Roca, W. 1983 El Cultivo de Tejidos Vegetales para la Conservación y. 382. World fournal of ... el Intercambio lnlemacional sobre Métodos de Cultivo de Tejidos.
( World Journal ol Microbiology & Biotechnology 11 . 37&-382
Ex situ conservation of plant germplasm using biotechnology V.M . Villalobos* and F. Engelmann
i 1
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 sita• storage centres throughout the World. The present review is of these ex situ collections and the 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 vitro slow growth is used routinely for conserving germplasm of plants such 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 and 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.
Key words: Artificial seeds, cryopreservabon, exisbng collections, germplasm charaderization, in vifro storage.
Plant genes, which have either been selected by nature or by man, through on-farm empírica! 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 variebes to be bred. In recent years, severa! factors, including the substitution of local genotypes with improved variebes and hybrids, the development of new land, forest deplebon, changes in agricultura! techniques, and the abuse of agrochemicals, have caused a rapid and profound erosion of this genetic resource, with the loss of potenbally valuable material which had barely been explored. Those constantly trying to increase food production have often neglected the value of proteding genetic resources and have often failed to make efficient use of those V.M. Vollalobos is with the Food and Agricultura Organization of the United Nations. Viale delle Terme di Caracalla. 00100 Rome. ltaly; fax: 522·56347. F. Engelmann is with the lnternational Plant Genetic Resources lnstitute (IPGAI). Via delle Sette Chiese 142. 00145 Roma. ltaly. ·corresponding author.
resources which are available. Such protection and exploitation require the corred collection, conservation, evaluation, documentaban and exchange of plant germplasm. Although germplasm conservaban is attracting more and more public concem as the only way to guarantee food supplies fo r future human generabons, it is not simple, since it involves culturaL economical, technical and political issues.
Existing Collections of Plant Genetic Resources 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 ex sifu (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 Food and Agriculture Organization (FAO) (Anon. 1994a), there 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
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V.M. Villa lobos and F. Engelmann Table 1. Ex situ collectlons by region. Reglon
Africa· Asia" Europe· Latin America North America Oceanía Subtotalt lnternational (CG IAR):j: Total
No. of accesslons
%o! total
265,000 971 ,500 1,344,000 441 ,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 88.4 11.6 100.0
• lncludes. for their respective regions. the collections of the Centro Agronomico Tropical de Investigación y Enseñanza (CATIE) and the Nordic Gene Bank (NGB), since these are controlled by, or service, the governments of the region. t From the World lnformation and Early Warning System on Plant Genetic Resources (WIEWS/ PGR) data-base. May 1994. :j: From the Stripe Study of Genetic Resources in the Consultative Group on lnternational Agricultura! Research (CGIAR). on lnternational 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).
A lthough the genetic diversity of many crops has been well preserved ex sifu, many o ther crops which are important at a national or local leve! are poorly represented in the existing colledions for a variety of reasons. For example, sorne important crops, such as mango, rubber, cocoa, coconut, coffee and oil palm produce recalcitrant seeds which are unable to withstand desiccation. Long-term sto rage of perennial plants, including trees, which have long juvenile periods before any seed is produced can also be difficult. On the other hand, sorne vegetatively propagated species, including many of those eaten as roots o r tubers (yams, potato, cassava, Xanfhosoma and sweet potato) and severa! fruits (bananas and plantains) are either sterile or do not have stable sexual reprodudion. Success in germplasm storage is determined by: the inherent longevity and physiological storage behavio ur 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 instituhons World-wide have sorne sort of ex sifu colledion. Of these, 308 institutes have the capacity for medium-term storage, 175 for lo ng-term storage and 119 have the facilities for
Table 2. Ex situ collections in May 1994, by crop group. Crop
No. of accessions
% o! total
National collections
CGIAR centres
Totals
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
317,200 118,150 50.900
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
Cereals Pulses Forages Vegetables Fruit Roots and tubers Oi l c rops Fibre crops Beverages Rubber Miscellaneous Sugarcane Narcotics and drugs Condiments. spices. flavourings, herbs Shelter crops Chocolate crops Ornamentals Medicinal plants Oyes Perfume crops Building materials Weeds Timber crops Unknown Banana· Multi-purpose trees· Totals
3,905,200
22,450
1500 300 510,500
46.82 16.27 9.63 7.62 3.95 4.07 2.03 1.59 0.97 0.69 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 0.0 1 100.0
• The CGIAR centres class bananas and multi-purpose trees as separate categories. In the case of national collections, they are s ubsumed into other categori es and not reported separately.
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Conservalion of planl germplasm Table 3. Ex situ collections by maintenance method. Maintenance regime
No. of accessions·
Short-term storage Medium-term storage Long-term storage In vilro storage Fieid collections
628,500 2,333,100 2.045,200 37,600 302,300
• These numbers shouid not be summed . The data were interpretad on the assumption that, when a mixture of categories was given, the crop is stored in aii the indicated manners. and this may have inflated sorne figures. To derive percentages wouid be misleading .
storage at temperature below - 18 ·e Both in vitro and field colledion.s are categorized as either short- or mediumterm storage.
The Contribution of Biotechnology to Plant Germplasm Conservation The biotechnological techniques recently applied to plants have great agricultura! potenbal. They provide new approaches to overcome the problems of plants in marginal environments, biobc stresses and pests and diseases, permitbng plants with unique gene combinations, greater pestresistance and/ or yields to be produced. They also have major applicabons in the field of plant germplasm conservaban. Since complete plants can now be produced from isolated cells, tissues or organs, germplasm banks based on plant bssue cultures can now be established. During the last 15 years, in vitro 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 mulbplication of plants which produce recalcitrant seeds and those which are usually propagated vegetatively (Thorpe el al. 1995). The development of in vitro technology has, in fact, been a strong motivation for the planning, research and development of alternative methods of conservation. In principie, 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 leve! 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 condibon 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 invilro c;onservation, 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 vilro techniques require the substitubon 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 sorne roots, tubers and other annuals, in vilro 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 et al. 1995). In the modern approach to conservaban and rabona! use of genebc diversity, in vitro conservabon should include the elimination of virus from the stored material and the ability to micro-propagate the germplasm in large quantities when necessary (Villalobos el al. 1991). Any tissue-culture system employed for in vilro conservation should fulfil two requirements: (1) 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 morphogenic potential, in comparison with other bssues and isolated cells. (2) 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 conservation/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 mid-term conservabon, germplasm is cultured under normal growth conditions or growth-limiting 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
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V.M. Villalobos and F. Engelmann
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Growth in vitro 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.
I/
Long-term Conservation or Cryopreservation
M
Figure 1. Schematic representation of the classical cryopreservation procedure employed for freezing carnation apices (from Galerne 1985). A-/n vitro mother plantlets [shoot t ips (m) are dissected and used for freezing and microcuttings (b) used for further multiplication] ; B-pregrowth of apices (24 h on sol id medium with 0.5 M sucrose); and D-(final concentration 5%) (add it ion of DMSO is performed in ice (g)); E-transfer of apices into cryovials; F~ryoprotective medium using pincets (p) precooled in liquid nitrogen; G~ontrolled slow cooling (by 0.5 m in) down lo - 40 ' e; H- i mmersion of cryovials into liquid nitrogen ; 1 and J-rapid thawing in a water-bath al 40 ·e; K to M- post-treatment and recovery, with elimination of cryoprotectants (K and L); M~ulture on standard medium .
e
·e¡
mid-term germplasm storage is to define the experimental conditions that favour minimal growth without alteration of genetic stability, with the mínimum 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 beneficia] effect on culture stability, since diminishing the cellular division rate should reduce the frequency of the mutations that occur during the duplication 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 (Roca 1983 ).
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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 N 2 ( - 196 •e). 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: (1) classical cryopreservation techniques; and (2) new cryopreservation techniques.
Classical Cryopreservation Techniques Classical cryopreservation processes have already been well reviewed (Withers 1985, 1992; Dereuddre & Engelmann 1987). Brief!y, the classical procedure involves pretreatment, slow cooling, storage at ultra-low temperatures, rapid thawing and post-treatment (Figure 1). 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 po lyethyleneglycol. 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 ·c / min, 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 •e, 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 Cryopreservation 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
Conservafion of planf germplasm methods was initially developed for the apices of severa] 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 ef al. 1992); sugar cane (Gonzalez-Arnao ef al. 1993; Paulet ef al. 1993); and coffee (Mari ef al. 1993). This technique was based on the technology developed for the produdion of synthetic seeds, in which embryos are encapsulated in a pellet of alginate. For cryopreservation using encapsulation-dehydration, apices are disseded and cultured overnight on standard medium to Jet them recover from the dissedion stress. They are then encapsulated in alginate pellets and precultured in ~iquid 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 severa! advantages when used for the cryopreservation of apices compared with classical techniques: the survival rates of the cryopreserved apices are usually high (up to lOO% 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-Arnao ef al. 1993). From a practica! point of view, the regrowth and freezing conditions are relatively simple as sucrose is the only cryoprotedant 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 asped 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 cryoprotedive 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 ha ve been developed for cell suspensions, somatic embryos and apices of severa] species (Sakai 1993). No controlled freezing apparatus is required but the cryoprotedive mixtures used are often highly toxic because of the very high concentrations, and the duration of the pretreatment and the progressive dilution of the cryoprotedants 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 cryoprotedants.
u
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 oC. Once temperature of the samples matches that of the freezer, they are rapidly immersed in liquid N 2 • This technique has been employed for freezing carrot and coffee somatic embryos (Lecouteux ef al. 1991; Abdelnour et al. 1992b), zygotic embryos of banana and plantain (Abdelnour et al. 1992a; Villalobos & Abdelnour 1993) and embryogenic cell suspensions and calluses of severa! varieties of Cifrus (Engelmann ef al. 1993). This method will be of great use in freezing those materials which do not require very precise freezing rates.
Desiccafion. Zygotic embryos or embryonic axes can be successfull y desiccated. The embryos are isolated from seeds, dehydrated in filtered air and frozen rapidly by direct immersion in liquid N 2 • 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 1S% to 20% (fresh wt). Dehydration must be sufficient to ensure survival after freezing but not so intense as to induce desiccation injury. Abdelnour ef al. (1992b), working with zygotic embryos of Coffea arabica, found that the embryos had an initial water content of 64% and, without treatment 100% viability but no tolerance of freezing in liquid N 2 . After 30 min of desiccation, however, water content dropped to 21%, viability of the unfrozen controls decreased to 80% but SO% of the embryos withstood cryopreservation. After l.S h of desiccation, survival of the unfrozen controls dropped to 2S%, due to excessive dehydration, and only 14% embryos survived freezing at - 196 oc. Pre-growth Desiccafion. Cryopreservation processes combining pre-growth on media with cryoprotedants and desiccation have been developed for severa! 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 ISO g glycerol/1. Freezing and thawing were performed rapidly. Recovery rates varied between 33% and 93%, depending on variety.
Genetic Stability of Preserved Material Shorf- and Medium-term Conservafion Results of quantitative studies on the genetic stability of seed-bank colledions, and in particular predidions of allele losses during conservation, have been published (Breese 1989; Cale & Lawrence 1984). However, very little is known about in vitro stored colledions. During in vitro propagation, heritable changes have been observed, the amount of variation being dependent on the interadion
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V.M. Villalobos and F. Engelmann 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 índices of somaclona! variation may be attributed to severa! types of genomic change. In particular, variant morphological trai ts 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 et al. (1985) also observed high mutation frequencies: 17% to 75% for Oryz.a saliva; 2.1% to 45.1% for Trilicum aestivum; 0.4% to 2.3% for Zea mays; 0.4% to 1.7% for Lycopersicon esculenlum; 1.4% to 5.6% for Lacluca saliva; and 1.8% for Apium graveolens. Similar estimates are not available for most tropical crops but high índices of somaclonal variation have been observed (Villalobos el al. 1991). Only a limited number of well-documented reports is available on the effect of in vilro 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 el al. 1990). In the case of organized structures, a comprehensive study has been performed recently, within the framework of a Centro Internacional de Agricultura Tropicallnternational Plant Genetic Resources Institute (CIATIPGRI) collaborative project, on various aspects of the establishment and operation of a pilot in vilro active genebank of cassava (Anon. 1994b). Based on morphological, biochemical (isoenzyme) and molecular descriptors, no observable changes were noted in the field material retrieved from in vitro 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-lerm Conservation using Cryopreservation 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). Severa! hundred oil-palms regenerated from cryopreserved somatic embryos have been planted in the field and none differed, in their vegetative and floral development, from unfrozen control palms (Engelmann et al. 1993). No modification was noted in the electrophoretic profiles of two isoenzymatic systems in plants regenerated from control and cryopreserved apices of
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