Background papers

Rationale for establishment of regional genebanks
Towards a coconut conservation strategy
Conservation priorities and guidelines on accessions to be conserved
Genebank design and activities
A multilateral system for the exchange of plant genetic resources
International movement of coconut cultivars

Rationale for establishment of regional genebanks

G.A. Santos

Div. Chief III, Breeding & Genetics Division, Philippines Coconut Authority, Zamboanga Research Center, San Ramon, Zamboanga City; Member, COGENT Steering Committee

Introduction

It is widely recognized that an efficient international R & D programme on coconut can only be achieved through the establishment and organization of a research network under the auspices of an international body which could further spearhead the progress made in the past by coconut researchers in the producing countries and advanced laboratories in the developed countries.

The importance of genetic resources is obvious in coconut varietal improvement. Hence, the conservation of these genetic resources is one of the main concerns of the recently organized and emerging International Coconut Genetic Resources Network (COGENT).

Following the Workshop on Coconut Genetic Resources which was held in Cipanas, Indonesia in October 1991, the Steering Committee (SC) of COGENT resolved, during its first organizational meeting in Singapore in December 1992, to undertake a series of consultations on the various concerns that were raised by the 15 country representatives at the Cipanas workshop. The 1992 meeting in Singapore emphasized the long-felt need for the conservation of coconut genetic resources as a priority owing to the increasing rate of genetic erosion and threats to the genetic diversity of coconut germplasm in national collections and those in farmers' fields in the growing areas.

The role of COGENT

As originally defined in the September 1993 meeting of the COGENT SC in Montpellier, France, the Network will function at the national, regional and global levels to strengthen the capacity of the national programmes to conserve and utilize coconut genetic resources.

Why regional genebanks?

With a few exceptions, the coconut varieties in most national collections were collected on the basis of popular knowledge and chosen on the merits of highly distinguishable morphological features often with minimum regard to accurate representation and possibilities for long-term conservation and use. This is quite understandable because coconut is a bulky plant requiring a large area. Additionally, it needs considerable financial support for experimentation and upkeep which most individual countries cannot and will not be able to sustain on their own.

Although beset with funding constraints, these national collections have at least fulfilled the pressing need of some countries for genetically improved planting materials which are now being used in replanting programmes. Therefore, the genetic base of these collections deserves to be restructured, kept and used (see related paper on priorities) under a scheme of shared responsibility among the users of the germplasm (as either producers or consumers) for their mutual advantage.

The general purpose and long-term aim of COGENT is to safeguard and secure the coconut germplasm of the world against permanent loss and genetic deterioration. Since this cannot be carried out in only one location without facing great risks, the SC, upon the recommendation of the task force, deemed it critical to keep the world collection in five regional genebanks (formerly called regional holding centres) and to adopt a commonly agreed conservation strategy.

The proposed Regional Genebanks, in collaboration with the technology sources, shall have the following specific tasks:

1. To study the diversity of coconut germplasm and conserve the identified diversity, per se

These functions will be of particular importance for coconut germplasm from countries which do not have the ability or capacity to conserve or study their own indigenous germplasm, in view of lack of area, financial and human resources, frequent weather disturbances, occurrence of pests and diseases and the tendency of some countries to shift to other crops. The Regional Genebank will, therefore, duplicate and multiply coconut varieties held in national programmes and preserve the germplasm in optimum numbers following the agreed procedure in the STANTECH manual.

The proposed strategy is to assemble all germplasm from countries within the Regional Genebank in the form of living collections with duplicates in the form of pollen and embryos in all of the other regional genebanks and hold them in trust in each regional genebank. For instance, in Indonesia, all accessions from Southeast Asia will be kept as living collection while all the rest from other regions shall be in the form of pollen and embryos. Since pollen can be used right away in breeding, the hybrids can be tested immediately in all regional genebanks as in a multilocational test. Take note that with this feature, the genebank also serves as a testing site which matches with the original idea of a germplasm research unit as indicated by Persley (1992).

2. To conduct performance evaluation and genetic trials

Apart from the germplasm to be conserved and secured, other interesting germplasm may be tested in the genebank for agronomic performance and genetic evaluation subject to a general agreement or special agreements among: (1) genebanks, (2) countries contributing to the genebank, (3) the genebank and each contributing country, or (4) combination of 2 and 3 with regards to sharing of costs and results.

3. To mass produce and distribute promising breeding materials to interested countries

Expectations are high that when biotechnology becomes finally usable for coconut, better and more efficient mass propagation techniques could be employed and more diversity will be discovered such that geneflow information to analyze the origin of the coconut would be available and lead to the selection of superior materials.

Regional genebanks established under agreed schemes of shared responsibility among developing countries will promote better cooperation among them and will encourage donor nations and agencies to continue supporting and mobilizing funds to ensure the success of the endeavour.

Potential problems of a regional coconut genebank

Past movements between countries of germplasm as seed, pollen and embryo have not caused any major pest and disease outbreaks during the last 50 years. Of late, however, even the use of embryos in transferring germplasm, which has been considered relatively safe and easier to be moved than seeds, needs to be carefully evaluated because of the implied problems that could be brought about by the reported existence of phytoplasmas in coconut embryos and the possible pathogenicity of viroid-like organisms.

A coconut genebank requires strong financial support in the initial years. Funding, maintenance and physical upkeep as well as the basic agreements for sharing research results and germplasm transfer require sufficient deliberations to establish responsibilities and to guarantee mutual benefits.

Our strength

Increased collaboration and information exchange on all coconut research has been made possible through COGENT. The Regional Genebanks in Asia and the Pacific regions will certainly benefit from the ongoing ADB-supported project on Coconut Genetic Resources Network in Asia and the Pacific (CGRNAP) wherein 13 countries are involved.

The availability of the Coconut Genetic Resources Database (CGRD) can be a good starting point in obtaining valuable information for the selection of the first materials to be planted in the genebank to establish a core collection or a full complement of the world's coconut germplasm.

Indications that precocity can be enhanced by good agronomic practices increase our chances for the expeditious evolution of modern varieties even through classical breeding. In the meantime, the rapid development of the applications of biotechnology, particularly molecular biology, on coconut could certainly facilitate genetic diversity studies and screening for exceptional and relevant genotypes.

Increasing knowledge on the means of preserving coconut tissues/embryos/pollen provides an avenue for more modern and practical conservation techniques leading to better and increased long-term protection of coconut genetic resources.

Reference

Persley, G.B. 1992. Replanting the Tree of Life. CABI in association with ICAR and TAC of CGIAR. CABI, Wallingford, UK.

Towards a coconut conservation strategy

V. Ramanatha Rao¹, K.W. Riley¹, J.M.M. Engels², F. Engelmann² and M. Diekmann²

¹ Senior Scientist (Genetic Diversity/Conservation), and Regional Director, IPGRI-APO, Singapore

² Director, Germplasm Maintenance and Use (GMU), Senior Scientist (In vitro Conservation), and Senior Scientist (Germplasm Health), GMU, IPGRI, Rome, Italy

Introduction

Coconut (Cocos nucifera) belongs to a monotypic genus and there are no known wild forms. The variability, in terms of what could be described as provincial/local types, is reported to be the highest in Southeast Asia (Whitehead 1976). The distribution of coconut palm is very wide, but restricted to 20° either side of the equator. It has not been possible to establish either the centre of diversity or the origin of coconut. Though there is still much debate, ethnological information as well as the diversity of local types indicate Southeast Asia to be the most probable centre of diversity and a Malesian origin has been advocated (Harries 1990). Nevertheless, it is generally agreed that coconuts have moved from Asia in both directions, to the Pacific and Americas in the east and to Africa in the west. Lengthy discussion is available in the literature on speculations of these movements. The bulk of the dispersal must have been through humans, although some nuts might have moved by flotation, judging by the coastal distribution of coconut (Burkill 1935; Foale 1987, 1992). Adding to the complexity, the relationship between American and Asian coconuts has not been established. In general, two types - tall and dwarf - are recognized, each with several variants in most of the regions of its distribution. More than 40 races have been recognized based on the height of the plant and the quantity of fruits (seednut yield) produced. These palms belonging to different races tend to intermate if grown together (Burkill 1935). Recent, though limited, studies using random amplified fragment length polymorphisms (RFLPs) appear to confirm the movement of coconut from Southeast Asia to the Pacific and then to the west coast of the Americas (Leburn et al. 1995). The West African ecotypes appeared to be similar to those from India and Sri Lanka. Though no wild forms have been described, Villareal et al. (1993) reported that some Atlantic coast populations showed a wild type fruit with angular shape, thick husk, thick shell, thick endosperm and low water content.

Several organizations, both national and international, contributed to collecting and conservation of coconut genetic resources around the world (van Sloten et al. 1993). The need for increased efforts on collecting and conservation of coconut genetic resources was first recognized during the Eighth Pacific Science Congress in Manila in 1953. This was followed by several meetings over the years and considerable efforts have been made by individual countries and organizations like the Food and Agricultural Organization of the United Nations (FAO), the South Pacific Commission (SPC), Institut the Recherches des Huiles et Oléagineux (IRHO), etc. (Ramanatha Rao and Riley 1995). FAO initiated a regional project on coconut which was completed in 1966. Various national and bilateral support efforts have resulted in coconut germplasm collecting and breeding taking place in countries such as Côte d'Ivoire, Tanzania, Philippines, India, Indonesia and Papua New Guinea (PNG). In 1975-76, the then International Board for Plant Genetic Resources (IBPGR) was requested to convene an Advisory Committee on Coconut Genetic Resources, in line with other Crop Advisory Committees. In 1991, owing to the global concern over the decline in production and erosion of genetic resources of coconuts, the International Plant Genetic Resources Institute (IPGRI), then IBPGR, was asked to initiate a network on coconut genetic resources.

This paper is an attempt to further develop the ideas presented at the Coconut Genetic Resources Network-Asian Development Bank (COGENT-ADB) Project and Steering Committee Meeting at the Central Plantation Crops Research Institute (CPCRI), Kasaragod, India on 11-15 September 1995. Several suggestions that came up during and after the meeting have been incorporated into this paper. The development of a final agreed strategy will require interaction and discussion with COGENT members and other collaborating institutions. This paper also briefly discusses the need for, and possible components of, a complementary conservation strategy including field genebanks, in vitro, pollen, cryopreservation and in situ conservation. The status of each as a practical conservation method for coconut will be briefly reviewed, followed by a detailed account of field genebanks.

Components of a complementary conservation strategy

It is now well recognized that for any given genepool, a number of different approaches and methods will be necessary for efficient and cost-effective conservation. Such a strategy is known as complementary conservation strategy (CCS). The two basic approaches to conservation of plant genetic resources (PGR) are termed ex situ and in situ. Ex situ approach involves conserving PGR outside their original habitat in the form of seed, embryos, tissues or plants. Methods of ex situ conservation can include cold storage, in vitro storage or field genebank, depending on the type of propagules used. In contrast, in situ conservation involves the maintenance of genetic diversity of a species or genepool in the habitat in which the diversity evolved. In the definition of the Convention on Biological Diversity, it includes the maintenance of diversity in farmers' fields and orchards; thus it includes the so-called on-farm conservation. It is important to emphasize that in situ and ex situ conservation are not mutually exclusive, but are indeed complementary. Any efficient conservation strategy for a genepool could employ a combination of methods, from nature reserves to genebanks. The appropriate strategy and the balance depend on factors such as the biological characteristics of the plants, their present management and use by humans, available resources for conservation, number of accessions in a given collection and geographic sites, the purpose of conservation, the availability of germplasm and political and administrative policies. The extent of a particular method used may differ from one genepool to another (Withers 1993). As coconut is monotypic, without any known wild form, we are concerned with the conservation of diversity within a single species. This species includes many ecotypes.

At this point, it will be appropriate to examine a broad range of conservation methods (within each approach) in an effort to evaluate the suitability of each method for the conservation of coconut genetic resources. An attempt will also be made to look at how coconut can fit into the scheme for efficient conservation as well as effective use of available resources. For obvious reasons, ex situ conservation of seeds is not possible in the case of coconut. Other methods that can be used are field genebanks, in vitro conservation, cryopreservation of tissue and/or pollen and in situ (on-farm) conservation.

Field genebanks (FGBs)

FGBs are presently the most feasible ex situ conservation method that can be used for coconut. Many important varieties of field and horticultural crops including coconuts are either difficult or impossible to conserve as seeds (i.e. no seeds are formed or if formed, the seeds are recalcitrant) or the species are vegetatively propagated. Hence, they are conserved in FGBs. FGBs may run a risk of being damaged by natural calamities, infection, neglect or abuse. Ex situ conservation of tree species using FGBs requires a substantial number of individual genotypes to be an effective conservation measure. Thus, FGBs require more space, especially for large plants such as coconut, and they may be relatively expensive to maintain depending upon the location and the complexity of alternative techniques available. However, FGBs provide easy and ready access to conserved material for research as well as for use. For a number of plant species, the alternative methods have not been fully developed so that they can be effectively used (Ramanatha Rao and Riley 1995). Thus, it is clear that the establishment of FGBs will play a major role in any conservation strategy for PGR. FGB is one of the options of a CCS for the conservation of germplasm of many plant species, including coconut. At the same time, efforts to develop and refine other methods, such as in vitro conservation and on-farm conservation, must go on.

There are many field collections of coconuts in various countries, usually connected with coconut research institutes. According to the FAO database on germplasm holdings, 950 accessions have been reported. Often these collections are quite old and may originally have developed in a somewhat haphazard manner. Exact origins of many of the palms in collections are often unknown. Although they represent a commendable effort, there is a need to update the collections in a scientific manner, with due regard to thorough documentation of populations, and correct and foolproof labelling. We will come back to FGB of coconuts a little later.

In vitro conservation

Another method of ex situ conservation of germplasm is through the use of tissue cultures. The use of in vitro culture techniques, including slow growth and cryopreservation, represents an important additional option for the medium- and long-term conservation of species like coconut. Slow growth techniques, which allow medium-term conservation, and cryopreservation techniques that allow long-term conservation, have been developed for reproductive and somatic tissues of a large number of species and are now either available or at various stages of development (Withers and Engelmann 1996; Engelmann and Ramanatha Rao 1996). A considerable amount of work has already been conducted in coconut for the development of in vitro collecting, culture, conservation and distribution techniques.

Coconut is one of the species with big seeds. Moreover, there is no dormancy period and germination of mature seed (fruit) starts within 2-3 weeks after it drops on the ground. These two characteristics drastically limit the amount of material which can be gathered during collecting missions. A simple and efficient in vitro field collecting technique has been established which involves extracting the embryos from the nuts and inoculating them directly onto culture medium (Assy-Bah et al. 1987). Embryos can be kept for 2 months before they are cultured in a controlled laboratory environment (Engelmann and Assy-Bah 1992). This technique is used routinely to collect coconut germplasm. In vitro culture is fully functional for coconut zygotic embryos (Assy-Bah et al. 1989); plants can be regenerated on a one to one basis with ease, even though hardening of young plants in field conditions can be problematic in circumstances where experience in handling in vitro cultured material is limited. Problems arise at the stage of clonal propagation onwards. Mass propagation by means of somatic embryogenesis is under development and several clonal plantlets have already been produced for some genotypes (Verdeil and Buffard-Morel 1995). However, extensive additional research is needed before this technique can be routinely employed for the large-scale propagation of elite genotypes in different locations and countries.

Typical slow growth protocols are not currently available for coconut owing to the difficulties encountered with propagation techniques for this crop. However, short-term conservation of zygotic embryos has been achieved by defining culture conditions which delay their germination for 12 months (Assy-Bah and Engelmann 1993). For long-term conservation, preliminary experiments have led to the development of a cryopreservation protocol which has been successfully applied to zygotic embryos of four different genotypes (Assy-Bah and Engelmann 1992). The difficulties encountered in hardening in vitro cultured plantlets need to be resolved. Additional work is required to refine the cryopreservation technique and to carry out experiments with additional genotypes.

In vitro techniques have been used extensively for exchanging coconut germplasm in the form of excised embryos inoculated in vitro. The FAO/IBPGR Technical Guidelines for the Safe Movement of Coconut Germplasm recommend that coconut germplasm be distributed in this form to reduce chances of introducing diseased material into disease-free areas (Frison et al. 1993).

Additional research is needed to refine in vitro techniques and to make this method fully operational for the medium- and long-term conservation of coconut. However, these techniques have a great potential for conservation purposes and are expected to play a greater role in a not too distant future in the overall approach to conservation of coconut germplasm. Involvement of a greater number of experienced researchers in different countries is critical in refining in vitro techniques to be used routinely.

Conservation of pollen

This is another important area that needs to be explored as a component of the conservation strategy for coconut genetic resources. Pollen storage was mainly developed as a tool for controlled pollination of asynchronous flowering genotypes, especially in fruit tree species (Alexander and Ganeshan 1993). In addition, pollen storage has also been considered as an emerging technology for genetic conservation (Harrington 1970; Roberts 1975; Withers 1991). Even if it may not be considered to be a viable method for meaningful genetic conservation of genotypes, cryopreservation is likely to be more successful than other storage techniques routinely employed for pollen (e.g. under organic solvents, desiccation freeze drying, low temperature) in facilitating hybridization when flowering is asynchronous or for use in the next season. Thus, it can help in better utilization of available genetic resources. Pollen can be easily collected and cryopreserved in large quantities in a relatively small space. In addition, exchange of germplasm through pollen poses fewer quarantine problems compared with seed or other propagules.

In recent years, cryopreservation techniques have been developed for pollen of an increasing number of species (Towill 1985; Bhat and Seetharam 1993) and cryobanks of pollen have been established for fruit tree species in several countries (Alexander and Ganeshan 1993; Ganeshan and Rajasekharan 1995).

In the case of coconut, assisted pollination is routinely employed in hybridization. This requires that pollen be conserved until it is used for crosses. For this purpose, storage duration up to several months only is required. Pollen is usually dried down to 4-5% moisture content with silica gel and stored under vacuum, which is viable for more than 2 months at room temperature, and for 6 months or more in a domestic deep freezer (Rognon and De Nucé de Lamothe 1978). Experiments have been conducted with coconut pollen which resulted in no viability loss after 3 (Whitehead 1966) and 6 months (Bernard 1973) of storage at room temperature. More recently, preliminary experiments have demonstrated that coconut pollen could be successfully cryopreserved (Dr Assy-Bah, 1995, pers. comm.). Long-term storage of coconut pollen under cryopreservation would represent an important additional technique for genetic resources conservation. Additional research is needed to further develop and refine this technique.

In situ conservation

This type of conservation is dynamic as opposed to the semi-static nature of ex situ conservation. One of the reasons given for choosing in situ conservation over ex situ is the need to maintain the evolutionary potential of species and populations (Frankel and Bennett 1970; Frankel and Soulé 1981; Ledig 1988, 1992). However, as human activities can cause habitat destruction and loss of biodiversity in some cases, and the maintenance of biodiversity in other cases, the need to complement in situ with ex situ conservation effort is well recognized. In general, research and monitoring are needed at three levels for successful in situ conservation: the assay of genetic variation represented within a target species in a particular area (ideally by studies of intraspecific morphological and molecular variation and the diversity as recognized by local users, including farmers), regular inventory of species numbers (this does not apply in the case of coconut), and observation of general ecological condition and habitat alteration, including farming systems.

In the case of coconut, since most of the stands in South and Southeast Asia are in more or less intensively managed areas, the effects of growers' practices are of paramount importance. In almost all these areas, there is presently little information available on the status of the genetic diversity. It is now possible to monitor and estimate genetic diversity using molecular markers (Hodges 1991; Foale 1992; Hodgkin and Debouck 1992). However, the limited resources available for such work makes such a proposal difficult to implement. Measurement of genetic diversity in coconut depends largely on morphometric traits as described in the Coconut Descriptors (IBPGR 1992), but increasing use of molecular markers assists in better understanding of the structure of genetic diversity, both at a specific site and across regions (Ayad et al. 1997).

Systematic documentation of farmers' knowledge of coconut diversity and uses is needed. Sustainable in situ conservation will require community participation, control of land rights in local communities, education, extension and development of environmental awareness. Of equal importance is the principle that any in situ conservation programme must also benefit the local communities. Management by local communities can often be developed to effectively link conservation and use (McNeely 1994, 1996). It is important to consider indigenous knowledge, participation and cooperation between local people, researcher and conservationists and non-governmental organizations (NGOs). Additionally, it is important to consider the establishment of areas of intensive management or high-yielding plantations for long-term sustainability of any in situ conservation programmes. Conservation activities by commercial and private agencies can also be promoted as these groups have the capacity to fund such activities. Since it will be necessary to foster sustained conservation and use of resources to derive long-term benefits from the exploitation of the resources, we believe that the commercial sector and private agencies will be interested in activities mentioned above. This can lead to much-wanted linkages among public, community and private sectors in plant genetic resources conservation (Riley et al. 1995). It is likely that the major part of coconut diversity still remains in situ, in the yards or gardens of small farmers, and undisturbed tropical sea coasts and uninhabited islands. The methods of management and benefits to local communities in maintaining and using this diversity must be considered while implementing an in situ (or on-farm) conservation programme. Although programmes to establish in situ conservation of coconut have not yet been initiated, coconuts grown by small-scale farmers have several features that should make in situ conservation feasible. These include the long-term perenniality of the crop which increases the sustainability of conservation, the variety of types of coconut that farmers in community or groups of communities maintain, and the great value of coconut for multiple uses which can encourage growing different types by specific communities.

Conservation of coconut germplasm in field genebanks

It is clear from the above discussion that establishment of FGBs appears to be the immediate option to consider for the conservation and utilization of coconut genetic resources, especially from the researcher's point of view. In many annual crops like cereals and legumes, seed storage, multiplication and regeneration methods have been agreed upon (FAO/IPGRI 1994), but for crops like coconut, there are no such agreed methods. So there is a need to look closely at scientific and practical criteria to be considered in establishing, maintaining and managing a coconut field genebank. It must be recognized that a FGB is presently the first option in a CCS for coconut genetic resources. However, efforts to develop the other methods outlined above must be continued.

In the following sections, some of the issues involved in developing a successful field genebanking technique for coconut germplasm are discussed. Some of the genetic and agronomic issues discussed below tend to overlap, as it is difficult to separate them. Additionally, factors such as the floral biology, male-female phase of the palm (which do not usually overlap, especially in tall types) and the length of the time taken to fruit development compound the problem.

Genetic issues

Ensuring genetic integrity and maintenance of genetic diversity

Isolation. If genetic integrity has to be maintained in an outbreeding species like coconut, the accessions or populations need to be grown isolated from each other. However, in the case of FGB for coconut, different accessions may have to be planted together for practical reasons. Available information (see below) indicates that coconut pollen under natural conditions does not move over long distances. Leaving a border of a few metres around the plot may help to avoid pollen contamination.

Sampling and selecting entries. To establish a regional collection, accessions displaying a range of diversity need to be planted. For this reason and from the point of population genetics, the principle of random sampling of genotypes from a given source population should be followed. In practice, both elite material and genetic stocks are also maintained in FGBs. Therefore, as far as possible, even if the random sampling is not followed, it is essential to sample a range of diversity to be represented in the FGB. In addition, seedlings or clones of elite material may be included as selective samples. Thus, the FGB can include populations/genotypes representing a range of diversity, elite materials, genetic stocks and some unique materials.

Sample and plot size. The size of the plot depends mainly on the breeding system, and diversity in the sample and on the number of palms planted. It will be most appropriate if the material planted in a field genebank can be representative of the source population. For raising seedlings and transplanting, the best methods should be used to ensure maximum survival and vigour. Larger plot sizes with more plants are better for outcrossing species like coconuts. However, coconut FGBs are not really for very long-term conservation (compared with techniques like cryopreservation), with 3-5 generations being a realistic time frame. Hence, smaller plots may be acceptable.

It is well established that square plots will be better than row planting for reducing pollen contamination (Breese 1989). The minimum number required per entry is the number of seedlings/plants that represent the genetic diversity of the population while the maximum number is determined by the resources available for maintaining and managing the collection. The number of plants is also determined by the size of the original sample and the frequency of alleles to be conserved. It is assumed that the material will be conserved for the next 200 years (with a frequency of replanting in a FGB being 20 years for dwarfs and 25 years for tails) which gives about 8-10 regenerations (replanting). If the decision is to have a 90% probability of maintaining the alleles with a frequency of more than 5%, a population of about 40-60 will be needed per accession. This is also based on the assumption that the seedlings for exchange or replanting will be produced by hand-pollination of female parents with mixed pollen of plants of the accession/population in question. (For more information, see Gale and Lawrence 1984; Breese 1989; Gregorius 1991; Crossa and Vencovsky 1994). This number needs to be supplemented with a few additional palms to compensate for any losses that may occur. From a conservation point of view, it may be better to plant and maintain an equal number of plants for each accession, keeping in mind the need for representation of genetic variation. However, from the practical point of view, the number of trees of certain highly productive accessions may have to be increased to make the FGB commercially viable. Additional numbers of these accessions may be best planted in a separate area in the FGB.

Drift. Another consideration is avoidance of genetic drift or loss of rare but important genes or alleles from the population or accessions. We have seen that for coconut, 40 to 60 individuals will be required to maintain alleles with a frequency greater than 0.05 with a reasonable degree of confidence, and this helps to mitigate the effects of genetic drift (again depending on the original sample size) and to maintain genetic integrity. Assuming pollination is not controlled, there may be a need for some buffering or isolation to maximize random mating among the individuals of an accession/population. However, in reality, isolation between plots is not practised. Then only a small number of plants in the middle of the plot may represent the gene frequencies of original populations. This would imply, especially if the original population size is large, that the total number planted in one replication should be high enough to give sufficient number of plants to get an effective population size of at least 30, so as to sample/maintain alleles with frequency greater than 0.05. This would translate into a minimum of 100 palms in two replications of 50 each per accession, when no isolation or hand-pollination is practised for production of offspring generation (see below). It will be possible to have 60 palms in two replications of 30 each per accession, if isolation and/or hand-pollination is practised.

Plot management. Although, as a general principle, it is best not to mix conservation and evaluation, planting equal numbers of palms per plot assists in evaluation and characterization of the material as well. Evaluation for important traits which are affected by environmental variation can only be carried out using replicated plots. It must be kept in mind that the FGB needs to be protected from biotic and abiotic stresses, as far as possible, to avoid poor quality characterization and evaluation data. If evaluation is to be carried out in the FGB, it will be a good idea to plant each accession in at least two, and ideally four replications, depending on the total number of plants per accession. The accessions within each replication must be randomized. It is important to realize that, for purposes of conservation, planting sufficient number of plants (40-60) in a square block with a few metres of border will be appropriate to reduce the chances of pollen contamination. Additionally, if these are planted in at least two replications, then evaluation of differences between and within populations, as well as selection of superior palms may be combined with conservation.

Supply material from the FGB

There are a few genetic principles to be kept in mind while taking material out of the genebank for testing in another location or for exchange as this has implications on the size as well as management of the FGB. In either case, it is important to supply a sample that represents the population conserved. If progeny from an accession in the FGB is required, then hand-pollination among the plants of an accession to produce the required offspring material has to be carried out. Otherwise, from the standing population one will only get offspring resulting from open-pollination which may be contaminated from another accession. However, it has been reported that pollen of coconut can travel, even under favourable wind conditions, only to distances of about 350 m and the maximum height of dispersal was about 6 m (Mantriratne 1965). Coconuts are also known to be pollinated by insects such as bees, wasps and ants. However, it was observed that the insects tend to return to the same plant or to neighbouring plants, effecting mostly selfing or sibling, if the neighbouring plants belonged to the same population (Child 1964). Hence, if the plot size is fairly large, it should be possible to obtain fewer or uncontaminated (by foreign pollen) nuts from the middle of the plot. Nevertheless, it must be recognized that there have been very few studies and this may vary depending upon location-specific situations. Here two choices are available: hand-pollination and bagging vs. natural outcrossing and selecting mother plants from the centre of the plot. The decision probably depends on the capacity to carry out hand-pollination on a large scale or the ability to maintain a large population. It will also depend on the final use of the material. In any case the standard recommendation is to produce seednuts for planting by hand-pollination.

A number of genetic issues considered in this section must be taken into account when making decisions about the management of coconut FGB. It is also clear that one cannot make a “practical” decision without influencing the “genetic” impact of the practice on the populations maintained in FGB. It is important that the FGB curators or managers are aware of the pros and cons of the decisions made. One of the first to consider the genetic factors in maintaining living plant collections was Esser (1976). He concluded that true genetic conservation is not possible but, by knowing the boundaries and being able to channel plant conservation on the basis of the knowledge and application of genetic parameters, we are at least aware of our limits.

Agronomic considerations

Security from natural disasters, and safety-duplication

Despite the problems discussed above, FGB is the current method for medium- to long-term conservation and use. So the security of the site must be assessed (both in terms of outright destruction - say growth or industrialization, tenure, etc. - or likelihood of natural disasters). Whenever possible, from the point of view of safety (in numbers) as well as genetics, it may be appropriate to replicate the collections in more than one location as has been recommended for seed collections. At this point it may be appropriate to note that there are several advantages of establishing regional genebanks, including the safety-duplication of a national collection. That also improves the collaboration between the countries in a region, facilitating joint multilocational evaluation testing, exchange of germplasm, etc. As is common practice for seed collections, it will be important to establish (an) adequate safety duplicate collection(s) of the material maintained in the field genebank. Along with abiotic stresses like hurricanes, cyclones, drought, fire, etc., the biotic stresses such as pests and diseases can be continuous and serious threats to the germplasm being conserved. Therefore, the establishment of safety-duplicate collections should be regarded as routine and budgeted for accordingly.

From the safety point of view, the site should not be located in an area known for natural calamities or other disasters. This will help not only in effective monitoring and management of the FGB but also for the long-term safety of the material conserved.

Climatic adaptation

Adaptability of the species or the accession to the location may be an important point to consider when long-term conservation is involved, especially in the case of regional genebanks. However, often this may not be possible as a FGB may contain introduced or unadapted material. For efficient maintenance as well as use of the material conserved, it is important that the plants in the FGB be able to flower, fruit and set viable seed. If the site for the genebank is located in an area with (a potential for) commercial orchard plantation, then the value of the genebank would be even greater, in terms of use of the conserved material. The genebank can act as the nucleus and provide planting material for commercial plantations.

Minimal pest occurrence

It is essential to establish FGBs in areas that are relatively free from pests and diseases, especially those that are transmitted through propagules. This aspect will be discussed further under the section on germplasm health issues. Additionally, the site should be protected from animal pests such as wild pigs, porcupines and elephants.

Access to the FGB

To facilitate protection and management of the stands, continued access to them must be assured. The site chosen for the FGB should be easily accessible and preferably near a research station so that the material available can be effectively used and the material in the FGB can be monitored frequently. It should be possible for the genebank staff and other researchers to reach the site easily. From a practical point of view, this probably has an overriding importance.

Choice of material

Entries in regional genebank

The major factors that control the choice of material in any genebank are: firstly, to determine the needs of the users of that particular genebank, and secondly, to have as much representation of genetic diversity as possible and diverse ecotypes that are available in the region. This requires giving emphasis to regionally recognized accessions. Therefore, there may be a tendency to acquire/assemble mostly the well-known elite accessions. However, it is important to have a balance between elite lines and accessions that represent a broader range of genetic variation from within the country as well as from the region. Giving due regard to the points mentioned earlier, it is important at the time of FGB establishment to plan for the materials to be included and lay out sufficient space. Care must be taken to ensure that each accession is unique and is not a duplicate.

Need for a national collection

Decisions will have to be taken on which accessions are to be maintained regionally and which nationally. Some nationally important accessions, or accessions representing diversity at national level, may not be accommodated in the regional genebank, and must continue to be maintained in the national collection. In addition, it is essential to make sure that each accession is duplicated in another FGB, especially until the other methods of conservation become available. The situation in any genebank is dynamic as more new material may always be obtained for conservation. So it is essential to maintain space in the FGB for new material.

Policy and management issues

National, regional and international collections

Conservation of genetic resources is a long-term responsibility and requires long-term commitment of institutions and governments. It is for this reason that any conservation effort must be conducted, preferably within the framework of a national programme. Clear institutional responsibilities must be assigned as part of the national mandate and a reliable budget should be established for sustained funding. At the regional and international level, the situation is different since it is not easy to assign clear mandates and responsibilities for the conservation of genetic resources of a specific genepool commonly agreed upon and implemented. The international germplasm collections held by the International Agricultural Research Centres are an exception as they have been placed under the auspices of FAO as part of the International Network of Ex Situ Collections. Individual centres have accepted responsibility for conserving global genetic diversity for one or more specified genepools as part of their broader mandate to deal with the improvement of the so-called mandate crops.

Sustained commitment

It is important to critically examine the existing arrangements with regard to mandate and responsibilities at both the national and regional levels for genetic resource conservation in general and about the crop in question, in particular. An assessment of the level of governmental and institutional commitment (of the host country in the case of a coconut regional field genebank) to the maintenance of collections in field genebanks is necessary. Only when an effective governmental commitment exists should the establishment or extension of a collection be considered. This is especially so for crops which need a relatively large land area in order to plant sufficient number of plants/trees necessary to represent the genetic diversity. Initial establishment costs, which in some cases can be very high, and recurring costs for maintenance of the collection should be considered at the planning stage and should be provided for. In many cases, the latter is ignored and the collections can run into problems within a few years of their establishment. Given this background, establishment and maintenance of FGBs appears to be more easily organized at national level, as part of national PGR programmes, rather than at regional or international levels. In the case of regional or international efforts, it is essential to obtain the full support and commitment of the government of the country in which the FGB is to be set up and to obtain commitments from the individual member states of the network to financially support the effort. For any emergency situation, provisions have to be made as to how and where the collection can be duplicated, if so decided. The role of the cooperating/supporting international institutions/organizations needs to be defined as well.

Legal issues

Since the Convention on Biological Diversity (CBD) has come into force, countries now have the sovereign rights over the biological diversity present within their borders. Existing ex situ collections, established prior to the CBD, or germplasm acquired not in accordance with the CBD, do not fall under the legal framework of the Convention and no obligation exists to share the benefits derived from their use. The FAO has been requested to resolve this latter issue as part of the harmonization of the International Undertaking on Plant Genetic Resources (IUPGR). In view of this, a clear consensus must be reached by all the member countries of a given crop genetic resources network with regard to access to germplasm and sharing of the benefits derived from the germplasm conserved as well as on access conditions to the conserved germplasm and information related to it. The necessary agreements and mechanisms on access to germplasm should be in place prior to the establishment of a regional or international genebank, be it a seed genebank or a field genebank. Within these mechanisms, there might be a need to develop some form of material transfer agreement to accompany germplasm accessions being sent to researchers/breeders within and outside the network. These will be further elaborated in another presentation.

Considering the current legal situation with regard to genetic resources, one of the options for regional or global PGR networks is to consider the possibility of placing their germplasm collections under the auspices of FAO, thus becoming part of the FAO International Network of Ex Situ Collections. In doing so, the host country, which acts as trustee of the germplasm on behalf of all the member countries, agrees not to claim ownership over the germplasm and not to claim any form of intellectual property protection to the material or on any information related to it. The host country will also ensure that any further recipients of the germplasm are bound to the same conditions as mentioned above (FAO 1995).

Germplasm health issues

There are two reasons for establishing FGBs in areas free from important pests and diseases. One is the risk of the entire collection, or part thereof, being destroyed by pests or diseases. This could be one of the reasons for the failure of FGBs in the past. The other is the risk of spreading pests and pathogens to new areas, which may easily happen with germplasm (Hewitt and Chiarappa 1977). An effective quarantine system should act as a filter, and should not be a barrier to germplasm exchange. It should help keep pathogens out and allow germplasm to pass. However, as some countries have stronger controls than others, breeders and the germplasm community have a certain responsibility to give due attention to pathogens. For example, FGB managers should apply restrictions to the international movement of seednuts and choose instead the movement of embryo cultures even when local quarantine authorities do not impose such restrictions.

Table 1 gives a summary of areas from where important pathogens are reported. Obviously, before establishing a FGB a critical evaluation of the disease situation in the location concerned will be required. Often parts of countries are free from a reported pathogen, e.g. CCCVd is not reported from Mindanao and the northern part of Luzon in the Philippines (Hanold and Randles 1991a), or Kerala wilt is only reported to occur in parts of Kerala and Tamil Nadu (Frison et al. 1993). On the other hand, absence of evidence is not a guarantee for absence of the disease, i.e. a pathogen occurring in an area may not be reported because of lack of thorough surveys or to lack of reporting mechanisms. In the case of coconut, the situation is further complicated by the existence of diseases of uncertain etiology, i.e. symptoms affecting the plant which so far cannot be attributed to a causal agent such as a virus, a fungus, etc. A list of coconut diseases of uncertain etiology is given by Frison et al. (1993). An inverse case exists with the reports of viroid-like sequences in coconuts, which could not yet be linked with clear disease symptoms (Hanold and Randles 1991b; Fassil and Diekmann 1995).

Table 1. Coconut pathogens with limited geographical distribution (from Frison et al. 1993)

Pathogen	Reported to occur in:†
Coconut foliar decay virus (CFDV)	Vanuatu
Coconut cadang-cadang viroid (CCCVd)	Philippines
Coconut tinangaja viroid (CTiVd)	Guam
Blast (phytoplasma)	Africa
Lethal yellowing (phytoplasma)	Bahamas, Cayman Islands, Cuba, Dominican Republic, Haiti, Jamaica, Mexico, USA
Kerala wilt (phytoplasma)	India
Tatipaka disease (phytoplasma)	India
Marasmiellus cocophilus	Kenya, Tanzania, Solomon Islands
Phomopsis cocoina	Australia, Guam, India, Jamaica, Kenya, Malaysia, Mauritius, Nepal, Papua New Guinea, Puerto Rico, Seychelles, Solomon Islands, Sri Lanka, Trinidad & Tobago
Bipokiris incurvata	Australia, Central and South America, Pacific Islands, Seychelles
Phytophthora kasurae	Caribbean area, Hawaii, Vanuatu, West Africa
Phytomonas spp.	Brazil, Colombia, Costa Rica, Ecuador, Grenada, Guyana, French Guyana, Nicaragua, Peru, Surinam, Trinidad & Tobago, Venezuela
Bursaphelenchus cocophilus	Caribbean, Central America, Mexico, S. America

^† The countries where a pathogen was reported to occur are listed. Often parts of these countries are free from the respective pathogen.

Germplasm health aspects need to be considered not only at the point of exchange, but at any stage of germplasm management. During collecting, care must be taken that germplasm is collected only from healthy trees. In the regeneration and multiplication process, plant protection measures including pesticide application may be required. If an evaluation of traits like resistance to pathogens is done under conditions of high disease pressure, e.g. with artificial inoculation, a careful evaluation of the material with regard to its use in regeneration or exchange is essential.

Cooperation between breeders/germplasm curators and quarantine organizations is essential. Consultation should occur permanently, but particularly at early planning stages for collecting or establishing field genebanks. Germplasm should be exchanged only for immediate use or for conservation, including required safety-duplications.

Table 2. Summary of Technical Guidelines for the Safe Movement of Coconut Germplasm

General recommendation: to move embryo culture or pollen, not nuts

Pathogen	Specific recommendation
Coconut foliar decay virus (CFDV)	Indexing or exclusion of germplasm from Vanuatu
Coconut cadang cadang viroid (CCCVd)	Indexing or exclusion of germplasm from the Philippines
Tinangaja viroid (CTiVd)	Indexing or exclusion of germplasm from Guam
Viroid-like sequences	Indexing recommended for germplasm that is moved from countries where these sequences are known to occur to countries where they have not yet been reported. Recommendation under revision.
Lethal yellowing (Phytoplasma, MLO) Kerala wilt (Phytoplasma, MLO) Tatipaka disease (Phytoplasma, MLO)	Transmission through seed, embryo culture or pollen not reported
Blast (Phytoplasma, MLO)	A nursery disease which does not occur on adult trees
Marasmiellus spp. (bole rot, shoot rot)	Possibly seed-borne, can be eliminated in embryo culture
Phomopsis cocoina (leaf spot) Bipolaris incurvata (leaf blight)	May be dispersed on husks. The recommendations are: - embryo and pollen transfer should be carried out - healthy nuts should be partially de-husked and treated with an appropriate fungicide
Phytophthora palmivora, P. katsurae (bud rot, fruit rot)	Nuts may be infected internally, but then do not germinate. The recommendations are: - embryo and pollen transfer should be carried out - healthy nuts should be partially de-husked and treated with an appropriate fungicide

Summary and conclusions

In the discussion presented here, it has been assumed that the collecting has been effectively carried out keeping the sampling of genetic diversity in mind, and that all requirements have been completed. No attempt has been made to review extensively the information available on genetic diversity or quarantine problems and only brief description of conservation methods other than FGB has been provided, as appropriate to the occasion. Since other methods of conservation are not fully viable, the emphasis is on establishing and managing FGBs. Earlier discussions have defined the purpose of a regional genebank for coconut germplasm as: (1) to serve as a conservation centre for important coconut germplasm to the world, (2) in addition to the conservation efforts, genetic trials for and on behalf of contributing countries of COGENT can be conducted and promising breeding materials distributed to countries, and (3) to serve as holding centre for coconut germplasm from natural (original) countries of origin which do not yet have the ability or capacity to conserve their own such germplasm (Second Steering Committee Meeting, Montpellier 1993). Thus, any regional coconut FGB that is established should have at least the diversity from the region, should be able to evaluate the material as well as to hold germplasm from other countries for future repatriation.

So to establish, maintain and manage a FGB for coconut (as a Regional Collection), the following critical checklist of steps is suggested. This is by no means an exhaustive list of steps to be taken but only the important considerations that determine the effectiveness and sustainability of the field genebank are mentioned. These also assume that the issues considered in the earlier discussion were taken into account, decisions have been made and the consequences noted. Some of the principles of agronomy, nursery management etc., can be found in Santos et al. (1995).

1. Agreement on precise functions of the collection.

2. Selection of site, based on the established criteria.

3. Agreement on obligations and responsibilities of host countries, countries participating in the Network (i.e. COGENT) and international institutions, including funding agencies.

4. Establishment of infrastructure and facilities.

5. Legal aspects and exchange protocols (ownership, conditions of release, IPR issues, benefit sharing, use of MTA and other mechanisms) as agreed by all partners.

6. Establishment of a coconut FGB:

· Assure comprehensiveness of collection by including as much genetic diversity as appropriate from the subregion

· Consider carefully the sampling techniques (random vs. non-random, and the need for deviation)

· Assure that there is no duplication of accessions as this directly increases cost of FGB

· Determine the need for having replications, the number to be based on the objectives of FGB

· Determine through discussions and by actual visitations the accessions and the number of accessions to be included in FGB, and number of plants per plot

· Establish nursery of vigorous and healthy seedlings, determine planting conditions, etc., depending on the location of FGB

· Lay out square blocks of equal size

· Plan space for present and future accessions (as much as possible) to be randomized in the FGB

· Follow all protocols for safe movement of germplasm

· Ensure that embryo culture/tissue culture facilities can be put in place for exchange of material

· Accept more material into FGB as they become available by going through all the steps discussed.

· Take all the necessary agronomic and plant protection measures to maintain a healthy stand of coconut palms

· Take all the measures feasible to protect FGB from adverse environmental conditions, physical stresses etc.

· Make sure that safety-duplication is established and all the needs of health care are fulfilled

· Document all accessions as well as activities carried out in FGB by establishing and running an appropriate information management system

· Provide linkages to other methods of conservation, if any, such as in vitro conservation of zygotic embryos, pollen preservation, etc.

· Ensure physical availability of the material

· Keep the plants in healthy condition

· Facilitate nut/seedling production through hand-pollination for distributing germplasm as agreed at the time of establishment of the genebank

· Characterize/evaluate the material in FGB according to agreed criteria

· Sample populations from centre of each plot

· Make available the information on the material conserved in the FGB to all users

· Exchange material using embryo culture rather than seeds/nuts.

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Conservation priorities and guidelines on accessions to be conserved

G.A. Santos

Div. Chief III, Breeding & Genetics Division, Philippines Coconut Authority, Zamboanga Research Center, San Ramon, Zamboanga City; Member, COGENT Steering Committee

Introduction

Collecting a good representative sample of the different cultivars, ecotypes and/or strains from their original source is the main objective in the conservation and use of plant genetic resources (PGR). Depending on type and nature, these materials are kept either in a status of suspended growth and development such as seeds in cold storage or as a living collection, in the hope that someday, when the need arises, it will be possible to regenerate a good stand of the plant possessing a good replica of all the genetic attributes of the original parent or source plant together with all of the inherent variability in the case of preserved seeds or preserved tissues of sexually propagated crops. By nature, annual crops, particularly grains and legumes, lend themselves rather easily to artificial preservation techniques but perennials bearing large seeds are more difficult to handle for obvious reasons.

Purposive breeding necessarily follows the conservation of PGR. Without breeding, there is very little point conserving PGR. But owing to the limitations and constraints, and since we will be dealing with a perennial crop, we have to be selective and conscious in prioritizing what, and knowing how and where, to conserve and maintain the identified variation.

The reasons for keeping the world coconut collection in regional genebanks have already been discussed in another presentation. This discussion paper suggests a few guidelines and some basic practical considerations in deciding which varieties should we keep and how many shall we hold to ensure its practical usefulness in the future.

Which varieties do we keep?

A recent inventory of coconut varieties from 15 countries that have been registered in the COGENT CGR Database has come up with 738 accessions. It is expected to increase further as more countries join the network and provide data to the database. This big number does not exclude the possibility of similarities and close consanguinity in many of the entries in spite of the different names attributed to them and the fact that they may have come from different origins. For this reason, it is extremely important to prioritize the choice of materials for the genebank according to an agreed set of criteria. The list of suggested criteria is not exhaustive and is based on practical considerations.

1. They must be listed as a major variety, i.e. parents of existing hybrids and advanced generations of selected cultivars

This group appears to be the most important to conserve because their offspring are being utilized in replanting programmes around the world. The increasing vulnerability of these genotypes or ecotypes to human-made and natural causes in their countries of origin further justify the high priority that should be accorded to these materials, not only for safekeeping but also for more in-depth genetic analysis.

2. Varieties which are considered threatened in countries without the means for their conservation

Many islands and atolls in the Pacific and Indian Ocean islands depend very much on coconut for food and ecological security. However, most of these countries are poor and cannot bear the burden of keeping a good stock of these materials for conservation purposes.

3. Varieties with special traits or highly visible genetic markers

For the purpose of capturing diversity, it is important that such materials are also given high priority. These materials are easily identified and because of this, the genetic divergence of the resultant population(s) or generation(s) from the original can readily be detected, measured and rectified in future duplications or hybrid progenies, if needed. The value of such materials for practical training purposes and inheritance studies will be of prime importance.

4. Relevant genotypes in ongoing projects on molecular biology for diversity studies and tissue culture experiments

The recently concluded review and evaluation of an STD3-funded project on coconut tissue culture (TC) by somatic embryogenesis reinforces the need to conserve the identified coconut genotypes being used in coconut TC work since results indicate variable genotypic response to tissue culturing.

How will the varieties be kept?

Living collections

The size of the coconut seed and the absence of a dormancy period leave us with no choice but to keep and maintain living collections for several decades. In fact most national collections are living collections of coconut germplasm which have been established under different conditions, purposes and representation.

Being a perennial crop, coconut fits very well into a living collection which gives the advantage of outright utilization after evaluation. Thus, immediate improvement of the variety becomes possible through the recurrent selection technique. Initial costs involved in collecting, establishment and physical maintenance could be high, but with proper management, it is possible to make a coconut collection pay for itself in the long run.

Cryopreserved embryos and lyophilised pollen

Studies on cryopreservation of embryos and lyophilised pollen as means of conservation indicate that these techniques are possible. Cryopreservation will significantly reduce the need for a large space but it is potentially subject to some technical limitations like possible genetic shift due to changes in genetic fidelity of the preserved material as a result of the cold treatment and subsequent exposure to TC conditions. On the other hand, research on lyophilised pollen and cold storage has shown that, although coconut pollen can be kept for several years, its germinability is drastically affected soon after thawing.

How many varieties or ecotypes to keep?

After deducting unsuitable portions, the average size of the regional genebank which COGENT plans to maintain is roughly 400 ha. Following the Manual on Standard Coconut Breeding Techniques (STANTECH), the suggested number of palms per accession shall be 90 in order to make plot sizes of 30 palms replicated three times. Therefore, following the triangular system of planting, each accession will occupy an area of at least 0.5 ha (if planted at 8 × 8 m) to a maximum of 0.64 ha (if planted at 9 × 9 m). Each genebank could therefore accommodate around 250 accessions (in about 125-160 ha), another 200 ha for the genetic trials and seed multiplication blocks, while the rest shall be for roads, infrastructures and pollen barriers.

For how long?

While natural stands of coconut had been observed to survive long years with minimum or no care at all, it appears from our experience that in a genebank, palms should be renewed or rehabilitated once every 20 years for dwarfs and earlier for tails, say 15 years, because it becomes very difficult to work on them when the stem reaches a height of over 10 m. Rehabilitation of the “old stand” should be made through hand-pollination following two systems. For talls, where maximum heterozygosity is preferred, the pollination scheme must guarantee maximum intermating among the individuals representing the population or accession. For dwarf ecotypes wherein maximum homozygosity is favoured, maximum selfing should be arranged. The STANTECH manual will come in handy for this purpose as it describes the steps to be taken in hand-pollination and other research techniques.

Genebank design and activities

F.V. Rognon
Head, Coconut Programme, CIRAD-CP, Montpellier, France

Introduction

The rationale for establishing regional genebanks, the coconut conservation strategy, the conservation priorities and accessions to be conserved have been described in other presentations. This present paper deals with the genebank design in general, including different forms of conservation, and the activities where the main point is to establish clear limits rather than an exhaustive inventory of possibilities.

There is already a number of national collections of coconut germplasm, mostly utilized as sources of breeding material. For most of them, design is a weak point for different reasons (limited area, difficulties in obtaining accessions according to plan). The regional collections to be established under the advice of COGENT/IPGRI and supervision should take into account the existing experience.

Concerning the activities, planning should go beyond the strict germplasm conservation objectives to allow for future utilization of the genebanks as sources of breeding materials. In other words, the requirements of breeding have to be considered from the outset. During this presentation, I will not discuss technical details which are very clearly presented in the STANTECH manual.

The design

Forms of conservation

There are three main forms of conservation for coconut germplasm, each with its advantages and limitations.

Freeze-dried pollen. Freeze-drying pollen is an easy and cheap form of conservation. However, its only use will be for pollination and mostly for breeding purposes.

Cryopreserved embryos and tissues. Zygotic embryos, in vitro culture and somatic embryogenesis associated with cryopreservation are a very useful complement to field genebanks. Excised embryos or plantlets in test tubes are easy to transport. Cryopreservation can be used as a means of short-term conservation (a few months to a few years) while waiting for indexing or for checking the identity of the material with genetic markers. As it multiplies single genotypes, somatic embryogenesis may not be the most efficient way of preserving and propagating base populations. However, it will be well adapted when it comes to advanced breeding material.

Field genebanks. Field conservation is still the preferred method because it allows agronomic evaluation under controlled conditions and ensures the availability of pollens and seeds for breeding. Coconut is very demanding in planting area and its reproductive cycle is long but its long life span reduces the need for rejuvenation.

The genebank

The rest of this presentation will focus on field genebanks which are the main object of this workshop.

Environmental constraints

The field genebank requires good environmental conditions for the accessions to express their full potential. Ideally, the site should be quite flat with a uniform soil of suitable physical composition and texture. The chemical characteristics are more easily controlled through fertilizer applications.

The climatic conditions should be suitable for coconuts with a sufficient well-distributed rainfall of 1500-2500 mm per year, little variation in temperature (around 28°C average) and sufficient relative humidity of about 80%. Rainfall can, however, be supplemented by irrigation but it will be an additional cause of heterogeneity. The sites should not be in areas prone to hurricanes or other natural disasters. They should be in areas free of severe pests and diseases. As we will see later on, the proposed regional collection sites are far from ideal and will require careful management to overcome the problems encountered.

The last constraint is a sufficient area, which was estimated at 200 ha minimum, but this point is covered by each country's commitment.

Design

In many existing collections, design is a weak point. Accessions are planted side by side in the plots which were available for planting at the time of obtaining the seedlings. There are no controls (or standards), no replications, no randomization, no blocking and thus, no possibility to compare the accessions statistically. This is not a problem if the only objective is to conserve the accessions but if evaluation and comparisons are required, one will regret the lack of a proper design.

Choice of the accessions to be planted in the field genebank is closely related to the objectives. It has been discussed in an earlier presentation. An important point is that new techniques are now available to study the genetic diversity, mostly using genetic markers, and thus avoid or reduce duplications or over-representation of some types of material. Even if they may seem costly, they are much cheaper than planting, characterizing and evaluating the material for 12 years before realizing that it already exists in the genebank under a different name.

Choice of the controls is very important as it will allow comparisons between accessions in the same site and between sites. There should be some common controls for all four proposed collections. For dwarf types, one control should be enough and it could be the Malayan Yellow Dwarf (MYD) which is widely available. For tall types, we should agree on at least one international control for the four collections, say the RIT and one local type if possible different from the RIT, i.e. West African Tall (WAT) in Ivory Coast, West Coast Tall (WCT) in India, etc. The discussion is open for the two other sites (groups according to Bourdeix, that is, to divide the Tails in two groups with complementary characters to be improved against each other under the recurrent reciprocal selection scheme). The controls should be available every year during the development of the collections and occur in each replicated block in the case of a randomized complete block design. The design as proposed in the STANTECH manual is a simple randomized complete block design with replications. Each individual plot is made of six rows of five palms. With a triangular spacing, the plot shape is about square. The blocks themselves should be as square as possible. Each planting date should include controls of the same age as the accessions.

The number of palms representing an accession is 90 (3 × 30). This is the required sample size to have characters presenting a coefficient of variation ranging from 22.5% to 25% with a confidence interval of about 5% (CI_0.05). Such high coefficients of variation are easily found in heterogeneous tall populations. Dwarf populations are more homogeneous but maintaining the same number of palms per accession is advisable for standardizing the designs and data processing.

Dwarf and tall types must be planted separately as their optimum spacing is different: 180-205 palms/ha (8.0-7.5 m, triangle) for dwarfs and 143 palms/ha (9.0 m, triangle) for tall.

The activities

Planning

Planning is the first activity to start with and a very important one. Besides the general planning that is being discussed in this workshop, the annual planning will be a difficult exercise. It is not always easy to get all the accessions in due time with the corresponding controls. Starting from embryos will be an additional complication. Screening for diversity will also have to be arranged with specialized laboratories. The nursery and land area cleared should allow for some flexibility in case some unforeseen accession becomes suddenly available. Orders for germplasm exchanges and export are also difficult to plan as the seeds have to be produced by hand-pollination.

Technical activities

The technical activities are well described in the STANTECH manual but some of them are especially important. The first activity will be survey and delimitation of the selected sites to obtain all necessary information for planning, designing and budgeting the collections. The survey will concern:

· the soil types with a grading from highly suitable to unsuitable

· the topography with different recommendations according to the slopes: cover cropping, soil conservation with continuous terraces, individual platforms, drainage, etc.

· the vegetation, which has an impact on land-clearing costs.

Seedbeds and nurseries should be very well managed with the aim to keep the identity of each seed and plant, and reduce the environmental variability to a minimum. This implies separating seedbeds and nurseries, severe selection on the speed of germination, using polybags in the nursery.

Field planting should be carefully organized and monitored to avoid mistakes in the statistical design and identification. The use of colour codes may be helpful. If polybags have been used in the nursery, field control must be especially tight to ensure that they are not discarded during transportation or at the field site because the benefits of using polybags would be lost.

The field maintenance must be good but without excess. The use of fertilizers is recommended to let the palms express their full potential. Where rainfall is limiting, irrigation must be done. The palms must also be protected against pests and diseases. Where wild pigs cause damages, it is necessary to establish a strong wire mesh or electric fence.

Harvesting should be done under supervision by the workforce of the collection site and not by the surrounding villagers every time they need some additional income.

Scientific activities

A precise identity of the planted material must be kept at all times. Regular monitoring of losses and supply is essential. There are a number of documents to be established: registers, maps, listings, etc.

The observations to be secured are detailed in the list of descriptors and in the STANTECH manual. The characterization and evaluation data must be carefully controlled by random checks. The recapitulations and statistical analyses should be done early enough to be able to check in the field for abnormal values and discrepancies.

Germplasm exchange

When the field genebanks enter into production, they will become a source of germplasm and should therefore be equipped to conduct hand-pollination, pollen and embryo preparation and to send them through proper transport and quarantine procedures.

Genetic diversity utilization

The collecting strategy will differ depending on the objectives: will it be a core collection trying to represent the diversity and variability without being exhaustive?, a target collection trying to gather certain desired characters?, a safety duplicate collection for samples of threatened populations? Will the collection include elite material?

When the collection is established, characterized and evaluated, the most important part remains to be done and that is breeding.

Breeding may be associated with the collection and conducted as a joint activity among the countries of the region. It would imply designing a common breeding programme and distributing the breeding materials and hybrids produced on the collection to the cooperating countries for plant genetic trials.

It could be that national programmes would only order the breeding material and hybrids they require from the collection for their own use.

In both cases, the genebanks need to be able to cater to the requirements of the countries of the region or of other regions. They will have to muster the necessary skills and equipment.

Information exchange and training

If a regional breeding network can be established around each collection, sharing a common breeding programme and its results, the system should be very efficient. It would avoid unnecessary work and duplication. It would speed up the testing process. It would allow each participating country to benefit from the output of a much larger breeding programme than it could set up on its own.

The collection would also be a meeting point for all the breeders and their researchers of the region, where they could get training, exchange ideas and set up cooperative programmes.

Conclusions

Through the STANTECH meetings, COGENT has established a strong basis for cooperative work among the member countries.

The development of the regional collections will be a good practical application of the network development, beyond the establishment of the regional coconut breeding programmes. This is an open area for new cooperation.

A multilateral system for the exchange of plant genetic resources

J.M.M. Engels
Director, Germplasm Maintenance and Use, IPGRI, Rome, Italy

Introduction

Three broad options exist for systems to govern the exchange of plant genetic resources (PGR). These range from purely bilateral approaches, to informal multilateral arrangements to a structured multilateral exchange framework. In this presentation I have chosen to focus on the third option which describes a system which might well be suited to the development of regional genebanks, such as the coconut genebank under discussion today.

After a brief description of the current policy climate as regards genetic resources, the paper will specify the general requirements for an international exchange system. It will then explain why a multilateral exchange system is preferable to strictly bilateral arrangements and will describe the characteristics of such a system. The prerequisites for an agreement on access will be mentioned before drawing some general conclusions.

The PGR paradigm shift

For a better understanding of the current policy environment on access to genetic resources, it is important to note some key events and changes which have occurred over the past 10 years or so. Historically, PGR have been regarded as “common heritage” or as a “heritage of humankind”. This concept formed the cornerstone of the International Undertaking on Plant Genetic Resources, a legally non-binding agreement between more than 100 nations. However, starting in the second half of the 1980s, this concept began to change to reflect the following:

· extended capacities to exploit PGR
· rapid development of biotechnology
· increased application of intellectual property protection
· a changed perspective of the role of farmers in managing biodiversity
· the development of a strong environmental movement
· an increasing privatization of plant breeding.

Some of the major statements and articles of the CBD are cited here to illustrate its implications on questions of ownership and access.

“The objectives...are the conservation of biological diversity, the sustainable use of its components and the fair and equitable sharing of the benefits arising out of the utilization of genetic resources...”.

“Recognizing the sovereign rights of States over their natural resources, the authority to determine access to genetic resources rests with the national Governments and is subject to national legislation”.

“Each contracting party shall endeavour to create conditions to facilitate access to genetic resources... and not to impose restrictions that run counter to the objectives of this convention”.

“Access, where granted, shall be on mutually agreed terms...”.

“Access...shall be subject to prior informed consent, unless otherwise determined by that party”.

The requirements of an international PGR exchange system

Any international system for the exchange of plant genetic resources should meet the following requirements which form the key elements of the CBD:

· Ensure conservation
· Facilitate access
· Promote sustainable use
· Enable equitable benefit-sharing.

Why a multilateral exchange system?

A key question when deciding on an access regime for plant genetic resources for food and agriculture (PGRFA) will be whether to opt for a system based on bilateral or on multilateral arrangements. The following are some of the major reasons why a multilateral approach appears to have advantages over bilateral arrangements:

· complexity of ownership of PGRFA
· complexity of plant breeding
· the case of the international collections
· need for a non-bureaucratic but transparent system.

Before proceeding, it should be noted that, for the purposes of this paper, the term “genetic resource” encompasses the biological material (e.g. the seed, cutting or a gene), the information about the material, the knowledge of how to grow and process it and the technology to use it.

Ownership. The question of ownership over genetic resources is critical and the following points will be considered:

· types of “owners”
· methods of acquisition of PGRFA
· complexity of “country of origin” concept
· documentation on the origin of material.

· private ownership (e.g. material held by breeders for use in their crop improvement programmes)

· public ownership of material in private institutions (e.g. material in the public domain including germplasm bred or improved by the institution).

Acquisition of PGR. The recognition by the CBD of national sovereignty over genetic resources and the fact that ex situ collections established before the entry into force of the Convention on 29 December 1993 are not covered by the CBD, have a direct bearing on the status of the material. Consequently, the way in which germplasm accessions have been acquired by a genebank might be of direct significance for later decisions on their availability. For example, one could distinguish the following categories of PGRFA held in genebanks:

· material collected locally
· material collected in other country(ies) (pre- or post-CBD)
· material received from collection(s) in other country(ies) (pre- or post-CBD)
· material bred within or outside a given country.

Country of origin. One of the aspects which is of basic importance in the CBD is the “origin” of the material, especially in light of recognition of the sovereign rights of a nation over the genetic resources found within its borders. In this context, the following aspects should be considered: (1) much of the diversity belonging to genepools of crop species is naturally distributed over several countries, (2) over the centuries, secondary centres of diversity have developed through natural and human selection, (3) in the past, germplasm was exchanged freely among communities in different countries, (4) major improvements on many important crop species have been developed in countries other than where the species was domesticated, and (5) for many crops, the bulk of production takes place outside the areas where the species originally evolved.

These aspects may lead to difficulties in determining the precise origin of a particular crop species. If at all possible, this needs to be taken into account when deciding on an exchange system for PGR.

Complexity of plant breeding. The “origin” question can also be obscured by the process of plant breeding. This aspect is important if tracking of germplasm and the decision on a possible cut-off point for benefit-sharing becomes an issue. Apart from the difficulty in determining the accurate origin of the individual accessions (especially of those which have been collected in the past, on which information is frequently lacking), it is difficult to assess the contributions of individual accessions to a pedigree which has been developed in a crossing programme. In addition, if one would apportion, in general terms, the contributions of individual accessions the question regarding up to which generation the contributions should be calculated then becomes an issue. To understand the complexity of plant breeding with regard to the above questions from a PGR point of view, the following points should be considered:

· breeding requires a wide range of diversity

· pedigrees are growing increasingly more complex

· it is often extremely difficult to attribute specific contribution of a progenitor to an improved variety

· the sources of genes are frequently unknown

· apportioning benefits from different sources is difficult, if not impossible.

International collections. The germplasm collections maintained by the International Agricultural Research Centres of the CGIAR have a special legal character. Most of the germplasm they contain was assembled prior to the entry into force of the CBD. In almost every case, the holdings maintained by the Centres serve as global base collections for the respective mandate species. Collectively, they represent the world's largest germplasm collection of major food crops. The collections are held in trust under the auspices of FAO as part of the International Network of Ex Situ Collections. As such, the germplasm is available without restriction and the Centres have agreed not to take out intellectual property protection on the material itself or on related information. The Centres have also agreed to ensure that recipients of the germplasm are bound to the same conditions as the Centres.

The importance and role of these international collections can be illustrated by presenting some figures on the rice germplasm collection maintained by IRRI in 1994. The distribution data represent the activities carried out in 1994.

Total number of designated accessions	79 278
Countries that donated germplasm	75
Countries that received germplasm	33
Countries requesting improved germplasm	19
Germplasm accessions distributed	11 129
Number of improved lines distributed	1 367

Characteristics of a multilateral exchange system

After having mentioned several difficulties associated with exchanging germplasm, especially when this is carried out on a strictly bilateral basis, we now consider the characteristics of a multilateral system.

Of the options available for the exchange of PGR, bilateral or multilateral approaches can be considered. Bilateral arrangements might be most appropriate when a small number of countries are involved in the exchange of a specific crop or when highly expensive and advanced research is the basis for cooperation in a very competitive environment. This does not seem to be the case for coconut genetic resources. Multilateral arrangements might be most appropriate when many countries depend on widely spread diversity and when research and breeding efforts by individual nations are expensive, complex and difficult to handle. The latter seems to apply to coconut genetic resources, both on a global and a regional level.

The current informal PGR exchange system, as practised by the International Agricultural Research Centres of the CGIAR, is a flexible and cost-effective approach. Therefore, it could provide the basis for a multilateral system as envisaged for coconut. To reflect the current thinking with regard to access and better benefit-sharing, it should allow for more transparency in its operations and encourage more partners to participate. This can be achieved through the introduction of some basic ground rules for the exchange of PGR and the benefit-sharing which could be agreed upon by governments of countries that wish to participate in such a formalized multilateral exchange system. Provided that such a system is made simple and transparent, it can be expected that participation will be attractive to governments and institutions.

Scope of the multilateral system

Important questions to consider when deciding on the scope of a multilateral exchange system include the categories of plant genetic resources to be covered by the system; whether the system should define its scope according to an inclusive or exclusive list of species; whether conservation methods should be used to determine scope; whether the intended use of the germplasm by recipients might serve as a criterion for its inclusion in the scope, and whether the date of collecting should be a criterion (i.e. whether the material was collected before or after the entry into force of the CBD).

Operational aspects

The policy and organizational aspects of a structured multilateral system also need to be considered. These include matters such as: membership terms and conditions, governance and decision-making mechanisms, mechanisms for monitoring and enforcement, rules for interacting with non-members; terms of access to germplasm, mechanisms for multilateral benefit-sharing, and conditions under which bilateral benefit-sharing might be negotiated. In addition, a formal approach to multilateral exchange would require arrangements for the governance of the system, the rules for membership, rules and procedures for germplasm exchange and benefit-sharing, and finally the funding of the system.

Benefits of a multilateral exchange system

Probably the most important consideration of individual nations when deciding to join the multilateral system will be the benefits they can obtain by joining. Some advantages that might be assured are:

· better conservation strategies and shared responsibilities
· research partnerships and pooled research resources
· access to much more genetic diversity than contributed
· access to improved materials and technologies
· wider dissemination of germplasm in the system
· access to information (specific traits/genes/evaluation data)
· more efficient information management
· access to training
· possible sharing of financial benefits.

Prerequisites for agreements on access

For a multilateral exchange system to function properly, it will have to satisfy the four requirements mentioned earlier, as per the CBD:

· it must meet the interests of all partners

· it must be in accordance with international agreements

· the system should be as “unbureaucratic” as possible and allow as much transparency as possible

· it should allow transaction costs to be minimum and it should maximize efficiency and effectiveness.

Conclusion

As a general conclusion I would like to summarize the foregoing as follows. A multilateral system benefits all. It would encourage and increase the use of PGR. It would especially benefit small and/or poor countries since they can better exploit their own and other genetic resources through increased international cooperation. Although not directly related to a multilateral system, it should be noted that it would be very disadvantageous for the agricultural sector in general and, to the plant breeding sector in particular, if no agreement on the exchange of germplasm is reached in the very near future.

International movement of coconut cultivars¹

¹ Not presentated at the Workshop.

L. Baudouin
Breeder/Geneticist, CIRAD-CP, Montpellier, France

Two types of international germplasm movement are described. The first type is the collecting of germplasm in a foreign country. The Indian and Jamaican research centres were very active in this domain. The other type involves transfers from one research centre to another. In this case, the contribution of Marc Delorme research station in Côte d'Ivoire is clearly the most important.

For each movement, the position of the arrow indicates the origin of the parent population. For example, in Table 1, the Cameroon Red Dwarf was introduced from Cameroon to Côte d'Ivoire, and from there to seven other research centres. In Table 4, the Catigan Green Dwarf was first planted at Zamboanga, and directly reached Côte d'Ivoire, Vanuatu and Vietnam. But Tanzania received it from Côte d'Ivoire.

The cultivar names and codes are given according to the international coconut cultivar list which is recommended by IPGRI and evolved recently. It consists of 3 or 4 letters, the last one being a T or D to indicate the type (tall or dwarf). Subpopulations in a cultivar are differentiated by a two-digit number, for example, EAT is the East African Tall, EAT32 is the Kenyan subpopulation of EAT.

Codes used for countries in Tables 1 to 5

BEN	Benin
BRA	Brazil
CIV	Côte d'Ivoire
FJI	Fiji
IDN	Indonesia
IND	India
JAM	Jamaica
LKA	Sri Lanka
PHL	Philippines
PNG	Papua New Guinea
SLB	Solomon Islands
THA	Thailand
TZA	Tanzania
VNM	Viet Nam
VUT	Vanuatu

Table 1. African cultivars

CKT: Cameroon Kribi Tall
Cameroon
	® Marc Delorme (CIV)		52 (R82)
CMT: Comoro Moheli Tall
Comoro
	® Marc Delorme (CIV)		72
CRD: Cameroon Red Dwarf
Cameroon (probably imported from Pacific)
	® Marc Delorme (CIV) (R81, 2 accessions)		55
		® CPCRI (IND)	77
		® CRI (LKA)	85
		® EMBRAPA (BRA)	82
		® NCDP (TZA)	80
		® Zamboanga (PHL) (2 accessions)	78
		® Seme Podji (BEN)	62
		® Saraoutou (VUT)	82
EAT32: East African Tall Kenya
Kenya
	® CPCRI (IND)		56
EGD: Equatorial Guinea Green Dwarf
See BGD (America)
MZT: Mozambique Tall
Mozambica
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