Seed and Plant Genetic Resources Service
Food and Agriculture Organization of the United Nations
Rome, November 1997.
2. Need for Germplasm Conservation
4. Current Applications of Biotechnology
Organogenesis, Embryogenesis, In vitro Selection
5.2 Biotechnologies with Application to Conservation of Plant Genetic Resources
I gratefully acknowledge the contributions of three people who helped
with this manuscript: Professor Pierre Debergh for his comments on the
whole manuscript, Dr. Mick Graham for his suggestions and comments on sections
involving Molecular
Biology, and my wife Leone for many hours and late nights spent in editing
the manuscript.
R. A. Drew
The Seed and Plant Genetic Resources Service of the Food and Agriculture
Organization of the United Nations is greatly indebted to the author of
this technical paper Dr. R. A. Drew, and to all those scientists who collaborated
on this work.
U.G. Menini Chief,
Seed and Plant Genetic Resources Service
Plant Production and Protection Division.
As we rapidly approach the 21st century, with the promise of exciting new technologies and the concerns of overpopulation and destruction of our environment, we have become acutely aware of the state of the world's plant genetic resources for food and agriculture, and the need for urgent action. Three events have been pivotal in bringing these issues into sharp focus.
Firstly, in 1992, the Convention of Biological Diversity was signed. The aims included 'the conservation of biological diversity, the sustainable use of its components' and 'appropriate access to genetic resources and appropriate transfer of relevant technologies'. Secondly, the fourth International Technical Conference on Plant Genetic Resources for Food and Agriculture, held in Germany in 1996, focussed attention on two reports: The State of the World's Plant Genetic Resources, and The Global Plan of Action for the Conservation and Use of Plant Genetic Resources for Food and Agriculture. The Global Plan of Action was adopted. Thirdly, the technical meeting on the Methodology of the World Information and Early Warning System of Plant Genetic Resources (WIEWS) provided methods to monitor the state of the world's plant genetic resources and to facilitate implementation of The Global Plan of Action.
The conventional methods used to conserve plant material have been augmented in recent years by rapid developments in plant biotechnology. That these scientific developments have an important role to play has been recognised at sessions of the Commission for Plant Genetic Resources in the 1990's, where emphasis has been placed on the need to apply these modern biotechnologies to facilitate the conservation and use of plant genetic material (FAO-2, FAO-6). Both plant cell and tissue culture, and more recently, plant molecular biology, have made available techniques that will greatly assist in the evaluation, collection and storage of plant genetic material. The purpose of this document is to examine the role of plant biotechnology in the conservation and improvement of tropical and subtropical fruit species. The term 'plant biotechnology' is interpreted throughout as encompassing all aspects of plant cell and tissue culture as well as techniques arising from plant molecular biology.
Much of the research into biotechnology has been carried out in industrialised countries, resulting in the comparative neglect of regional crops in developing countries (FAO-2), where many of the tropical and subtropical fruit species are grown. In addition, since the task of establishing collections of the world's plant genetic material began, most activity has occurred, quite naturally, with the major food crops. Very little effort has been applied to the minor crops and their wild relatives. At the Commission on Plant Genetic Resources for Food and Agriculture in June 1995, the decision was taken to broaden the mandate to cover all components of biodiversity of relevance to food and agriculture (FAO-4). The Commission had already recognised in April 1991, that new biotechnologies should be applied to major local crops of social and economic importance, but of little international market importance, and that protocols worked out for temperate species may not apply to tropical species (FAO-2). Tropical and subtropical fruit species should benefit from both of these decisions. Many of these fruit crops are vital to local communities in terms of variety of available food, nutrition (particularly vitamins), and therefore quality of life. In addition, there exists potential to bring some of these crops to an expanded international market by applying new biotechnologies, particularly those which can enhance flavour and postharvest characteristics. In recent years some of the minor tropical fruits have already become more popular, resulting in increased exports to countries such as USA, EC, Singapore and Hong Kong (Appendix 1).
Tropical and subtropical fruit species are currently somewhat neglected as far as both germplasm collection and application of biotechnology are concerned. From a positive perspective, they represent a wealth of as yet untapped potential for valuable research effort and development. There is much that can be done using biotechnology techniques not only to conserve germplasm of these species but also to develop many of them as major fruit crops.
There exists in the field of plant biotechnology the prospect of some spectacular breakthroughs, for example, being able to construct and store individual genes as genomic DNA or DNA sequence information. It is proposed by some that traditional methods of plant breeding and germplasm collection will be superseded. This possibility is currently a long way from reality and both new and traditional technologies have unique and complementary roles to play in the evaluation, conservation and use of plant genetic resources.
This document briefly re-examines the need to conserve our plant genetic resources and summarises the strong arguments for a global approach to the task, as proposed at international meetings. The techniques involved in plant biotechnology are defined, illustrating the past and ongoing impact they are having on food crop improvement. The role of plant biotechnologies in improving and conserving plant genetic resources is then thoroughly examined. Existing applications and future potentials for tropical and subtropical fruit species are encorporated. Conclusions and recommendations are then presented. To help establish the current status of tropical and subtropical fruit crops, attached as appendices, are both worldwide production figures and individual case studies of fruit crops of thirteen genera, which are either rapidly developing in importance or exhibit potential for the future.
2. Need for Germplasm Conservation
The survival of mankind has always depended upon the use of the earth's resources. It has been only in recent decades, as these resources have come under threat from a rapidly increasing population and from natural and manmade disasters, that the world generally has started to focus on the notion that we who take and use may be in some way responsible for the ongoing care and management of these precious resources. Since food and its production is one of the essential ingredients of mankind's existence, conservation of our resources is a particularly vital concept for those working in the field of plant science.
There is evidence that as early as Egyptian times mankind has sought to select and conserve crop species with superior genetic qualities. Over the centuries there has been a natural ongoing process of crop improvement as production has been threatened or even devastated by pests, diseases or other cultural and socio-economic factors. Our world environment is not static and so mankind has learnt to 'adjust' food production by exploiting the genetic diversity that occurs naturally. Genotypes which by virtue of their genetic composition are able to flourish in adverse conditions, whether in the cold, or in salty conditions or in the presence of insects or diseases, have been introduced or crossed with other lines to overcome problems in production. In the twentieth century this process has become a science as plant breeders equipped with greater knowledge have set about systematically developing elite lines and maximising crop yields.
With the advent of plant biotechnology and the rapid development of gene transfer techniques, the potential to introduce desirable character traits is no longer restricted to those occurring in close relatives. In theory, genes, once identified, isolated and cloned may be introduced to a plant from any organism. There are still many constraints and limitations involved in this process, which may be overcome in the future, and so the need for the conventional approach is not negated. Rather, the entire discussion serves to bring into sharp focus the value of genetic diversity as a natural resource and highlights the need to value and conserve it for future use.
The escalation in the world population and the implications this has for food production has been a matter of concern for some time. It is probably the single most urgently motivating factor when considering the need for germplasm conservation. In 1992, the Convention of Biological Diversity made a foundational move to establish awareness in this area by focussing on the conservation of genetic diversity and the universal access to plant genetic resources, while acknowledging sovereignty over genetic resources. The principal reason for doing so was stated as being the need for increased productivity to feed current and future populations (Tao & Anishetty 1995). Genetic resources are the raw materials which scientists use to alter plant performance, and with which they hope to achieve the dramatic increase in crop production that will be required in the decades ahead.
Another factor of concern when considering biodiversity has been the emphasis on increasing yields by large scale production of superior clones. An obvious consequence of crop uniformity and neglect of wild related species, which often contain many useful traits, is a narrow gene pool and resultant vulnerability. The fruit species papaya (Carica papaya) is an excellent example of a crop that is extremely vulnerable, but which is economically important in many tropical countries (Appendix 1). In some of these countries, for example The Philippines, many farmers are already struggling with a subsistence existence and so the loss incurred by the failure of a cash crop causes real hardship. The crop is threatened worldwide by a number of serious diseases, including viruses, namely Papaya Ringspot Virus (PRSV-P), fungal diseases including Phytophthora root, stem and fruit rots, and mycoplasmas. PRSV-P has devastated crop production in many regions and may cause many landraces or useful genotypes to be lost completely. Extensive conventional crop breeding has achieved significantly improved fruit size and flavour and has been responsible for large improvements in crop yields but has been unsuccessful in addressing the current disease threats. However, the wild Carica relatives contain a huge pool of useful genes and valuable natural resistances to major papaya diseases have been observed. Carica cauliflora, C. quercifolia, C. pubescens and C. stipulata have resistance to PRSV-P; Phytophthora-resistance has been found in C. goudotiana (Zee, pers comm.); and C. parviflora appears resistant to two mycoplasma diseases in field plantings at Redlands Research Station in Southeast Queensland, Australia (author unpublished). Although the focus in collecting germplasm has been placed on plant species that are valuable as food sources, the case of papaya illustrates the value of other genetic resources. Documentation of wild relatives, landraces and their useful characteristics and in-situ or ex-situ collection requires much time, effort and funds. However, the final benefits may be substantial, particularly as useful traits, such as resistance to a particular disease (eg. Phytophthora), potentially can be transferred to more than one crop species by gene transfer technology.
The importance of germplasm conservation is being increasingly realized and many collections already exist. The report on the State of World's Plant Genetic Resources records some of the difficulties being encountered, for example 'much of the plant genetic resources held in ex-situ collections are insufficiently and/or poorly documented' and 'globally, governments and donor agencies have made insufficient provisions for on-going maintenance costs of conservation infrastructure' resulting in a 'steady deterioration of many facilities and their ability to perform even basic conservation functions', and, 'a high percentage of accessions that are held in these banks are in need of regeneration'. These and other problems led to the adoption of The Global Plan of Action at the fourth International Technical Conference on Plant Genetic Resources for Food and Agriculture in June 1996 (FAO-6).
Any attempt to systematically conserve genetic material on a worldwide basis will of course necessitate extensive government cooperation and funding. The task at hand is a daunting one but is complicated by the prevailing attitude of many governments in western countries to agricultural research. Although they are signatories to all relevant conventions there is a growing reluctance to fund agricultural research. As funds dry up, competition causes available funding to go to those research areas promising significant results in the short term. Germplasm storage is not offering spectacular short term outcomes and is motivated by the need to take responsibility for the long term.
All countries need to value and be responsible for the conservation of genetic diversity. It is a natural resource, the value of which is still to be fully appreciated as science continues to unravel the mysteries of plant function at a cellular level and employ them to meet the constant stream of new and ongoing pressures and difficulties facing crop production. There is an urgent need to increase research into technologies that facilitate conservation of plant genetic resources. This is consistent with The Global Plan of Action (FAO-6).
2.1 Status of Germplasm Storage
Since the 1970's there has been a conscientious effort to collect germplasm (FAO-6). In 1995, Tao and Anishetty reported that worldwide more than 4 million accessions of crop plants and their relatives were stored in genebanks and that there were 256 genebanks with long-term storage facilities and 1227 institutions with ex-situ collections.
However, these statistics are rendered less impressive by two considerations. Firstly, 40% of these accessions are cereals (wheat represents 14%). No consistent effort has been applied to the collection of other major crops, while minor crops including fruit crops, have been largely neglected (FAO-8). Secondly, more than 90% of the accessions are stored as seed (Tao and Anishetty 1995). As a consequence, vegetatively propagated species and species with recalcitrant seeds are greatly underrepresented. In addition, if we are to preserve diversity, collections must also include wild relatives of crop species. As gene transfer techniques overcome the incompatibility between species, there is also a need to be mindful that non-crop species may also represent a valuable source of useful genes such as disease and insect resistance.
Although seed storage has been the conventional method of conserving germplasm, pollen storage and field collections have also been employed. Scientific advances in the field of cell and tissue culture systems have made available, as alternatives, conservation in vitro in either slow growth systems or cryopreservation. More recently, developments in molecular biology offer the future prospect of DNA storage as libraries of either DNA sequence information or of DNA constructs (Rao and Riley 1994).
As reported in the State of the World's Plant Genetic Resources (FAO-6), one of the major concerns with germplasm storage is the maintenance of collections. Stored germplasm requires careful characterisation as well as periodic regeneration to ensure viability. Techniques resulting from biotechnology offer improvements in this area. Molecular marker techniques enable characterisation at the DNA level, and an advantage of in vitro conservation is that viability is easily assessed visually. As regular regeneration, usually annually, is essential, it is less likely to be overlooked.
A major effort is still required in the evaluation and conservation of tropical and subtropical fruit species.
3. Need for Regional and Global Networks
The development and maintenance of germplasm banks is of limited usefulness if the genetic material is not distributed and used worldwide to develop new and improved varieties of food plants. Hence The Global Plan of Action (FAO-6) promoted the development of networks for the effective exchange and utilisation of germplasm. It states that 'networks are important platforms for scientific exchange, information sharing, technology transfer, research collaboration, and for the determination and sharing of responsibilities for such activities as collecting, conservation, distribution, evaluation and genetic enhancement'.
FAO has supported and collaborated in the development of networks in the 1990's. Networks have been established on both a crop (mushroom, cactus pear, olive, citrus, nut tree) and regional basis (Southern African, West Asian and North African, Andean, Amazonian, and Central American). These networks and their aims are summarised in appendix 1 of FAO document FAO-5. As stated in this report 'the innovative aspect of the newly launched networks is the promotion of a coordinated approach to identifying, evaluating and conserving the genetic variability of selected crop species, with the aim of its utilisation for the improvement of cultivars and their adaptation to farmer's needs'. There exists the potential to strengthen existing networks, develop new networks and to coordinate these regional and crop-based networks into an effective global system.
The chief aims of The Global Plan of Action are the conservation and sustainable use of genetic resources, by developing more effective conservation strategies at both the national and international level (FAO-8). A major advantage of crop-based networks is that they can facilitate linkages between conservation and sustainable utilisation of crop genetic resources (FAO-4). A number of factors highlight the importance of a global approach. Plant Genetic Resources (PGR) are a common concern of humanity and all nations are both donors and users of PGR (FAO-3). The effective use of a wide range of PGR in all countries is an incentive to ensure their conservation and maintenance (FAO-3). Thus the international community as a whole has both an interest in and a responsibility to conserve PGR and this is the basis for an effective integrated and rational Global Plan of Action (FAO-6).
A related development which emphasises the need for a global approach is the proposed World Information and Early Warning System on Plant Genetic Resources (WIEWS). The technical meeting on the methodology of WIEWS recommended the establishment of a global network, on a voluntary basis, under the auspices of FAO (FAO-7). WIEWS would provide a vehicle to register cases of genetic erosion both in in situ populations and ex situ collections. However, it would require an accurate information base on the current status of PGR including crop species, local varieties and wild relatives (FAO-7). The Global Network would provide 'timely and precise information on PGR conservation and utilisation', contribute to the implementation and updating of The Global Plan of Action, provide an information flow between countries and develop an effective early warning system (FAO-7). One of the recommended ways to achieve these aims is to develop and improve links with existing networks.
Two recent documents detail the advantages of a worldwide approach, which coordinates regional and crop-based networks. They are the reports detailing The Global Plan of Action that was presented at the International Technical Conference on Plant Genetic Resources in Leipzig (June 1996) (FAO-6) and the IPGRI report - Options for Access to Plant Genetic Resources and the Equitable Sharing of Benefits Arising from their Use - which was presented to the Sixth Session of the Commission on Genetic Resources for Food and Agriculture in Rome (December 1996) (FAO-8). The advantages that have been detailed in the above documents can be summarised as follows:
The enormity of the task of conserving our plant genetic resources begins to come clear only as the many steps involved are defined. In addition, there will be difficulties encountered at each stage, simply because of the enormous diversity within the plant kingdom. It is difficult to see how progress can be achieved without a concerted, coordinated and combined effort.
4. Current Applications of Biotechnology
The sixties and seventies witnessed a new era in plant biotechnology, ie. the rapid development of techniques for plant cell and tissue culture. The number of published reports on tissue culture technology and their application to a wide range of species has increased dramatically over the past 20 years. It is now possible to regenerate plants from a range of explant types of many plant species, including most of the important crop, fruit and ornamental species. Technologies include isolation, culture and regeneration of tissues, cells, protoplasts, organs, embryos, ovules, anthers and microspores.
In the eighties and nineties another major development has occurred in the field of plant biotechnology, which has combined rapid advances in the understanding of molecular biology with the relatively new science of cell and tissue culture. The result has been genetically engineered plants and the development of molecular markers and gene mapping.
Plant biotechnologies have continued to evolve in response to the constant challenge that is presented by the incredible range of biological diversity in plant species. As a result of each technique, valuable and fascinating breakthroughs have been achieved. This chapter provides an overview of plant biotechnologies giving some historic perspective and examples of the practical way in which they have been employed to improve crops. In the latter section, a review is provided of the specific application of these biotechnologies to tropical and subtropical fruit crops.
Micropropagation system based on growth of microcuttings from axillary buds of apically dominant plants. From left: Apically dominant papaya plant; growth of axillary shoots from nodal sections; dissected axillary shoots on rooting medium; and small rooted shoot.
Micropropagation has been defined as 'in vitro regeneration of plants from organs, tissues, cells or protoplasts' (Beversdorf 1990) and 'the true-to-type propagation of a selected genotype using in vitro culture techniques' (Debergh and Read 1991). True-to-type propagation has important benefits for plants that have no named varieties, such as Australian dioecious papaw genotypes (Drew 1988, Drew 1992) where traditional plant breeding has failed to produce stable lines. Micropropagation has also been useful for the rapid initial release of new varieties prior to multiplication by conventional methods, eg. pineapple (Drew 1980) and strawberry (Damiano et al. 1983, Smith and Drew 1990a), and germplasm storage for maintenance of disease-free stock both in controlled environment conditions (Wilkins and Dodds 1983, Withers 1989) and long term via cryopreservation (Kartha 1985).
However, the ability to propagate plants in vitro free of genetic off-types is dependant on the technique used for micropropagation. Failure to understand this principle has resulted in disastrous consequences with some species, for example the commercial production of banana clones containing 90% dwarfs (Smith and Drew 1990b). Protocols which have been developed for in vitro propagation of plant species can be divided into three systems (Drew 1992). System 1 is based on callus culture followed by organogenesis or embryogenesis. System 2 comprises proliferation of axillary buds and/or adventitious buds, resulting from repeated subculture on multiplication media containing cytokinin. In system 3, micro-cuttings obtained from axillary buds of apically dominant shoots are grown on hormone-free or low cytokinin media. System 3 has been used in the author's laboratory for clonal propagation of papaw (Drew 1992), neem (Drew 1993), coffee (Drew 1991) and passionfruit (Drew 1995) and is not prone to production of genetic off-types.
Somaclonal Variation and In vitro Selection
Example of somaclonal variation; a dwarf banana after micropropagation.
Exploitation of the variation that exists in populations has led to the development of many commercial varieties and hybrids. With the rapid expansion of tissue culture technologies came the observation that genetic variation was occurring in plants regenerated from somatic cells and this was seen as a novel source of variation (Larkin and Scowcroft 1981). The frequency of off-types varies with species, culture type and number of sub-cultures and has been attributed to a number of variations within cultured cells (Scowcroft et al. 1987). However, the cause of somaclonal variation is not fully understood (Phillips et al. 1990); therefore, it cannot be controlled and changes can be epigenetic and unstable (Maretzki 1987). Variation can be promoted by the use of radiation or chemical mutagens (Micke 1987) and the use of colchicine to change ploidy (Heinz and Mee 1970). The potential of somaclonal variation involves the ability to change one or a few characters without altering the remaining part of a genotype.
Initial reports of potentially useful variation included increases in cane and sugar yield, and resistance to eye-spot disease in sugar cane (Heinz et al. 1977); improved tuber shape, colour, and late blight resistance in potato; and, increased solids and Fusarium resistance in tomato (Evans and Sharp 1983, Evans 1989).
In vitro selection involves the screening of cell cultures that are exhibiting genetic variation, for tolerance or resistance to pathogens, herbicides, low or high temperatures, metals and salt (Tomes and Swanson 1982, Chaleff 1983). It necessitates a reliable method of regeneration from callus. For successful application of the technique, tolerance at the cellular level must also occur in the regenerated plant in the field and be transferable to other plants via conventional plant breeding.
The use of in vitro screening to select for disease resistance is most effective for diseases which produce toxins. This was initially demonstrated by Carlson (1973) who regenerated tobacco plants resistant to Pseudomonas tabaci, from haploid cells resistant to methionine sulfoxamine, an analogue of the disease toxin. Similar successes have been reported with a range of diseases including Phoma lingam in canola (Sacristan 1982), Helminthosporium oryzae in rice (Vidhyasekaran et al. 1990), Pseudomonas and Alternaria in tobacco (Thanutong et al. 1983) and Pseudomonas solanacearum in tomato (Toyoda et al. 1989).
Addition of herbicides to culture is an ideal system for in vitro screening as defined concentrations can be used. However, effectiveness is dependent on the mode of action of a herbicide on the whole plant being similar at the cellular level, and is therefore better suited to herbicides that interfere with basic metabolic functions (Chaleff 1986). Resistance or tolerance has been developed in vitro to a range of herbicides including paraquat in tomato (Thomas and Pratt 1982), picloram (Chaleff 1986) amitrole (Singer and McDaniel 1984) and triazine (Cseplo et al. 1985) in tobacco and 2,4-D in Lotus corniculatus (Swanson and Tomes 1983). Carrot suspension cultures which were resistant to 35mM glyphosate in solution were selected after 8 subcultures on low concentrations of glyphosate (0.3 to 0.6 mM) (Shyr and Widholm 1990). Carrot plants regenerated from these cultures were resistant to glyphosate sprays in field plantings. A similar stepwise selection procedure for gene amplification produced tobacco cell lines with high tolerance to glufosinate (Ishida et al. 1989).
Cell cultures that are resistant to increased salt (NaCl) in vitro do not necessarily produce regenerants which tolerate high salinity levels in soil. For example, the use of somatic cell cultures of rice did not lead to heritable salinity tolerance in the field; however, this was eventually achieved after in vitro selection of callus cultures initiated from immature ovaries (Winicov 1990) and anther culture-derived lines (Zapata et al. 1991).
Tomes (1990) documented examples of genotypes which had been released or were in field trials in the USA and Canada. Many of his references were patent applications or personal communications. These included chlorsulfuron and imidazilinone resistance in canola, imidazilinone resistance in corn, high solids and Fusarium race 2 resistance in tomato, a white flowered form of lucerne (Bingham et al. 1988), potato virus Y resistance in tobacco and fall armyworm resistance in sorghum.
Potentially useful somaclonal variants have been identified in a number of fruit crops. These include Phytophthora resistance in papaya (Sharma and Skidmore 1988) and apple (Utkhede 1986), Xanthomonas resistance in peach (Hammerschlag 1988), Erwinia resistance in pear (Brisset et al. 1988) and salt tolerance (Ben-Hayyim and Goffer 1989) and 2, 4-D tolerance (Spiegel Roy et al. 1983) in orange.
The production of haploids via gametic embryogenesis is one of the major contributions of plant tissue culture research to crop improvement. Natural occurrence of haploids is rare and is confined to a few species (Kimber and Riley 1963). Haploids facilitate the detection of mutations which are usually recessive and difficult to detect (Bajaj 1983) and consequently have played an important role in the isolation of resistant cell lines via in vitro selection (Mazur and Falco 1989). They can significantly reduce time required for plant breeding as they offer a technique for rapid production of homozygous lines (Chase 1974). Although doubled haploids have been obtained via gynogenesis (Bossoutrot and Hosemans 1983, Keller 1990, Zhu and Wu 1979), the majority of published successes have resulted from anther and microspore culture.
The first success of embryogenesis from anther culture was in Datura (Guha and Maheshwari 1964) and most early progress with anther culture was on Solanaceous species (Hu and Zeng 1984). Difficulties associated with anther culture are low frequencies of haploids, difficulty in distinguishing spontaneously doubled haploids from diploids which regenerate from somatic tissue and the considerable time and labour which may be needed to generate numbers required for critical evaluation in a breeding program (Smith and Drew 1990a).
The potential of microspore culture has been demonstrated in brassicas, particularly canola (Chuong et al. 1988, Pechan and Keller 1989, Polsoni et al. 1988). Many brassica pollen spores are embryogenic, producing haploids or homozygous diploids spontaneously. Mature seed can be produced from plants derived from pollen grains in only nine months (Beversdorf 1990). This procedure was used to study a number of traits including herbicide resistance, yield, oil and protein content and lodging (Beversdorf 1990).
Haploidy has made a significant contribution to improvement of important cereal species including barley, corn, rice and wheat. Good progress has been made with barley using the 'bulbosum' technique which was first described by Kasha and Kao (1970). Haploids are produced when Hordeum vulgare is fertilised with Hordeum bulbosum pollen, as the bulbosum chromosomes are preferentially eliminated. Maternal haploids have also been produced using Solanum phureja pollen to fertilise potato (Evans et al. 1984) and Nicotiana africana pollen on tobacco (Burk et al. 1979). A more recent development of this procedure, which has led to the production of wheat haploids in high frequency, is the fertilisation of wheat ovules with maize pollen (Laurie and Bennett 1986, 1988; Laurie and Reymondie 1991). This technique has the potential to provide doubled haploids for wheat breeding programs. Anther culture is affected by genotype, stage of development, pre-treatment conditions and numerous cultural conditions (Tomes 1990). However, significant progress is achievable given a large research effort, as has been demonstrated with several cereal crops which until recently had been difficult to culture, eg. rice (Zapata et al. 1991), maize (Mitchell and Petolino 1990, Pescitelli et al. 1989, Pescitelli et al. 1990, Wan et al. 1989) and wheat (Hassawi and Liang 1990, Luckett and Darvey 1992, McBeath et al. 1990, Ziauddin et al. 1992).
Plants have been produced following anther culture of a number of fruit species. Success has been achieved with custard apple (Nair et al. 1983), papaw (Litz and Conover 1978), four citrus species (Chen 1985), longan (Yang and Wei 1984), lychees (Fu and Tang 1983), apples (Zhang et al. 1990), cherry (Seirlis et al. 1979), pear (Jordan 1975) and grape (Rajasekaran and Mullins 1979).
Embryo culture was probably the first tissue culture technique to be applied to plant improvement. Hannig (1904) cultured mature embryos of Raphanus and Cochlearia using aseptic techniques. Principal applications of embryo culture are rescuing embryos after interspecific hybridisation, clonal propagation of families such as Gramineae and conifers which contain recalcitrant species, and overcoming seed dormancy and seed sterility (Hu and Wang 1986).
Interspecific crosses are often attempted to transfer desired traits such as disease resistance, stress tolerance or high yield from wild species into important crop species, eg. cotton (Liang et al. 1978), soybean (Broue et al. 1982) and papaya (Manshardt and Wenslaff 1989). Incompatibility after these crosses normally results in embryo abortion and this is often caused by breakdown of the endosperm (Hu and Wang 1986) or embryo-endosperm incompatibility (Emsweller and Uhring 1962). In vitro culture of hybrid embryos often successfully bypasses postzygotic incompatibilities.
There are numerous references of embryo rescue of incompatible hybrids. Hu and Wang (1986) documented reports of 65 different combinations in a period from 1980 to 1984.
Another approach to wide hybridisation is in vitro fertilisation of cultured ovules and ovaries (Tilton and Russell 1984, Zenkteler 1984). This was achieved initially with opium poppy (Kanta et al. 1962). The major application of this technology in monocotyledonous crop species is in maize (Gengenbach 1984, Kranz et al. 1990).
In an extensive research program funded by the Australian Centre for International Agricultural Research (ACIAR) techniques have been developed for interspecific hybridisation and embryo rescue of papaya and wild relatives (Drew et al. 1995). Hybrids have been produced between C. papaya and C. cauliflora, C. quercifolia, C. pubescens, C. parviflora and C. goudotiana. Useful characteristics of the wild species include papaya ringspot virus resistance, Phytophthora resistance, high sugar content, and ornamental characteristics.
Non-sexual gene transfer was initially made possible by Carlson et al. (1972) who recorded the first successful protoplast fusion between Nicotiana species. The first example of a somatic hybrid which could not be produced sexually was from fusion of tomato and potato protoplasts (Melchers et al. 1978). Although this technique was initially expected to have a large impact in the development of new varieties, its application has been limited by the difficulty in regenerating plants from fused protoplasts (Evans 1983).
Practical applications have resulted from somatic hybridisation between closely related species (Vasil 1990). Atrazine tolerance has been produced following Brassica spp. hybridisation (Barsby et al. 1987) and protoplast fusion of haploid cells has been used to combine cytoplasmic male sterility and cytoplasmic atrazine resistance in canola (Chuong et al. 1988). Cytoplasmic male sterile cybrids of rice have been produced and used in hybrid seed production (Kyozuka et al. 1989). Hybridisation between Nicotiana tabacum and N. rustica has produced tobacco plants which varied in nicotine and tar content and in resistance to blue mould and black root rot (Pandeya et al. 1986).
Interspecific hybridisation using protoplast fusion has been used extensively with citrus species (Gmitter et al. 1992) including the production of hybrids between the two sexually incompatible species Citrus reticulata and Citropsis gilletiana (Grosser et al. 1990). Hybrids have also been produced between apple species (Saito et al. 1989), between papaya and a wild relative (Jordan et al. 1986) and between pear and cherry (Ochatt et al. 1989).
Rapid and spectacular advances in molecular biology are driving a new revolution in plant science. Many years of research have culminated in an increasingly detailed understanding of biology and genetics at the molecular level. Combined with tissue culture technology, this knowledge is being applied to the modification of plant genomes.
Because of the universality of the genetic code, potentially useful genes can be transferred to plants from any organism (virus, bacteria, insect etc.). This has lead to the development of new and improved genotypes by the addition of single genes that code for traits such as insect or disease resistance, or by the inactivation of single gene faults. Future potential includes the possibility of transferring larger DNA constructs that code for multiple genes conferring more complex traits.
Applications of gene transfer are increasing rapidly, driven by an extensive research effort worldwide. Initial successes were in the development of disease, insect and herbicide resistant crops. More recently, gene transfer is resulting in control of plant development in individual species (Newbigin et al. 1995). Inhibition of expression of polygalacturonase in a tomato genotype lead to the first commercial release of a genetically engineered plant - the FLAVR-SAVR tomato. Extensive research is being directed towards gene inactivation techniques for control of ethylene production, and thus ripening, in climacteric fruits. Gene transfer techniques have also lead to the development of carnations with increased vase life and new colour forms (Mol et al. 1995). Isolation of a flower-meristem-identity gene from Arabidopsis has the potential to dramatically reduce time to flowering and hence length of the juvenile phase in plant species (Weigel and Nilsson 1995). Developing applications include transformed plants which will produce more or increased levels of valuable oils, proteins and starches (Flavell 1995), and plants that produce vaccines and can be used for oral immunization against a range of serious human diseases (Manson and Arntzen 1995).
Useful agricultural applications of gene transfer necessitates incorporation of a foreign DNA construct into a plant genome, the regeneration of transformed plants, the stable expression of the introduced gene (Walden and Wingender 1995) and inheritance in subsequent generations.
The successful application of plant transformation techniques is dependent on plant tissue culture protocols to regenerate transformed plants. Since the first reports of gene transfer with species that are relatively easy to tissue culture, petunia and tobacco (Bevan and Flavell 1983, Herrera-Estrella et al. 1983, Horsch et al. 1983), transformation procedures have been published for a wide range of species including tree crops, such as apples (James and Dandekar 1991) and papaws (Fitch et al. 1992). Transgenic plants of a number of fruit species have been produced, including kiwi-fruit (Uematsu et al. 1991), citrus (Vardi et al. 1990), strawberry (Nehra et al. 1990), grape (Mullins et al. 1990), cranberry (Serres et al. 1990), peach (Hammerslag 1988) and plum (Mante et al. 1991).
Numerous DNA delivery systems have been reported. Direct gene transfer systems such as microinjection, electroporation and polyethylene glycol have been used to transform protoplasts. Protoplasts are easily damaged and regeneration of plants from them is very difficult with many species. Consequently, such systems are not widely used. The popular method of direct gene transfer is particle bombardment. DNA coated microparticles are bombarded into tissues, and major crop species have been transformed using this technique, for example monocots such as wheat, barley, rice and recalcitrant dicot species such as cotton (Finer and McMullen 1990), and soybean (McCabe et al. 1988). The advantages of the particle gun in terms of tissue culture regeneration is that it can be used on callus and suspension cultures and on organised tissue of both monocots and dicots. Increasing the number of options for tissue culture technology increases the probability of successful regeneration.
The preferred method of gene transfer for dicots is Agrobacterium mediated transfer. Co-cultivation of callus, suspension cultures or leaf discs with Agrobacterium has been used to successfully transform many species (Komari 1989), for example efficient systems have been described for major crop species such as potato (Visser 1991), tomato (McCormick 1991) and sugar beet (Lindsay et al. 1991). Virus, insect and herbicide resistance in a range of species including tobacco, potato, tomato, lucerne and soybean have been successfully trialled in field conditions and are in the early stages of commercialization.
Transformation of cereals, which are the world's most important food, has been by direct DNA uptake systems, particularly biolistics. Embryogenic cultures in cereals have generally been developed from immature embryos and inflorescences and subsequently transformed in the form of protoplast, suspension or callus cultures. The most recalcitrant of these species is wheat, where difficulty in developing reliable regeneration systems (Vasil et al. 1990) is exacerbated by low frequency of transformation (Vasil 1992). Other problems associated with tissue culture of cereals are loss of regenerative capacity with increasing number of subcultures (Rhodes and Gray 1992) and occurrence of abnormalities such as sterility in regenerated plants (Vasil 1990, Zapata and Abrigo 1986). Agrobacterium was thought incapable of infecting monocots, however recent reports describe Agrobacterium mediated transformation of rice, maize and barley which offer considerable potential.
Systems have been reported for transformation of haploids via particle bombardment of pollen (Twell et al. 1991) and via microinjection of DNA into microspore derived embryoids of canola (Neuhaus et al. 1987). More recently, maize has been transformed by pollen which was mixed with Agrobacterium containing binary plasmids (Donowati et al. 1997). Such systems may provide a method that eliminates the need for in vitro regeneration (Heberle-Bors et al.1990), which is a major limiting step for some species and is prone to somaclonal variation.
Although in theory tissue of any species can be transformed, it is currently not possible to regenerate transformed plants at high frequencies for many species. Current methods of gene transfer and selection can be disruptive to growth of in vitro cultures, thus it can be difficult to develop efficient transformation procedures for species that are recalcitrant in vitro. In addition, there is a need to be able to deliver gene constructs to tissue that is competent both for transformation and regeneration. Limitations of current technology include low frequency of transformation, high frequency of undesired genetic aberrants, unpredicitability of transgene expression, the need for techniques to transfer large DNA sequences coding for multiple genes (Birch 1997) and the absence of efficient repeatable regeneration protocols for many species in vitro. According to Birch (1997) the key to progress with recalcitrant species in the future is in the development of new methods which will expose large numbers of regenerable cells to nondestructive DNA delivery systems. Widespread future application of this technology will also depend on various regulatory, patent and environmental issues, as well as the cost and consumer acceptance of genetically engineered products.
One of the recent applications of new techniques of molecular biology is the rapid development in molecular markers and gene mapping. The use of DNA-based markers is allowing researchers to mark genes or chromosome regions that are related to genetic traits such as disease resistance or fruit colour. A particular advantage of such techniques is that complex polygenic traits can be analysed. Mapping techniques can also be used to isolate genes based entirely on their genetic behaviour. Once these have been identified, sequenced and cloned, gene transfer techniques can be used to transfer them to other species.
Various techniques have been used to mark individual characters in segregating populations and construct genetic maps. Initially isozymes were used as markers. Isozymes are enzyme products of particular genes. Individuals which vary in their genetic characteristics are separated by polymorphisms (variability) in isozyme banding patterns on gel plates. Recently, the more precise marker techniques such as RFLPs, RAPDs, AFLPs and VNTRs are being preferred. Restriction Fragment length Polymorphisms (RFLPs) are short fragments of DNA generated by restriction enzymes (bacterial enzymes that cut DNA at specific coding sequences). The fragments are separated by electrophoresis and are identified with radioactive probes. Random Amplified Polymorphic DNA (RAPDs) are produced by randomly amplifying DNA sequences using PCR technology and primers (with arbitrary known DNA sequences). AFLPs (Amplified Fragment Length Polymorphisms) are a related PCR-based technique. Highly polymorphic markers which also depend on PCR technology are VNTRs (Variable Numbers of Tandem Repeats). These are tandem repeats of short DNA sequences and include minisatellites (10-45 bp) and microsatellites (2-6 bp). The use of RAPDs, AFLPs and VNTRs is much quicker than RFLPs. Using these techniques it is possible to link markers on a genetic map with traits of interest in a particular species.
Once markers are defined, they can be used directly in breeding programs since they allow researchers to predict phenotypes based on the presence or absence of the marker. Alternatively, once genes for particular character traits have been identified by marker techniques, they can be cloned. Although currently a difficult task, the development of automated systems will likely make such 'map-based cloning' fairly routine in the future. Major research efforts are underway to sequence, or partially sequence, all genes in model species such Arabidopsis or rice. Once this is achieved in a major dicot and monocot species, it will be easier to locate particular genes in other species. Genes that are located together in one species are likely to be similarly located in another species (Flavell 1995). Alternatively, a gene which codes for a similar function in a related species may be found by identification of homologous DNA sequences.
Although much of the initial research effort has been applied to important vegetable and cereal species, these techniques will have a large impact on fruit tree breeding. For example, the long juvenile period required by many fruit tree species has made the process of breeding and selection of superior genotypes a long term effort. If a genetic marker is obtained for a trait of interest, plants could be selected at the seedling stage.
4.2 Applications and Potential in Tropical and Subtropical Fruit Species
A review of available literature shows that applications of biotechnology to tropical and subtropical fruit species is limited. There has been some research effort directed to this field in the 1990's, however, before this period little information was available, with the exception of a few species (citrus, banana, papaya, pineapple). Relative to the effort that has been applied to research of temperate species, biotechnology of tropical and subtropical species is a vastly unexplored frontier. A notable exception is Citrus spp., which have been the subject of considerable in vitro research, and are a model for application to other tropical and subtropical species. Much information is available in a number of reviews (eg Gmitter et al. 1992).
More information is available on micropropagation than on any other biotechnology applied to tropical and subtropical fruits. Micropropagation from apical or axillary bud explants (Table 1) has been separated in this review from regeneration systems based on organogenesis or embryogenesis (Table 2). Regeneration via callus is prone to somaclonal variation, whereas the meristematic tissue in buds is inherently the most genetically stable tissue in plants. Genetic stability in a micropropagation system is very important when applied to elite genotypes, particularly if the aim is germplasm conservation.
Micrografting, an innovative technique which should be applicable to other tropical and subtropical species, was developed in 1972 for Citrus. Micrografting of Citrus results in virus-free plants that are not juvenile (Murashige et al. 1972).
A summary of micropropagation protocols is presented for fruit species comprising 15 genera in Table 1. Most of this research is at a preliminary stage and this is demonstrated by two factors. Firstly, with a few exceptions (citrus, banana, papaya, pineapple), most of the successes have been achieved using juvenile bud explants, and in many cases seedling tissue. Secondly, only 50% of the reports document acclimatisation of plantlets in soil. However, the increase in research effort in this area in 1990's and published results on a wide range of tropical fruit species is encouraging and justifies a larger research effort.
Table 1: Micropropagation of Tropical Fruit Species
|
|
|
|
|
|
| Ananas comosus | pineapple | lateral buds | high multiplication rates
mature plants* |
Rangan (1984) ** |
| Annona atemoya | atemoya
custard apple |
nodal cuttings | rooted plantlets* | Rasai et al. (1994) |
| Annona cherimola | cherimoya
custard apple |
nodal sections
nodal sections |
multiple shoots
rooted plantlets * |
Jordan et al. (1991)
Encina et al. (1994) |
| Annona squamosa | sugar apple
custard apple |
nodal sections | rooted plantlets* | Lemos et al. (1996b) |
| Artocarpus heterophyllus | jack fruit | shoot apices
shoot buds |
rooted plantlets
rooted plantlets * |
Rajmohan et al. (1988)
Roy et al. (1993) |
| Arerrhoa carambola | carambola
five corner fruit |
nodal sections | rooted plantlets * | Khalekuzzaman et al. (1995) |
| Carica papaya | papaya
papaw |
apical and lateral shoots | mature plants * | Drew (1992) |
| Carica pubesans | mountain papaya | shoot tips | rooted plants | Jordan (1992) |
| Eugenia javanica | java apple | shoot tips | rooted plantlets | Katooka et al. (1993) |
| Feijoa sellowiana | feijoa
pineapple guava |
seedlings
explants |
shoots
shoots |
Bhojwani et al. (1987)
Bertoni et al. (1996) |
| Litchi chinensis | lychee | shoot segments | surviving shoots grafted to seedlings | Yu (1991) |
| Musa spp. | banana | shoot tips | mature plants* | Teisson et al. (1997)** |
| Passiflora spp. | passionfruit | apical or lateral buds | rooted plants* | Drew (1997)** |
| Persea americana | avocado | nodal segments | rooted shoots | Biasi et al. 1994 |
| Psidium guajava | guava | nodal segments
shoot tips shoot tips shoot tips stem nodes |
leaf development
shoots, roots rooted shoots* shoot growth rooted plantlets* |
Broodrijk (1989)
Fitchet-Purnell (1990) Papadatou et al. (1990) Khattak (1990) Mohamed-Yasseen et al. (1995) |
| Syzgium cumini | jambu
java plum |
nodal and shoot tips | rooted plantlets* | Yadav et al. (1990) |
| Tamarindus indica | Tamarind | nodal segments | rooted plantlets | Kopp et al. (1992) |
| Ziziphus sativa | jujube | microcuttings | shoots, roots | Vashakidze et al. (1988) |
| Ziziphus mauritana | indian jujube | nodal explants | rooted plantlets | Rathore et al. (1992) |
** Review - not necessarily achieved by this author
Organogenesis, Embryogenesis, In vitro
Selection
Regeneration via embryogenesis from callus derived from an immature zygotic embryo of papaya.
Regeneration of plantlets from callus can follow two distinct pathways. Organogenesis involves the development of meristematic tissue which produces either shoots or roots, with vascular connections to the parental callus. Somatic embryos progress through characteristic developmental stages (globular, heart, torpedo and mature), have both a shoot and root meristem (bipolar) and become independent from the parental callus.
As with most species, tropical and subtropical fruit species tend to regenerate by either organogenesis or embryogenesis (Table 2), although occasionally there are reports of regeneration via both pathways (eg. Carica papaya). As with micropropagation, most of the published successes via regeneration from callus has been achieved using juvenile tissue explants. Most tropical fruit species are woody perennial dicots, which in general are a difficult group to culture in vitro. Tissue culture of recalcitrant species has been achieved in many cases using immature embryos or very juvenile seedling explants, as these often represent the most regenerative tissue in a plant.
Much of the documented research on recalcitrant species entails a thorough evaluation of a range of media components and concentrations. However, recent innovative protocols for embryogenesis and regeneration of Musa spp. from male flower explants (Teisson et al. 1997) demonstrate the importance of testing a range of explants as well as media when working with untried or 'difficult' species.
However, few species are truly recalcitrant, and most can be regenerated provided they can be subjected to a concentrated research effort. In vitro culture of papaya has been researched extensively worldwide and highly refined protocols have been developed. Efficient methods for embryogenesis from immature zygotic embryos have been used to develop transgenic plants (Fitch et al. 1990) and encapsulated artificial seeds (Ye et al. 1993). Recent reports of culture of integuments of immature seeds and the development of embryos of maternal origin (Monmarson et al. 1995) should have important implications for rapid propagation and production of transformed 'elite' papaya genotypes. Regeneration of plantlets via embryogenesis following culture of nucellar tissue of mango, another recalcitrant species, (de Wald et al. 1989 a, b) demonstrates what can be achieved by innovative research. There are two other noteworthy achievements with regeneration of tropical fruit species. Plantlets have been produced after embryogenesis from mature leaf tissue of mangosteen (Goh et al. 1990), and seedless triploid pummelo plants were produced following culture of endosperm (Wang and Chang et al. 1978).
Regeneration via organogenesis or embryogenesis is seen as having three major applications: micropropagation, cryopreservation and gene transfer. However, regeneration from callus can be prone to production of genetic off-types, thus any micropropagation technique should not be assumed to be clonal until large scale field testing has been undertaken to evaluate a protocol for genetic stability. Similarly, embryogenic cultures are well suited to cryopreservation studies, however an assessment of genetic stability after large scale field evaluation is also necessary for each species and protocol, before it can be endorsed as a method of germplasm conservation. Regeneration protocols are basic to the development of transformation systems, as transformed plantlets have to be grown from single transformed cells. Thus a large research effort is warranted for many tropical and subtropical fruits, if
Table 2: Caulogenesis, rhizogenesis and embryogenesis following callus
induction from various tissue explants of tropical fruit species.
|
|
|
|
|
|
| Annona atemoya | atemoya
custard apple |
leaf
hypocotyls |
shoots
rooted shoots* |
Nair et al. (1984)
Rasai et al. (1994) |
| Annona cherimola | cherimoya
custard apple |
petioles
zygotic embryos |
shoots, roots
shoots |
Jordan (1988)
Jordan et al. (1991) |
| Annona muricata | soursop | hypocotyl | rooted shoots* | Bejoy et al. (1992) |
| Annona squamosa | sugar apple
custard apple |
hypocotyls | rooted shoots * | Lemos et al. (1996a) |
| Averrhoa carambola | carambola
five corner fruit |
leaf
root root hypocotyl and cotyledon cotyledons |
shoots
rooted shoots rooted shoots* rooted shoots rooted shoots* |
Litz et al. (1989)
Rashid et al. (1992) Kantharajah et al. (1992) Amin et al. (1993) Khalekuzzaman et al. (1995b) |
| Carica papaya | papaya
papaw |
seedling stem and cotyledon
immature zygotic embryos embryogenic suspension culture integumens of immature seeds |
caulogenesis
embryogenesis, plantlets encapsalated artificial seeds embryos of maternal origin |
Litz (1986)**
Fitch et al. (1990) Ye et al. (1993) Monmarson et al. (1995) |
| Carica pubescens | mountain papaya | axillary buds | embryogenesis | Jordan et al. (1996) |
| Citrus grandis | pummelo | endosperm
epicotyl & root |
triploid plantlets
rooted shoots |
Wang et al. (1978)
Goh et al. (1995) |
| Euphoria longan | longan | nodes | embryogenesis
plantlets |
Celo et al. (1997) |
| Feijoa sellowiana | feijoa
pineapple guava |
cotyledons
cotyledons |
embryogenesis
embryogenesis |
Cruz et al. (1990)
Canhoto etal (1996) |
| Feronia limonia | elephant apple | cotyledon | rooted shoots* | Hossain et al. (1994) |
| Garsinia mangostana | mangosteen | leaf
seed sections seed segments |
rooted shoots*
shoots, roots rooted shoots* |
Goh et al. (1990 &
1994)
Teo (1992) Normah et al. (1992, 1995) |
| Litchi chinensis | lychee | immature embryos | embryogenesis, plantlets | Zhou et al. (1993) |
| Mangifera indica | mango | nucelli from immature seed | embryogenesis plantlets* | Mathews et al. (1992) ** |
| Musa spp. | banana | young male flowers | embryogenesis, plantlets | Tesson et al. (1997)** |
| Passiflora spp. | passionfruit | stem and leaf sections | rooted shoots | Drew (1997)** |
| Persea americana | avocado | immature zygotic embryos | embryogenesis | Pliego-Alfaro et al. 1988 |
| Psidium guajava | guava | fruit | callus | Madhavi et al. (1992) |
| Tomarindus indica | tamarind | cotyledons | shoots, roots | Jaiwal et al. (1991) |
Regeneration of haploid plants following anther or ovule culture has application to plant breeding and gene transfer programs. However, there are limited applications to woody perennial species.
There are several published reports of successes with tropical fruit species. Plantlets have been regenerated following androgenesis with four species: Annona squamosa (Nair et al. 1983) Carica papaya (Tsay and Su 1985), Euphoria longan (Yang and Wei 1984) and Litchi chinensis (Fu and Tang 1983). Embryogenic callus has been produced from anthers of Feijoa sellowiana (Canhoto and Cruz 1994), Pouteria lucama (Jordan et al. 1994) and Psidium guajava (Babbar and Gupta 1986).
Although haploid plantlets have been regenerated from anthers of Carica papaya (Litz and Conover 1978, Tsay and Su 1985) work in the authors laboratory has resulted in a very low success rate with many papaya genotypes (1:1000 anthers producing embryogenic callus). Consequently, anther culture can be labour intensive, requiring plating of large numbers of explants, and a thorough investigation of factors that affect androgenesis. The stage of development of the microspore (usually best at the uninucleate stage), pretreatment of buds or
anthers (eg. cold treatment) as well as media factors and incubation conditions (light/dark treatment, daylength, temperature) should be considered when developing protocols for anther culture.
Considering the limited application of this technology to tropical and subtropical fruit species, the successes reported above are sufficient to suggest that this could be both a novel and rewarding field of research for further studies with these species. The potential to detect and exploit recessive character traits in one generation via production of doubled haploids has enormous potential for species such as woody perennial fruit species with long juvenile periods. This potential also applies to gene transfer projects where transformed homozygous diploids could be produced in one generation, or flowers could be fertilised with transformed pollen.
Production of plants from mature embryos is one of the most straight forward in vitro techniques. Immature embryos represent the most regenerable tissue for many species. Consequently, culture of immature embryos has facilitated both in vitro regeneration of recalcitrant species and rescue of interspecific hybrids.
The potential of embryo culture for the regeneration of recalcitrant fruit species has been demonstrated with Litchi chinensis. Lychee is one of the most difficult tropical fruit species to establish and grow in vitro. Kantharajah et al. (1992) cultured immature embryos of lychee as small as 3mm in length. Ability to produce adventitious buds varied with genotype, however cultivar 'Bengal' produced 15 shoots after pretreatment of immature embryos with BAP. Root formation was achieved with 65% of adventitious shoots, and resulting plantlets acclimatised.
Other reports of regeneration via embryogenesis of immature zygotic embryo explants have been reported (Table 2), and include avocado, custard apple and papaya. The potential for further research with embryo culture is obvious, given the success already achieved with recalcitrant tropical fruit species.
Protoplast Culture, Somatic Hybridisation
Although protoplasts can be isolated from a range of tissues of almost any plant species, regeneration of plants from protoplasts is one of the most difficult in vitro techniques. Reports of success with recalcitrant woody species are limited, and applications to tropical and subtropical fruit species are rare. The only exception is with Citrus species. These reports have been reviewed by Ochatt et al. (1992).
There have been reports of protoplast isolation of non-woody tropical fruit species: papaw (Litz 1986) and banana (Krikorian et al. 1988), although sustained cell division was not achieved.
The principal uses of protoplasts are as targets for direct methods of gene transfer and to facilitate interspecific hybridisation. Direct methods of gene transfer requiring protoplasts are becoming obsolete. Although protoplast fusion may provide a method of interspecific hybridisation, the difficulties in achieving sustained cell division and plantlet regeneration in many species generally outweigh the advantages. In reality, protoplast fusion is only an advantage when prezygotic barriers prevent interspecific hybridisation. If zygotic embryos are produced, embryo rescue and culture is a superior technique. A comparison of these techniques has been demonstrated with interspecific hybridisation of Carica papaya and related species. An efficient method has been developed for embryo rescue, embryogenesis and plantlet production for hybridisation between C. papaya and C. cauliflora (Magdalita et al. 1996) and between C. papaya and C. pubescens, C. quercifolia, C. parviflora and C. goudotiana (Drew et al. 1997). By contrast, attempts to hybridise C. papaya and C. pubescens have resulted in callus growth but no plants (Jordan 1992). Similar difficulties have been experienced in the authors laboratory when protoplasts of C. papaya were fused with those of C. pubescens, C. quercifolia and C. stipulata.
As with most difficult aspects of in vitro culture, intensive funding and research effort usually result in success. Protoplast culture of tropical and subtropical fruit species is no exception and, given sufficient attention, new and improved protocols would probably be developed. However, at this stage, it would be preferable to direct funds towards other aspects of in vitro culture that appear to hold greater potential.
The development of molecular markers is a rapidly expanding field. To date a major effort has been applied to model species such as Arabidopsis, and to major crops such as tomato, rice and maize. There has been some application of isozyme markers to tropical and subtropical fruit crops eg. Musa spp. (Jarret and Litz 1986), mango (Degani et al. 1990), papaya (Manshardt and Wenslaff 1989 a, b) and pineapple (De Wald et al. 1988). By comparison there are few reports of the use of DNA markers in tropical and subtropical fruit species. Cultivar identification has been achieved by use of satellite probes in avocado, mango and papaya (Sharon et al. 1991) and by simple sequence repeats and minisatellite probes in avocado, lychee, mango and papaya (Sharon et al. 1995). Magdalita et al. (1996) used RAPD analysis to confirm interspecific hybrids between Carica species.
There is great potential for the application of genetic markers to tropical, subtropical and indeed all perennial fruit crops. Firstly, DNA fingerprinting allows accurate identification and characterisation of genotypes and species. The application for germplasm conservation and collections is discussed in chapter 5. Secondly, DNA markers provide a rapid method of analysis of genetic variation in a population. Analysis of genetic variation between Carica papaya and related species facilitated a breeding program to introgress disease resistance genes from wild relatives into papaya (Jobin-Decor et al. 1996). Off-types, resulting from genetic variation in in vitro regeneration systems, can be identified by DNA markers in some instances. A RAPD marker has been developed to identify dwarf off-types in banana (Damasco et al. 1996). Dwarf off-types have been a major problem in micropropagation of elite banana cultivars. Potential applications for PGR are discussed in chapter 5.
Thirdly, the use of molecular markers can facilitate plant breeding programs. Moore and Durham (1992) report that breeding of most fruit species is complicated by factors including self-incompatibility, apomixis, dioecy, seedlessness, embryo maturity, heterozygosity, and long juvenile periods. Consequently, conventional breeding and assessment based on morphological markers can be a difficult and slow process with perennial fruit species. A combination of in vitro techniques and use of molecular markers may help overcome many individual problems. Marker assisted breeding could be particularly useful for gene introgression (Moore and Durham 1992), breeding for multigene resistance, and resistance to a disease not yet in a region or country (Henry 1996). Somsri et al. (1997) has recently developed DAF markers to identify sex types (male, female, hermaphrodite) in papaya. An example of the savings that could be made in terms of time and funds, is the potential to select, at the seedling stage, for recessive characters such as red female dioecious papayas. The advantages for fruit species with much longer juvenile phases is obvious.
Currently, the high cost of this research is preventing large scale application in tropical and subtropcial fruit species. However, it represents a wide open field of research and the potential gains warrant a large investment of funds.
First large scale evaluation of micropropagated papaya plants. These are some of 12,000 plants established on a commercial plantation at Yandina in Southeast Queensland, Australia.
As with marker technology there is enormous potential in applying recombinant DNA technology to tropical fruit species. Gene transfer offers the potential to add one or two genes to an elite genotype. Most tropical fruit species are highly heterozygous. They often have difficulties that interfere with traditional plant breeding programs (Moore and Durham 1992) and have long juvenile periods. A good example is papaya industries that are based on dioecious female genotypes. In Australia, breeding programs over 20 years have produced elite female genotypes after years of concentrated effort, however the genotypes have not been maintained in future seed generations. Plant biotechnology offers the potential to add one characteristic (eg. Papaya Ringspot Virus Resistance, PRSV-P) to an elite genotype, which could then be micropropagated in vitro.
Papaya has become the model crop for application of transformation technology to tropical fruit crops. Transgenic papaws resistant to PRSV-P have been produced (Fitch et al. 1992) and are currently being tested in large scale field plantings in Hawaii (Fitch et al. 1997). There are also a few preliminary reports on transformation of Musa spp. (May et al. 1995, Sagi et al. 1995), passionfruit (Manders et al. 1994) and pineapple (Ko et al. 1997) with marker and reporter genes. The only published reports on transformation of a woody perennial tropical fruit species are in mango (Matthews et al. 1993).
Transformation of other tropical and subtropical fruit species is warranted and could result in considerable benefits. Potential includes reduced juvenility in tropical fruit species (eg. mangosteen), disease resistance (eg. Phytophthora resistance in durian) and insect resistance (eg. resistance to fruit fly). Recent advances in understanding and cloning of promoters provides the potential of tissue specific expression of genes, hence insect resistance could be targeted to meristems, roots, or epidermal tissue.
There have been numerous recent reports on transformation of temperate woody perennial fruit species (Dandekar 1992). This demonstrates that transformation of woody species is achievable. In addition, in the last decade, in vitro regeneration systems, which underpin transformation technology, have been developed for some tropical and subtropical fruit species (as described previously in this chapter). Further progress will be dependent on the degree to which this research is funded in the next century.
5. Applications of Biotechnology to Conservation and Use of Plant Genetic Resources
There has been an increasing realisation in the 1990's that biotechnology can make major contributions, not only to crop improvement, but also to the conservation and use of plant genetic resources. The emphasis at sessions of the Commission on Plant Genetic Resources has been on the potential of biotechnology to contribute to increasing food production, to promote sustainable agriculture, to promote the conservation of plant genetic resources and biodiversity, and to effect free exchange of scientific information and plant genetic resources (FAO-1, FAO-3). Consequently, the Commission agreed to the need for a Code of Conduct to address (i) the promotion of sustainable use of biotechnology in the conservation and use of plant genetic resources; (ii) the promotion of access to plant genetic resources; (iii) the promotion of biosafety to minimise environmental risks throughout the world; and (iv) the equitable sharing of the benefits of biotechnology between the owners of the technology and the donors of the germplasm (FAO-3). A draft international code of conduct for plant biotechnology was submitted to the fifth session of the Commission in April 1993. This session also recommended actions to maximise positive effects and minimise possible negative effects of biotechnology, including action by governments to establish national research programs in appropriate biotechnologies. Appropriate technologies were defined as being 'technically feasible, bringing tangible benefits to the users' while being 'environmentally safe, socio-economically and culturally acceptable' (FAO-3).
The impact of biotechnology on crop improvement has been extensive, and its potential influence on the process of conservation of plant genetic resources and biodiversity is profound. The relationship between plant biotechnology and the conservation of genetic resources is a reciprocal one. Biotechnology offers many novel techniques that will help maximise efforts to conserve genetic resources, while the conservation of genetic resources is vital for future research and development of biotechnologies. The world's genetic resources are the raw materials, not only for plant breeders, but also for developing biotechnology industries (FAO-1). This chapter provides an overview of biotechnologies that can contribute to the conservation process, followed by their specific application to tropical and subtropical fruit species and areas of future potential.
5.2 Biotechnologies with Application to Conservation of Plant Genetic Resources
This topic has been the subject of some excellent reviews in recent years (Kartha 1985, Chen and Kartha 1988, Bajaj 1991, Withers 1992, Fay 1994, Engelmann 1997). The subjects of in vitro storage systems and in vitro collecting have also been extensively reviewed. In addition, a status report on the application of in vitro techniques for the conservation and use of plant genetic resources has recently been produced by IPGRI (Ashmore 1997). Consequently, this section is not presented as an exhaustive review or as new information, but rather as a brief overview of available techniques.
In vitro collecting involves initial disinfestation and placement of plant explants in sterile culture medium, before transport to a tissue culture laboratory for further in vitro procedures. In vitro collection is particularly useful for species that are vegetatively propagated and for those with recalcitrant seeds or embryos which deteriorate rapidly. The technique has much potential to facilitate the collection of germplasm of tropical and subtropical fruit species, as has already been demonstrated with Cassava (FAO-8) and coconut (Assay-Bah et al. 1987). Recently, 300 Musa accessions were collected in Papua New Guinea using this technique, before being transported to a collection in Australia (Hamill et al. 1993). An added advantage of this exercise is that it complied with quarantine regulations that are in place to stop the spread of Fusarium and other diseases.
In vitro culture offers some major advantages for conservation and use of plant genetic resources. In vitro cultures can be established from disease-free parent material and maintained in a disease-free state. Alternatively, meristem culture, in combination with treatments such as chemo- or thermotherapy, is a proven technique for elimination of specific viral and other diseases. Consequently, disease-free germplasm can be safely and rapidly transferred between countries. However, it is not safe to assume in vitro cultures are free of specific viruses (Drew et al. 1989), unless the initial parent material has been cleared by appropriate ELISA or equally stringent testing. Caution should always be exercised when dealing with export/import of plant tissue cultures.
In vitro culture has been used to facilitate international collaboration in a research project between Australia and the Philippines on development of PRSV-P resistance in papaya. Sterile cultures of Carica species, interspecific hybrids and embryogenic cultures were regularly transferred between the two countries and contained in quarantine approved laboratories. For example, cultures from the Philippines have been transported to Australia, subjected to in vitro procedures in the authors laboratory and returned to the Philippines. Australian quarantine restrictions that prevent early field release of this material are not contravened.
Finally, in vitro banks can be used for safe storage of plant germplasm.
Ninety percent of all accessions held in germplasm collections are stored as seeds (Tao and Anishetty 1995). Withers (1992) listed three limitations of seed storage. Firstly, it is not applicable to species with no seeds eg. Musa species and Artocarpus altilis (breadfruit). Secondly, for highly heterozygous species, eg. dioecious papaya, it is preferable to conserve vegetative or clonal material. Thirdly, seeds of recalcitrant species are sensitive to desiccation and/or chilling, and tropical recalcitrant species often lack a natural dormancy mechanism, eg. Mangifera indica (mango), Nephelium lappaceum (rambutan) and Cocus nucifera (coconut).
In vitro storage also represents a viable alternative to field collections for species included in the three groups listed by Withers (1992), or for rare and endangered species. Field collections require regular maintenance and are prone to damage from disease and insect attacks, extremes of weather and natural disasters. By comparison in vitro collections are safe from these problems, although failures in environmental control can result in loss of cultures. Thus duplicate collections are advisable. Another advantage of in vitro storage over field collections is that large numbers of cultures can be stored in a small volume in a growth room.
In vitro collections are generally maintained in either slow growth storage or by cryopreservation. Slow growth storage employs various methods to reduce sub-culture frequency, and thus labour and media costs. They have been categorised by Bajaj (1991) into five groups: (i) low temperature (2-8� C) and low light; (ii) mineral oil overlay; (iii) low atmosphere pressure/low oxygen; (iv) desiccation (embryos or embryogenic callus) and (v) minimal medium and growth retardants. Bajaj (1991) reports storage of Chrysanthemum and Petunia for six years without subculture using low temperature storage.
Cryopreservation involves long term growth suspension in liquid nitrogen (-196� C). Cryopreservation was achieved initially using cryoprotectants and controlled cooling. This classic technique has been successfully applied to cell suspension and callus cultures (Kartha 1985). Newer methods involve dehydration of cultures before rapid freezing by immersion in liquid nitrogen. This method shows potential with organised tissue, including embryos and meristems. There exist a number of different dehydration/freezing procedures, including vitrification, encapsulation - vitrification, encapsulation - dehydration, desiccation, pregrowth and droplet freezing (Ashmore 1997).
As advances in the field of molecular biology continue to identify, isolate and clone individual genes, the storage of DNA in vitro is being proposed (Rao and Riley 1994). This may be achieved in the future, as genomic DNA, tissues from which DNA can be extracted, or as DNA sequence information.
Micropropagation has positive and negative aspects in terms of conservation and use of plant germplasm. Although clonal propagation of elite genotypes offers increased yields of high quality fruit crops, by definition 'clonal' is counter to the concept of genetic diversity. Large scale clonal propagation increases the possibility of genetic erosion and crop vulnerability (FAO-1). On the positive side, micropropagation can facilitate regeneration, multiplication and international distribution of useful germplasm in a disease-free condition.
A disadvantage of some micropropagation systems is that they are prone to the production of genetic off-types. Some species exhibit high levels of genetic instability in in vitro culture while others are inherently more stable. Genetic instability is obviously undesirable for conservation of elite germplasm. An improved micropropagation protocol, which is not prone to production of genetic off-types, has been developed in the author's laboratory and applied to tropical plant species (Drew 1996). Multiplication is achieved by subculture of nodal sections from apically dominant plantlets which have been grown in vitro. Shoots that develop from axillary buds of these nodes are rooted as microcuttings. New plants are produced only from axillary or apical buds. Meristematic tissue in buds is genetically the most stable tissue in a plant. A further improvement of this system has been to optimise duration of exposure of shoots to auxin before transfer to hormone-free medium, thereby increasing rooting percentages and quality of root systems (Drew 1991). Resultant plants can be remultiplied or acclimatised. An advantage of this protocol is that exposure of cultures to plant growth regulators, which have been linked to off-type production, is minimised. The protocol has been highly refined for papaya (Drew 1992), where a large range of genotypes can be rooted and acclimatised with >95% success rate. Micropropagated papaya plants have been tested on a commercial scale with 12,000 of an elite female line, generated from one cutting, being grown on a plantation in Southeast Queensland. This planting was free of genetic off-types. The protocol has also been used to micropropagate and field test Passiflora spp. (passionfruit), Coffea arabica (coffee) and Azadirachta indica (neem) (Drew 1996). This protocol has much potential for stable in vitro preservation of woody perennial fruit species.
In vitro Regeneration and Somaclonal Variation
Protocols based on regeneration of plantlets via organogenesis or embryogenesis can provide very high multiplication rates, but can also be prone to genetic instability or somaclonal variation. In recalcitrant species, regeneration has, on occasions, been achieved via culture of immature embryos, as these can represent the most regenerative tissue in a plant. In Litchi chinensis (lychee), immature embryos have produced embryogenic cultures and plants (Zhou et al. 1993), but there are no reports of successful shoot or bud culture.
For conservation purposes, and in cases where these regeneration systems are the only alternative, it would still be preferable to collect and conserve germplasm and risk production of some off-types, than to lose a species completely. Many somaclonal variants have alterations in only one gene. Researchers in molecular biology have already developed a number of gene silencing techniques (eg. antisense, ribozyme, insertional inactivation, cre-lox systems), which have the potential to overcome single gene faults. Alternatively, future technology will be more efficient at taking genes of interest from a conserved species and inserting it in another species. The presence of a single gene fault in a donor species may not be relevant. However, where possible, priority should still be given to in vitro systems that are genetically stable.
A recent development in the research on embryogenesis is artificial seed technology. Artificial seeds developed from embryos of somatic tissue show much potential for germplasm storage, particularly if combined with cryopreservation. However, most current applications are to species that are easy to tissue culture, and these systems may still be prone to genetic off-type production. Consequently, short term application to germplasm conservation remains limited.
The major application of anther culture is generally seen to be the production of doubled haploids to facilitate breeding programs. However, it is also known that haploids facilitate the detection of recessive character traits (Bajaj 1983), which can remain masked in heterozygous diploids. Useful characteristics may be present, but unknown, in germplasm collections. More research effort into haploid production and evaluation is warranted.
Germplasm can also be conserved as microspores or pollen. Production of haploid plants from conserved pollen is potentially an extremely efficient means of conserving and accessing plant genetic resources.
Embryo rescue and culture is a relatively simple in vitro technique and is useful when embryos are slow to develop relative to the fruit, in cases of seed dormancy, or where embryos remain immature. An excellent example of what can be achieved is the production of macapuno coconut plants at the University of the Philippines at Los Banos. The embryo within the coconut remains immature, precluding conventional methods of propagation. However, large scale propagation has been achieved using embryo rescue and subsequent growth of plantlets in vitro. In terms of conservation of germplasm, Tao and Anishetty (1995) report that genebanks have saved valuable accessions using embryo rescue and in vitro culture.
Molecular markers provide an extremely useful technology for conservation and use of plant genetic resources. This technology will facilitate analyses and monitoring of biodiversity, evaluation and characterisation of germplasm which is being collected, and assessment of variation in current collections. Traditionally, morphological markers and, more recently, isozymes were used for these evaluations, but both have limitations. The rapid development of molecular markers (RFLPs, RAPDs, AFLPs, VNTRs) is revolutionising the approach to these tasks.
Initially, molecular markers will be valuable in assessing genetic diversity and stability. This technology represents an unprecedented opportunity to provide information on the variation that exists for particular species within local regions and between countries. This will serve as a valuable guide for effective collection and use of genetic resources (Henry 1996). Characterisation and evaluation of germplasm when it is collected is considered to be as important as collecting, maintenance and storage (FAO-1), otherwise it is likely to be 'lost' in collections and not used effectively. Molecular markers provide an extremely accurate and objective method of identifying genotypes and marking character traits. This could be particularly useful for accurate dissemination of information in regional and global networks.
Finally, molecular markers provide an effective and efficient way to evaluate current in vitro collections, in terms of variation that exists and in identification of unintended and unnecessary duplications. One aim of The Global Plan of Action is to reduce the number of such duplicates (FAO-6). Molecular markers also provide the potential to identify somaclonal variants, both in current collections and those arising in developing protocols for new species. Although the presence of a marker can identify an off-type (Damasco et al. 1996), the absence of a marker does not necessarily prove that variation has not occurred.
Despite this potential, the current limitations of molecular marker technology are that it requires expensive equipment, trained personnel and a large research effort to provide wide applicability to a range of species.
Gene transfer technology encompasses an increasing range of molecular techniques used to locate, isolate, clone, transfer and express foreign genes in plants. As all organisms are now a potential source of useful genes for plant improvement, this technology has profound implications for the conservation and use of genetic resources (FAO-1).
5.3 Relevance of Biotechnology to Conservation of Tropical and Subtropical Fruit Species
Biotechnology encompasses a wide range of techniques that have broad application to conservation and use of genetic resources. However, in reality there have been few applications of these techniques to many tropical and subtropical species. No publications were obtained on Durio zibethinus (durian) or Nephelium lappaceum (rambutan), which are two of the most popular fruits in Asia. Other species such as Litchi chinensis (lychee), Feijoa sellowiana (feijoa) and Pouteria spp. are recalcitrant in vitro, and progress has been slow. By contrast, three genera have been extensively researched: Musa spp. (banana), Carica spp. (papaya) and Citrus spp. Recent successes with embryogenesis and regeneration of banana plantlets show what can be achieved with a recalcitrant species if enough research effort is applied to the difficult in vitro stages. However, the bulk of the work with tropical and subtropical species remains untouched, and there is a great need to start by applying the more basic biotechnologies to these species.
In vitro collecting uses basic techniques and requires minimal training. Its use could facilitate collection of tropical fruit species in remote areas, as many have large seeds which have high moisture content, low viability and short life, eg. Garcinia mangostana, Durio zibethinus. In vitro culture could also aid in the rapid exchange of disease-free germplasm between countries.
Although relatively expensive compared with seed collections, in vitro storage will be essential for germplasm collections of tropical fruit species, as many have recalcitrant seeds. The status report on the development and application of in vitro techniques to the conservation and use of plant genetic resources (Ashmore 1997) produced by IPGRI, presents an extensive review of countries with facilities for in vitro storage, and the status of in vitro conservation for a range of species. The only tropical fruit species listed in this report as having well developed and practised in vitro conservation techniques is Musa spp. A collection of papaya genotypes and related Carica species is held in the author's laboratory in Southeast Queensland. Other collections of tropical fruit species exist as field collections. In the Philippines, the Institute for Plant Breeding at the University of the Philippines, Los Banos, launched a nationwide biodiversity conservation program in 1976. An example of their effectiveness is the recent report of 256 accessions of Mangifera species in a field collection (Coronel et al. 1997). In another program in the Philippines, undertaken by Bureau of Plant Industry staff, 1066 accessions representing 31 fruit species and 21 genera of 13 families, with potential in tropical highland regions, have been collected and planted in a field collection (Boncato et al. 1997).
However, there are a number of factors that make field collections vulnerable. The threat of natural disaster in many tropical countries is constantly present in the form of typhoons, earthquakes and volcanic activity. Inconsistencies in government funding may also result in the loss of valuable field collections. Many Pacific Island economies struggle to support research, while changes in policy direction in Australia recently resulted in the sale and destruction of an excellent field collection at Kamerunga Research Station in North Queensland. Many tropical and subtropical fruit species are slow growing and have long juvenile periods, making their establishment in field collections a long term project. It is therefore particularly unfortunate to lose collections of mature trees.
There are few reports of attempts to apply slow growth storage systems to tropical fruit species. Tropical species are cold sensitive and it is unlikely that many would survive for long at incubation temperatures of 2-6� C. Musa spp. (banana and plantain) were stored for 15 months at 15� C (Banerjee and De Langhe 1985). These results suggest that other tropical species may survive in storage at intermediate temperatures (10-20� C). Alternatively, growth media may be altered to slow growth. Rooted apically dominant plantlets of papaya were maintained for 12 months without subculture when 10 gl-1 fructose was substituted for sucrose in the growth medium (Drew 1992).
The importance of genetic stability is a major factor when considering medium term storage using slow growth techniques (Withers 1992). Some species are more prone to genetic instability in vitro. Musa spp. (banana) and Ananas comosus (pineapple) can produce high numbers of off-types in culture (Smith and Drew 1990a). Pineapple plants can also have a 'carry-over' effect of in vitro culture when established in the field. After multiplication in vitro high numbers of suckers and slips were observed in the first generation in field plantings at Redlands Research Station (author unpublished), however these effects diminished in the second field generation. With both bananas and pineapples, off-type production and carry-over effect were minimized by using low BAP concentrations in vitro, which also resulted in lower multiplication rates in vitro (Smith and Drew 1990a).
Although genetic instability can be minimised in these species, it cannot be eliminated. There is evidence to suggest that slow growth systems may apply an in vitro selection system which selects for off-types (Withers 1992), eg. low temperature incubation may select for cold tolerant lines. This would be an undesirable outcome if it occurred during conservation of tropical species. However, any discussion on somaclonal variation should be balanced to some extent by two factors. Firstly, if combined with a directed selection pressure, it may be useful in providing novel genotypes of species where reproduction is from apomictic seeds and is clonal (Mangifera indica, Garsinia mangostana), or for species that produce no seeds (Musa spp.). Secondly, conservation of a species and production of some genetic instability is preferable to loss of a species.
To achieve medium term storage and genetic stability, the micropropagation system based on growth of microcuttings from axillary buds of apically dominant plants should be considered (Drew 1996). This system has been highly refined and used to store Carica papaya genotypes and related Carica species in the author's laboratory. The system uses standard incubation conditions. A disadvantage is the need for regular transfer, however, plantlets in the apically dominant plantlet stage can be held for 6-12 months for papaw, coffee, neem and passionfruit without transfer (author unpublished). Although leaf drop occurs, as long as the axillary buds and stem of the plants are alive, they can be re-multiplied. A further advantage of this system is that much of the preliminary research with shoot cultures of tropical fruit species (Table 1) could be used in adapting them to this system. It would also be well suited to experimentation with a medium range of incubation temperatures (10-20� C). In summary, this improved micropropagation technique could be used for valuable medium term storage in response to The Global Plan of Action, and until cryopreservation technology is developed to a stage where it can be applied routinely.
As with short term storage, there have been very few attempts to apply cryopreservation techniques to tropical and subtropical fruit species, with the exception of Musa spp. (Panis et al. 1997) and Citrus spp. (Perez et al. 1997). Withers (1992), in a review article, reports successful cryopreservation of the recalcitrant tropical species Theobroma cacao (cocoa), Artocarpus heterophyllus (jackfruit), Cocos nucifera (coconut), and Nephelium lappaceum (rambutan), but provided no details. Very low survival rates have been reported when excised embryos from seeds of jackfruit, rambutan and coconut were cryopreserved (Chin et al. 1988). No survival was achieved when excised embryos from partially dehydrated seeds of rambutan, durian and chempedak (Artocarpus integer) were cryopreserved (Hor et al. 1990). However, before cryopreservation can be universally applied to woody perennial fruit species, there is still much research, development and field testing that needs to be done. For example, the issue of genetic stability is rarely mentioned. Because growth is suspended, the potential to store material for long periods without genetic variation is assumed. However, any system based on cell suspension or callus (including embryogenesis) is prone to somaclonal variation and should be field tested before being accepted unreservedly. Field testing of tropical and subtropical fruits should be continued through to the fruiting stage, as fruit production is the primary reason for their collection and use. Unfortunately this requires long term projects for many species. Another limitation of cryopreservation at this stage is cost. Tao and Anishetty (1995) quote a review on the USA National Seed Storage Laboratory and report that the capital and operational costs of a cryogenic seed storage system could be 365 times higher than conventional storage. Nevertheless a research effort into cryopreservation of tropical and subtropical fruit species should be encouraged because of its potential for long term preservation of germplasm. At the current rate of development, it is reasonable to assume that routine protocols for cryopreservation and subsequent regeneration of explants will eventually become available for most plant species. However, protocols must be repeatable and result in high percentages of preserved tissue being viable after thawing, before they can be used routinely for storage of germplasm.
Cryopreservation may be overtaken or supplemented by DNA storage systems for long term storage (Rao and Riley 1994). However, it should be noted that mapping and sequencing of plant genomes is still in its infancy. Arabidopsis, which is a model species and has the smallest plant genome, is currently the subject of extensive worldwide research. Even so, only 25% of its genes have been partially sequenced and two thirds of these code for proteins with an unknown function (Birch pers comm.). By comparison, genomes of tropical fruit species are virtually unexplored, although some progress is being made with Musa spp. In the short term, DNA storage will be limited to gene constructs that code for useful characteristics such as disease or insect resistance.
Another biotechnology with application to conservation and use of plant genetic resources is artificial seed technology. Currently, the only application to a tropical fruit species is in papaya (Ye et al. 1993). Development of protocols for other species and subsequent field testing is required before this technology can make a significant contribution. As with other technologies, there is also a need for more information on somaclonal variation. Although there is significant progress in regeneration of tropical and subtropical fruit species from callus (Table 2), there is little information on field or molecular evaluation for off-types (Musa spp. excepted). The need for more research is apparent.
Progress with anther culture of tropical fruit species (Ch. 4) is encouraging, particularly in recalcitrant species such as Litchi chinesnsis (Fu and Tang 1983). More research is warranted in this field and further developments could facilitate in vitro culture of recalcitrant species, as well as identification and use of recessive character traits in development of new genotypes. Successful culture of anthers may hold greater potential for storage of pollen.
The almost unlimited potential of molecular marker technology to assist in the conservation and use of plant genetic resources (described previously Ch 5.2) equally applies to tropical and subtropical fruit species. Recent evaluation of the genetic relationships between longan, lychee and alupag (Sotto et al. 1997) demonstrates the effectiveness of marker technology. Development of DAF markers for sex determination in papaya (Somsri et al. 1997) demonstrates a particular application to in vitro conservation of tropical fruit species with dioecious forms. Many of the current in vitro protocols for tropical and subtropical species depend on the use of juvenile tissue where sex may be unknown. The ability to determine sex of individuals in in vitro collections, particularly in species with long juvenile periods would greatly facilitate storage and international exchange of germplasm. Much of the initial molecular marker technology has been applied to high value and/or temperate species. The rapid development of quicker, automatic systems could have a large impact when applied to tropical and subtropical fruit species. Currently, this technology requires expensive equipment and trained personnel, thus funds are required for increased use in tropical countries.
Gene transfer technology is being applied to the tropical species papaw, mango, banana, and pineapple as described previously (Ch. 4). The next decade should see widespread applications to other tropical and subtropical fruit species. In vitro regeneration protocols underpin this technology, and a number of recent successes (Table 2) suggest that gene transfer systems will follow. Although there is great potential, it should be noted that current applications are still restricted to the addition or deletion of single useful genes, and that in the past, single gene resistance produced by conventional plant breeding programs has broken down when field testing has exposed the plants to the pressures of environmental factors. Any single gene resistance produced by gene transfer will be thoroughly tested in tropical regions, as crop production in these areas faces many pressures that are unique. For example, the natural control of insect pests and disease organisms occurring during the low temperatures of winter in temperate areas, does not occur in tropical regions. In addition, many problems are exacerbated by regional environmental and social factors, such as inability to fund expensive farming practices in poorer tropical countries. Application may also be limited if intellectual property rights result in expensive farming systems or buyer resistance to increased prices of produce.
This decade has witnessed unprecedented attention worldwide on the issue of plant genetic resources and their conservation. Key meetings and conventions have focussed on the major issues, decisions have been taken and commitments made by many nations, and regional and crop-based networks have been established. The rapid development of new biotechnologies combined with existing techniques have facilitated implementation of this action.
Biotechnology now offers a wide range of techniques, including cell and tissue culture and molecular biology. It is not only a valuable tool in the process of evaluation and conservation of plant species, but its rapid development continues to offer new and improved applications for the use of this germplasm.
Despite good progress, there is still much that can be done. Tropical and subtropical fruit species have been the subject of only minimal research effort, in terms of both conservation of genetic diversity and application of biotechnologies. Surprisingly, there are several commercially important species on which no biotechnology research reports were found. By contrast, citrus, papaya, and banana have been the subject of intensive research effort, and they are the models for further application to a range of species.
Plant biotechnology is an exciting field of science. It has the ability to bring practical and sometimes dramatic improvement to both food crop production and germplasm conservation. However, many of the more sophisticated techniques are still in developmental stages. In addition, they require expensive equipment and highly trained staff. When considering the issue of conservation of tropical and subtropical fruit species, we need to focus on what can and should be done in the short term, and assess what is immediately achievable using biotechnology as a tool. It is therefore concluded that greater benefits are to be derived by the application of the more basic and proven techniques.
Priority in basic cell and tissue culture techniques should be given to research on untried species, and those where very few reports are available. There are no reports of in vitro studies on two of the most popular fruit species in Southeast Asia: Durio zibethinus (durian) and Nephelium lappaceum (rambutan).
Further research on regeneration systems, particularly embryogenesis, is necessary. These techniques are vital for longer term applications such as cryopreservation, gene transfer techniques and artificial seed technology. All have application to conservation and use of plant genetic resources. Funds should also be directed to field evaluation of regenerated plants to assess genetic stability. This important final stage can be expensive and time consuming, and is often overlooked. However, it must be established that the procedures employed in the conservation of genetic material are reliable and result in the production of genetically stable material.
Of the minor tissue culture techniques, more research would be valuable on haploidy and embryo culture of tropical and subtropical fruit species. Embryo rescue and culture protocols are not difficult to develop and would be a valuable asset for any team involved in in vitro collecting of tropical and subtropical fruit species. If highly efficient protocols were developed for plantlet regeneration from pollen, pollen storage may represent the most cost and space efficient way to conserve many species. Protoplast culture has limited application and represents a very low priority for research.
As tropical and subtropical fruit species will be relatively expensive to preserve in vitro, a study of biodiversity both within and between countries will help prevent needless duplication. Although there are currently limitations to its use, molecular marker technology will provide a method to characteristise plant material in a detailed and accurate manner never before available. Many tropical and subtropical fruit species are highly heterozygous, exhibiting considerable genetic diversity. This is a great asset. Firstly, there is a need to document the diversity that exists in the wild, under cultivation and in collections. Secondly, further collection and preservation of unique germplasm should be encouraged, along with its distribution and use. There are excellent ongoing programs in some countries aimed at collection and evaluation of genetic diversity, and these programs should be encouraged and extended to other tropical countries. Almost all collections of woody perennial fruit species are held in field collections, as most of these species have recalcitrant seeds or other problems preventing standard seed collection and storage. However, field collections are vulnerable and there is a need to establish in vitro collections.
Cryopreservation and DNA storage may eventually provide long term storage capabilities, however they cannot be applied in the short term. It would be beneficial to extend research efforts in more basic techniques of in vitro collecting, micropropagation based on shoot and bud explants, and slow growth techniques for shoot cultures of tropical and subtropical fruit species. The improved micropropagation technique described earlier (Drew 1996) should be tested and developed for a range of species and used for slow growth studies.
Until recently, many of the woody perennial fruit species were considered to be recalcitrant in vitro. However, in the last decade there have been significant advances with recalcitrant temperate species when concentrated research effort has been applied. Given sufficient attention, it would seem that few, if any, species are recalcitrant. The work that has been done to date with tropical and subtropical fruit species is sufficient to demonstrate great hope for further valuable successes, especially as the field attracts more workers and funding.
The development of regional networks for tropical and subtropical fruit species, in line with recommendations of WIEWS and The Global Plan of Action, would both encourage individual groups of researchers and facilitate many of the recommended research priorities as detailed above. Their establishment would also promote conservation and use of this relatively neglected genetic resource.
Finally, it is recommended that this document be read in conjunction with the IPGRI Status Report on the Development and Application of In Vitro Techniques for the Conservation and Use of Plant Genetic Resources (Ashmore 1997), as it provides an extensive review of countries with in vitro storage facilities, and the status of in vitro conservation for a range of tropical species.
Tropical Fruit Production and Statistics
Total world production of tropical fruits was estimated at 42 million tonnes in 1995 (FAO-11). Mango, pineapple, papaya and avocado accounted for 92% of this production and minor tropical fruits represent 8%. However, it is difficult to obtain comprehensive data, particularly on production of the minor tropical fruits. The attached tables (3 and 4) show that production of the minor fruits has increased, dramatically in some cases, and this is matched by export and import figures (FAO-12). Specific examples of growing import markets are durian and lychees into USA and rambutan and guava into EC. Seventy-five percent of exports are from Malaysia, Thailand, Indonesia, China, Pakistan and India, while Hong Kong, Singapore and Taiwan are the major importers in Asia (FAO-11). Brazil is the major producer of passionfruit.
By comparison, in the 10 years from 1985 to 1995, there has been a more modest increase in the production of major fruits of approximately 20-25% (Table 5). This category comprises mango, pineapple, papaya and avocado, but by far the most outstanding tropical fruit crop in terms of production is banana. Commercial production figures of banana exceed those of all other tropical fruits combined, and in 1993 global production was 53.7 million tonnes.
Tables 3 and 4 are copied with permission from the document FAO-12. They provide data for global production, export and import of minor tropical fruit crops between 1980 and 1993. Table 5 presents a summary of trends in global production of the major tropical fruit crops, mango, pineapple, papaya and avocado, for the ten year period, 1985-1995. This data is adapted from the document FAO-12.
Table 3 Fresh tropical fruits: Production and exports
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| GUAVA | ||||||||||||
| Brazil | --- | 208930 b | 230380 | 213920 | --- | --- | 55 | 328 | --- | --- | --- | --- |
| China (Prov of Taiwan) | --- | --- | --- | --- | --- | --- | --- | --- | 140 | 14 | 97 | 76 |
| India | --- | --- | --- | --- | --- | --- | --- | 25 | 741 | 439 | 148 | --- |
| Indonesia | 296931 a | 306501 b | 366888 | 341552 | --- | --- | --- | 3 | 23 | 7 | 8 | 10 |
| Malaysia | --- | --- | --- | 24884 | 31450 | --- | --- | --- | --- | 1373 | 1554 | --- |
| Pakistan | --- | --- | 347254 | 355166 | 373115 | --- | --- | 63 | --- | --- | --- | --- |
| Thailand | --- | --- | --- | --- | --- | --- | --- | --- | 155 | 67 | 29 | 27 |
| MANGOSTEEN | ||||||||||||
| Indonesia | --- | --- | --- | --- | --- | --- | --- | --- | 358 | 452 | 1905 | 1074 |
| Malaysia | --- | --- | --- | 17000 | 17880 | --- | --- | --- | --- | 1325 | 1813 | --- |
| Thailand | --- | --- | --- | --- | --- | 23556 | --- | --- | 1077 | 353 | 1116 | 2062 |
| LIMES | ||||||||||||
| USA | --- | --- | --- | 64000 | 40000 | 8000 | --- | --- | --- | 5000 | 4000 | 3000 |
| Brazil | --- | 568620 b | 560830 | 559110 | --- | --- | 883 | 1747 | 6290 | 3310 | 32550 | --- |
| CARAMBOLA | ||||||||||||
| Malaysia | --- | --- | --- | 24250 | 30930 | --- | --- | 13002 | --- | 7901 | 7058 | --- |
| LONGAN | ||||||||||||
| China (Prov of Taiwan) | --- | --- | --- | --- | --- | --- | --- | --- | 216 | 761 | 211 | 156 |
| Thailand | --- | --- | --- | --- | --- | 57066 | 3684 | 4942 | 14355 | 7618 | 12811 | 21310 |
| RAMBUTAN | ||||||||||||
| Indonesia | 184643 a | 226850 b | 270686 | 335792 | --- | --- | --- | 5 | 108 | 109 | 265 | 202 |
| Malaysia | --- | --- | --- | 101100 | 106635 | --- | --- | --- | --- | 1725 | 1389 | --- |
| Thailand | --- | --- | --- | --- | --- | 77349 | 116 | 1169 | 2219 | 871 | 1700 | 1196 |
| DURIAN | ||||||||||||
| Indonesia | 199361 a | 139200 b | 242585 | 205389 | --- | --- | --- | 192 | 272 | 46 | 277 | 331 |
| Malaysia | --- | --- | --- | 180715 | 210380 | --- | --- | --- | --- | 34903 | 26676 | --- |
| Thailand | --- | --- | --- | --- | --- | 486644 | 3516 | 9784 | 14392 | 7978 | 15116 | 18641 |
| LYCHEES | ||||||||||||
| China (Prov of Taiwan) | 64000 | 98200 | 186800 | --- | 306700 | --- | 4797 | 8973 | 6191 | 12381 | 2999 | 6989 |
| Thailand | 15000 | --- | --- | --- | --- | 23556 | --- | 184 | --- | 563 | 1447 | 1862 |
| PASSIONFRUIT | ||||||||||||
| Brazil | --- | 256210 b | 317240 | 380170 | --- | --- | --- | --- | --- | --- | --- | --- |
b = 1988
Table 4 Fresh tropical fruits: Imports
|
|
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|
|
|
|
|
|
|
|
|
|
||||||
| GUAVA | ||||||
| E.C. | 57 | 147 | 162 | 170 | 150 | 154 |
| C.I.S. | --- | --- | 523 | 52 | 81 | --- |
| Canada | --- | 64 | 110 | 110 | 91 | 58 |
| Japan | --- | --- | 3 | 6 | 2 | 15 |
| Hong Kong | --- | --- | 132 | 64 | 18 | 56 |
| Singapore | --- | --- | 24 | 31 | 20 | 11 |
| Saudi Arabia | 2 | 3 | 23 | 208 | 13 | --- |
| MANGOSTEEN | ||||||
| E.C. | --- | --- | 104 | 70 | 91 | 75 |
| Canada | --- | --- | 45 | 7 | 3 | 1 |
| Japan | --- | --- | 25 | 2 | 77 | 40 |
| Hong Kong | --- | --- | 694 | 346 | 438 | 769 |
| Singapore | --- | --- | 5 | 6 | 2 | 2 |
| China (Prov. of Taiwan) | --- | --- | 349 | 235 | 2114 | 2109 |
| Saudi Arabia | --- | --- | 47 | 18 | 30 | 19 |
| LIMES | ||||||
| E.C. | 9 | 1285 | 6260 | 3260 | 32090 | --- |
| U.S. | --- | --- | --- | 81000 | 86000 | 112000 |
| LONGAN | ||||||
| E.C. | 14 | 76 | 214 | 134 | 243 | 206 |
| Canada | --- | 15 | 349 | 739 | 211 | 1420 |
| Hong Kong | 2221 | 2569 | 10168 | 5583 | 7281 | 13116 |
| Singapore | 1385 | 1780 | 1890 | 988 | 1974 | 3039 |
| RAMBUTAN | ||||||
| E.C. | 59 | 121 | 80 | 48 | 133 | 116 |
| U.S. | --- | 56 | 5 | 7 | 6 | 0 |
| Canada | --- | 29 | 27 | 7 | 0 | 0 |
| Japan | --- | 9 | 14 | 5 | 8 | 8 |
| Hong Kong | 24 | 664 | 363 | 27 | 90 | 45 |
| Singapore | 0 | 150 | 12 | 20 | 551 | 355 |
| China (Prov. of Taiwan) | --- | --- | 139 | 4 | 76 | 49 |
| U.A.E. | --- | 22 | 70 | 53 | 160 | 86 |
| DURIAN | ||||||
| E.C. | 81 | 119 | 166 | 25 | 41 | 40 |
| U.S. | 3 | 48 | 178 | 136 | 119 | 208 |
| Canada | --- | 51 | 114 | 158 | 115 | 260 |
| Japan | 15 | 37 | 50 | 45 | 54 | 60 |
| Hong Kong | 3136 | 8313 | 10971 | 5859 | 7371 | 10057 |
| Singapore | 227 | 632 | 399 | 128 | 396 | 457 |
| LYCHEES | ||||||
| E.C. | --- | 25 | 5 | 19 | 27 | 16 |
| Canada | 69 | 884 | 1282 | 1762 | 596 | 1248 |
| Japan | 413 | 859 | 1238 | 1261 | 890 | 1227 |
| Hong Kong | 9641 | 3096 | 4776 | 7103 | --- | --- |
| Philippines | 78 | --- | 1264 | 1649 | 657 | 1061 |
| Singapore | 3022 | 3769 | 1002 | 1960 | 882 | 1303 |
| PASSIONFRUIT | ||||||
| E.C. | --- | 392 | 918 | 966 | 1012 | --- |
Table 5: Global production of mango, pineapple, papaya and avocado for 1985 and 1995 and percentage increase in production for the ten year period. Data adapted from document FAO-12.
| FRUIT CROP | Production (1985)
'000 tonnes |
Production (1995)
'000 tonnes |
% increase in
production |
| Mango
Pineapple Papaya Avocado |
15 977
9 379 4 321 1 710 |
19 392
11 847 5 245 2 094 |
21
26 21 22 |
| TOTAL | 31 392 | 38 578 | 23 |
Individual Case Studies on Tropical Fruits.
Display of tropical fruits, Malaysia.
The following case studies document information on botany and importance, distribution and constraints, genetic diversity and the current state of biotechnology of fruit crops representing thirteen genera, which are either rapidly developing in importance or have much potential. The major tropical fruits (avocado, banana, citrus, mango, papaya and pineapple) have been thoroughly reviewed in books on biotechnology and are not covered in this appendix.
Annona muricata (soursop) at a roadside stall at Los Banos in the Philippines.
| Annona atemoya (A. cherimola x
A. squamosa)
Common name: atemoya, custard apple |
Annona cherimola
Common name: cherimoya, custard apple |
| Annona squamosa
Common name: sugar apple, custard apple |
Annona muricata
Common name: soursop |
Annona includes about 100 species of small trees that bear edible fruit and belongs to the family Annonaceae. Custard apples are grown commercially in many countries. A. cherimola is more cold tolerant than the other commercially used species and is grown in subtropical and Mediterranean regions. A. squamosa is more popular in tropical countries. A. atemoya, thought to be a hybrid between A. cherimola and A. squamosa, produces large sweet fruit, and is grown in tropical and subtropical regions. Annona alkaloids have been reported to have anti-inflammatory and antimicrobial properties (Jordan and Botti 1992).
Distribution and Constraints
The tropical Annona species originated in the tropical regions of northern South America, West Indies and Mexico, however A. cherimola originated in the tropical highlands of the Andes mountains in Equador and Peru (George 1984). At least one hybrid of atemoya originated in Florida (Jordan and Botti 1992). The main environmental factors affecting fruit production and therefore commercial distribution are temperature, humidity, rainfall and wind (George 1984). Conventional breeding programs have been generally ineffective due to high levels of heterogeneity and long juvenile periods (5-8 years) (Jordan and Botti 1992).
Genetic Diversity
There is a high level of genetic diversity both within and between species resulting to some extent from the wide geographical range of centres or origin in South and Central America (Jordan and Botti 1992). A related species Asimina triloba, also called pawpaw, originated in North America. Much diversity is also present in the large range of wild species.
Biotechnology Applications
There are reports of micropropagation and plantlet production using nodal explants of A. atemoya, A. cherimola and A. squamosa (Table 1). Organogenesis and rooted shoots have been obtained from hypocotyls of A. atemoya, A. muricata, and A. squamosa (Table 2). Organogenesis has also been reported from petioles and zygotic embryos of A. cherimola (Table 2). Plantlets have been obtained following anther culture of A. squamosa (Nair et al. 1983). Protocols for regeneration via organogenesis could form the basis for the development of a transformation system for Annona spp. Molecular markers would be useful both for documenting genetic diversity and elucidating the confusion surrounding the naming and origin of many commercial cultivars (Jordan and Botti 1992).
Jackfruit (Artocarpus heterophyllus) is the largest of all tropical fruits. Photographed at Kamerunga in North Queensland, Australia.
| Artocarpus altilis
Common name: breadfruit, breadnut |
Artocarpus heterophyllus
Common name: jackfruit |
| Artocarpus integer
Common name: chempedak |
Artocarpus odoratissima
Common name: marang |
Artocarpus belongs to the family Moraceae and comprises about 50 species of monoecious evergreen trees. Compound fruits are derived from swollen flower heads (Purseglove 1968). The jackfruit is the largest of all cultivated fruits bearing fruit up to 90cm in length on the trunk and older branches (Sedgley 1984a). Chempedak produces smaller fruit than jackfruit and has long wiry brown leaf hairs on the leaves, however they may belong to the same species (Sedgley 1984a). Chempedak has a strong smell like durian, and the pulp is eaten fresh. Young fruits and seeds are used in cooking (Purseglove 1968). The breadfruit is an important food in the Pacific region where it is cooked in a variety of ways (Purseglove 1968). Bark cloth was made from A. elastica in Malaysia and A. tamaran in Borneo. Jackfruit is used as a fruit or vegetable and in soups and pickles. In India, jackfruit seeds are cooked and eaten.
Distribution and Constraints
The breadfruit originated in the South Pacific Region and grows in hot humid tropical lowlands (Purseglove 1968). Bligh transported breadfruit trees from Tahiti to St Vincent and Jamaica in the West Indies in 1792. Centre of origin of jackfruit is India, however the champedak occurs wild in Malaysia. Breadfruit, chempedak and marang prefer hot humid climates and will not grow at temperatures below 5� C, however jackfruit are more cold tolerant (Sedgley 1984a) and will grow in a range of subtropical and temperate regions. All species prefer good rainfall.
Genetic Diversity
All Artocarpus species display high levels of variability within and between species and this is evident in the wide range of local genotypes. Breadfruit varieties are triploid and seedless (Barrau 1976) and a collection of South Pacific genotypes was established in Western Samoa in the 1970's.
Biotechnology Applications
There are reports of micropropagation and rooted plantlets of A. heterophyllus (jackfruit) using apical and axillary buds of juvenile tissue as explants (Table 1). Research is required on applications of other biotechnologies to Artocarpus spp.
Averrhoa bilimbi
Common name: pickle fruit, bilimbi
Averrhoa carambola
Common name: starfruit, five corner fruit, carambola
Botany and Importance
Averrhoa belongs to the family Oxalidaceae. Carambola produces bright yellow star shaped fruit, however bilimbi bears more cylindrical fruit. The fruit vary in flavour from acidic to very sweet and are high in vitamin C. Carambola is increasing in popularity as a fresh fruit in tropical countries with large scale plantings in Malaysia.
Distribution and Constraints
Carambola and bilimbi originated in Southeast Asia. It grows in tropical and subtropical regions, however young trees are more cold sensitive than older trees. Carambola is affected by water stress, which causes restrictions in vegetative growth and promotion of flowering (Ismail et al. 1996). Carambola is distylous, comprising trees with pin (long styled) and thrum (short styled) flowers. Flowers with similar style types are compatible with each other but those with different style types are self and intramorph incompatible (Wong 1996). For pollination and fruit production, a mixture of the two flower types is recommended although self compatible pin morphs have been identified (Wong 1996).
Genetic Diversity
Both species exhibit high levels of genetic variability. This variation is apparent in fruit quality and flavour of genotypes that are grown in different countries, and variation in flower forms. Seventeen different clones (B1-B17) are registered by the Department of Agriculture in Malaysia, and B10 and B17 are grown as commercial clones (Zabedah and Izham 1996).
Biotechnology Applications
There is a recent report of micropropagation of Averrhoa carambola from nodal explants (Khalekuzzaman 1995a) and a few reports of regeneration of rooted plants in vitro via organogenesis from root, hypocotyl and cotyledon explants (Table 2). Isozyme analysis has been used to distinguish 12 carambola clones in Malaysia (Yunus et al. 1996). There is a need for research on application of other biotechnologies to Averrhoa species.
| Citrus grandis
Common name: pummelo, shaddock |
Citrus madurensis (x Citrofortunella
mitis)
Common name: calamansi |
Citrus grandis is a member of the family Rutaceae and is a spreading spiney tree [5-15m high] which produces very large, round or pear shaped fruit (up to 30 cm in diameter and 1-2 kg in weight) with very sweet yellow or pink pulp (Purseglove 1968). It is grown in many tropical countries, particularly in Southeast Asia although some varieties are grown in sub tropical regions including Australia and Florida. Calamansi is considered to be a Citrus x Fortunella hybrid. It is a thornless, upright and high yielding tree. The fruit are small and round with smooth, thin, deep orange coloured rind enclosing orange coloured flesh (Patena et al. 1978). They contain numerous small seeds and produce sour, highly acidic juice which is used as a fresh juice and as a food flavouring. It is the most important citrus crop in Southeast Asia, with production of 51,291 tonnes from 11,420 ha. in the Philippines in 1987 (Avenido et al. 1991).
Distribution and Constraints
Purseglove (1968) suggested that pummelo is a native of Thailand and Malaysia and spread to China, India and Persia. However, Jorgensen (1984) contends that although it occurs wild in Indonesia and Malaysia, it originally occurred in China where it was recorded in 2200 BC. It was taken to Barbados in the 17th Century by Shaddock and is called shaddock in the West Indies and United States (Purseglove 1968). Although a range of varieties grow in tropical and subtropical regions, best quality in terms of flavour and sweetness is obtained in tropical varieties, although there is limited germplasm in subtropical countries. In temperate regions the fruit produces excess acid (Jorgensen 1984). Calamansi originated in China but has been distributed throughout Asia and is popular in the Philippines and in Indonesia. It is highly cold tolerant.
Genetic Diversity
Citrus grandis seedlings exhibit high levels of variability and many varieties occur wild in Southeast Asia. Pummelo is monoembryonic and in most cases, self-incompatible (Soost 1964), and these factors contribute to the variability of seedlings (Jorgensen 1984). There are many named and cultivated clones in Southeast Asia, United States and Australia. Calamansi is polyembryonic and widely distributed in Asia.
Biotechnology Applications
There have been few applications of biotechnology to pummelo. The most interesting application was in the production of seedless triploid plants following endosperm culture by Wang et al. (1978). Patena et al. (1996) also report embryogenesis following culture of nucellar-endosperm explants of pummelo and calamansi (Citrofortunella mitis). Because calamansi fruit contain numerous small and bitter seeds there have been attempts to produce a seedless triploid in the Philippines where it is popular as a fruit juice. Rooted shoots were produced following culture of epicotyl and root explants of pummelo (Goh et al. 1995). There is a need to apply other biotechnologies to pummelo and calamansi, particularly the use of molecular markers to study biodiversity.
Durian (Durio zibethinus) is very popular in Southeast Asia. Trees photographed at Mindanao in the Philippines.
Durian fruit is famous for its strong smell and flavour. Photograph taken in Malaysia.
Durio zibethinus
Common name: durian
Botany and Importance
Durio belongs to the family Bombacaceae and contains 27 species of which six produce edible fruit (Watson 1984a). Durio zibethinus is a large tree growing up to 40m in height and producing large fruit with a rough spiney rind and a cream or yellow pulp. It is famous for its strong smell and flavour. Durian is arguably the most popular fruit crop in Southeast Asia, and although there have been extensive plantings in recent years, demand is greater than supply. Hassan et al. (1996) states that 83,000 hectares were planted to durian in Malaysia in 1993; and there are large plantings in the Philippines, Indonesia and Thailand.
Distribution and Constraints
Durian is naturally distributed in Malaysia, southern Thailand, Indonesia and Borneo (Watson 1984a). It grows well in tropical regions with high rainfall and cannot withstand more than 3 months drought (Malo and Martin 1979). Trees will tolerate low mean temperatures and it is grown at altitudes of 600m in Java; however, temperatures below 22� C limit growth and below 10� C leaf abscission occurs (Watson 1984a). The major limiting factor of production is patch canker caused by Phytophthora palmivora. The organism initially attacks the roots and subsequently causes a stem canker on the trunk at or just above ground level. It is not prone to insect attack, however durian seed borer (Mudaria luteileprosa) occurred in Thailand in 1987 and is spreading rapidly (Buara 1996).
Genetic Diversity
Watson (1984a) reports that durian are highly heterozygous in all attributes causing variability in: tree form, vigour and leaf size; flowering position, flower shape and fruit number per cluster; peduncle length; fruit size and shape; rind colour and thickness, acid volume, colour, aroma, texture and flavour; and seed number and size. Large numbers of cultivars exist in many Asian countries. Related wild species also occur in Southeast Asia, and are a potential source of many useful traits, including Phytophthora resistance (Somsri pers comm.).
Biotechnology Applications
No reports are available on any biotechnology applications in Durio zibethinus. This is surprising given the popularity of the fruit. Durian seeds lose viability very rapidly thus in vitro collecting and embryo culture may be useful methods to initiate in vitro cultures. In vitro regeneration studies are necessary and could facilitate development of transformation protocols which may lead to production of phytophthora resistant clones.
Euphoria longan
Common name: longan
Botany and Importance
Longan (Euphoria longan) belongs to the family Sapindaceae and is related to lychee (Litchi chinensis) and alupag (L. philippinensis). The flesh of longan fruit is similar in texture to that of lychee, however it has a smooth yellow-brown skin when ripe and takes 5-7 months to mature. Yield varies from 6 to 400 kg per tree depending on variety and season, and biennial bearing a common (Watson 1984b).
Distribution and Constraints
The centre of origin of longan is Sri Lanka, southern India, and China however it is now grown in many Asian countries, Australia, Hawaii and Florida (Watson 1984b). It is a popular fruit in Thailand and Taiwan. Longans grow well in subtropical climates and prefer a mild winter followed by hot spring and summer temperatures. They grow well in areas with high rainfall and high soil moisture is necessary for good fruit set and fruit production (Watson 1984b). Longan trees will tolerate wind.
Genetic Diversity
Seedling populations are highly heterozygous. Commercial cultivars have been selected for large fruit size, pulp colour, small seeds, flavour, high sugar content and high yield. Although many named cultivars are known in a range of countries, Watson (1984b) suggested that some may be identical but have different names in different countries.
Biotechnology Application
There are few reports on the application of biotechnology to Euphoria longan. Plantlets have been regenerated via embryogenesis following culture of nodes (Celo et al. 1997) and anthers (Yang and Wei 1984). A study of genetic variability that exists both within and between countries using molecular marker technology would be useful in assessing and conserving biodiversity of longan.
Feijoa sellowiana
Common name: feijoa, pineapple guava
Botany and Importance
Feijoa sellowiana belongs to the family Myrtaceae and is a small dense attractive tree which is often used for windbreaks. It produces attractive flowers with bright red stamens and a small round or oval fruit, green in colour. It grows in subtropical and temperate climates and is cultivated for its fruit in Australia and New Zealand.
Distribution and Constraints
Feijoa sellowiana is native to South America but is grown in many subtropical and temperate regions (Purseglove 1968). It is frost tolerant (tolerating temperatures down to -9� C) and drought resistant, and grows well in New Zealand and California, however it prefers a climate without extremes of temperature (Batten 1984). The major pests in Australia are scale insects and fruit fly.
Genetic Diversity
As with many perennial fruit trees, seedling populations exhibit high levels of heterozygosity. Commercial varieties have been selected for consistent fruit size and shape, large fruit and pulp volume, flavour, yield, good post harvest qualities and upright bush habit (Batten 1984). A range of varieties exist in many countries.
Biotechnology Applications
The limited information that is available on Feijoa sellowiana suggests that it is recalcitrant in vitro and production of whole plantlets in vitro is very difficult. Shoots have been grown in vitro from seedling explants (Bhojwani et al. 1987, Bertoni and Biricolti 1996). Embryogenesis has been reported following culture of cotyledons (Cruz et al. 1990, Canhoto 1996) and anthers (Canhoto and Cruz 1994). Practical application of biotechnologies will probably require a considerable research effort.
Flowers and immature fruit of mangosteen (Garcinia mangostana) considered by many to be the most delicious and under exploited of all tropical fruits.
Garcinia mangostana
Common name: mangosteen
Garsinia is a large genus of evergreen trees, belonging to the family Guttiferae. Garsinia mangostana is a slow growing, glabrous tree to 15 m in height with dark green leaves and a distinct yellow latex (Purseglove 1968). Although dioecious it usually occurs as female trees with infertile staminodes, producing apomictic seeds. Males are extremely rare (Purseglove 1968). The fruit have tough thick purple pericarp surrounding a white pulp comprising 4-8 segments with a delicate sweet flavour. Mangosteen is considered by many to be the most delicious and most under exploited of all tropical fruits.
Distribution and Constraints
Garsinia mangostana is native of Malaysia but is now grown in many tropical regions. It prefers tropical climates with high consistent rainfall, high consistent temperatures and high humidity. Growth slows below 20� C and trees die below 5� C (Alexander 1984). Commercial production has been limited by slow growth, long juvenile periods (10-15 years), difficulties experienced in budding and grafting (Wiebel et al. 1992), and short shelf life of fruit when mature. Seed has low viability and short life and must be planted within a few days (Purseglove 1968).
Genetic Diversity
Because males are rare and seeds are formed from nucellar tissue, plantlet production from seeds is clonal and genetic diversity is rare; however, Alexander (1984) records variation in genotypes between countries eg. those in Asia and Central America.
Biotechnology Applications
There have been no reports on micropropagation of Garcinia mangostana from bud explants however, multiple plantlets have been produced in vitro via organogenesis from leaf and seed explants (Table 2). A study of genetic variability (using molecular markers) to determine how much diversity exists between countries would be very useful. Gene transfer could be useful to reduce the juvenile phase and extend shelf life, however these would be long term projects.
Litci chinensis
Common name: lychee
Litchi philippinensis
Common name: alupag
Botany and Importance
Litchi is a genera in the Sapindaceae or soapberry family which contains about 140 genera and 2000 species (Chapman 1984). Litchi chinensis is a dense polygamous evergreen tree (Purseglove 1968) which grows 10-15 m in height. It produces clusters of small round fruit , covered with tubercles, red when ripe and containing a delicious sweet white aril, similar in flavour to a sweet grape. It is a popular fruit crop in many tropical and subtropical countries. In addition to its popularity as a fresh fruit, lychees are popular as a dessert when canned or frozen, or in China are dried to produce brown 'litchi nuts'. A related species Litchi philippinensis has small fruit and a low flesh content and grows in highland areas of the Philippines.
Distribution and Constraints
Litchi chinensis originated in southern China where it was extensively selected, however, L. philippinensis is native of the highland regions of the Philippines. Lychee varieties are an important fruit crop in many tropical and subtropical countries including most Asian countries, Australia, United States, Central America and South Africa. Although they grow well in lowland tropics, they require cool dry winters (<10� C) to bear fruit. For good growth and fruit production lychees require consistently high temperatures, humidity and rainfall (or irrigation).
Genetic Diversity
Compared with most tropical fruit species, genetic diversity of lychee is limited. Most varieties originated and were selected in China and are vegetatively propagated. The occurrence of wild genotypes is rare although Hsu et al. (1964) reported that they do occur in China. L. philippinensis is confined to the Philippines.
Biotechnology Applications
Published reports show that Litchi chinensis is recalcitrant in vitro. Shoot or bud culture is very difficult to achieve. Yu (1991) was able to sustain shoot cultures of lychee in vitro and then graft them onto seedlings, however subculture or multiplication was not achieved. Embryogenesis and plantlets have been obtained by culture of anthers (Fu and Tang 1983) and immature embryos (Zhou et al. 1993) and these protocols would be the best basis for further in vitro studies on Litchi spp.
Rambutan (Nephelium lappaceum) is a popular sweet fruit in Southeast Asia and tropical Australia. Fruit are coloured red or yellow, are covered in soft spines, and have a single seed covered with a soft juicy aril. Photographed in North Queensland, Australia.
Nephelium lappaceum
Common name: rambutan
Botany and Importance
Nephelium lappaceum is a member of the family Sapindaceae and is an evergreen bushy tree up to 20m high (Purseglove 1968). It is dioecious however three flower forms have been reported: male, hermaphrodite where flowers are functionally female; and hermaphrodite where flowers are functionally female or male (Valmayor et al. 1970). Fruit is produced in clusters, coloured red or yellow, covered with soft spines, and contain a single seed covered with a white juicy aril. It is highly esteemed for its flavour although fruit quality varies with genotype. It is a popular fruit crop in Southeast Asia and tropical Australia. There is also a large canning industry in Malaysia and Thailand and unlike many tropical fruits it has a reasonable shelf life, given optimum conditions, and can be transported (Watson 1984b).
Distribution and Constraints
Rambutan is native to Malaysia but is common in many tropical regions of Southeast Asia. It prefers tropical climates (within 15� C of the equator) with mean yearly temperature range of 22� to 30� C and high annual rainfall (Watson 1984b). It is intolerant of wind and low humidity, and low temperatures cause leaf tip die-back and marginal necrosis (Watson 1984b).
Genetic Diversity
Nephelium lappaceum is highly heterozygous in terms of tree form and growth, leaf colour and size, flower type and number per panicle, fruit colour, thickness and length, aril colour, thickness, flavour and texture, adherence of aril to testa (freestone varieties exist), seed size and form, and susceptibility to pests, diseases and drought (Watson 1984b). Numerous commercial cultivars and wild relatives exist in many Asian countries and in Australia. A related species, Nephelium mutabile (common name: pulasan) is also native of Malaysia and is grown in Southeast Asia. It also produces an excellent fruit similar to rambutan but with shorter spines.
Biotechnology Applications
There are no reports on applications of biotechnology to Nephelium spp. This is surprising considering its popularity as a fruit and its potential for development and export.
Passionfruit hybrid (P. edulis x P. edulis f. flavicarpa ) photographed in Southeast Queensland, Australia.
| Passiflora edulis, P. edulis f. flavicarpa
Common name: passionfruit |
Passiflora quadrangularis
Common name: granadilla |
| Passiflora ligularis
Common name: sweet granadilla |
Passiflora mollissima
Common name: banana passionfruit |
| Passiflora laurifolia
Common name: water lemon |
The genus Passiflora belongs to the family Passifloraceae and comprises 400 species, of which 50 or 60 bear edible fruit (Martin and Nakasone 1970). They are herbaceous and woody vines and have tendrils to aid their climbing habit. Passiflora species have bright coloured flowers and fruit which are capsules or berries containing numerous seeds surrounded by a fleshy aril. Commercial passionfruit production is based on the purple passionfruit (P. edulis), the yellow passionfruit (P. edulis f. flavicarpa) and hybrids between the two. Passionfruit have a piquant flavour, a pleasant aroma and are strongly acidic. They have a long shelf life and are exported to many countries and are a popular component of fruit juices.
Distribution and Constraints
Most Passiflora species originated in Central and South America although 40 are native to Asia and South Pacific islands (Beal and Farlow 1984). P. edulis is indigenous to Brazil and is well suited to tropical and subtropical regions with high rainfall. Temperatures below 18� C reduce growth, flowering and pollen germination. P. edulis f. flavicarpa (flavicarpa), P. quadrangularis (granadilla) and P. laurifolia (water lemon) are better adapted to tropical climates and are grown in many tropical countries. P. mollissima (banana passionfruit) and P. ligularis (sweet granadilla) are indigenous to highland areas in Central and South America. Because of their cold tolerance they are grown at high altitudes or in countries with cooler climates such as New Zealand.
Genetic Diversity
Extensive genetic diversity exists in Passiflora and many species grow wild throughout tropical and subtropical regions. Wild species have been reported to contain many useful characteristics including vigour, high fruit production, climatic and ecological adaptability, fruit quality, colour and flavour, and resistance to many diseases including passionfruit woodiness virus, Fusarium and Phytophthora (Beal and Farlow 1984).
Biotechnology Applications
There have been numerous reports on micropropagation of Passiflora spp. and on in vitro regeneration of plantlets via organogenesis (Drew 1997). Passiflora spp. are much more responsive in vitro when juvenile explants are cultured. There have been recent reports of regeneration of transgenic plants from flavicarpa cotyledons, however there are no reports on the use of molecular markers with Passiflora spp.
Pouteria caimito
Common name: abiu
Pouteria sapota
Common name: mamey sapote
Botany and Importance
Pouteria spp. belong to the family Sapotaceae. Pouteria sapota is a large tree (25-30 m high when mature) with large dark green leaves. It produces large (up to 3-4 kg) fruit with a rough brown skin and sweet spicy pink flesh surrounding 1-5 seeds (Scholefield 1984). Mamey sapote is a popular fruit in Central America. P. caimito is a smaller tree, which produces fruit with a bright yellow skin and sweet white flesh surrounding 2-3 seeds (Scholefield 1984).
Distribution and Constraints
Pouteria spp. are indigenous to Central and northern South America. P. sapote prefers tropical climates however P. caimito will grown in tropical and subtropical regions and is being grown in United States and Australia. They grow well in areas of high temperature and humidity, and high rainfall.
Genetic Diversity
Both Pouteria spp. are highly heterozygous in seedling populations and extensive genetic diversity exists.
Biotechnology Applications
Little information is available on application of biotechnology to Pouteria spp. Campbell and Lara (1992) review attempts to culture mamey sapote in two laboratories in the United States. Callus was produced with considerable difficulty from bud, stem and leaf explants, however no regeneration was achieved. There has also been a report of embryogenic callus from anthers of Pouteria lucama (Jordan et al. 1994).
Psidium quajava
Common name: quava
Botany and Importance
Psidium belongs to the family Myrtaceae and comprises approximately 150 species of trees and shrubs, many of which have edible fruits. P. quajava is a small tree with many branches. It produces large numbers of small fruit which vary between genotypes in colour, size and flavour (tart to sweet), and has a characteristic musky odour. It is widely distributed throughout the world and is grown for its fruit in many tropical and subtropical countries. The fruit is high in vitamin C and is popular canned and in jams and juice.
Distribution and Constraints
Psidium quajava is indigenous to tropical regions of America and wild varieties occur at both lowland and elevated regions (Purseglove 1968). Because of the diversity of genotypes it will grow in a wide range of environments and is cultivated throughout Asia, India, United States, South Africa and Australia. It is cold tolerant but frost susceptible and does not respond well to prolonged exposure to low temperatures. Prolonged high summer temperatures are essential for flowering, fruit set, fruit maturation and flavour (Batten 1984). Consistent rainfall or irrigation is required following flowering or yield and fruit quality are poor (Shigeura 1973).
Genetic Diversity
Extensive genetic diversity exists and genotypes are adapted to a wide range of soil and environmental conditions (Purseglove 1968). Diversity exists in fruit size, shape, pulp to seed ratios, seed characteristics; flavour, texture and colour of flesh, aroma, ascorbic acid content of fruit; susceptibility to pests, diseases, frost and fruit cracking; growth, habit, flowering and fruiting characteristics; and post harvest characteristics (Batten 1984). Extensive variability also exists between cultivated genotypes in different countries.
Biotechnology Applications
There have been reports of successful micropropagation and plantlet production from apical and axillary bud explants (Table 1). Callus has been produced from fruit tissue (Madhavi et al. 1992), however regeneration of organs or embryos or plantlets from callus appears to be more difficult, although embryogenic callus has been developed from anthers (Babbar and Gupta 1986). To date, there are no applications of molecular biology to Psidium species.
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2,4-D 2,4-Dichlorophenoxy Acetic Acid
AFLP Amplified Fragment Length Polymorphism
DAF DNA Amplification Fingerprinting
DNA Deoxyribose Nucleic Acid
FAO Food and Agriculture Organisation of United Nations
GPA Global Plan of Action
IPGRI International Plant Genetic Resources Institute
NaCl Sodium Chloride
PCR Polymerase Chain Reaction
PGR Plant Genetic Resources
PRSV-P Papaya Ringspot Virus form P
RAPD Random Amplified Polymorphic DNA
RFLP Restriction Fragment Length Polymorphism
VNTR Variable Numbers of Tandem Repeats
WIEWS World Information and Early Warning System
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FAO 1997
