BIOTECHNOLOGY AND PLANT GENETIC RESOURCES Conservation and Use Biotechnology and Plant Genetic Resources Conservation and Use Edited by J.A. Callow, B.V. Ford-Lloyd and H.J. Newbury School of Biological Sciences University of Birmingham UK CAB INTERNATIONAL CAB International CAB International Wallingford 198 Madison Avenue Oxon OX10 8DE New York, NY 10016-4341 UK USA Tel: +44 (0)1491 832111 Tel: +1 212 726 6490 Fax: +44 (0)1491 833508 Fax: +1 212 686 7993 E-mail: cabi@cabi.org E-mail: cabi-nao@cabi.org © CAB INTERNATIONAL 1997. All rights reserved. No part of this publication may be reproduced in any form or by any means, electronically, mechanically, by photocopying, recording or otherwise, without the prior permission of the copyright owners. A catalogue record for this book is available from the British Library, London, UK A catalogue record for this book is available from the library of Congress, Washington DC, USA ISBN 0 85199 142 4 Typeset in 10/12pt Palatino by Columns Design Ltd., Reading Printed and bound in the UK by Biddles Ltd, Guildford and King’s Lynn Contents Contributors vii Preface ix Foreword xi G.C. Hawtin 1. Overview 1 J.A. Callow, B.V. Ford-Lloyd and H.J. Newbury 2. Use of molecular marker techniques for description of 9 plant genetic variation A.L. Westman and S. Kresovich 3. Genetic diversity – population structure and conservation 49 M.D. Hayward and N.R. Sackville Hamilton 4. Genomic relationships, conserved synteny and wide-hybrids 77 D.A. Laurie, G.J. Bryan and J.W. Snape 5. Molecular markers and the management of genetic resources 103 in seed genebanks: a case study of rice. B.V. Ford-Lloyd, M.T. Jackson and H.J. Newbury 6. In vitro conservation methods 119 F. Engelmann 7. Conservation of DNA: DNA banking 163 R.P. Adams v 8. Genetic resources and plant breeding – identification, mapping and manipulation of simple and complex traits 175 M.J. Kearsey 9. Gene identification, isolation and transfer 203 I.D. Godwin 10. Importance of biotechnology for germplasm health and 235 quarantine H. Barker and L. Torrance 11. Biodiversity for bioindustries 255 G. Tamayo, W.F. Nader and A. Sittenfeld 12. Internet resources for the biologist 281 M.L. Anderson and S.W. Cartinhour Index 301 vi Contents Biodiversity for Bioindustries G. Tamayo, W.F. Nader and A. Sittenfeld Instituto Nacional de Biodiversidad (INBio), Apartado Postal 22-3100, Santo Domingo, Heredia, Costa Rica 11.1. Biodiversity and its Benefits Biodiversity refers to the variety and variability of living material and eco- logical complexes in a given area and comprises species, genetic and ecosystem diversity. Biodiversity is not only the basis of life on earth, but also provides the goods and services essential to support every type of human endeavour. Accordingly, biodiversity enables societies to adapt to different needs and situations (US National Research Council, 1992). Biodiversity generates economic value in different ways. Populations are interconnected, for instance where predators and disease organisms control populations of their prey, or when pollinators and seed dispensers promote the growth of plant populations. Thus agriculture directly bene- fits from a functioning ecosystem, allowing the extensive use of agro- chemicals to be avoided. Biodiversity also generates economic value from extractable products obtained from individual species (Wilson, 1992). For centuries biodiversity has provided fuels, medicines, materials for shelter, food and energy. The use of compounds, genes and species is essential to meet industry needs. Furthermore, ecosystems contribute to climate regu- lation, maintenance of hydrological cycles and nitrification of soils. In addition, recreation, science and education also figure among the vast array of social, ethical, spiritual, cultural and economic goods and services provided by biodiversity that are recognized as fundamental for human livelihoods and aspirations. Bioprospecting links biodiversity and industry. Previously, this activ- ity generated benefits almost exclusively for industry, leaving biodiversity © CAB INTERNATIONAL 1997. Biotechnology and Plant Genetic Resources (eds J.A. Callow, B.V. Ford-Lloyd and H.J. Newbury) 255 11 conservation and source countries to generate benefits and returns else- where. The rapid loss of biological diversity, with the extinction of 30 to 300 species per day (Japan Economic Newswire, 1995), has initiated a new attitude towards the exploration of natural resources. Costa Rica’s Instituto Nacional de Biodiversidad (the National Biodiversity Institute, INBio) has pioneered a new concept of bioprospecting that integrates product dis- covery with financial and intellectual returns to ‘nature’. INBio’s Biodiversity Prospecting Department links the understanding and non- damaging exploration of biodiversity to conservation activities and eco- nomic development of the countries where bioresources were first obtained (Sittenfeld and Villers, 1994). The exploration and conservation of the world’s biotic resources require an approach involving bioindus- tries, research centres and developing countries, all collaborating towards a common goal, each participant benefiting from the relationship. Presently, a natural resource conservation strategy based on the three over- lapping steps – saving, knowing and using biodiversity – is paving the way towards implementing joint activities for the benefit of industry, bio- diversity conservation and source countries (Global Biodiversity Strategy, 1992; Janzen and INBio, 1992). Gene technology opens a new dimension for bioprospecting. However, because in its current stage of development it represents more of a threat than a benefit to the primarily agricultural societies of the devel- oping world, new strategies must be implemented to combine benefits to the biotech industry with biodiversity conservation and the development of biodiversity-rich nations. 11.2. Industry and Biodiversity 11.2.1. Development of drugs and pesticides Up until the present, the primary beneficiaries of biodiversity have been the pharmaceutical and agricultural industries. Sales of drugs based on natural products from plants were estimated at $US43 billion in 1985 worldwide, accounting for approximately 40% of the total drug market (Principe, 1989). Earnings from a single new successful pharmaceutical on the market can be in the range of a billion dollars. The value of yet undis- covered pharmaceuticals in tropical forests is estimated at $US3–4 billion for a private pharmaceutical company, and as much as $US47 billion to society as a whole (Mendelsohn and Balick, 1995). On the other hand, sales of pesticides in the US by 18 major suppliers were estimated at $US6.5 billion in 1992 (Frost & Sullivan Inc., 1992). Although humans have traditionally used plant products like rotenone or nicotine as agricultural insecticides, most industrial pesticides on the 256 G. Tamayo et al. market have not been derived from natural compounds (Wink, 1993). However, the toxicity and ecological hazards created by most of these chemicals have initiated intensive research for more specific and biodegradable pesticides, including new biological pest control agents based on natural compounds and microorganisms. The first results of these screening efforts are already on the market. For example, aver- mectins are macrolides derived from Streptomyces avermitilis and possess- ing insecticidal properties (Babu, 1988). Dehydration of avermectins yields the even more efficient ivermectins, which generate approximately $US1 billion in annual sales, and are useful not only for the treatment of infesta- tions of parasitic worms and insects in livestock, but also in humans. Pyrethroids, on the other hand, were derived by chemical synthesis from pyrethrins as lead structures as natural insecticides found in chrysanthe- mum flowers (Wink, 1993). Pyrethrins are more stable and active than their natural precursors (by a factor of 1000), but also produce more side-effects in mammals. These side-effects have led Germany to consider banning pyrethrins from the market (Wink, 1993). Still undergoing development is a nematocide, the pyrrolidine alkaloid 2R,5R-dihydroxymethyl-3R,4R- dihydroxypyrrolidine from Lonchocarpus species, developed through a col- laboration between the British Technology Group, the Royal Botanical Garden at Kew and INBio (Janzen et al., 1990; Birch et al., 1993). Many of the technical aspects of screening and developmental processes involved in creating new drugs or pesticides are related to more general issues like biodiversity, technology transfer and biodiversity con- servation, and will be described in more detail below. Sources for new drugs or pesticides Three major sources for the screening of new compounds, suitable for drug or pesticide development, are available: (i) molecule libraries created by combinatorial chemistry; (ii) fermentation broths of microorganisms; and (iii) plant and animal extracts. Modern automated methods have cre- ated high-throughput screening, allowing thousands of substances to be tested for their biological activity rapidly and inexpensively. Therefore, access to these three sources can be regarded as a prerequisite rather than a privilege. The goal is the discovery of a ‘leading structure’ that will guide the development of new drugs, pesticides or fine chemicals. Although combinatorial chemistry plays an increasingly important role, half of the ten best-selling drugs are derived from secondary metabo- lites originally isolated from microorganisms or plants (O’Neill and Lewis, 1993). Obviously, organic chemistry has not yet caught up with the capac- ity of nature to create new structures with a complex molecular diversity (for review see Ecker and Crooke, 1995). Screening for natural compounds has traditionally concentrated on plants and microorganisms, identifying a huge variety of pharmacologically Biodiversity for Bioindustries 257 active alkaloids, terpenoids, aromatics and glycosides. Fungi and bacteria can easily be isolated from soil samples and other sources. Once in a strain col- lection, microorganisms and their products are readily accessible. Plants, on the other hand, are more difficult to collect, but offer a higher molecular com- plexity and diversity. Marine organisms yield new structures with high mol- ecular diversity and important biological activities (König et al., 1994). For example, briostatin and didemnin B are compounds found in molluscs and shown to have strong antitumour activity; they have reached preclinical and clinical trials, respectively. Shark and tunicate alkaloids are also currently undergoing intensive investigations (Moore et al., 1993; Research Foundation of the State University of New York, 1994). In contrast, insects, spiders and other invertebrates have mainly been examined for their potential to produce bioactive peptides and proteins rather than small molecules (see below). The same applies to vertebrates like frogs, snakes and bats. Certain alkaloids obtained from the skin of frogs (see below), and insect hormones and pheromones useful for pest control are the exceptions. Nevertheless, drug research is also heading in this direction. In collaboration with Merck & Co. and Bristol-Myers Squibb (within an International Cooperative Biodiversity Group together with Cornell University), INBio is screening insects for small bioactive compounds (Sittenfeld and Lovejoy, 1995). However, years of research may fail if the initial collection and docu- mentation of biological material are not done properly. Problems may arise if further material from the same species or subspecies, from the same environment or even from the same location, is not available for later investigation. Calanolides, for example, were isolated from Calophyllum lanigerum var. austrocoriaceum (Kashman et al., 1992). These compounds inhibit HIV in vitro. Although fully characterized (Cardellina et al., 1993a) material from the same tree is now unavailable because the tree was sub- sequently cut down. There is a lesson to be learned from this event as other specimens from the same area did not yield even trace amounts of the desired compounds. The collection strategy Natural compounds can be accessed through ethnobiological information, evaluation of chemotaxonomic relationships, random sampling or bio- rational observations. Approximately 3000 million people use traditional medicines (Balick, 1994). Many of today’s multinational giants in the pharmaceutical indus- try made their first millions with products derived from ethnobotany. Prior to Bayer’s aspirin, American and Eurasian peoples treated fevers, inflam- mation and pain with salicin-containing plants like willows and poplars. The worldwide market for phytomedicines derived from ethnobotany is estimated at $US12.4 billion, headed by products derived from ginseng, ginkgo, garlic, horse chestnut and echinacea (Grünwald, 1995). Today 258 G. Tamayo et al. ethnobotanical information is readily accessible through Internet’s data- bases, e.g. NAPRALERT or AGIS. But most companies avoid the search for new compounds on the basis of ethnobiological information. An evalua- tion of ‘hits’ during the Natural Products Drug Discovery Program at the National Cancer Institute revealed no appreciable differences between samples collected at random and those screened on the basis of ethno- botanical leads (Cragg et al., 1994). These results were obtained after dereplication, which eliminates a substantial number of substances. Interpretation of illness descriptions from the ‘native pharmacologists’ is also problematic. However, ethnobotanical information can be extremely useful when applied to diseases that can be translated into the language of Western medicine, e.g. diabetes, skin infections, wounds, etc. Aspects of intellectual property rights in relation to this approach are discussed in more detail in a recent study of the Rural Advancement Foundation International (1994). Chemotaxonomy is based on the assumption that related species from the same genus or family produce the same type of secondary metabolites. For example, most members of the Asteraceae produce sesquiterpenelac- tones. An example is artemisinin, isolated from Artemisia annua on the basis of ethnobotanical information. Likewise, members of the Rubiaceae may produce alkaloids (quina alkaloids, for example), and species of the genus Taxus may contain taxanes, etc. Most large pharmaceutical companies, not limited by their screening capacity, collect biological material randomly. They may show a preference for certain plant families, but in general only the taxonomic identification and exact documentation of the collection site of the sample are required. The biorational approach requires the systematic study of interactions between organisms within the ecosystem. The leaf-cutter ant, Atta cephalotes, for example, avoids feeding the symbiotic fungus found in its nest with leaves from Hymenaea courbaril (Harborne, 1989). The tree con- tains a terpenoid (caryophillene epoxide) that inhibits the growth of the fungus. Observations like this may provide decisive information leading to the discovery of antifungal or formicide compounds. Evaluation of larvae or adult insect feeding preferences may lead to the discovery of new insec- ticides. Technological aspects The amount of material that can be taken from a particular environment without causing damage is a primary concern for biodiversity conserva- tion. Bioassays can be performed on 10–100 mg of a compound mixture, the usual yield from 10 g of dry plant material. For confirmatory assays and further fractionation and isolation, amounts in the range 100–1000 g must be collected. Depending on a given compound’s yield, preclinical trials to evaluate toxic side-effects and effectiveness in animals may require Biodiversity for Bioindustries 259 large amounts (tonnes) of dried plant material if the compound is too com- plex to be synthesized. There is no doubt that the survival of certain species has been threatened in the past by drug researchers. The exploita- tion of pilocarpine, for example, threatened the survival of Pilocarpus pignatifolios, P. microfilla and P. jaburandi species in South America (Balick, 1994). In another case, clinical trials with taxol have affected the survival of Taxus brevifolia in its natural habitat. When considering the possibility of countering this danger through sustainable breeding or planting, one must keep in mind that cultivation of a desired species may lead to a loss of the desired compound. The iso- lation and identification of epibatidine from Epipedobates tricolor, a frog used by indigenous people to poison arrowheads, has been described (Sapnde et al., 1992). Epibatidine is a remarkably simple, but highly effi- cient alkaloid that is 200 to 500 times more potent than morphine in anal- gesic assay systems. However, investigation of the alkaloid required the skins of 750 frogs collected from the wild. Attempts to breed these frogs in an artificial environment led to a loss of epibatidine in the skin of the sec- ond generation of frogs. Therefore, the compound is probably produced by complex interactions with other organisms in the environment. Extraction of plant material is a philosophy in its own right. An effi- cient protocol for organic extraction was developed at the National Cancer Institute (McCloud et al., 1988). The decision whether to use raw extracts, prepurified fractions or even randomly isolated pure compounds for bioassay depends on a drug company’s philosophy. Depending on the bioassay, natural compounds like polysaccharides, tannins, saponins or fatty acid esters must be removed prior to testing. This is of particular importance for bioassays involving cell cultures (Cardellina et al., 1993b). In recent years, drug bioassays have become increasingly specific, rapid, reproducible and sensitive. At the same time, they have become less susceptible to matrix and other effects that tend to cause false positive or negative reactions. Even during the 1970s, companies were screening with receptors isolated from cell cultures, a technique that yielded various top- selling drugs. Today, gene technology allows the cloning and expression of receptors, enzymes and other proteins important for signal transduction, metabolic conversions, or cell or viral structures. Once produced on a large scale, they can be immobilized on ELISA (enzyme-linked immunosorbent assay) plates and integrated into a chromogenic assay to measure lig- and–receptor interactions. The end result is the introduction of a huge vari- ety of new assays, which has increased the industry’s screening activities and the overall demand for new compounds. Nevertheless, the develop- ment of drugs against diseases not investigated down to the molecular level still requires complex cell culture assays or even screening in animal models. This is the case for most types of anticancer drug. Drug development, from the collection and taxonomic classification of 260 G. Tamayo et al. biological material to compound extraction and fractionation, bioassays, structure elucidation and final clinical testing, is usually not performed by a single drug company, but by various research partners, working under contract. Therefore, the transfer of related technology to developing-world source countries is a possible, and even logical, approach for the techno- logical development of these regions. Collection, taxonomic classification and extraction are not trivial tasks, but require a high standard of docu- mentation and reliable and reproducible work that can easily be conducted in Southern nations with the aid of technology transfer. Drug companies would benefit from carrying out further bioassaying and chemical struc- ture elucidation directly in the source countries, not only because of devel- opmental issues and cheaper labour costs, but also because it would ensure these companies gained more direct access to important markets. 11.2.2. Prospecting for genes Genetic engineering opens a totally new dimension for bioprospecting. The search for new genes and their applications is the primary objective of the biotech industry. Today’s biotech products, already on the market, are based on genes from humans, domesticated animals and cultivated plants. Examples are human cytokines and growth factors like interfer- ons, colony-stimulating factors, erythropoietin and bovine somatotropin, and also Calgene’s Sav-R-Flavr tomato. The market value of erythropoi- etin alone amounts to several billion dollars per year worldwide. Hence the tremendous appetite of this new industry for novel genes; indeed, the hunt for them in tropical rainforests is already on. In contrast to random screening in natural compound research (see above), gene technology allows a more straightforward approach, as illustrated by the following examples. The pharmaceutical biotech industry and biodiversity Biodiversity and protein engineering. Minor changes to the amino acid sequences of pharmaceutical proteins and peptides (biologics) and enzymes may lead to new or improved activities. Computer simulation in combination with site-directed mutagenesis are the basis for a new tech- nology, called protein engineering, which creates its own ‘molecular diversity’. Nevertheless, the first successful examples of amino acid sequence improvement are the result of screening genes from the wild. Calcitonin is a peptide hormone that inhibits the release of calcium ions and phosphate from the bones, and has therapeutic uses for osteoporosis (MacIntyre et al., 1987). The investigation of related hormones from ani- mals revealed that the calcitonin from salmon is more active and has a longer half-life within the human body than the human peptide structure Biodiversity for Bioindustries 261 (Epand et al., 1986). Today, chemically synthesized ‘salcatonin’ is on the market under tradenames including Calsynar and Miacalcic. Industrial enzymes, used to catalyse chemical processes, can be improved to increase their heat stability, activity and specificity. Naturally thermophilic bacteria have become a useful source of industrial enzymes. Hydantoinase, for example, catalyses the conversion of chemically syn- thesized hydantoins to precursors of D-amino acids. D-Amino acids, like D-hydroxyphenyglycine and D-phenylglycine, are needed to derive amox- icillin and ampicillin from penicillin, just as D-serine is a precursor for pes- ticide production. The first enzymes to be used for this conversion on an industrial scale were isolated from common soil bacteria. The characteris- tics of these enzymes limited the maximal temperature for the catalysis to 40°C (Kanegafuchi Co., 1978; Yamada et al., 1978), conditions which do not permit hydantoins to dissolve well in water. Researchers at BASF screened thermophilic bacteria from Yellowstone geysers and found two new hydantoinases with much improved heat stability and specificity charac- teristics (BASF AG, 1987). Through recombinant DNA technology, these enzymes are now produced in Escherichia coli and already on the market. The catalytic process can be performed more efficiently and more compet- itively at temperatures reaching 75°C. Protein engineering based on computer simulation is doubtless a very powerful tool. However, the screening of natural products for improved principles still has a higher success rate, proving again that the computer cannot yet rival Mother Nature. Animal defence and attack mechanisms as a source for biologics. For sev- eral decades now spider, snake, frog and bee venoms and squid and leech salivas have been investigated for pharmaceutically active peptides and proteins. Leeches (e.g. Hirudo medicinalis) have been used in traditional medicine to treat thrombosis since ancient times. The active principle from their saliva, the protein hirudin is now an ingredient of numerous oint- ments and gels and thus used against varicosis and haemorrhoids. Hirudin was one of the first proteins isolated from wild biodiversity. Recombinant hirudin has now been produced in Escherichia coli (Fortkamp et al., 1986). Other leech species are currently under investigation to dis- cover new hirudin variants with improved therapeutic applications (Sacheri et al., 1993). Promotion of blood clotting during wound healing can be achieved using proteins from snake venom (e.g. from the Egyptian sand viper) that induce platelet aggregation (Baheer et al., 1995). The mammalian enzyme tissue plasminogen activator (tPA) dissolves thrombotic blood clots. Recombinant human tPA, developed and patented by Genentech, has been approved as a therapeutic agent against heart attack in the USA and Europe. Researchers at Schering AG found four sim- ilar proteins in the saliva of Desmodus rotundus, the common vampire bat, 262 G. Tamayo et al. that are more efficient and safer for therapeutic application than their human counterparts (Schleuning et al., 1992). These products are presently undergoing preclinical studies. Eledoisin is a hendecapeptide isolated from the salivary gland of cer- tain squids (Eledone spp.). Its physiological action resembles that of other tachykinins. The peptide stimulates extravascular smooth muscles; it is a potent vasodilator and hypotensive agent (Pisano, 1968), and has potential therapeutic use to counter dry-eye syndrome. Through combining common biological knowledge with simple obser- vation and commonsense, a new biotech. company with millions of dol- lars in venture capital may be formed. In the following case, common knowledge of frogs and the Gram-negative bacteria found in wet environ- ments was sufficient to launch a successful search for antibiotics. Although frogs live in ponds infested with Gram-negative bacteria, they rarely become infected by these pathogens. Based on their research, the biotech company Magainin Sciences Inc. is named after a peptide (magainin) that is highly effective against Gram-negative bacteria and occurs naturally in the skin of frogs. Effective antibiotics against this type of bacteria are rare and as a result this peptide is currently undergoing clinical trials (Jacob and Zasloff, 1994). This same way of thinking led to the discovery of the steroid squalamine, which has antibiotic properties and occurs in the stom- ach, liver and other organs of the shark (Moore et al., 1993). These few examples indicate that there is a whole new world to be found in wild biodiversity, accessible by gene technology, and merely awaiting exploration by the pharmaceutical biotech industry. The agricultural biotech industry and biodiversity Recombinant genes found in wild biodiversity may be even more impor- tant for agriculture than for the pharmaceutical industry. Classical breed- ing has quite successfully used genes from wild ancestors of cultivated plants to promote pest resistance and develop new and improved crop variations. Gene technology now enables humans to integrate revolution- ary new properties into cultured plants through interspecific gene trans- fer. As with recombinant pharmaceuticals, research and development does not require random screening, but is rather a product-orientated engi- neering approach. Exploiting natural defence and attack mechanisms for pest control. Plants protect themselves against pathogens through various enzymes, enzyme inhibitors and lectins. For example, the basic chitinases from rice and the acidic β-1,3-glucanase from alfalfa are directed against the cell walls of fungi. Transfer of the genes encoding these compounds into tobacco has yielded resistance against these pests (Zhu and Lamb, 1991; Maher et al., 1994; Zhu et al., 1994). The α-amylase inhibitor in the common bean makes Biodiversity for Bioindustries 263 the starch of the seed indigestible for insects, and this property can be transferred into the garden pea (Pisum sativum) (Shade et al., 1994). The transfer into plants of genes from viruses, bacteria and animals is becoming a standard procedure in crop protection. Numerous successful field trials with recombinant crops expressing genes for the δ-endotoxins of Bacillus thuringiensis prompted intensive screening of bacterial species with insecticidal proteins (e.g. Koziel et al., 1993). Researchers at Monsanto found a cholesterol oxidase in a streptomycete which lyses the midgut epithelium of pest insects; expression of the gene in transgenic plants could promote insect resistance (Corbin et al., 1994). Spider, scorpion and mite venoms contain neurotoxic peptides which specifically kill insects. The expression of their respective genes in plants also leads to resistance against insect pests (FMC Corporation, 1993). Resistance against bacterial infections can be achieved by the produc- tion of peptides with antibiotic activities, such as cecropin B produced by wounded silk moths (Florack et al., 1995). The production of viral coat or nonstructural proteins in plants protects the plant not only against infec- tion from that same virus, but also against related virus types (Murray et al., 1993). Even mammalian enzymes can be useful. For instance, 2’-5’- oligoadenylate synthetase from rats, when produced in potatoes, protects the plant against virus X under field conditions (Truve et al., 1993). Finally, resistance against herbicides can also be achieved through het- erologous gene expression. A detoxification pathway for 2,4-dichlorophen- oxyacetic acid, an agonist of indoleacetic acid, can be created in plants through the expression of a monooxygenase gene from the bacterium Alcaligenes eutrophus (Lyon et al., 1989). Although only few of these examples deal with the transfer of genes from wild biodiversity, there is no doubt that tropical environments, espe- cially tropical rainforests, engender a multitude of defence and attack mechanisms among their inhabitants. This might lead us to suspect that these survival mechanisms must be highly sophisticated, and may repre- sent a rewarding resource for the genetic engineer. It can be expected that the investigation of novel defence mechanisms will increase dramatically in the future as pest resistance develops to counter the first generation of recombinant plant variations. However, DNA sequences coding for defence proteins and peptides can be patented, and this may give cause for socioeconomic problems in the source countries, many of which are devel- oping countries. This issue will be discussed below. Engineering of metaboIic pathways. Expression of recombinant genes in cultivated plants is under intensive investigation to improve oils, proteins, starch and other polymers for the food industry. A comprehensive overview of the state of the art is given by Beck and Ulrich (1993). However, it is not solely the food industry that stands to benefit: paper, 264 G. Tamayo et al. packaging and chemical industries will also be greatly affected. For exam- ple, Zeneca’s method (Zeneca Ltd., 1995) to suppress cinnamyl alcohol dehydrogenase through antisense technology in trees facilitates the removal of lignin from cellulose, and will therefore have an impact on the aforementioned industries. In most cases, new plant variations are engi- neered by interspecific transfer of genes, coding for enzymes, which alter metabolic pathways. Examples based on genes from the wild biodiversity are still rare, but already quite impressive. For the production of soaps, chocolate and candies, medium-chain fatty acids found in coconut and palm kernel oil have great economic importance. In 1992, the US alone imported 600 000 tons of these oils, which contain up to 50% of trilaurin, a medium-chain dodecanoic unsaturated fatty acid. Recently, the USDA approved a high-laurate canola oil developed by the US ag-biotech com- pany Calgene (PR Newswire, 1994). Calgene researchers investigated the synthesis of lauric acid in certain plant families and found a thioesterase which prematurely hydrolyses the growing acyl thioester of the fatty acid with an acyl-carrier protein in the wild Californian bay (Umbellularia cali- fornica). This 12:0-acyl-carrier protein thioesterase from the bay’s develop- ing oilseeds was expressed in transgenic Arabidopsis thaliana and Brassica napus ssp. napus, with the result that laurate and stearate became the most abundant types of fatty acid found in the oil of these plants (Voelker et al., 1992). Three of Calgene’s patents cover the purified enzyme, the recombi- nant nucleic acid construct with the gene for the enzyme, and a method to produce laurate in recombinant Brassica seeds (Calgene Inc., 1994a,b,c). The socioeconomic consequences of these patents will be discussed below. Long-chain wax esters are required for a variety of industrial applica- tions including pharmaceuticals, cosmetics, detergents, plastics and lubri- cants. Such products, especially long-chain wax esters, have previously been available from endangered species such as the sperm whale, or more recently, from the desert shrub, jojoba (Simmondsia chinensis or S. californica). Waxes are fatty alcohol and fatty acid esters, and their synthesis requires a fatty acid reductase as well as a synthase. The jojoba genes for the wax synthase (fatty acyl-CoA:fatty alcohol acyltransferase) and the fatty acyl reductase have been cloned and patented by Calgene (Calgene Inc., 1995a,b), and as a result, wax may be produced in rape seed in the future. New biodegradable plastics produced from the bacterial storage com- pound polyhydroxybutyrate have been developed and are already on the market. However, production of the compound through fermentation is not cost efficient. In recent development, the transfer of the entire anabolic pathway, consisting of three enzymes from the bacterium Alcaligenes eutro- phus, into Arabidopsis thalania (Nawrath et al., 1994) may transform the farm field into a chemical factory for plastics in the near future. Examples for pathway engineering do not yet involve genes from the tropical rainforest, but a thorough and product-orientated survey of oils, fats, waxes and Biodiversity for Bioindustries 265 other polymers (formerly not of commercial interest because of low pro- ductivity or abundance) from tropical plants, animals and microorganisms may lead to new compounds for industrial applications. These new compounds would have the advantage of being both biodegradable and available for farm production through genetic engineering. Gene technology will also lead to the more efficient production of nat- ural compounds presently used for pharmaceuticals and pesticides. The chrysanthemyl diphosphate synthase gene from Chrysanthemum cinerariae- folium was patented by Agridyne Technologies Inc. (1995) and promises to open new paths for producing insecticidal pyrethrins, pyrethroids and their derivatives with greater efficiency and higher purity levels in culti- vated plants. Metabolic engineering of medicinal plants has been performed suc- cessfully with Atropa belladonna to produce the alkaloid scopolamine. The naturally occurring alkaloid in this plant is hyoscyamine, known for its anticholinergic activity, and an active ingredient in eye drops, antidotes and spasmolytics (Yun et al., 1992). Scopolamine, found throughout the Solanaceae, is an epoxy-derivative of hyoscyamine but with a broader ther- apeutic spectrum, including, e.g., antiemetics and hypnotics. Expression of the hyoscyamine 6-β-hydroxylase gene from Hyoscyamus niger in Atropa led to an almost exclusive accumulation of scopolamine in the plant’s leaf and stem. Flux through a pathway to a plant secondary product can be ele- vated by genetic engineering. For example, over-expression of the yeast ornithine decarboxylase gene in transgenic roots of Nicotiana rustica led to enhanced nicotine accumulation (Hamill et al., 1990). The same effect can be obtained to produce sterols in plants by over-expressing 3-hydroxy-3- methylglutaryl CoA reductase, which catalyses the production of the sterol building block mevalonate (Amoco Corporation, 1994). 11.3. Modern Bioprospecting: Linking Industry, Biodiversity Conservation and Developing Country Technology Acquisition The rapid loss of biological diversity – indicated by the extinction of an estimated 30 to 300 species per day (Japan Economic Newswire, 1995) – together with the potential opportunities and threats of gene technology, led to the United Nations Convention on Biological Diversity (UNEP, 1992). In light of the vast potential of biotic materials and the need to ensure their survival, in addition to measures taken to improve biodiver- sity conservation activities, it is imperative that industries move from the passive role of simple users to the more active one of reinvesting part of their revenues into conservation efforts. Companies should be aware that 266 G. Tamayo et al. they are among the first to lose as a consequence of species extinction, and indeed such an awareness is growing. The principle of this modern approach to bioprospecting may be sim- ple, but the link between biodiversity conservation and its sustainable use requires a careful design and strategic planning. The goals are to maximize those uses which generate information, and to reinvest part of the benefits obtained from bioproducts into acquiring knowledge and improving bio- logical resource management. As a consequence, wildland biodiversity can be developed as part of a country’s national economy at the same time as its preservation into perpetuity is guaranteed. The bioindustries are thereby encouraged to initiate relationships with partners in biodiversity- rich countries. Following the guidelines of the Biodiversity Convention such partnership can facilitate sustainable and nondamaging biological and genetic resource use for research and development, while taking care to share economic and intellectual benefits with the owners of the biolog- ical resources. The process of collecting bioresources, extracting and testing con- stituents (either chemicals or genes) for biological activity, and the further development of a product is long and expensive (Reid et al., 1993). Based on the research and development costs of 93 randomly selected new chem- ical entities during the period 1970 to 1982, the development of a single drug to the point of market approval was estimated at $US114 million for the USA (DiMasi et al., 1991). Although rewards might be high, the chances of failure are equally so: of 10 000 different products tested, only one will make it to the market (Farnsworth, 1994). However, the real challenge for this new generation of bioprospectors is to find a way to capture part of the financial revenues for the source country’s biodiversity conservation efforts and economic development. As an example of this innovative approach to prospecting activities, INBio is negotiating agreements with scientific research centres, universities and private enterprise that are mutually beneficial to all parties (Sittenfeld and Lovejoy, 1994). These pioneering agreements provide significant returns to Costa Rica while simultaneously assigning economic value to natural resources, and providing a new source of income to support the mainte- nance and development of the country’s Conservation Areas (Sittenfeld and Lovejoy, 1995). 11.3.1. Bioprospecting frameworks Modern biodiversity prospecting requires the creation of appropriate frameworks and the cooperation and involvement of governments, inter- mediary institutions, private enterprise, academia, and local communities and entities. This activity also requires the involvement of lawyers, Biodiversity for Bioindustries 267 lawmakers, scientists, managers and economists from developing and developed countries (Sittenfeld and Lovejoy, 1995). The fundamental point of departure for a biodiversity prospecting framework is macro-policy, the set of governmental and international reg- ulations, laws and economic incentives that determine land use patterns, access to and control of biological resources, intellectual property rights regimes, technology promotion, and industrial development. Macro-poli- cies are formed on the international, national and social levels. On the international level, agreements, conventions and other mechanisms estab- lish the relationships and protocols for sharing biological resources between countries. Documents considered important in providing the guidelines and regulations for biological resource use include: the Biodiversity Convention, the Trade Related Intellectual Property Rights (TRIPs) of the General Agreement on Tariffs and Trade (GATT), the Draft Declaration on Indigenous Rights of the United Nations Working Group on Indigenous Populations, and also subregional agreements, such as the North American Free Trade Agreement (NAFTA), the Amazonian Treaty, and the Pacto Andino. Nevertheless, conventions, agreements and organizations still leave open the major responsibilities of designing adequate legislation and reg- ulations regarding land ownership, land tenure rights, the creation of pro- tected areas, the use of biological resources, nationally recognized intellectual property rights, the definition of public-domain resources, and the creation of market incentives or deterrents for private enterprise and research investments to each individual country. Such legislation and reg- ulations promote stability and manoeuvrability of in-country partners, characteristics considered attractive to private industry and academic research counterparts. Deterrents, such as national policy vacuums or legislation drafted out- side the framework of the Biodiversity Convention, still exist in many countries and continue to create obstacles to establishing collaborations with academic and industrial research partners. In general, changes in laws and policies governing the ownership of and access to genetic resources are needed as well as changes in the way bio-business has evolved to date. The importance of favourable national policies, regula- tions and laws becomes obvious when considering international intellec- tual property rights. Drug research within the source country itself is an important step towards national economic development, but will only be attractive to the industrial partner if results can be patented. It is for this reason more than any other that international patent laws should be rec- ognized by national law. At the same time, there is concern within the industrial sector that countries, spurred by the Biodiversity Convention, may promulgate new laws restricting access to biological and genetic resources, and reducing 268 G. Tamayo et al. renewed enthusiasm for natural products (Putterman, 1994). Yet the recog- nition by the Convention of sovereign rights of nations over their genetic resources is intended to encourage world trade in genetic resources, since it commits countries to facilitate access, based on mutually agreed terms (Putterman, 1994). National governments should implement rules, regu- lations and policies that take advantage of Articles 15 and 16 of the Convention. These Articles encourage source-country participation in researching their own biological resources, transferring technologies to uti- lize these resources and reaping a fair share of the benefits from their com- mercial exploitation (Sittenfeld and Lovejoy, 1995). Finally, on the social level, heavy investment in education and other social services has created a scientific environment of qualified institutions, researchers and educated personnel in Costa Rica. Such an environment is a prerequisite for research collaborations with private enterprise and is essential for integrating biodiversity into economic development (Sittenfeld and Lovejoy, 1995). 11.3.2. Inventories, business development and technology access Supported by a favourable international and national macro-policy, three basic elements guide the rational and productive use of biological resources in prospecting agreements: (i) biodiversity inventories and infor- mation handling; (ii) business development; and (iii) technology access. Inventories and information management As pointed out in Section 11.2.1, screening for drugs and pesticides will only be successful through the development and management of biologi- cal, ecological, taxonomic, and related systematic information on living species and systems. Even with these data, further information is required for the more systematic screening approach used in gene technology. For example, biochemical data must be evaluated for, e.g., the occurrence of certain biopolymers, metabolic pathways, enzymes and defence or attack mechanisms. Biodiversity inventories create catalogues of available resources and their location. They prevent damage to ecosystems, areas, species and populations by indicating what resources are available, and where they can be collected without damaging the environment (Raven and Wilson, 1992). Simultaneously, the source-country collaborator becomes a more attractive, knowledgeable and reliable business partner because the inventory-generated information reduces the uncertainties of collecting further material should this prove necessary. Business development Building upon inventory-generated knowledge, business development defines markets, market needs, major players, and national capacities in Biodiversity for Bioindustries 269 science and technology as well as institutional strategies and goals. Important requirements include knowledge of one’s assets and drawbacks, market surveys and evaluation of conservation needs. The key to business development is interacting with international industry in order to approach the market in a realistic and practical way. Because bioprospect- ing should promote source-country economic development, business development must encourage the sustainable use of biodiversity by local entrepreneurs. However, this is a considerable challenge in developing countries where industry normally cannot take the financial risk of apply- ing innovative technologies, let alone those that are sustainable and non- damaging to the environment. Technology transfer One of the major issues discussed in the Biodiversity Convention refers to technology transfer, allowing source countries to convert raw biological materials into products of greater value in exchange for access to their bio- diversity (Putterman, 1994). This issue is of tremendous importance, par- ticularly in a decade of patentable genes, and will be discussed in more detail below. In the near future, genes isolated from tropical biodiversity may provide the farmers in developed countries with advantages over the farmers of the source countries. These advantages stem from the bioin- dustries’ historical development and their physical proximity to the devel- oped agricultural economies of the North. This may lead biotechnological research and development to concentrate solely on improving the proper- ties of crop and livestock in the North (for discussion, see Shand, 1993). Technology transfer may enable source countries to keep pace with the developed countries, and avoid being left out of important agricultural developments (Lesser and Krattiger, 1993). This scenario is realistic because gene technology, in contrast to natural compound chemistry, does not particularly rely on expensive investments in laboratory equipment, and would therefore be easier to implement. 11.3.3. Contract negotiations In general, contract negotiation is divided into three basic sets of issues: sci- entific issues, business issues and legal issues. To negotiate, an organization must have a good sense of its own fundamental needs and those of its potential collaborator. The typical source-country needs are: the generation of income to support protected areas and conservation management activi- ties through direct contributions as well as royalties; the transfer of process- ing technologies and a guaranteed future profit-sharing if commercial products are forthcoming. Sampling must be done under best ecological 270 G. Tamayo et al. practice without damaging the ecosystem. For bilateral contracts with indus- trial partners, exclusivity and time limitations are further requirements. In summary, modern bioprospecting requires that the source country: 1. Creates an infrastructure guaranteeing a reliable supply of natural prod- ucts (including correct taxonomic identification, quality control, full support from government and adherence to national or local regula- tions on access to resources). 2. Acquires technology that adds value to natural products wherever pos- sible (from extracts to partially purified or pure compounds or gene sequences). 3. Takes advantage of local capabilities using all types of organisms as bio- logical resources attractive to industry (from plants and microbial resources through marine or freshwater life forms to arthropods). 4. Develops a reputation as a reliable business partner over the time. 5. Reinvests part of the revenues in improving biodiversity management and conservation. In exchange for access to biological resources, the industrial partner must agree to: 1. The fair and equitable sharing of benefits, both in intellectual and mon- etary terms. 2. The implementation of collection and production methods with mini- mum effects on biodiversity. 3. The use of equitable bioprospecting practices for further research on tropical diseases and problems specifically associated with developing countries. 11.4. How to Face the New Challenge of Gene Technology With few exceptions classical bioprospecting for drugs has not proved eco- nomically beneficial for developing nations, but nor has it directly dam- aged these economies. In contrast, bioprospecting for genes may soon pose a real threat to the economic survival of these biodiversity-rich countries. The farmers of the North are currently suffering under low prices and overproduction of certain traditional crops and livestock. As a result they are looking for new markets and products. Modern biotechnology promises to aid Northern farmers in this endeavour, eliminating or displacing traditional export commodities from developing countries and transferring production or substitutes from the farm fields of the South to those of the North. Quite possibly the transfer may even skip the Northern farms and jump straight into bioreactors. In Africa alone, US$ 10 billion in exports are vulnerable to industry-induced Biodiversity for Bioindustries 271 changes in raw material prices and requirements (Shand, 1993). Most developments in plant biotechnology have been achieved with crops cultivated mainly in industrialized countries. This denies the farmers of developing countries the chance to benefit from the new agricultural opportunities of gene technology. As mentioned above, Calgene’s high-laurate canola oil may displace coconut and palm kernel oil, posing a threat to the economic survival of millions of farm families in the South. In the Golfito region of Costa Rica the government has started a programme to grow oil palms on banana fields deserted by the US fruit multinationals. All these efforts, which are partially financed with Northern developmental aid, may vanish into thin air as a result of Calgene’s new rape seed. Thaumatin, a sweet-tasting basic protein from the tropical plant Thaumatococcus, has been traditionally used in West Africa as a sweetener. With the collection of Thaumatococcus fruits for the British food industry, the population in this region earned a large part of its income. However, the thaumatin gene has now been cloned and the sweetening protein can be produced by large-scale fermentation of brewer’s yeast at low cost (Lee et al., 1988). The same applies to natural compounds like indigo, which can now be produced by the fermentation of Escherichia coli engineered with genes from a toluol-degrading sub- species of the soil bacterium Pseudomonas putida (Ensley et al., 1983). Products like vanilla, pyrethrum and rubber may follow this same path. Along the same lines, the extension of patent laws to the developing nations through the GATT could mean that the biotech industry obtains a monopoly on genetically engineered livestock and crops, which farmers in developing countries must cultivate under constraint in order to remain competitive. For example, US-Patent No. 5 159 135 of Agracetus (a sub- sidiary of W.R. Grace & Co.) covers all genetically engineered cotton. This patent is a warning of potential future problems, and has already caused an outcry in developing countries like India (Kidd and Dvorak, 1994). If the biotech industry continues developing without adequate controls, the consumers and farmers of developing countries may even end up paying royalties on biotech products that were originally developed from their very own resources and knowledge. As a step in the right direction, the US Patent and Trademark Office reversed its decision to grant the Agracetus patent at the end of 1994, but primarily as a result of pressure from the US biotech industry which argued that patents like this will inhibit research and development (AGWEEK, 1994). Such issues bring important questions to light: How can developing nations be motivated to conserve their biodiversity under these threatening circumstances? Are the regulations and tools of modern bioprospecting, as described above, sufficient to face this challenge? The Biodiversity Convention attempts to address this new threat by requiring that access to biodiversity’s genetic potential be combined with 272 G. Tamayo et al. the biotechnology transfer to the South in order for those countries to develop their own methods of sustainable biodiversity utilization. Nevertheless, the Convention suffers from three major drawbacks. 1. The treaty was not ratified by the USA, the leading country in biotech- nological research, development and application. 2. It specifically excludes (under US pressure) ex situ genebank material collected before the enactment of the treaty (Shand, 1993). As a result, huge stocks of germplasm collected by the North, mostly in tropical and subtropical countries, are not restricted by the Convention. The recent transfer by the Consultative Group on International Agricultural Research of its 12 genebanks to the auspices of the UN must be the first step in keeping the access of developing countries open to their own resources (Madeley, 1994). 3. It still remains nearly impossible to control the illegal transfer of genetic material into the North. Genes can be cloned from minute amounts of DNA or RNA and isolated from biological material that easily fits into an airmail envelope. Genes do not have tags designating their country of origin, and once they are cloned, they are no longer controlled by their source country. This is quite different from the isolation of natural compounds from plants, where larger amounts of plant material must be collected and, for the process of isolation and structure elucidation, must be re-collected. In this last case, controlling the flow of biological material is possible simply because industry will eventually require sample resupply at a given point, and for this the industry needs reli- able partners in countries of origin. These issues must be approached in an active and more aggressive manner than traditionally used. During collaborations with traditional pharmaceutical and biotech companies like Merck & Co., Bristol-Myers Squibb and the British Technology Group, INBio used such an approach, proving to industry that fair partnerships are mandatory and conducive to success. More importantly, INBio demonstrated that reliable applied research is possible in a developing country, and that technology transfer to acquire necessary know-how and equipment works to the advantage of the industrial partner as well. The same applies to the biotech industry. In this case, the transfer of gene technology is not critical, in contrast to natural compound chemistry, because it does not require million-dollar investments in laboratory infra- structure. Rather, the industry relies on the know-how already existing in many source countries. Gene technology also represents a promising development opportunity for countries that do not have large research budgets at their disposal. Moreover, gene technology is a very straight- forward approach, relying on natural history observations (e.g. whether certain plants show natural resistance to pathogens), and not involving Biodiversity for Bioindustries 273 the automated random screening of thousands of samples. Biodiversity inventories, which are already in place in countries like Costa Rica, are a reasonable and advantageous prerequisite for successful ‘gene prospecting’. Costa Rica’s aggressive strategy to foster collaborations with the international industry and academic institutions in drug research, gene technology and agriculture actively seeks to develop and patent natural compounds, proteins and genes in Costa Rica based on a foundation of national research. Collaborations of this nature will help launch Costa Rica onto a scientific and technological plane that offers services and goods that are both competitive and compatible with those of industrial nations. Simultaneously, INBio works within this strategy to increase knowl- edge about Costa Rican biodiversity in general, access revenues for further conservation efforts, and to assign biodiversity a higher value than it has had in the past. 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Biodiversity for Bioindustries 279 Index 301 Ac/Ds 217–219 ACEDB 293, 295 see also database active/working collections 105, 111 adaptation 62, 65 Advanced Technology Program (ATP) 34, 36, 37 AFLP see amplified fragment length polymorphisms AGIS 289 Agrobacterium 204, 207–213, 222 agroecogeographic analysis, 60 Agropyron elongatum 204 Alcaligenes eutrophus 264, 265 alfalfa 126, 263 alkaloid 257–260, 266 allele diversity 36, 88, 89, 112–113, 177, 179 alleles 49, 52 Allium 138 see also garlic, onion allopolyploid 78, 89, 92 see also polyploid amplified fragment length polymorphism (AFLP) 26, 27, 30, 104, 80, 88, 89, 180, 220 ancient DNA 9, 23 antibiotics 205, 263 antibody 15 see also monoclonal antibodies antifreeze proteins 205 antifungal 259 antigen 15 Antirrhinum 217 antisense RNA see RNA antiserum 15 see polyclonal antisera apple see Malus domestica Arabidopsis 14, 77, 83, 84, 182, 204, 218, 222–223, 265, 288, 290, 292–293 Armoracia 139 Artemisia annua 259 asparagus 138, 140, 141, 209 Asteraceae 259 Atropa belladonna 266 Avena 57, 208, 212, 285 avocado 122, 241 B-chromosomes 53 bacterial artificial chromosome (BAC) 86, 206, 221 Bacillus thuringiensis 264 banana 5, 120, 123, 125, 136, 140, 142, 144, 204, 208, 272 see also Musa base collections 105 barley see Hordeum bean 140, 242, 243, 263 see also mungbean, Phaseolus, Vicia faba beet see sugarbeet Bermuda grass 57 bioassays 260 Biodiversity Convention 268, 270, 272 biodiversity 58–59, 61, 67, 72–73, 88, 255, 256, 261, 263, 266–269, 271, 272, 283 biolistic see microprojectile bioprospecting 6, 255–256, 261–269, 271 Brassica 28, 35, 77, 82–84, 90, 93, 186, 204, 222, 240 Brassica napus 35, 140, 141, 143, 204, 208, 215, 216, 265 Brassica oleracea, 182, 185, 191, 193, 204 Brazil nut 214 bread wheat 182 breeding system 51, 52, 54, 56, 57, 66, 73 bulked segregant analysis 188, 190, 220 cabbage see Brassica oleracea cacao see Theobroma cacao Calophyllum lanigerum 258 canola see Brassica napus CAP see cleaved amplified polymorphism Capsella bursa-pastoris 140 carnation 128, 136, 139 carrot 126, 127 cassava see Manihot Castanea 125, 140 Catharanthus 126, 130, 144 centipedegrass 26 centres of diversity 63, 235 centromeres 12, 14, 28 Cf-9 gene 218, 219 see also resistance genes chemotherapy 237 chestnut see Castanea chiasma 53, 181–184, 186–187 chickpea 207, 209 chloroplast 12, 13, 17, 22, 25, 29, 32 chromosome bacterial artificial chromosome (BAC) 86, 206, 221 B-chromosomes 53 chromosome banding 16, 20, 31 chromosome landing 81 chromosome painting 28 chromosome pairing 78, 92, 181–184, 197, 219–222 chromosome walking 81, 86 yeast artificial chromosome (YAC) 82, 86, 206, 220–222 see also centromeres and telomeres Chrysanthemum 126, 143, 257, 266 Citrus 54, 122, 138 sweet orange 143 navel orange 144 orange 208, 209 Cladosporium fulvum 218 classification 104, 260, 261 cleaved amplified polymorphism (CAP) 24, 180 clover see Trifolium repens co-linearity see comparative mapping co-suppression 215 coconut see Cocos nucifera Cocos nucifera 119, 122, 140–141, 208, 265, 272 Coffea 112, 120, 122, 124, 126, 136, 139, 140, 141 coffee see Coffea colchicine 190 collections active/working collections 105, 111 base collections 105 collecting procedures 59, 169, 258 core collections 11, 105, 111–114 duplicate safety collections 105 see also safety storage ex situ collections, conservation and genebanks 3–5, 11, 33, 49, 59, 68–70, 103, 115, 146, 273 see also seeds in situ conservation 3–4, 11, 33, 49, 50, 71, 146, 235 in vitro collections 103, 121, 122 plant collections 169 see also genebanks Colocasia esculenta 124 comparative mapping see mapping conservation 267, 271 Cordyline 125 core collections 11, 105, 111–114 coriander 216 cotton 122, 204, 208, 209 cottonseed 215 cowpea see Vigna crossability 6, 106, 107, 108 cryopreservation 121, 127–143, 145, 165, 168, 172 see also preservation in liquid nitrogen Cucurbita 290 Cynodon 57 DAF see DNA amplification fingerprint database 25, 36, 292, 295 see also ACEDB date palm 140, 141 dehydration 127–128, 130–137, 138, 139–140, 141 denaturing gradient gel electrophoresis (DGGE) 27 desiccation see dehydration Desmodus rotundus 262 digoxigenin (DIG) 241, 242 Dioscorea 120 see also yam disease resistance genes 218–219, 221 see also resistance genes DNA DNA amplification fingerprint (DAF) 24, 25, 57 302 Index see also random amplified polymorphic DNA DNA banks 3, 5, 163–72 DNA content 19–20, 78, 181 DNA–DNA hybridization 32 DNA fingerprinting 80, 88, 281 DNA libraries 21, 25, 206, 214, 221 DNA markers 56, 81 DNA reassociation 19, 20 DNA repeated sequences 12–13, 19, 22, 24, 27, 78–79, 81, 222 DNA satellites 12, 14, 19, 20, 22, 27, 28 DNA sequencing 9, 17, 28–30, 31, 32, 33, 34, 110, 292, 295 sequencing by hybridization (SBH) 36, 37 T-DNA 207–209 see also Tm dot and slot blot hybridization 17, 27, 28, 30–31, 241, 246 double antibody sandwich (DAS) ELISA see ELISA double-stranded RNA (dsRNA) see RNA doubled haploid 178, 184, 190–191, 193 drift see genetic drift Drosophila 87, 187, 217 duplicate safety collections 105 see also safety storage duplicate accessions 11, 105, 107, 108, 110, 111, 112 Echinacea 258 ecogeographical 78 ecological distribution 63 effective population size 51 Elaeis guineensis 120, 124, 126, 129, 140, 141, 143, 145, 215 electronic mail 282–283 electrophoresis 16–18, 20–21, 25, 31, 212, 213, 240 electroporation 210, 212 ELISA 247, 248, 260 double antibody sandwich (DAS) 246–247 enzyme-linked immunosorbent assay 18, 238, 243–246 triple antibody sandwich (TAS) 246 EMBL 25, 290 embryos embryo culture 90 embryo rescue 90 somatic embryo 126, 129, 130, 137, 139, 140–141, 143, 144–145 zygotic embryos 131–135, 139, 140 encapsulation 130, 136, 137, 138–139 environmental diversity 62 enzyme-linked immunosorbent assay see ELISA Epipedobates tricolor 260 epistasis 90 Escherichia coli 217, 262, 272 EST see expressed sequence tag ethnobotany 258–259 eucalyptus 136 evolution 50, 63–64, 77, 78, 80, 87 ex situ collections, conservation and genebanks 3–5, 11, 33, 49, 59, 68–70, 103, 115, 146, 273 see also seeds expressed sequence tag (EST) 36, 86 extinction 6 fatty acids 215–216 see also oleic acid and petroselinic acid Festuca pratensis 53 field genebanks 120 File Transfer Protocol (FTP) 283–284 fingerprinting see DNA fingerprinting flow cytometry 16, 19–20, 31–32 fluorescent in situ hybridization (FISH) see hybridization fluorochrome 19 Fragaria 124, 143 Frequently Asked Questions 283 FTP see File Transfer Protocol fungi Cladosporium fulvum 218 Helminthosporium maydis 163 Phytophthora infestans 163 Rhynchosporium oryzeae 112 yeast 87 garlic 258 see also Allium GenBank 25, 290 gene flow 55, 64, 69, 71 gene-for-gene interaction 218 see also resistance genes herbicide resistance genes 205, 214 major genes 187, 193 polygenes 175, 189 pool 5, 6, 66, 89, 91, 106, 107, 213 prospecting 6, 261 see also bioprospecting rbcL gene 29 transfer 164, 203–223, 263 virulence genes 207–210 see also marker-assisted gene transfer Index 303 genebank 121, 146 genetic genetic diversity 49, 50, 51, 59–62, 65–66, 72–73, 163 genetic drift 11, 64, 68, 70 genetic erosion 59–60 genetic mapping 77, 80–85, 281, 292, 295 see also linkage groups genetic stability 142 genetic variation 51, 72 genome analysis 77 see also genetic mapping genome 78 see also DNA geographical distribution 63 germplasm catalogue 292 exchange 123 utilization 87 ginger 126 ginkgo 258 ginseng see Panax ginseng gladiolus 242 glycosides 257 Gopher 284–287 Gramineae 54 grape 122, 126, 129, 136, 137, 138, 208, 209, 240, 241 Green Revolution 203 GRIN 288, 295 groundnut 242 Hardy-Weinberg 51, 61 heat shock protein 30 Helminthosporium maydis 163 herbicides 260 herbicide resistance and resistance genes 205, 214 heterochromatin 12, 14, 20 heterosis 178 heterozygosity 26, 54, 180 Hevea 140 Hirudo medicinalis 262 Hordeum 57 barley 54, 80, 82, 84, 86, 89, 91, 112, 208, 213, 247 Hordeum bulbosum 90, 91 Hordeum chilense 92 Hordeum spontaneum 89 Hordeum vulgare 77 horse chestnut 258 Howea 140 human 25, 35 Human Genome Project (HGP) 24, 33, 34, 36, 37 human genotyping kits 34 hybridization DNA–DNA hybridization 13, 16, 19, 21, 27, 32, 241 dot and slot blot hybridization 17, 27, 28, 30, 31, 241, 246 fluorescent in situ hybridization (FISH) 28, 80 in situ hybridization (ISH) 13, 17, 27, 28, 31, 32, 93 nucleic acid spot hybridization (NASH) 241 see also DNA sequencing by hybridization and DNA reassociation kinetics sexual hybridization 80, 89–92 Hymenaea courbaril 259 Hyoscyamus niger 266 IGS see ribosomal RNA immunocapture PCR see polymerase chain reaction immunocapture RT-PCR see polymerase chain reaction immunoelectrophoretic assays 18 immunosorbent electron microscopy-ISEM 239 in situ conservation 3–4, 11, 33, 49, 50, 71, 146, 235 hybridization (ISH) see hybridization population 11 in vitro assays 6 collections 103, 121, 122 conservtion 119–146 culture 5, 38, 120, 142, 207, 235 see also tissue culture fertilization 91 incompatibility 53 informatics 7, 281–296 information management 269 insect resistance 214, 215 insecticidal 257, 264, 266 intergenic spacers (IGS) see ribosomal RNA internal transcribed spacers (ITS) see ribosomal RNA Internet 7, 281–282 inter-simple sequence repeat 220 inter-repeat PCR see polymerase chain reaction introgression 177, 194–196, 203 introns 213 Ipomea batatas 120, 123, 138, 208 isozyme 4, 9, 18, 30–32, 52, 56, 57, 106, 112, 113, 142, 185 304 Index ITS see ribosomal RNA internal transribed spacers JOINMAP 185 jojoba 265 Juniperus 170 karyotypes 20, 27, 32 Kiwi fruit 124 landraces 2, 50, 71, 103, 235 lectin 215 Lens culinaris (lentil) 83 lily 139 linkage linkage disequilibrium 88, 114 linkage drag 177, 194, 195 linkage groups, 180, 185, 186, 187 see also genetic mapping Lolium 53, 57, 61, 64 Lycopersicon 52, 54, 77, 81, 82, 182, 203, 204, 205, 208, 218–221, 290 MAbs see monoclonal antibodies maintenance 68 maize see Zea mays major genes see genes Malus domestica 124, 136, 138, 208, 209 Manihot 112, 120, 123, 124–125, 142, 143, 144, 208 mapping comparative mapping 4, 22, 81–85, 87, 93, 222 JOINMAP 185 map-based cloning 81, 84, 197, 216, 219–221 map length 181, 182, 183, 184, 186, 187, 197 MAPMAKER 185 mapping functions 183, 185 physical mapping 28, 31, 93, 220–222, 281, 292, 295 see also genetic mapping and linkage groups marker-assisted marker-assisted selection 175–176, 196, 213 marker-assisted gene transfer 194 marker-assisted prediction of performance 114–115 melon 140, 141 microcomplement fixation (MCF), 15, 16, 31, 32 microprojectile 207, 208, 211–212, 213 microsatellites see simple sequence repeats microspore or ovule culture, 178 millet 82 minisatellites see variable number of tandem repeats mint 138 mitochondrial 13, 17, 25 molecular diversity 258 molecular markers 81, 83, 88 see amplified fragment length polymorphism see cleaved amplified polymorphism see DNA amplification fingerprint see expressed sequence tag see inter-simple sequence repeats see random amplified polymorphic DNA see restriction fragment length polymorphism see sequence tagged sites see simple sequence repeat see simple sequence length polymor- phisms see single-stranded conformation polymorphism see variable number of tandem repeats monoclonal antibodies 16, 18, 30, 31, 244–245, 247 see also antibodies morphine 260 mulberry 127, 136, 138 multiplex 35 mungbean 83 Musa 120, 122, 123, 124 see also banana and plantain Mylodon (ground sloths) 9 navel orange see Citrus near isogenic lines (NILs) 197, 220 nematocide 257 Nephrolepsis 125 nested PCR see polymerase chain reaction Netscape Navigator 286 newsgroups 283 Nicotiana Nicotiana rustica 189, 266 Nicotiana tabacum 126, 204, 211, 213, 214, 215, 216, 218, 223, 263 nucleic acid spot hybridization (NASH) see hybridization nucleolar organizing regions (NORs) 20 see also ribosomal RNA oak see Quercus Index 305 oats see Avena Oenothera 66 oil-palm see Elaeis guineensis oilseed rape see Brassica napus oleic acid 215 oligonucleotide ligation assay (OLA) 35 onion 209, 211 see also Allium orange see Citrus orthodox seeds see orthodox seeds Oryza sativa 4, 77, 82, 84, 86, 103–112, 114–115, 126, 182, 197, 207, 208, 209, 210, 211, 215, 222, 223, 263 palm oil 265, 272 Panax ginseng 126, 144, 258 Panicum maximum 54 passport data 106, 108, 110, 112–113 pathogens 50, 263 PCR see polymerase chain reaction pea see Pisum sativum peach 126 peanut 215 pear 136–137, 138 pearl millet 91 pesticide 256–257, 262 petroselinic acid 216 phage-display 248 pharmaceutical 2, 164, 256, 263 Phaseolus 112, 209, 246 see also bean phenotypic variation 4, 188, 192, 193 phylogeny 63, 77, 165 physical mapping see mapping phytochrome 30 Phytophthora infestans 163 Picea abies 143 pilocarpine 259 Pilocarpus pignatifolios 259 Pisum sativum 83,140, 141, 236, 246, 263 plant breeding 2, 5, 175 plant collections 169 Plantago 57 plantain 120, 204 see also Musa pleiotropic effects 179, 214 plum 243 see also Prunus polyclonal antisera 244, 247 see antiserum polygalacturonase 205 polygenes 175, 189 polygenic traits 176 polymerase chain reaction (PCR) 9, 13, 16, 22, 23–27, 29, 31, 32, 80, 88, 104, 238 anchored PCR 24, 26 arbitrarily primed PCR (AP-PCR) 25 immunocapture PCR 243 immunocapture RT-PCR 247 inter-repeat PCR 24, 26 nested PCR 24, 26 RT-PCR 242–244 see also Random amplified polymorphic DNA, DNA amplification fingerprint and amplified fragment length polymorphism polyploidy 53, 54, 57, 178, 204 see also allopolyploid population structure 49 potato see Solanum tuberosum preservation in liquid nitrogen see cryopreservation protein engineering 261 protoplasts 135, 210, 212 Prunus 122, 124 see also plum and wild cherry Pseudomonas 221, 272 Pto locus 221 see also resistance genes pyrethroids 257 quality control 178 quantitative trait loci (QTLs) 4, 51–52, 58, 81, 84, 88, 115, 189–194, 197, 222 quantitative traits 4, 51, 114, 175, 176, 188, 191–192, 196, 213 Quercus 125, 140 random amplified polymorphic DNA (RAPD) 24, 25, 27, 30, 32, 57, 88, 89, 104, 107–109, 111, 180, 220, 281 see also DNA amplification fingerprinting rape see Brassica napus Rauvolfia serpentina 125 rubisco large subunit (rbcL) gene 29 recalcitrant seeds see seeds recombinant inbred lines 184, 190 recombination 53, 66, 181, 186, 222 recombination frequency 183–187, 190–191, 194 regeneration in culture 206–207, 209–210 regulatory sequences 213–214, 223 repeated DNA sequences see DNA resistance 1, 106, 112 resistance genes 204 see also Cf9, Pto, disease resistance genes, herbicide resistance genes and insect resistance genes restriction 21–22, 24, 26, 30, 186 306 Index endonucleases 206 fragment analysis 9 fragment length polymorphism (RFLP) 16, 21–22, 28, 30–32, 57, 80, 81, 84, 89, 104, 107, 143, 144, 179, 180, 185, 220, 281 Rhynchosporium oryzeae 112 ribosomal RNA see RNA rice see Oryza RNA antisense RNA 215, 264 double-stranded RNA (dsRNA) 240 ribosomal RNA 13–15, 30 intergenic spacers (IGS) 13, 14, 15, 30 internal transcribed spacers (ITS) 30 nucleolar organizing regions (NORs) 20 Rosaceae 54 rRNA see RNA Rubiaceae 259 rye see Secale cereale Saccharum 82, 120, 142, 143–144, 204, 208 safety storage 105 see also duplicate safety collections sampling procedures 61, 66, 67 satellite DNA see DNA Secale cereale 77, 79, 86, 90, 93, 138, 181, 204, 212 secondary metabolites 259 see also alkaloid, terpenoid, pyrethroid and pilocarpine seeds ex situ seed collections 103 see also ex situ conservation seed multiplication 69, 70 orthodox seeds 119, 120, 121 recalcitrant seeds 41, 119, 121 synthetic seed 126 see also encapsulation selection 53, 55, 68 sequence tagged sites (STS) 24, 31, 33, 35, 89 sequencing see DNA sequencing Simmondsia 265 simple sequence length polymorphisms (SSLPs) 14 simple sequence repeat (SSRs) 13, 14, 25, 30, 31, 34, 35, 80, 104, 107, 180, 206, 220 single-stranded conformation polymorphism (SSCP) 27 slot blots 28 see also dot blot slow-growth storage 124 snowdrop 215 Solanum tuberosum 5, 55, 77, 82, 108, 110, 120, 123, 129, 138, 141, 143, 144-145, 163, 208, 215, 220, 238, 242, 243, 245, 246–247, 264 somaclonal variation 121, 210, 212 somatic embryo see embryos sorghum 35, 77, 82, 91, 112, 208, 209, 210, 222 soybean 82, 207, 208, 209, 215, 216 Spartina anglica 55 species diversity 58 SSLP see simple sequence length polymorphism SSCP see single-stranded conformation polymorphism SSRs see simple sequence repeat STS see sequence tagged sites steroid 263 stratified sampling 65–66, 112, 113 strawberry see Fragaria Streptomyces avennitilis 257 sugar-beet 137, 208, 209 sugar-cane see Saccharum sunflower 215 super binary plasmid 209 sustainable 272 sweet orange see Citrus sweet potato see Ipomea batatas synteny see comparative mapping synthetic seed see seeds T-DNA tagging 216, 222–223 T-DNA 207–209 taro 124 Taxodium (bald cypress) 9 taxol 260 taxonomic identification 4, 106, 107, 109 Taxus 259–260 tea 138 technology transfer 270 Telnet 284 telomeres 14 teosinte 88, 89 see also Zea mays terpenoids 257 Thaumatococcus 272 Theobroma cacao 64, 119, 122 thermotherapy 237 tissue culture 5, 38, 120, 142, 235 cryopreservation 121, 127–143, 145, 165, 168, 172 embryo culture 90 embryo rescue 90 in vitro assays 6 Index 307 tissue culture continued in vitro collections 103, 121, 122 in vitro fertilization 91 microspore or ovule culture, 178 protoplasts 135, 210, 212 regeneration in culture 206–207, 209, 210, 211 slow-growth storage 124 somaclonal variation 121, 210, 212 somatic embryo 126, 129, 130, 137, 139, 140, 141, 143, 144, 145 vitrification 128, 130, 131–135, 137, 138, 139, 145 Tm 19 tobacco see Nicotiana tomato see Lycopersicon transcription factors 87 transformation 165, 204–213, 222, 223 transgenic plants see transformation, DNA transfer and Agrobacterium transposon tagging 86, 216, 217, 219 see also Ac/Ds Trifolium repens 60, 61, 65, 240 triple antibody sandwich (TAS-ELISA) see ELISA Triticeae 285 Triticum aestivum 54, 77, 79, 80, 82, 86, 89, 90, 91, 92, 93, 112, 140, 203, 204, 208, 210, 221, 222, 285 Tritordeum 92 trypsin inhibitor 214 turnip see Brassica oleracea Umbellularia californica 265 universal (uniform) resource locator (URL) 287 utilization 91 variable number of tandem repeats (VNTR) 12, 13, 14, 22, 25, 30, 32, 104 Valeriana wallichii 126 Veitchia 140 Vicia faba 240 see also bean Vigna 83, 164, 214 viroids see viruses virulence genes 207–210 virus 236–249 bioassays 239 resistance 205 viroids 236, 237, 241, 242 virus-free plants 120 vitrification 128, 130, 131–135, 137–139, 145 VNTR see variable number of tandem repeats wasabi 138, 139 Western blots 248 wheat see Triticum white spruce 125 wild cherry 125 see also Prunus World Wide Web (WWW) 285–296 YAC see yeast artificial chromosome yam 120, 123, 209 see also Dioscorea yeast 87 yeast artificial chromosome (YAC) 82, 86, 206, 220–222 Zea mays 28, 35, 77, 79, 82, 84, 86, 88, 89, 91, 140, 163, 182, 187, 197, 208, 209, 210, 213, 217, 221, 222, 288, 293 see also teosinte zygotic embryos see embryos 308 Index Callow00 Callow11 Callow13