strength one (Matsumoto and Sakai, 2003). This procedure has been developed for apices, cell suspensions, embryogenic tissues, and somatic embryos of numerous species (Cyr, 2000; Sakai, 2000; Sakai et al., 2002).
Encapsulation – vitrification. Encapsulation – vitrification is a combination of encapsulation – dehydration and vitrification pro-cedures, where samples are encapsulated in alginate beads, then subjected to freezing by vitrification. It has been applied to apices of an increasing number of species (Sakai, 2000; Sakai et al., 2002). Dehydration. Dehydration is the simplest procedure since it consists of dehydrating explants, then freezing them rapidly by direct immersion in liquid nitrogen. This technique is mainly used with zygotic embryos or embryonic axes extracted from seeds. It has been applied to embryos of a large number of recalcitrant and intermediate species (Engelmann, 1997a, 2000). Desiccation is usually performed in the air current of a laminar airflow cabinet, but more precise and reproducible dehydration conditions are achieved by using a flow of sterile compressed air or silica gel. Ultra-rapid drying in a stream of compressed dry air (a process called ‘flash drying’ developed by Prof. Berjak’s group in South Africa) allows freezing of samples with a relatively high water content, thus reducing desiccation injury (Berjak et al., 1989). Optimal survival is generally obtained when samples are frozen with a water content between 10 and 20% (fresh weight basis).
Pregrowth. The pregrowth technique consists of cultivating samples in the presence of cryoprotectants, then freezing them rapidly by direct immersion in liquid nitrogen. The pregrowth technique has been developed for Musa meristematic cultures (Panis et al., 2002).
Pregrowth – dehydration. In a pregrowth – dehydration pro-cedure, explants are pregrown in the presence of cryoprotectants, dehydrated under the laminar airflow cabinet or with silica gel, and then frozen rapidly. This method has been applied notably to asparagus stem segments, oil palm polyembryonic cultures, and coconut zygotic embryos (Uragami et al., 1990; Assy-Bah and Engelmann, 1992; Dumet et al., 1993).
Droplet freezing. The droplet-freezing technique has presently been applied to potato (Scha¨fer-Menuhr, 1996), asparagus (Mix-Wagner et al., 2000), and apple apices (Zhao et al., 1999). Apices are pretreated with liquid cryoprotective medium, then placed on an aluminum foil in minute droplets of cryoprotectant and frozen slowly (apple) or rapidly (potato) in liquid nitrogen.
Cryopreservation of Vegetatively Propagated and Recalcitrant Seed Species
Vegetatively Propagated Species
Several review papers have been published recently, which provide lists of species which have been successfully cryopreserved (Engelmann, 1997a, b; Cyr, 2000; Engelmann and Takagi, 2000; Sakai et al., 2002). For vegetatively propagated species, cryopreservation has a wide applicability both in terms of species
coverage, since protocols have been successfully established for root and tubers, fruit trees, ornamentals, forestry and plantation crops, from temperate and tropical origin, and in terms of numbers of genotypes/varieties within a given species. With a few exceptions (e.g., potato, pear, mulberry), vitrification-based protocols have been employed. It is also interesting to note that in many cases different protocols can be employed for a given species and produce comparable results. Survival is generally high to very high and up to 100% survival could be achieved in some cases, e.g.,Allium, yam, potato, and conifers. Regeneration is rapid and direct, and callusing is observed only in cases where the technique is not optimized.
Different reasons can be mentioned to explain these positive results (Engelmann, 2000). The meristematic zone of apices, from which organized growth originates, is composed of a relatively homogeneous population of small, actively dividing cells, with few small vacuoles and a high nucleo-cytoplasmic ratio. Cells of somatic embryogenic tissues display the same characteristics. These characteristics make such cells more likely to withstand desiccation than highly vacuolated and differentiated cells. No ice formation takes place in vitrification-based procedures, thus allowing direct, organized regrowth of frozen explants. Other reasons for the good results obtained are linked with tissue culture protocols. Many vegetatively propagated species successfully cryopreserved until now are cultivated crops, often of great commercial importance, for which cultural practices, including in vitro propagation, are well established. In addition,in vitromaterial is ‘synchronized’ by the tissue culture and pregrowth procedures. Relatively homogeneous samples in terms of size, cellular composition, physiological state, and growth response are employed for freezing, thus increasing the chances of positive and uniform response to treatments. Finally, vitrification-based procedures allow the use of samples of relatively large size (shoot tips of 0.5 – 3 mm) which can regrow directly without difficulty.
Freezing techniques are now operational for large-scale experimentation and commercialization with an increasing number of vegetatively propagated plants. In view of the wide range of efficient and operationally simple techniques available, any vegetatively propagated species should be amenable to cryopre-servation, provided that the tissue culture protocol is sufficiently operational for this species.
Recalcitrant Seed Species
stored using classical or new storage techniques (Engelmann, 2000).
In comparison to the results obtained with vegetatively propagated species, research is still at a very preliminary stage for recalcitrant seeds. The desiccation technique is mainly employed for freezing embryos and embryonic axes. Survival is extremely variable, regeneration is frequently restricted to callusing or incomplete development of plantlets, and the number of accessions tested per species is generally very low. A number of reasons explain the limited development of cryopreservation for recalcitrant seed species (Engelmann, 2000). A huge number of species have recalcitrant or suspected recalcitrant seeds and the majority are wild species. As a consequence, little or nothing is known of their seed biology, and even less on seed storage behavior. In cases where information on seed storage behaviour is available, tissue culture protocols, including inoculationin vitro, germination and growth of plantlets, propagation and acclimatization which are needed for regrowth of embryos and embryonic axes after freezing, are often non-existent or not fully operational (Engelmann, 1999). Seeds and embryos of recalcitrant species also display very large variations in moisture content and maturity stage between provenances, between and among seed lots, as well as between successive harvests, thus making their cryopreservation difficult.
Seeds of many species are too large to be frozen directly and embryos or embryonic axes are thus successfully employed for cryopreservation. However, embryos are often of very complex tissue composition which display differential sensitivity to desiccation and freezing, the root pole seeming more resistant than the shoot pole. In some species, embryos are extremely sensitive to desiccation and even a minor reduction in their moisture content – down to levels much too high to obtain survival after freezing – leads to irreparable structural damage, as observed notably with cacao (Chandel et al., 1995). Finally, embryos of some species are too large to envisage using them for cryopreservation, and seeds of some species do not contain well-defined embryos.
Various options can be considered for improving storage of non-orthodox seeds. With some species, very precisely controlled desiccation (e.g., using saturated salt solutions) and cooling conditions may allow freezing of whole seeds, as demonstrated recently with various coffee species (Dussert et al., 1997). There is scope for technical improvements in the current cryopreservation protocols for embryos and embryonic axes. Pregrowth on media containing cryoprotective substances may confer increased tolerance to the tissues for further desiccation and reduce the heterogeneity of the material. Flash drying followed by ultra-rapid freezing has also been very effective for cryopreservation of several species (Berjak et al., 1989; Wesley-Smith et al., 1992). Other cryopreservation techniques, including pregrowth – desiccation, encapsulation – dehydration, vitrification, and encapsulation – vitri-fication which have seldom been employed so far, should be investigated (Engelmann, 2000). Finally, selecting embryos at the right developmental stage is of critical importance for the success of any cryopreservation experiment (Engelmann et al., 1995). However, in these cases, basic protocols for disinfection, inoculation in vitro, germination of embryos or embryonic axes, plantlet development, and possibly limited propagation will have to be established prior to any cryopreservation experiment.
With species for which attempts to freeze whole embryos or embryonic axes have proven unsuccessful, it has been suggested
using shoot apices sampled from embryos, adventitious buds, or somatic embryos induced from the embryonic tissues (Pence, 1995). This might be the only solution for species which do not have well-defined embryos; however, this will require more sophisticated tissue culture procedures to be developed and mastered.
Large-scale Utilization of Cryopreservation for Germplasm Conservation
Even though its routine use is still limited, there are a growing number of examples where cryopreservation is employed on a large scale for different types of materials, which are, or are not, tolerant to dehydration.
In the case of orthodox seed species, cryopreservation is used mainly for storing seeds with limited longevity and of rare or endangered species (Pritchard, 1995; Gonzales-Benito et al., 1999; Pence, 1999). The National Center for Genetic Resources Preservation (NCGRP, Fort Collins, CO) conserves 37 654 accessions (a total of over 360 629 seeds) over the vapors of liquid nitrogen (Towill et al., 1998). The National Bureau for Plant Genetic Resources (NBPGR, New Delhi, India) conserves 1200 accessions from 50 different species, consisting mainly of endangered medicinal plants (Mandal, 2000). This technique is also used in several botanic gardens. Over 110 accessions of rare or threatened species are stored under cryopreservation at the Perth Royal Botanic Garden in Australia (Touchell and Dixon, 1994) and the Cincinnati Botanic Garden in the USA conserves seeds of rare and endangered native species in liquid nitrogen (Pence, 1991).
Cryopreservation is also applied to intermediate seeds which are tolerant to freezing. In Catie (Tropical Agricultural Research and Higher Education Center, Costa Rica), seeds of 80 accessions of Coffea arabica, which constitute the core collection of the field collection, have been stored in liquid nitrogen after controlled dehydration and freezing (Dussert et al., 1997, 1999). In France, under the framework of a national grape genetic resources conservation project, seeds of several hundred accessions are being cryopreserved after partial desiccation (Dussert, personal communication).
In the case of dormant buds, the 2200 accessions of the US apple germplasm field collection are duplicated under cryopreservation (Forsline et al., 1999). In Japan, the mulberry field collection maintained at the National Institute of Agrobiological Resources (NIAR, Yamagata) includes a total of 756 accessions, 420 of which have been cryopreserved (Niino, 1995) and freezing of the whole collections will be completed within 3 yr. Dormant buds of over 300 European elm accessions are conserved in liquid nitrogen by Afocel (Nangis, France) and research is under way in France (IRD, Montpellier) and the USA (NCGRP, Fort Collins) on freezing of grape dormant buds for germplasm conservation.
and Rajashekaran, 2000). In the USA, the NCGRP conserves pollen of 13 pear cultivars and 24Pyrusspecies (Reed et al., 2000).
Cryopreservation is also applied to biotechnology products. Over 1000 callus strains of species of pharmaceutical interest are stored at21968C in the UK (Spencer, 1999) as well as several thousand conifer embryogenic cell lines employed in large-scale clonal planting programs in Canada (Cyr, 2000). In France, cryopreserva-tion is systematically employed for storing all the new embryogenic cell lines of coffee and cacao produced by the Biotechnology Laboratory of the Nestle´ Company (Florin et al., 1999) and the banana lines produced in Vitropic, a private tissue culture laboratory. Polyembryonic cultures of around 80 oil palm accessions have been cryopreserved and stored at IRD (Montpellier, France) (Dumet, 1994).
Finally, cryopreservation is being applied in genebanks for long-term storage of genetic resources of vegetatively propagated species, using apices sampled fromin vitroplantlets. Potato cryopreserva-tion is currently the most advanced, since 519 old potato varieties are cryostored in Germany at the Institute for Crop and Grassland Science in Braunschweig (Mix-Wagner et al., 2003) and over 200 accessions at the International Potato Center (CIP, Lima, Peru) (Golmirzaie and Panta, 2000). A duplicate of around 100 accessions of thePyrusfield collection of the US National Clonal Germplasm Repository (NCGR, Corvallis, OR) is cryostored at NCGR, with another duplicate at the NCGRP in Fort Collins (Reed et al., 2000). At the INIBAP (International Network for the Improvement of Banana and Plantain) Transit Centre (ITC), based at KU Leuven, Belgium, 100 accessions of the international Musa germplasm collection have already been cryopreserved. Cryopreservation of the remaining 1036 accessions of the collection is under way (Panis, personal communication). Large-scale testing of cryopreservation protocols is also performed notably with cassava and strawberry (Roca et al., 2000; Matsumoto, personal communication).
Large-scale utilization of cryopreservation implies scaling-up of the amount of material to be handled and stored, from one or a few genotypes in the laboratory to several tens, hundreds, or even thousands in the cryobank, which requires the establishment of specific procedures for their management. With this aim, probabilistic tools have been developed recently to assist genebank curators in the establishment and management of cryopreserved germplasm collections (Dussert et al., 2003).
Cryopreservation imposes a series of stresses to plant material, which is susceptible to induced modifications in cryopreserved cultures and regenerated plants. It is thus important to verify that the genetic stability of cryopreserved material is not altered before routinely using this technique for long-term conservation of plant germplasm. The number of papers published on this aspect has been increasing recently (see, e.g., Dixit et al., 2003; Gagliardi et al., 2003; Zhai et al., 2003). No modification has been observed at the phenotypical, biochemical, chromosomal, or molecular level which could be attributed to cryopreservation (Engelmann, 1997b). The few studies comparing the vegetative and floral development in the field of plants originating from control and cryopreserved material performed with oil palm (Engelmann, 1991), potato (Mix-Wagner et al., 2003), sugarcane (Gonzalez Arnao, 1996), and banana (Coˆte et al., 2000) did not reveal any differences in the characters studied. Studies performed on the cost of cryopreservation are still fragmentary, but the first estimates tend to confirm the interest of this technique also from a financial perspective. Hummer and Reed
(2000) indicate that, at the NCGR, the annual maintenance cost of one temperate fruit tree accession is US$77 in the field, US$23 under in vitroslow growth storage, and only US$1 under cryopreservation, to which US$50 – 60 should be added once for cryopreserving this accession. Roca (personal communication) evaluates the annual maintenance cost of CIAT’s (International Center for Tropical Agriculture, Cali, Colombia) cassava collection, which includes 5000 accessions, at around US$5000 under cryopreservation, against US$30 000 underin vitroslow growth storage.
Additional Uses of Cryopreservation
Cryopreservation is also employed for uses other than germplasm conservation. One such use is cryoselection, i.e., the selection of samples with special properties in the frozen population. For example, when lavender cell suspensions were submitted to successive freeze – thaw cycles, the number of colonies recovered from cryopreserved cells increased with the number of freeze – thaw cycles (Watanabe et al., 1985). However, no modifications were noted in the biosynthetic capacities of cryopreserved cells, suggesting a change in population structure rather than genetic change. A similar observation was made by Bercetche et al. (1990) who noted that cryopreserved Picea abies embryogenic calluses recovered faster than frozen controls. For these authors, non-embryogenic tissues were killed by freezing, thus leading to the production of a more homogeneous population, with a higher embryogenic potential. This suggests that cryopreservation could be used as a tool to ‘rejuvenate’ cultures when their proliferation capacities are decreasing. In the case of wheat, Kendall et al. (1990) were able to regenerate plants with increased cold tolerance from calluses submitted to repeated freeze – thaw cycles. A different protein pattern was associated with these cold-tolerant plants.
More recently, cryopreservation has been used for cryotherapy, i.e., for eliminating viruses from infected plants, as a substitute or complementary to classical virus eradication techniques such as meristem culture and cryotherapy. After cryopreservation of plum shoot tips sampled fromin vitroplantlets infected by the plum pox virus, 50% of plants obtained were virus-free against only 20% through meristem culture (Brison et al., 1997). With infected banana meristematic cultures, Helliot et al. (2002) obtained 30% and 90% virus eradication after cryopreservation, for cucumber mosaic virus (CMV) and banana streak virus (BSV), respectively. Finally, Wang et al. (2003) have shown that cryopreservation led to 97% elimination of grape virus A, against only 12% by meristem culture. Cryotherapy is thus based on selective cell destruction by cryopreservation. The differentiated cells of apices which contain viruses also have a high water content; they are killed by the formation of ice crystals during freezing. By contrast, meristematic cells, the multiplication of which leads to growth of apices, have a more concentrated cytoplasm and withstand freezing.
Finally, in large-scale propagation programs of conifers based on somatic embryogenesis, the ability to maintain donor tissue juvenility through cryopreservation represents an immeasurable advantage over propagation programs based on rooted cuttings (Cyr, 2000).
Conclusion
vitrification-based freezing techniques has made its application to a broad range of species possible. A significant advantage of these new techniques is their operational simplicity, since they will be mainly applied in developing tropical countries where the largest part of genetic resources of problem species is located. For many vegetatively propagated species, cryopreservation techniques are sufficiently advanced to envisage their immediate utilization for large-scale experimentation in genebanks. Research is much less advanced for recalcitrant seed species. This is due to the large number of mainly wild species, with very different characteristics, which fall within this category, and to the comparatively limited level of research activities aiming at improving the conservation of these species. However, there are various technical approaches to explore to improve the efficiency and increase the applicability of cryopreservation techniques to recalcitrant species. In addition, research is actively performed by various groups worldwide to improve the knowledge of biological mechanisms underlying seed recalcitrance. It is hoped that new findings on critical issues such as understanding and control of desiccation sensitivity will contribute significantly to the development of improved cryopreservation techniques for recalcitrant seed species. A very positive development in the establishment of cryopreservation protocols is that an increasing number of researchers make systematic use of analytical tools (e.g., histo-cytology, differential scanning calori-metry) which provide a scientific basis to the success/failure of an experimental treatment. Such a rational and scientific approach should greatly facilitate the establishment of freezing protocols, especially for problem species.
It is important to stress, however, that cryopreservation is not seen as a replacement for conventionalex situapproaches (Withers and Engelmann, 1998). Cryopreservation offers genebank curators an additional tool to allow them to improve the conservation of germplasm collections placed under their responsibility. In conclusion, it can be realistically expected that in the coming years, cryopreservation will become more frequently employed for long-term conservation of plant genetic resources.
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