World Journal

of Microbfology

& Biotechnology

11, 375382

Ex situ conservation using biotechnology V.M. Villalobos*

of plant germplasm

and F. Engelmann

Conservation of plant genetic resources attracts more and more public interest as the only way to guarantee adequate food supplies for future human generations. However, the conservation and subsequent use of such resources are complicated by cultural, economical, technical and political issues. (Over the last 30 years, there have been significant increases in the number of plant collections and in accessions in ex situ storage centres throughout the World. The present review is of these er sift collections and thLe contribution biotechnology has made and can make to conservation of plant germplasm. The applications and limitations of the new, molecular approaches to germplasm characterization are discussed. In z&o slow growth is used routinely for conserving germplasm of plants suc,h as banana, plantain, cassava and potato. More recently, cryopreservation procedures have become more accessible for long-term storage. New cryopreservation techniques, such as encapsulation-dehydration, vitrification antd desiccation, lengthen the list of plant species that can not only tolerate low temperatures but also give normal growth on recovery. Extensive research is still needed if these techniques are to be fully exploited. fiy words: Artificial

seeds, cryopreservation,

existing collections, germplasm

Plant genes, which have either been selected by nature or by man, through on-farm empirical improvement or more sophisticated plant-breeding techniques, are dispersed throughout domesticated and wild plant populations. Many improvements in crops and in agriculture generally could not have taken place without the diversity which occurs in these genes. This diversity is the limited natural resource that permits improved plant varieties to be bred. In recent years, several factors, including the substitution of local genotypes with improved varieties and hybrids, the development of new land, forest depletion, changes in agricultural techniques, and the abuse of agrochemicals, have caused a rapid and profound erosion of this genetic resource, with the loss of potentially valuable material which had barely been explored. Those constantly trying to increase food production have often neglected the value of protecting genetic resources and have often failed to make efficient use of those V.M. Villalobos is with the Food and Agriculture Organization of the United Nations, Viale delle Terme di Caracaila, 00100 Rome, Italy; fax: 522-56347. F. Engelmann is with the International Plant Genetic Resources Institute (IPGRI), Via delle Seth? Chiese 142, 00145 Rome, Italy. “Corresponding author. @ 1995 Rapid

Communications

of Oxford

characterization,

in uifro storage.

resources which are available. Such protection and exploitation require the correct collection, conservation, evaluation, documentation and exchange of plant germplasm. Although germplasm conservation is attracting more and more public concern as the only way to guarantee food supplies for future human generations, it is not simple, since it involves cultural, economical, technical and political issues.

Existing Collections Resources

of Plant Genetic

Since the early 1960s there has been a significant increase in the number of collections in which plant material is preserved, basically for subsequent use in plant improvement, in a highly protected state er sift (i.e. out of its natural habitat). There has been a consequent increase in the number of samples stored in seed banks and in field collections. According to the F(ood and Agriculture Organization (FAO) (Anon. 1994a), tlhere are 4.41 million accessions currently in ex sifu storage. Of these, 50% are maintained in industrialized countries, 38% are in developing countries and the remaining 12% are held by the Consultative Group

Ltd World Jmml

of Microbiology

6 Biofechnology, !/al 11, 1995

375

V.M. Villalobos and F. Engelmarm Table

1. Ex situ collections

by region. No. of accessions

Africa* Asia* Europe* Latin America North America Oceania SubtotalT International (CGlAR)$ Total

% of total

265,000 971,500 1,344,ooo 441,500 750,700 132,500 3,905,200 510,500 4,415,700

6.0 22.0 30.4 10.0 17.0 3.0 8%.4 11.6 100.0

* Includes, for their respective regions, the collections of the Centro Agronomic0 Tropical de lnvestigacion y Ensefianza (CATIE) and the Nordic Gene Bank (NGB), since these are controlled by, or service, the governments of the region. t From the World Information and Early Warning System on Plant Genetic Resources (WlEWS/PGR) data-base, May 1994. $ From the Stripe Study of Genetic Resources in the Consultative Group on International Agricultural Research (CGIAR).

on International Agriculture Research Centres (CGIAR). Table 1 shows the number of accessions by region. Most of the holdings are cereals (47%) and pulses (16%) (Table 2). Table

2. Ex situ

collections

in May 1994, by crop

Although the genetic diversity of many crops has been well preserved ex sift, many other crops which are important at a national or local level are poorly represented in the existing collections for a variety of reasons. For example, some important crops, such as mango, rubber, cocoa, coconut, coffee and oil palm produce recalcitrant seeds which are unable to withstand desiccation. Long-term storage of perennial plants, including trees, which have long juvenile periods before any seed is produced can also be difficult. On the other hand, some vegetatively propagated species, including many of those eaten as roots or tubers (yams, potato, cassava, Xatzfhosoma and sweet potato) and several fruits (bananas and plantains) are either sterile or do not have stable sexual reproduction. Success in germplasm storage is determined by: the inherent longevity and physiological storage behaviour of the species (i.e. whether it is orthodox, recalcitrant or intermediate); the initial quality (e.g. moisture content) of the material stored; and the storage methods and conditions used (Anon. 1993; see Table 3). Over 1200 institutions World-wide have some sort of ex situ collection. Of these, 308 institutes have the capacity for medium-term storage, 175 for long-term storage and 119 have the facilities for

group.

Crop

No. of accessions National

collections

Cereals Pulses Forages Vegetables Fruit Roots and tubers Oil crops Fibre crops Beverages Rubber Miscellaneous Sugar cane Narcotics and drugs Condiments, spices, Shelter crops Chocolate crops Ornamentals Medicinal plants Dyes Perfume crops Building materials Weeds Timber crops Unknown Banana* Multi-purpose trees* Totals

flavourings,

* The CGIAR centres subsumed into other

class bananas and multi-purpose trees categories and not reported separately.

376

herbs

1,750,200 600,200 374,450 336,600 174,400 157,400 89,750 70,300 42,900 30,500 17,350 16,700 14,650 10,050 9600 8750 4550 2950 1023 550 400 17 10 191,900

centres

317,200 118,150 50,900 22,450 1500 300 510,500

3,905,200

World Journal of Microbiology & Biotechnology, Vol 1I, 19%

CGIAR

% of total

as separate

categories.

In the case

Totals 2,067,400 718,350 425,350 336,600 174,400 179,850 89,750 70,300 42,900 30,500 17,350 16,700 14,650 10,050 9600 8750 4550 2950 1023 550 400 17 10 191,900 1500 300 4,415,700 of national

collections,

46.82 16.27 9.63 7.62 3.95 4.07 2.03 1.59 0.97 0.89 0.39 0.38 0.33 0.23 0.22 0.20 0.10 0.07 0.02 0.01 0.01 0.00 0.00 4.34 0.03 0.01 100.0 they

are

Conservafion of planf germplasm Table

3. Ex situ

Maintenance

collections

by maintenance

regime

Short-term storage Medium-term storage Long-term storage /n V&O storage Field collections

method.

No. of accessions* 628,500 2,333,lOO 2,04.5,200 37,600 302,300

These numbers should not be summed. The data were interpreted on the assumption that, when a mixture of categories was given, the crop is stored in all the indicated manners, and this may have inflated some figures. To derive percentages would be misleading.

l

storage at temperature below field collections are categorized term storage.

- 18 ‘C. Both iti vifro and as either short- or medium-

The Contribution of Biotechnology Germplasm Conservation

to Plant

The biotechnological techniques recently applied to plants have great agricultural potential. They provide new approaches to overcome the problems of plants in marginal environments, biotic stresses and pests and diseases, permitting plants with unique gene combinations, greater pestresistance and/or yields to be produced. They also have major applications in the field of plant germplasm conservation. Since complete plants can now be produced from isolated cells, tissues or organs, germplasm banks based on plant tissue cultures can now be established. During the last 15 years, in vifro culture techniques have been developed for more that 1000 plant species, including annuals and perennials. The use of these techniques is even more important for the conservation and multiplication of plants which produce recalcitrant seeds and those which are usually propagated vegetatively (Thorpe ef al. 1995). The development of in vifro technology has, in fact, been a strong motivation for the planning, research and development of alternative methods of conservation. In principle, tissue-culture techniques are appropriate for conservation, the development of a complete plant being the expected result in all cases; for this reason, it is logical to maintain an elevated level of tissue organization during storage. One of the major objectives of germplasm conservation is to maintain the genetic diversity of a species in a stable condition and so the storage techniques used should not endanger plant genetic stability. For this reason, it will always be more appropriate to use cultures of shoot apices or zygotic embryos, which minimize the risks of variation in comparison with other culture systems. Although tissue culture techniques have been used for imifro conservation, at present only 37,600 accessions are

stored using this system (Anon. 1994a). Such collections must be maintained in a carefully controlled environment and there are other problems, particularly of microbial contamination and maintaining genetic stability. In vifro techniques require the substitution of natural conditions for artificial conditions, permitting the control of light and temperature and storage in relatively small volumes. In many cases, for plants with short reproductive cycles, such as some roots, tubers and other annuals, k vifro transfer intervals are less frequent than the harvest cycle in the field. Another important advantage is the possibility of producing virus-free plants with a high multiplication rate, independent of climatic conditions (see Thorpe ef al. 19%). In the modern approach to conservation and rational use of genetic diversity,- iz vifro conservation should include the elimination of virus from the stored material and the ability to micro-propagate the germplasm in large quantities when necessary (Villalobos ef al. 1991). Any tissue-culture system employed for in vifro conservation should fulfil two requirements: the genetic stability of the material to be preserved should be guaranteed. This is usually achieved by culture of apical meristems, which are ideal tissues for storage because of their stability and morphogenie potential, in comparison with other tissues and isolated cells. well-defined protocols should be implemented, guaranteeing a high percentage of plant recovery and an acceptable grade of efficiency from the stored tissues. The factors that determine a good response in plant regeneration are environmental, physical and genotypic. Conservation of germplasm using tissue-culture techniques can be envisaged either as short- and medium-term conservation or as long-term c:onservation/cryopreservation. Both techniques have their own characteristics, advantages and disadvantages.

Short-

and Medium-term

Conservation

In short- and medium-term conservation, stored material is sub-cultured at regular intervals. Currently, the periods between sub-cultures tend to be kept as long as is possible without endangering the germplasm (3 to 9 months), basically because frequent transfers to fresh culture medium are costly and increase the risks of contamination, other technical errors and changes in genotype due to genetic instability. In short- and midterm conservation, germplasm is cultured under normal growth conditions or growth-hmiting conditions. It is known that, during culture, mutations and selections occur and these could lead to atypical progeny or even the loss of totipotency. The most obvious goal in short- and

V.M. Villalobos and F. Engelmann Growth in vifro can be limited during the conservation period by various physical and chemical factors, such as reduction of temperature and/or light intensity, the dilution of the nutritive elements in the culture medium and the use of osmotic agents and chemical growth inhibitors.

Long-term



I

hl

Figure 1. Schematic representation of the classical cryopreservation procedure employed for freezing carnation apices (from Galerne 1985). A-h vitro mother plantlets [shoot tips (m) are dissected and used for freezing and microcutGngs (b) used for further mukiplication]; B-pregrowth of apices (24 h on solid medium with 0.5 M sucrose); C and D-(final concenWation 5%) (addition of DMSO is performed in ice (g)); E-transfer of apices into cryovials; F-cryoprotecGve medium using pincets (p) precooled in liquid nitrogen; G-controlled slow cooling (by 0.5 ‘C/ min) down to -40X; H-immersion of cryovials into liquid nitrogen; I and J-rapid thawing in a water-bath at 40 “C; K to M-posWeatment and recovery, with elimination of cryoprotectants (K and L); M-culture on standard medium.

mid-term germplasm storage is to define the experimental conditions that favour minimal growth without alteration of genetic stability, with the minimum possible use of subcultures. Culture requirements can sometimes be reduced by limiting the growth rate of the stored material, thus extending the transfer intervals. This can also have a beneficial effect on culture stability, since diminishing the cellular division rate should reduce the frequency of the mutations that occur during th e d u pl’lea t ion of DNA in the mitotic phase of the cellular cycle (Henshaw 1982). Nevertheless, the risk of such mutation cannot be totally eliminated and growth-limiting conditions can introduce a new selection hazard: the inevitable physiological stress. It is important that the procedures applied to minimize growth are also capable of maintaining maximal viability in the cultures; plant recovery should be feasible whenever necessary. Although, in theory, it should be sufficient to recover just one plant from each culture, a high regeneration efficiency facilitates the propagation needed to initiate another cycle of conservation, reduces the possibility of selection imposed by sub-optimal storage conditions, and facilitates germplasm exchange and utilization (Rota 1983).

Conservation

or Cryopreservation

Cryopreservation is based on the reduction and subsequent arrest of all metabolic functions in the explants, including cellular division. This is accomplished when the material is brought to an ultra-low temperature, usually that of liquid N2 (- 196 ‘C). Once sufficiently chilled, the germplasm may be stored and maintained for virtually indefinite periods of time and its genetic stability is guaranteed (Ashwood-Smith & Friedmann 1979) Various cryopreservation techniques have been devised for cell suspensions, calli, shoot apices and somatic and zygotic embryos. They can be divided into two major categories: (I) classical cryopreservation techniques; and (2) new cryopreservation techniques. Classical Cyopreservafion Techniques Classical cryopreservation processes have already been well reviewed (Withers 1985, 1992; Dereuddre & Engelmann 1987). Briefly, the classical procedure involves pretreatment, slow cooling, storage at ultra-low temperatures, rapid thawing and post-treatment (Figure I). Pretreatment involves the cultivation of the biological material to be stored in the presence of a cryoprotective agent such as sucrose, sorbitol, mannitol, DMSO or polyethyleneglycol. These substances may only have an osmotic action (non-penetrating agents) or may also protect membranes, proteins and enzymatic binding sites from the freezing stress (penetrating agents). Freezing is carried out slowly, cooling at a rate of 0.1 to several ‘Urnin, using a controlled freezing apparatus. The adjustment of two parameters, freezing rate and prefreezing temperature, allows the modification of the amount of residual intracellular water and thus a reduction in the damage caused by this water’s crystallization. After storage at - 196 22, samples are usually thawed rapidly and placed for a transitory period in recovery conditions different from the standard culture conditions, in order to stimulate regrowth. New Cryopreservafion Techniques During recent years, there have been modifications in the classical cryopreservation techniques for plants coming from various ecological conditions and entirely new methods, such as the use of synthetic seeds, have been developed. The most successful of these techniques are reviewed below. Encapsulation-dehydration.

The

encapsulation-dehydration

Conservation of plant germplasm methods was initially developed for the apices of several temperate species and the somatic embryos of carrot (Dereuddre 1992). Recently, however, it has been applied to the apices of three tropical crops: cassava (Benson et al. 1992); sugar cane (Gonzalez-Arnao et al. 1993; Paulet ef al. 1993); and coffee (Mari et al. 1993). This technique was based on the technology developed for the production of synthetic seeds, in which embryos are encapsulated in a pellet of alginate. For cryopreservation using encapsulation-dehydration, apices are dissected and cultured overnight on standard medium to let them recover from the dissection stress. They are then encapsulated in alginate pellets and precultured in &quid medium with a high sucrose concentration. Preculture treatment has to be determined experimentally. Encapsulated apices are then dehydrated by exposing them to filtered dried air and frozen rapidly. The technique has several advantages when used for the cryopreservation of apices compared with classical techniques: the survival rates of the cryopreserved apices are usually high (up to 100% in the case of sugar cane); recovery is very rapid; and renewed growth generally takes place directly, without a transitory callus phase, because most of the meristematic cells remain alive after freezing (Gonzalez-Amao et al. 1993). From a practical point of view, the regrowth and freezing conditions are relatively simple as sucrose is the only cryoprotectant employed and a programmable freezing apparatus is not necessary. Moreover, manipulation of apices is greatly facilitated when they are encapsulated. Encapsulation-dehydration is therefore receiving great interest for the cryopreservation of apices and is expected to be applied to a larger number of plant species. Another important aspect to take into consideration is the greatly reduced risk of instability when apices are used in comparison with other explants. Vitrification. The vitrification process consists of placing the samples for pretreatment in an extremely concentrated cryoprotective solution and then freezing them ultra-rapidly. Under these conditions, the intracellular water vitrifies, forming an amorphous glass, and none of the intracellular ice crystals which are detrimental to cell survival develop. Vitrification procedures have been developed for cell suspensions, somatic embryos and apices of several species (Sakai 1993). No controlled freezing apparatus is required but the cryoprotective mixtures used are often highly toxic because of the very high concentrations, and the duration of the pretreatment and the progressive dilution of the cryoprotectants after thawing have to be precisely controlled. This technique is therefore far from easy to use with a large range of materials, particularly if the materials are sensitive to cryoprotectants. Simplified Freezing Process. To simplify

the freezing

process,

the controlled freezing apparatus used in the classical method can sometimes be replaced with a standard commercial freezer running at - 20 or - 40 ‘C. Once temperature of the samples matches that of the freezer, they are rapidly immersed in liquid Nz. This technique has been employed for freezing carrot and coffee somatic embryos (Lecouteux et al. 1991; Abdelnour ef al. 1992b), zygotic embryos of banana and plantain (Abdelnour et al. 1992a; Villalobos & Abdelnour 1993) and embryogenic cell suspensions and calluses of several varieties of Citrus (Engelmann et al. 1993). This method will be of great use in freezing those materials which do not require very precise freezing rates. Desiccation. Zygotic embryos or embryonic axes can be successfully desiccated~ The embryos are isolated from seeds, dehydrated in filtered air and frozen rapidly by direct immersion in liquid Nz. The duration of the desiccation period varies, mainly depending on the initial water content and size of the ‘embryos. Usually, the water content ensuring maximal survival of embryos after freezing is around 15% to 20% (fresh wt). Dehydration must be sufficient to ensure survival after freezing but not so intense as to induce desiccation injury. Abdelnour et al. (1992b), working with zygotic embryos of Coffea arabica, found that the embryos had an mitial water content of 64% and, without treatment, 100% viability but no tolerance of freezing in liquid Nz. After 30 min of desiccation, however, water content dropped to 21%, viability of the unfrozen controls decreased to 80% but 50% of the embryos withstood cryopreservation. After 1.5 h of desiccation, survival of the unfrozen controls dropped to 25%, due to excessive dehydration, and only 14% embryos survived freezing at - 196

‘C.

Pre-growtk Desiccation. Cryopreservation processes combming pre-growth on media with cryoprotectants and desiccation have been developed for several species, notably for zygotic embryos of coconut (Assy-Bah & Engelmann 1992). Mature embryos of four commercial varieties were desiccated for 4 h in filtered air, then placed for 11 to 20 h on a medium containing 600 g glucose and 150 g glycerol/l. Freezing and thawing were performed rapidly. Recovery rates varied between 33% and 93%, depending on variety.

Genetic

Stability

of Preserved

Material

Skort- and Medium-term Conservation Results of quantitative studies on the genetic stability of seed-bank collections, and in particular predictions of allele losses during conservation, have been published (Breese 1989; Gale & Lawrence 1984). However, very little is known about in vitro zstored collections. During in vitro propagation, heritable ‘changes have been observed, the amount of variation being dependent on the interaction

World Jownul of Mimobiology 6 Biotechnology, Vol 11, 1995

379

VM

V~~lalobos atid F. ~ngelma~n

between the tissue-culture process, genotype and the source of explant used. Relatively high genetic stability is associated with tissue cultures of plantlets, embryos or shoots, whereas unorganized explants, such as protoplasts, cells and calli, are usually associated with higher instability. High indices of somaclonal variation may be attributed to several types of genomic change. In particular, variant mo~hological traits may be related to chromosome imprinting or mutations in genes with pleiotropic effects on development (Walbot & Cullis 1985). Orton (1984) estimated somaclonal mutation frequencies on the basis of the seed progeny, obtaining surprisingly high figures. Scrowcroft ef al. (1985) also observed high mutation frequencies: 17% to 75% for 0ryze safiva; 2.1% to 45.1% for T~~~~# ~esfjv~~; 0.4% to 2.3% for Zoa mays; 0.4% to 1.7% for ~ycopersico~ esc~le~f~m~ 1.4% to 5.6% for Lacfuca safiva; and 1.8% for Apium graveolens. Similar estim mates are not available for most tropical crops but high indices of somaclonal variation have been observed (Villalobos ef al. 1991). Only a limited number of well-documented reports is available on the effect of in z&a slow-growth storage on the genetic stability of the plant material conserved. There is evidence of genetic instability in callus cultures conserved under slow growth, even after short periods (Mannonen ef ul. 1990). In the case of organized structures, a comprehensive study has been performed recently, within the framework of a Centro International de Agricultura TropicalInternational Plant Genetic Resources Institute (CIATIPGRI) collaborative project, on various aspects of the establishment and operation of a pilot in r&u active genebank of cassava (Anon. 199ab). Based on morphological, biochemical (isoenzyme) and molecular descriptors, no observable changes were noted in the field material retrieved from iti &ro shoot cultures after three successive slow-growth storage cycles. However, it is possible that extended storage of plant material under slow growth may lead to a progressive selection of genotypes better adapted to these sub-optimal conditions. Long-fern2 Conservaficw using C~opreservafion The possible modifications of plant material induced by cryopreservation have been studied at various levels. No modification which could be attributed to cryopreservation has been reported. Plants regenerated from cryopreserved apices of strawberry and cassava were phenotypically normal (Bajaj 1985). Several hundred oil-palms regenerated from cryopreserved somatic embryos have been planted in the field and none differed, in their vegetative and flokal development, from unfrozen control palms (Engelmann ef al. 1993). No modification was noted in the electrophoretic profiles of two isoenzymatic systems in plants regenerated from control and cryopreserved apices of

sugar cane (Paulet ef al. 1993). Flow cytometric analyses revealed that cryopreservation did not appear to induce ploidy changes in sensitive dihaploids of potato (Ward ef al. 199.3). Finally, the results of restriction fragment length polymorphism (RFLP) analyses performed with shoot tips of potato (Harding 1991) and cell suspensions of sugar cane (Chowdury & Vasil, 1993) appeared identical before and after cryopre~~ation. The effects of storage duration on the recovery of cryopreserved material has been much less studied in plant tissues than in animal. However, no changes in survival were ever noted with extended storage periods by Engelmann (1991) and apices of potato and cassava were stored for up to 4 years without any modification in their recovery rate (Bajaj 1985).

Germplasm Techniques

Characterization

by Molecular

During the last two decades, molecular techniques have been developed that enable characterization of genetic material. These techniques can be divided into three categories: (1) techniques that describe a genotype without looking at any trait (usually generating complex banding patterns); (2) techniques that detect DNA linked to a trait of interest (often generating a more simple banding pattern and usually not detecting the DNA encoding the trait itself); and (5) techniques that characterize the gene of interest itself. All the methods described below are capable of detecting differences between genotypes, making them suitable for characterization of the variety. The techniques used are based on three different detection principles: Southern hybridizaiion; PCR: and DNA sequencing. Soufhern Hybridizafion Southern blots display differences in the lengths of restriction fragments. Polymorphisms detected by this technique, RFLP, are examined using single- or multi-locus probes. The use of RFLP analysis with single-locus probes is widespread in plant breeding. The current main application of the technique is the const~~tion of molecular maps, which are now available for major crop plants. RFLP can be linked to traits of interest, such as resistance to pests. Once such a linkage is established, RFLP can be used as an indirect selecting marker for the gene conferring the trait. Multilocus probes detect several restriction fragments which may be dispersed throughout the whole genome. A multilocus RFLP pattern, often called a DNA fingerprint, can be obtained by using mini- or micro-satellite sequences as probes. Polymerase Chain Reacfion Methods based on PCR display differences in the length of amplified DNA fragments: the amplified fragment length

polymovhisms (AFLP). The fragments amplified are determined by the primer(s) used for the amplification process. A multilocus AFLP pattern can be obtained without the need for sequence information for designing primers. The primers are designed randomly and only a single primer is used. The methods that generate such patterns are known by the acronyms RAPD (random amplified polymorphic DNA), AI’-PCR (arbitrarily primer-polymerase chain reaciion) and DAF {DNA ampli~cation ~ge~~n~). Primers that show polymorphism between genotypes are selected. It is also possible to link these polymorphisms with a trait of interest and they can then be used similarly to the single-locus RFLF, although the bands generated by RAPD, AP-PCA and DAF are dominant. DNA

sequencing

DNA sequencing is a well established nique, Once the sequence is known it is sequence without affecting the function composition of the protein encoded Herrera-Mrella & Simpson 1995).

and reliable techeasy to change this or the amino-acid by the gene (see

Conclusions On a global Ievel, the use of ~#-~j~# techniques for the conservation of the existing collections of plant genetic resources is still marginal. Such techniques should be explaited more fully. An additional important application of tissue culture is in the international exchange of germplasm, since plant material produced by tissue culture can be exchanged in a virus-free condition and in very small volumes. ln vifro slow-growth storage is already used routinely for conserving germplasm of some crop species (banana, plantain, potato and cassava). In terms of genetic stability, organized structures stored in sub-optimal conditions for intermediate periods should be regarded as safe. However, there is a need to monitor the relatively long-term effects of growth limitation upon genetic stability and physiology. Cryopreservation of plant germplasm is still a relatively unexplored technique. Extensive research is still needed to develop new freezing techniques, to apply existing ones to additional plant species and to experiment with the methods on a larger scale,

Acknowledgements The authors gratefully acknowledge J. Suttie, J. Serwinski, K. Ringlund and H. Silva for providing suggestions on the manuscript and some of the information in it.

References Abdelnour, A., Mora, A. & Villalobos, V.M. 1992a Cryopreservation of zygotic embryos of A&sa acwninafa (AA) and h& bar~jsia~a (BB). Cryo hffers 13, 159-164. Abdelnour, A., Villalobos, V.M. & Engelmann, F. 1992b Cryopreservation of zygoti’c embryos of Co& spp. Cryo Letfers 13, 297-302. Anon. 1993 Ex Situ Storage oj Seeds, Pollen and in Vitro Cultures of Perennial Woody Planf Species. Forestry Paper 113. Rome: Food and Agriculture Organization. Anon. 1994a Survey of Existing Data on ex Situ Collections of Planf Genetic Resources for Food and Agriculfure. Rome: Food and Agriculture Organization, Anon. 1994b Esf&ishmenf and Operation of a Pilaf in Vitro Active Gene&& Cali: Centro Intemacional de Agricultura TropicaI; and Rome: IntemationaI Plant Genetic Resources Institute. Ashwood-Smith, M.J. & Friedmann, G.N. 1979 Lethal and chromosomal effects of freezing, thawing, storage time and X-irradiation on mammalian cells preserved at - I96 ‘C in dimethylsulfovide. Cryobiology 16, 132-140. Assy-Bah, B. & Engelmann, F. 1992 Cryopreservation of mature embryos of coconut (Cocostiucifera L.) and subsequent regeneration of plantlets. Cryo Hefters 13, 117-126. Bajaj, Y.P.S. 1985 Cryopreservation of germplasm of potato (Sokm fxberoswz L.) and cassava (Manikof esculenta Crantz) viability of excised meristems cryopreserved up to four years. Indian Journal

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Ex situ conservation of plant germplasm using biotechnology.

Conservation of plant genetic resources attracts more and more public interest as the only way to guarantee adequate food supplies for future human ge...
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