As detailed elsewhere in this report, micropropagation procedures have been developed for over 1 000 plant species, many of which are micropropagated commercially. Rapid multiplication, involving rapid growth and frequent subculture, generally is an objective of commercial micropropagation. The basis of successful storage of cultures is an increase in the interval between subcultures by reduction of growth. Methods employed to this end have been well reviewed by Engelmann (1991), Withers (1992) and Wang et al. (1993)1. Alternatives are:
Storage of cultures has involved explants of various types. Shoots with roots have demonstrated better survival than those without, and microtubers are suited to storage for potato (Engelmann 1991). An innovation which holds promise for some species is the encapsulation of explants in alginate beads. In work reviewed by Engelmann (1991), encapsulated mulberry buds and sandalwood embryos survived for 45 days at 4 degrees, and resumed growth after storage.
Withers (1992) tabulates some 30 species for which successful slow growth storage, from several months to several years, has been reported. Success has been achieved with several woody species, including Pinus radiata, Alnus glutinosa, and species of Eucalyptus and Populus. Research aimed at developing in vitro storage protocols is largely empirical, involving the testing of various treatments and environmental conditions. It is likely though that, with sufficient effort, in vitro storage procedures could be developed for many of the tree species for which micropropagation protocols exist. Some tropical species may be difficult, however, to store in this way.
In vitro storage is now routinely used for germplasm storage of some crops. The in vitro cassava gene bank at CIAT, Colombia, comprises nearly 5 000 clones, in an area of 50 square metres, with transfer (subculture) intervals of 12–14 months (Escobar et al. 1992). While in vitro storage thus offers some advantages over field genebanks, the management of large collections remains problematical, due to the requirement for periodic subculture. The possible introduction of genetic variants during culture may be a risk with some types of cultures (Withers 1992).
1 See Wang et al. (1993) for additional information on the potential use of in vitro storage and cryopreservation as a method of ex situ conservation.
Although some microorganisms and higher plant pollen can be freeze dried, suspension of growth in higher plant somatic tissues can be achieved only by transfer to ultra low temperatures such as that of liquid nitrogen (Withers et al. 1990, Wang et al. 1993). At these temperatures, metabolism is suspended, and free radical damage caused by ionizing background radiation is the only factor potentially causing deterioration (Withers 1992). Results indicate that exposure to such temperatures need not be intrinsically damaging, although transitions must be carefully managed. Cryopreservation procedures thus involve the successive steps of choice of material, pretreatment, freezing, storage, thawing, and post-treatment handling, for which optima must be defined for each species (Engelmann 1991).
Growth recovery in cryopreserved cell suspensions takes place if a certain percentage of cells survives (Charrier et al. 1991, Dereuddre 1992), and reports of successful cryopreservation most commonly concern cell suspensions. Maintenance of structural integrity is more important with organized tissues such as meristem and somatic, pollinic or zygotic embryos, and reports of successful cryopreservation of these are less common. Meristematic cells are the most resilient, and young somatic embryos or immature zygotic embryos are preferred (Engelmann 1992). In oil palm, somatic embryos of only a specific morphological type can be cryopreserved (Engelmann 1991).
Pretreatment comprise the application of a cryoprotectant, dehydration, or a combination of both. Pretreatment with cryoprotectants such as sucrose, sorbitol, mannitol, dimethylsulfoxide, or polyethylene glycol dehydrates tissues, but may also act by protecting membranes and enzymatic binding sites from injury. The appropriate type, concentration and duration of treatment varies with species (Engelmann 1991). The optimal duration of desiccation treatment varies from two to four hours, during which water content is reduced to 10–16% (Engelmann 1992).
A two stage cooling process is most commonly employed. Cultures are cooled gradually down to a specific temperature, and then immersed in liquid nitrogen. Species vary in their requirements with respect to both the rate at which this cooling is conducted, and the temperature to be reached, e.g. Citrus embryos have to be cooled at a very specific rate down to -42°C, while those of oil palm can withstand a wide range of freezing rates (Engelmann 1992). With this approach, ice formation occurs first in the external medium, and water flows out of the cells. Cells reach a level of dehydration at which crystallization of the remaining water causes no damage (Engelmann 1991). Programmable freezing apparatus is required for this work. For certain tissues of some species, a one step rapid freezing, accomplished by direct immersion in liquid nitrogen, is satisfactory. In this case, intracellular ice crystallizes as microcrystals which are not harmful to cell components (Engelmann 1991). A third alternative is “vitrification”, whereby water forms an amorphous glassy structure, without a crystallization phase. This is achieved by the use of very high concentrations of cryoprotectants and very rapid cooling. This process has been applied successfully to a range of cell suspension, protoplast and embryogenic cultures, and also to shoot cultures of asparagus, mint, orange, carnation, potato and white clover (Dereuddre 1992, Withers 1992).
Thawing after storage is generally rapid, to avoid recrystallization. After the freeze-thaw cycle, culture conditions different from those of the standard procedure may have to be defined in order to stimulate the regrowth of the material.
A recent innovation has been the incorporation of alginate bead encapsulation methods into cryopreservation procedures (Dereuddre 1992). This method offers protection from adverse effects of some of the treatments. Somatic embryos or shoot tips are suspended in culture medium supplemented with 3% Na-alginate. The mixture is then dispensed from a pipette. Beads of about 4 mm diameter containing 1 to 3 embryos or shoot tips are precultured for several days in media supplemented with sucrose. Coated organs are dried under sterile air flow at ambient temperature and humidity for up to six hours. After dehydration, artificial seeds are transferred into cryovials without liquid medium for cooling, using either a one or two step procedure. The encapsulation-dehydration procedure has been applied to cryopreservation of somatic embryos of carrot and shoot tips of pear, potato, grape and carnation.
Cryopreservation has been applied successfully to over 70 plant species (Engelmann 1992, Withers 1992), including tropical crops such as coconut, rubber, cocoa, oil palm, peanut, rice, banana, cassava, and coffee. Forest tree material for which successful cryopreservation has been reported includes embryos of Quercus petraea, Fagus sylvatica, Aesculus hippocastanum (Jorgensen 1990), Araucaria excelsa, Castanea (Engelmann 1992), Artocarpus, and Juglans (Withers 1992); embryogenic cell lines of Picea glauca, Acer pseudoplatanus (Withers 1992), Picea abies (Durzan 1988, Bercetche et al. 1990, Gupta et al. 1987) and Pinus taeda (Gupta et al. 1987, Durzan 1988), seeds of Abies alba, Sequoiadendron giganteum, Larix decidua, Pseudotsuga menziesii, Picea abies, Pinus sylvestris (but not Quercus petraea) (Jorgensen 1990), pollen of Betula pendula, B.pubescens, Larix decidua, L.kaempferi, Pseudotsuga menziesii, Picea abies, Pinus sylvestris, Quercus petraea and Q. robur (Jorgensen 1990), and encapsulated shoot tips of Eucalyptus gunnii (Monod et al. 1992).
The expectation is that, once cryopreserved, material can be stored indefinitely in liquid nitrogen without further loss. Strawberry and peanut meristem were capable of regeneration after two years storage, and 1.5 years for oil palm, while potato and cassava meristem survived for four years (Engelmann 1991). Cell lines of Picea glauca have shown no loss of viability after over two years of storage (Attree & Fowke 1991).
The main emphasis of most cryopreservation research programmes lies in defining non-damaging procedures for cooling and rewarming the cultures, for which species have differing requirements. Rapid progress is being made with this work, and it seems likely that, given a sufficiently intensive research effort, prospects for developing a suitable protocol for tissue preservation for any chosen species within the short term would be reasonably good. One obstacle to application for many species lies in the need to regenerate plants from the tissues or organs after the period of cryopreservation.
Three broad areas of application of cryopreservation and in vitro storage techniques to forest tree improvement warrant discussion:
In vitro storage is now routinely used for germplasm storage of cassava, at CIAT (Escobar et al. 1992, Chavez et al. and for many Solanum genotypes at CIP, in Peru (Withers et al. 1990). ORSTOM, at Montpellier, is developing methods for the preservation of coffee by in vitro culture of immature zygotic embryos (Charrier et al. 1991). Work aimed at the storage of somatic embryos of mango is underway (IBPGR 1991). In the IBPGR in vitro program, high priority is being given to the development of good in vitro culture procedures for sweet potato, taro, cocoyam, banana and plantain, sugarcane, cocoa, citrus and forage grasses (Withers et al. 1990). With respect to cryopreservation, preservation of a substantial number of genotypes of oil palm by ORSTOM is the closest to routine application (Engelmann 1991), while the development of routine procedures for banana and plantain (at CATIE), cassava (at CIAT) and coconut is underway (IBPGR 1991).
A number of features characterize the above crops targeted for in vitro or cryopreservation approaches to gene conservation:
They are limited in number, and are all of major, widespread importance.
Most are characterized by a long history of domestication and use.
Biological and agronomic features are relatively well known.
Several are vegetatively propagated.
Some have recalcitrant seeds.
Gene conservation in these crops is to some extent then directed towards:
The collection of known genotypes from many different areas of use. These genotypes have been evaluated to some degree, and it is desirable gene combinations, rather than individual genes, which are the target of preservation.
Preservation of threatened wild relatives, characteristics of which would also be partially known.
Under these circumstances, conservation priorities are reasonably easily set. For these crops, in vitro storage and cryopreservation offer alternatives to the traditional dependence on field genebanks. For agricultural crops where seed collection and storage are possible, however, seed storage is the recommended approach to gene conservation (Withers 1992).
Undoubtedly, urgent gene conservation and preservation problems exist for forest trees species, mainly among the tropical hardwoods and non-industrial species. Compared to the crop species discussed above, the following features characterize these tree species:
There is a large number of species of known or potential interest. Many may never be used commercially, and some will be important only locally.
Biological features of many are poorly known.
Species distributions, and those of relatives, are poorly known.
Seeds of some species will be in the recalcitrant category, but many are orthodox.
Major impediments to the preservation of these gene pools are:
Resources available are sufficient for only a very small fraction of the survey and collection work which would be required.
Seed storage facilities in many tropical countries are unreliable, such that satisfactory storage of seed collected now cannot be guaranteed (Ng 1985, Reid et al. 1991).
Wang et al. (1993) correctly drew attention to the potential use of in vitro storage and cryopreservation as a complementary method for the ex situ conservation of long-lived perennial species. This potential is undoubtedly real. It is of value though to carry this a little further, to consider specifically to which groups of tree species the technology might be applicable in the near future. It can be observed that replacement of existing seed storage facilities with more sophisticated technology is not likely to make a positive impact on the gene conservation problem with tropical tree species. Even for the recalcitrant species, activities should favour the establishment of ex situ plantings which will enable evaluation of the material. Cryopreservation and in vitro storage techniques thus offer no solution to erosion of tropical forest gene pools. More concerted efforts at documentation and in situ conservation are required.
Of established industrial species, cryopreservation and in vitro storage of vegetative material are not advantageous approaches to gene conservation in most of the conifers and eucalypts, for which seed can be stored satisfactorily. Poplars display some of the features characteristic of the fruit tree species described above - problematic seed storage, and breeding and commercial use based substantially on known clones. In vitro storage or cryopreservation may have a role as a back-up measure in large poplar breeding programmes. Similarly also for other species with seed production and storage problems, e.g. Juglans. It should be noted though that, for most forest tree species, introduction of in vitro or cryopreserved material into a breeding program will involve a delay of several years to flowering, and generally also for evaluation. Flowering genotypes in field clone banks can be much more quickly incorporated. An exception to the flowering obstacle is stored pollen, which can be rapidly incorporated into breeding programmes, and pollen storage should be more prominent in gene conservation programmes with industrial species. Cryopreservation may facilitate the broader use of pollen storage in these programmes.
It should be noted also that, as pointed out by Jackson and Coleman (1991) and Wang et al. (1993), the use of cryopreservation and in vitro storage could involve selection for genotypes which tolerate the conditions imposed (e.g. freezing and thawing), and which are capable of regeneration following such treatment. The conserved material is thus not necessarily a random sample of the genetic material collected.
Cryopreservation is perhaps the most promising approach currently on offer for maintenance of the juvenile state and capture of genetic gains through clonal forestry with industrial species (Haines 1992). The technology is applicable mainly in cases where good breeding programmes are in place and clonal forestry is a realistic goal. In particular, this applies to some programmes with conifers. Although some good breeding programmes are in place for the eucalypts, the need is not so urgent for these because rejuvenation can be achieved by other means. The approach ultimately may have some application to other tropical hardwoods, as good breeding programmes are established for these. Maturation is a much less important issue with many of the non- industrials, in particular those which flower precociously, can be selected early, and which can still be readily propagated at selection age.
The use of plants, organs or cells cultured in vitro circumvents to some extent the limitations which quarantine regulations place on the import of vegetative material (in particular) into many countries. International germplasm exchanges are becoming very important in the breeding of many crops, in particular to and from international genebanks at the International Agricultural Research Centres (IARCs) and other centres. In vitro exchanges are becoming common for some of these, e.g. for potato, coconut, banana and oil palm (Charrier et al. 1991, Withers 1992). In addition, in vitro collection, overcoming problems of inadequate or immature seeds and deterioration of vegetative material in transit, is now being used. Simple field procedures have been devised for cocoa, avocado, coconut, citrus, cassava, cotton, and forage grasses. (Withers et al. 1990, Withers 1992).
With the increasing emphasis on cooperative breeding of both industrial and non-industrial forest tree species in tropical countries, international movement of material will become increasingly important. In the short term, such movement is likely to be mainly in the form of seed for species and provenance testing. Movement of vegetative material, however, may become more important as breeding programmes advance, and in vitro approaches could find application in the longer term.
In vitro storage and cryopreservation of vegetative material is likely to have limited application to gene conservation activities for forest tree species, with minor use as a backup strategy for species displaying seed storage problems the most likely possibility. These technologies offer no solution to the urgent problem of genetic erosion of tropical forest gene pools. Cryopreservation of pollen is deserving of more attention as a method for medium and - maybe - in long-term gene conservation with industrial plantation species.
Cryopreservation warrants much more attention as a means of maintaining juvenility and capturing gains offered by clonal forestry with industrial species, but only for plantation programmes where good breeding programmes are in place and clonal forestry is a realistic goal. In particular, this applies to some conifers and eucalypts.
In the longer term, the use of in vitro germplasm transport procedures may become useful for species which are the subject of cooperative regional breeding programmes.