E PLANT CELL CULTURE. Mary Taylor South Pacific Commission, Suva, Fiji. Contents

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1 E PLANT CELL CULTURE Mary Taylor South Pacific Commission, Suva, Fiji Contents 1. Introduction 2. The Basics of Plant Cell Culture 3. Propagation of Plant Material 3.1. Micropropagation 3.2. Somatic Embryogenesis 4. Plant Improvement 4.1. Callus and Suspension Cultures 4.2. Protoplast Fusion 4.3. Haploid Culture 4.4. Embryo Rescue 5. Conservation 5.1. Embryo Culture 5.2. Germplasm Storage In Vitro 6. Utilization of Plant Germplasm 6.1. Germplasm Exchange 6.2. Production of Secondary Metabolites 7. Conclusion Glossary Adventitious : Development of organs (roots, buds, shoots, flowers etc) or embryos from unusual points of origin, including callus. If organs develop from organ initials, organ primordia, or embryos develop from zygotes, the term adventitious cannot be used. Agar: A vegetable product (made from algae) used to solidify nutrient media. Aseptic: Free from all microorganisms. Auxins: Group of plant hormones, which induce cell elongation, or in some cases cell division; often inducing adventitious roots and inhibiting adventitious shoots. Axillary: Originating in the axils of the leaves. Callus : Actively dividing non-organized tissues of undifferentiated and differentiated cells often developing from injury or in tissue culture. Cytokinins: A group of plant hormones, which induce cell division and often adventitious shoots, and in most cases inhibit adventitious roots. Cytokinins decrease apical dominance. Differentiation: The development of cells or tissues with a specific function, and/or the regeneration of organs or organ-like structures. Explant: An excized piece of tissue or organ taken from the plant, used to initiate a culture. Ex situ: When a plant is grown outside of its natural habitat. This can refer to field, seed or in vitro genebanks. Hyperhydricity: This is the term used to describe a condition that can occur with shoots in culture. Affected shoots are often swollen and pale green, and their leaves have a translucent, watery or glass-like appearance. Such shoots can be difficult to propagate and fatalities can occur when transferring to a soil environment. In situ: When a plant is grown in its natural habitat.

2 In Vitro: When a plant is grown as a tissue culture plant in a culture container of some kind. Leaf primordia: Initials of a leaf. Macronutrients: Group of essential elements such as N, P, K, Ca and Mg, normally required in relatively large quantities by the plant. Meristem: Collection of dividing cells in the tip of a root, shoot (apical meristem), in the intercalary cambium of buds, leaves and flowers. Micronutrients: Group of elements such as Fe, B, Zn, Mo, Mn important in relatively small quantities for inorganic nutrition of plants. Nutrient medium: Mixture of substances on/in which cells, tissues or organs can grow, with or without agar. TDZ: 1-phenyl-3-(1,2,3-thiadiazol-5-yl) urea or thidiazuron. A compound that shows strong cytokinin activity, and encourages shoot initiation and proliferation. Virus-indexed: Virus-indexed plants have been assayed for the presence of known diseases according to standard testing procedures. Abbreviations 2,4-D: 2,4-dichlorophenoxyacetic acid. CIAT: Centro International de Agricultura Tropical. This is a Centre that is part of the International Agricultural Research Centres (IARCs), and is responsible for the conservation of, and research into cassava genetic resources. CIP: Centro International de Papa. This is also a member of the IARCs, and is responsible for the conservation of, and research into, primarily potato, but also sweet potato. ECS: embryogenic cell suspensions FAO: Food and Agriculture Organization of the United Nations, Rome. IBPGR: International Board of Plant Genetic Resources, now known as IPGRI, the International Plant Genetic Resources Institute. Psi: Pounds per square inch. SE: Somatic embryos TDZ: 1-phenyl-3-(1,2,3-thiadiazol-5-yl) urea or thidiazuron. PRSV-P: papaya ringspot virus-type P USDA: United States Development Agency Summary Plant cell culture is the basis of many different technologies that are now proving to be of great benefit to many disciplines. The ability to culture plant cells or tissues is essential to the success of all of these techniques. This paper introduces the basics of plant cell culture and discusses the techniques, which utilize the ability of plant cells to be cultured. All of these techniques have an agricultural application and are used throughout the world to improve agricultural productivity. Many of the techniques discussed here are crucial for genetic transformation research. These techniques range from the ability to produce plant cells in a form in which they can be transformed, to the regeneration of those transformed cells. The effective and efficient use of such techniques all require a basic understanding of plant cell culture. 1. Introduction The origin of plant cell culture is derived from an interest in determining how cells would behave when isolated from the whole plant. In the wild certain plants are capable of regeneration from small pieces of severed tissue. For example, dandelions proliferate from isolated roots, and Begonia plantlets directly from leaf tissue. These observations aroused an interest in the plasticity of plant

3 development and the potential for cell development if removed from the control of the whole plant (see also BG Cell Biology). In 1902, Gottlieb Haberlandt spoke of his vision for cell biology in the future: To my knowledge, no systematically organized attempts to culture isolated vegetative cells from higher plants in simple nutrient solutions have been made. Yet the results of such culture experiments should give some interesting insight into the properties and potentialities which the cell, as an elementary organism, possesses. Moreover it would provide information about the inter-relationships and complementary influences to which cells within the multicellular whole organism are exposed. Haberlandt was never able to induce cell division in vitro, but in 1934, White cultured tomato roots on a basic medium of inorganic salts, sucrose and yeast extract. In the same year, Gautheret found that cambial tissue of Salix capraea and Populus alba could proliferate for several months once aseptically isolated, but growth was limited. In 1939, as a result of recognizing the importance of B vitamins and the auxin, indole-3-acetic acid, Gautheret reported on the unlimited growth of a cell culture of carrot, which resulted in the production of viable callus. From this early work developed the concept of plant cell culture, enabling scientists, through the manipulation of plant cells, to develop techniques that would be of great benefit to a range of industries, such as agriculture and the pharmaceutical industry. Plant cell culture is a generic description used to describe the growth of microbe-free plant material in an aseptic (sterile) environment, such as sterilized nutrient medium in a test tube. From this early work in the twentieth century plant cell culture has developed far beyond what was first thought possible, and with the introduction of genetically modified organisms agriculture (see also BG Nitrogen fixation biotechnology) we are seeing the ultimate in manipulation of plant cell culture. During the development of this area, numerous techniques have been established which have, in many cases, resulted in practical application. These techniques can be categorized according to whether they can be used for propagation, improvement, conservation and utilization of plant germplasm (see also BG Genetic engineering of plant cells; BG Traditional Plant Breeding for Yield Improvement and Pest Resistance). 2. The Basics of Plant Cell Culture In plant cell culture, plant tissues and organs are grown in vitro on artificial media, which supply the nutrients necessary for growth. The precise composition of the culture medium will depend on what is required. Cell proliferation has different requirements to cell differentiation. However, basic growth is supported by a basic medium, which is generally composed of water, macro and micronutrients, and a carbohydrate source, usually sucrose, to replace the carbon, which the plant normally fixes from the atmosphere by photosynthesis. To improve growth, media can include trace amounts of certain organic compounds, such as vitamins, amino acids and plant growth regulators. Culture media can also contain what are known as undefined components. These include fruit juices, plant extracts, yeast extract etcetera. One of the most well known of these undefined components is coconut milk, which has been a popular addition for orchid culture. Although good results can be achieved using these ingredients, their use is not encouraged as their composition is not consistent and can vary each time they are used. Depending on whether or not the culture medium is to be solid or liquid, a gelling agent is added. Ninety-five percent of a culture medium is water, and therefore the quality of the water is important. It is recommended that the water is always distilled and in some cases, double distilled. Macro and

4 micronutrients are essential for growth, and so are found in all culture media. When the nutrient requirements of a plant are unknown, the nutrient composition as defined by Murashige and Skoog, can be used, providing that the plant is not sensitive to salt. Sugar is a very important component in any culture medium. A concentration of one to five percent saccharose (a disaccharide) is usually used, as this sugar is synthesized and transported naturally by the plant. The gelling agent usually used in culture media is agar. Agar, a seaweed derivative, is a polysaccharide with a high molecular mass. For medium of the optimum solidity, agar is usually added at a concentration between 0.6 to 0.8 percent. Growth regulators are added according to what is required from the culture, but also depends on the type of explant and the plant species. For example, eggplants which themselves produce sufficient auxin do not need extra auxin for cell extension and/or division. Similarly, with explants producing enough cytokinin, cytokinin will not have to be added to the medium. There are general rules that apply with the use of growth regulators but the individuality of the explant will always have some influence. Generally, auxins cause cell elongation and expansion, cell division and the formation of adventitious roots, inhibition of adventitious and axillary shoot formation, and embryogenesis in suspension cultures. With low auxin concentration, adventitious root formation occurs whereas if the auxin concentration is high, callus formation is a possibility. Cytokinins stimulate growth and development. In high concentrations they can be used to induce adventitious shoot formation, but root formation is generally inhibited. Care should be taken in the use of growth regulators, as there is evidence in the literature that excessive use of certain growth regulators can lead to a large number of mutations. Once the medium has been prepared it has to be at the correct ph. The ph used is between 5.5 and 6.0. If the ph is too high, this can stop growth and development. With a ph that is too low, gelling can be affected, as well as the uptake of some of the components. Finally the medium has to be sterilized, and this usually takes place in an autoclave. Providing exposure is sufficient, pressurized steam can destroy all microorganisms. The conditions for sterilization are 20 minutes at 121 C and 15psi. Figure 1. Sterile room, Regional Germplasm Centre, Secretariat of the Pacific Community, Fiji. The rate at which cultures grow can be influenced by the physical nature of the medium. Liquid medium is often chosen because it can result in faster rates of growth (see also BG Microbial Cell Culture). This is because a greater surface area of the explant is in contact with a liquid, and, providing the medium is agitated, the diffusion gradients between it and the explant are reduced. With some cultures, transfer to liquid medium after an initial stage of establishment on a solid medium works well. Bioreactors can be used with liquid medium when a mass propagation system is required. (See somatic embryogenesis). Some success has been achieved in the use of these, but there can be problems with hyperhydricity. 3. Propagation of Plant Material 3.1. Micropropagation A range of tissues can be taken and used as source material for micropropagation. These are: (a) Shoot meristems and stem segments with axillary buds; (b) Tissues that will either form (i) adventitious shoots and/or (ii) adventitious embryos either directly on explants or indirectly via organized or partly organized callus.

5 The aim of micropropagation is usually to produce clonal plants that are true-to-type, and therefore any system which utilizes an unorganized callus phase is usually avoided as this can result in the production of variants (see the basics of plant cell culture). Therefore, in practice, most micropropagation is achieved through multiplication of shoot tips and axillary buds, thereby maintaining organized tissues. The most common method used in micropropagation is the excision of apical or lateral shoots, containing meristems. These are then cultured on media, which will suppress apical dominance, and encourage the development of axillary shoots. The medium usually contains cytokinins, or cytokinin like substances such as TDZ. Culture on this medium can be repeated many times depending on the number of plants required. In some cases, roots will be produced on the same medium, but often the shoots have to be transferred to a medium lacking in cytokinins so that roots are induced. This multiplication technique is used with many important crops such as strawberry, banana, taro, to name but a few. Multiplication can also be achieved through the subculture of stem nodes. An individual stem node forms the explant and this is cultured until it has grown to produce several nodes. Then the whole process is repeated again. Potato, cassava, sweet potato, yams are some of the crops where this multiplication technique is used. Ornamentals are a group of plants where micropropagation has had a tremendous impact, possibly because of the high intrinsic value of the final product. Orchids were probably the first ornamental to be propagated through tissue culture utilizing the method developed by Morel, who introduced meristem culture as a means of vegetative propagation. His method found almost immediate commercial use, and a whole new market developed which placed orchids within economic reach of the average person. Tropical orchids are predominantly produced by tissue culture, with Thailand being the largest supplier. Other ornamentals in which tissue culture has had a significant impact on their production are Gerbera, Ficus, Anthurium, to name but a few. Micropropagation has also been useful in the production of temperate fruit and nut trees. With these crops, such as raspberry and blueberry, micropropagation brings the benefits of plant vigor, early and heavy cropping, and the possibility of disease-free plants. The technique has proved valuable when there has been a limited supply of rootstocks, for example, GF 677 rootstocks for peaches. Tropical fruit have also benefited from tissue culture techniques. Banana is the most important tissue cultured plant worldwide. In Taiwan, the micropropagation system for bananas is now well established, with an annual production of over two million plantlets. Similarly, in 1989, about 50 percent of the new banana plantations in Israel, were planted with micropropagated material. Micropropagated banana and plantain planting material is capable of performing equal to or superior to conventional planting material. Figure 2. Sweet potato and banana accessions in cool temperature growth room (20 C) Generally, micropropagated plants establish more quickly, are more vigorous, and taller, and have a shorter and more uniform production cycle (see also BG Traditional Plant Breeding for Yield Improvement and Pest Resistance). In addition, they produce higher yields than conventional propagules 3.2. Somatic Embryogenesis Somatic embryogenesis is another system whereby whole plants can be regenerated from plant tissues. Somatic embryogenesis is the production of embryo-like structures from somatic cells. A somatic embryo (SE) is an independent bipolar structure, not physically attached to the original

6 tissue that develops in the same way as a zygotic embryo. Two pathways of development are possible for SEs; they can either develop directly from the tissue being cultured or indirectly from callus. The latter method, indirect embryogenesis from liquid cell suspensions is desirable for micropropagation, providing clonal integrity can be guaranteed. Using this system, large numbers of SEs can potentially be produced in small volumes of culture media in a synchronous manner, thereby allowing partial mechanization and reduced labor costs. Somatic embryos can be easily managed using bioreactors. These are widely used in Cuba for commercial micropropagation, where success has been reported with coffee. The system can produce approximately 3,000 somatic embryos ready for hardening in a one-liter vessel over a period of six months. Using this technology, costs are reduced by a factor of ten, and there is an improvement in the quality of the embryos such that rapid development takes place. Immature zygotic embryos are common explants for the initiation of somatic embryogenesis, but they are of limited value in banana as edible cultivars rarely set seed. Therefore, in the case of banana the focus has been on the use of somatic tissues such as corm slices and leaf bases, immature male inflorescences and highly proliferating meristem cultures. The development of Musa embryogenic cell suspensions is a time consuming task but cell suspensions remain the optimum material for protoplast culture and the genetic manipulation of the banana plant (see protoplast fusion). They can also be stored for unlimited periods through cryopreservation. At the Laboratory of Tropical Crop Improvement in Leuven, Belgium, embryogenic cell suspensions are derived from scalps that consist of the upper 3 to 5mm of highly proliferating in vitro meristem cultures. They can be generated from any given banana or plantain cultivar within one to ten months. This protocol can be applied to a wide range of Musa genome groups and types. Established cell suspensions have been successfully used for the production of transgenic banana and regenerable protoplasts. Somatic embryogenesis is a technique that lends itself to the production of synthetic seeds. Synthetic or artificial seeds have been defined as somatic embryos engineered for use in the commercial propagation of plants. Through the combination of vegetative propagation, long-term storage, and clonal propagation, synthetic seeds can have many applications in agriculture. In the early days synthetic seeds were simply hydrated somatic embryos produced from vegetative cells in plant tissue culture. These had the advantages of rapid multiplication but the system was labor intensive, costly and the propagules were very delicate. To improve on this a technique was developed in which a single embryo is enclosed in an alginate capsule. Prolonged storage of these synthetic seeds is now possible as the somatic embryos can be dried to moisture content of less than 20 percent. This technology has been developed for alfalfa, and embryos can be dried down to 15 percent moisture and can then be stored for a year or more with good viability. However, it is not applicable to a wide range of cultivars because somatic embryogenesis is a genetic trait that is sexually heritable in alfalfa, and encoded by two independent dominant genes. Recurrent selection breeding programs are now on-going to generate lines that will have the required in vitro and field characteristics. 4. Plant Improvement 4.1. Callus and Suspension Cultures Callus is an unorganized, proliferative mass of differentiated plant cells, and usually occurs naturally as wound response. It can be induced through culture of plant tissue on a medium usually containing relatively high levels of auxin, especially 2,4-D. (see the basics of plant cell culture).

7 However, because of the phase of disorganization that occurs, plants regenerating from callus, can be prone to genetic change. In addition, it is often difficult to induce regeneration from callus. Callus has been used for mutation breeding, in which callus cultures are subjected to radiation, and then the regenerated plants are examined for changes. As a general rule, mutation breeding is difficult to control and frequently yields mosaics and deleterious changes. However, in some cases, beneficial changes can occur, for example, in sugar cane, plants were obtained that had resistance to the Fiji disease, which was a significant disease in the production of sugar cane. Suspension cultures can be produced from non- embryogenic or embryogenic callus, and are commonly used these days by molecular biologists for transformation research. Suspension cultures derived from non-embryogenic callus can be grown in the presence of an herbicide and plants regenerated from that callus can be tested for herbicide resistance. For example, with asparagus this technique was used to generate plants that had resistance to the herbicide, chlorsulfuron. More commonly, embryogenic suspension cultures are used. With many species the development of regenerable embryogenic cell suspensions (ECS) has provided the opportunity for the production of transgenic plants (see ; Transgenic Plants). The technique depends on the production of ECS and then particle bombardment of the ECS followed by plant regeneration. This technique is proving to be very successful with banana. One of the most promising developments is the expression in banana and plantain of antimicrobial peptides, some of which have been shown to exert in vitro fungistatic activity to Mycosphaerella fijiensis and Fusarium oxysporum, the causal agents of Black Sigatoka and Panama wilt disease, respectively Protoplast Fusion Protoplasts are produced by the enzymatic removal of the cell wall, through the use of mixtures of fungal cellulases, pectinases and hemicellulases in a solution of high osmotic potential. Removal of the cell wall then facilitates genetic manipulation of some kind. This can be a combination of two complete genomes; partial genome transfer from a donor to a recipient protoplast, to produce partial asymmetric hybrids; and transfer of organelles for the transfer of properties, such as herbicide resistance and cytoplasmic male sterility. Fusion of protoplasts can be induced chemically or by electrofusion. After fusion has occurred the resulting culture usually contains a mixture of fusion products and parental types, and so some method has to be adopted to distinguish between these two. There are a number of methods but the most commonly used are the complementation selection of mutants and fluorescence-activated cell sorters. Protoplast fusion and the resulting production of somatic hybrids have been of benefit in the improvement of potato. The main characteristics incorporated in potatoes via protoplast fusion include resistance to nematodes, virus, Erwinia, and also to low temperatures, originating from Solanum brevidens, a diploid species sexually incompatible with S. tuberosum. Protoplast fusion has also assisted in the improvement of commercial species of Brassica and Citrus. In cabbages, most of the work has focussed on the transfer of cytoplasmic inheritance characters, such as male sterility and resistance to herbicides. Plants with a crown and root system from different genotypes frequently form commercial plantations of Citrus. The tangerine, Cleopatra (Citrus reticulata) is a stock of great importance in Florida, because it is tolerant to a number of diseases including tristeza, and to stress caused by low temperatures and saline soils. However, it is not tolerant to nematodes and collar rot and so these susceptible traits limit its use. Recently there have been reports of somatic hybrids formed from protoplasts isolated from C. reticulata and Citropsis gilletiana, a species resistant to collar rot and to nematodes.

8 4.3. Haploid Culture Haploid plants, plants with gametic chromosome numbers, are particularly useful in plant breeding, both for the rapid production of homozygous lines following chromosome doubling to the original ploidy level, and for the detection and selection of recessive mutants. Although haploids occur naturally, in vitro techniques can be used for the routine production of haploids, and involves the regeneration of plants from cultured anthers, or immature pollen grains (microspores). However, isolated ovules can also be used, for example, in wheat and rice. Haploid plants have been produced in more than 50 species, with the majority in Gramineae, Solanaceae and Cruciferae. The conditions and procedures vary with the species, but certain factors are important for the efficient production of haploid plants. These are: growth of donor plants under optimum conditions; adequate sample of different genotypes; choice of correct stage of pollen development; pretreatment conditions for the anthers; and the effect of additives to the culture medium. The majority of plants resulting from an anther-derived culture are doubled haploids. In some crops this has been extremely beneficial. For example, in barley, the production of doubled haploid pure lines is far more rapid than the conventional method of repeated selfing, and has had a significant impact on the production of new barley varieties. The Chinese, who have produced varieties of rice, wheat and tobacco by anther culture, have exploited the quick production of inbred lines, following chromosome doubling of haploids. One of the most widely grown wheat varieties in China is a doubled haploid. Anther culture is being used in Egypt for selecting potato plants tolerant of salinity. Anthers are cultured in the presence or absence of sodium chloride, and the resulting embryoids were also cultured in the same way. The mean salinity tolerance index showed that the selected genotypes were more tolerant than the non-selected genotypes Embryo Rescue In sexual crosses where the parents are taxonomically distinct and distant, in vitro culture can be used to overcome the barriers that prevent successful hybrid production. In the cases where fertilization is successful but the embryo fails to develop, the immature embryo can be excized and cultured, and hybrid plants regenerated. Several diseases limit pawpaw production in Australia but the main concern is papaya ringspot virus-type P (PRSV-P). In an attempt to produce PRSV-P resistant varieties, procedures have been developed to hybridise papaya with the Carica species that are PRSV-P resistant. Cross-pollination takes place in a shadehouse and embryos are removed from immature fruits, harvested 90 to 120 days after pollination. A very efficient protocol has been developed for both the rescuing and the germination of the embryos. Most of the rescued embryos produce embryogenic callus and multiple plantlets. Hybrid plants have been produced between C. papaya and PRSV-P resistant species, C.quercifolia and C. pubescens. Using embryo rescue techniques unique hybrids have been obtained from crosses within the Tulipa species. Embryo rescue techniques have also been developed for chickpea, and hybrid plants between C. arietinum and C. pinnatifidum have been produced.

9 5. Conservation 5.1. Embryo Culture In vitro culture techniques have important applications for the collecting, exchange and conservation of coconut germplasm. Because of its large size, and its immediate germination after seed maturation, coconut-collecting missions can be problematic. Simple and efficient in vitro field collecting techniques have been established, which involve extracting the embryos from the nuts and inoculating them directly in vitro. For germplasm exchange, the FAO/IBPGR Technical Guidelines for the Safe Movement of Coconut Germplasm recommend that coconut germplasm be distributed as zygotic embryos in vitro to reduce chances of introducing diseased material into disease-free areas (see germplasm exchange) For conservation of coconut germplasm it has been shown that zygotic embryos can be stored in vitro for one year with no deleterious effect on viability. The feasibility of storing embryos on a long-term basis in liquid nitrogen has also been demonstrated (see germplasm storage in vitro) 5.2. Germplasm Storage In Vitro The conservation of plant genetic resources of crop plants and related species is particularly important to ensure future access to valuable genes for plant improvement programmes. Germplasm may be stored in situ in nature reserves or on-farm collections, or ex situ in seed banks, field genebanks, and in vitro genebanks. Ideally, for each species or gene pool, an integrated conservation strategy should be developed, involving more than one approach. The risks associated with field genebanks are many. These include exposure to pathogens, pests, climatic extremes and human error. The most economical way of storing germplasm is through seeds, however, some crops do not produce seeds, some seeds have a limited storage life, and some are heterozygous. For these crops/plants seed banks are not an option. In vitro storage is a possible strategy for many vegetatively propagated crops. This can either be achieved using slow growth storage or cryopreservation. Slow growth storage involves the culture of plants in conditions that result in a reduced rate of growth. Storage at reduced temperatures has been used with strawberry, vines, potato, grasses, legumes, for example. Another possibility is modification of the culture medium by inducing osmotic stress through the inclusion of osmoticums, retarding growth through the addition of growth retardants, or by reducing the carbon source supply. Its success is illustrated by its use in some large in vitro genebanks. These include international collections of over 5,000 accessions of cassava at CIAT (Centro Internacional de Agricultura Tropical), and over 4,000 accessions of potato at CIP (Centro Internacional de la Papa). However, despite the apparent success in the application of slow growth techniques, a number of concerns remain. One of the main concerns is the problem of genetic stability. Long-term storage using cryopreservation offers an alternative to slow growth. Cryopreservation completely arrests growth as material is held at a temperature of -196C. It is attractive because of the potential reduction in maintenance costs and reduced exposure of germplasm to contamination and genetic change. In addition, it is not very demanding on space in that very large collections can occupy relatively small areas. The National Clonal Germplasm Repository (USDA) in Oregon has cryopreserved collections of Corylus, Fragaria, Pyrus, Ribes and Rubes. Changli Horticultural Institute has a small collection of cryopreserved apple cultivars, which was initiated in the early 1990s. The technology for cryopreservation has advanced significantly in the last decade, especially

10 for tropical crops. For crops like cassava, banana, taro, protocols have been established, but these have yet to be applied to large collections. Laboratories are still trying to optimize techniques so that they can be applied to a wide range of genotypes, and to generate evidence that genetic integrity is maintained. 6. Utilization of Plant Germplasm 6.1. Germplasm Exchange Virus diseases can be found in most crop plants and depending on the severity of the infection, can cause serious losses. This also effects germplasm exchange in that plant material often cannot cross borders from one country to another because of the possibility of spreading disease. This can have a limiting effect on agricultural productivity. For example, in Samoa in the South Pacific, taro was not only the major staple; it was also the main export thus being a significant source of foreign exchange. In the mid-90s the crop was wiped out with the introduction of Phytophthora colocasiae, (taro leaf blight). With the importation of taro leaf blight resistant material to Samoa, taro is now being grown again (see germplasm storage in vitro). This introduction was made possible by importing resistant material as virus-indexed tissue cultures. Figure 3. Pacific taro collection in normal temperature growth room (25 C). Viruses can be eliminated from many plants through meristem culture. This requires the excision of the apical dome and adjacent leaf primordia and then the culture of this tissue under appropriate conditions so that the meristem can regenerate into a plant. The proposed theory is that there are far fewer virus particles in this area of the plant. In addition, the cells of the meristematic tissue divide quickly and so can out-divide the virus cells. With some plants meristem culture has to be accompanied by either heat treatment or chemotherapy. The former just requires exposure of the plant to relatively high temperatures for a period of time (10 to 20 days), prior to excision of the meristem. The latter requires treatment with chemical agents, either before excision or in the meristem culture medium. The virus status of plants regenerated from the meristems is then tested using various techniques. Not only does meristem culture and virus-indexed plants facilitate germplasm exchange, there is also growing evidence that meristem culture, and presumably the removal/reduction in viral charge results in plants with more vigor. In addition, these plants can produce higher and more significant yields over a two/three year period than plants that have not been treated in this way. Figure 4. Part of the pathogen-tested sweet potato collection; these accessions are available for germplasm exchange Production of Secondary Metabolites Secondary metabolites are molecules produced by plants, but not considered as essential for the primary metabolic function of plants [see also BG Secondary Products in Tissue Culture]. The possibility of using cultured cells, rather than the intact plant for their production has been recognized for over 30 years. Certain classes of secondary metabolites are produced in a stable form in undifferentiated plant cells. One notable example is the commercial production of the red pigment shikonin from cultured cells of Lithospermum erythrorhizon. Organized culture systems can also be used. Many compounds that are scarcely synthesized in undifferentiated cells are produced at higher levels in cultured roots. These roots grow well in the presence of auxin or when

11 transformed with Agrobacterium rhizogenes. Root cultures of Panax ginseng have been grown successfully in Japan. In many of the cases studied, cultured cells appear to have low levels of metabolites compared to the intact plant. It would seem that there are two factors, which effect production levels. The first relates to the heterogeneity and instability of gene expression and metabolic activity within a population of cultured cells, and the second to the inverse relationship that exists between secondary metabolite production and the rate of cell division. The successful production of shikonin from culturing the cells of L. erythrhizon demonstrates this. With shikonin, there is a two-phase production process. In the first phase the cells are grown up in bulk, and in the second, they are transferred to a production medium, which favors shikonin biosynthesis at the expense of cell division. Organized plant tissues, such as roots and shoots, generally produce higher levels of secondary metabolites and have greater genotypic and phenotypic stability than suspended plant cells. However, because the synthesis of many plant metabolites requires the participation of both the roots and shoots, the range of products that can be formed using single organ cultures is restricted. At the University of New South Wales a co-culture technique has been developed. Genetically transformed roots and shoots are cultured in liquid medium so that precursor compounds produced in one organ can be translocated through the medium, taken up by the other organ, and converted into the final product. 7. Conclusion Plant cell culture has made great advances since the early 1900s and the time of Haberlandt and White. The achievements made have surpassed what was imagined and the future appears to hold great promise. Perhaps the most significant role that plant cell culture has to play in the future will be in its association with transgenic plants. The system most commonly used in this area of research is still the production of callus/suspension cultures (usually embryogenic) and then regeneration from that culture. However, the more basic uses of plant cell culture should not be forgotten as these uses can often be of more value to the countries where the needs are the greatest. The ability to accelerate the conventional multiplication rate can be of great benefit to many countries where a disease or some climatic disaster wipes out crops, and urgent attention is required to prevent hunger and nutritional problems. The loss of genetic resources is a common story when germplasm is held in field genebanks. Slow growth in vitro storage and cryopreservation are being proposed as solutions to the problems inherent in field genebanks (see also BG Culture collections and genebanks). If possible, they can be used with field genebanks, thus providing a secure duplicate collection. They are the means by which future generations will be able to have access to genetic resources for simple conventional breeding programmes, or for the more complex genetic transformation work. Germplasm exchange, as already discussed is facilitated through tissue culture, from the ability to move virus-indexed germplasm without any quarantine implications together with the more physical advantage of alleviating transportation restrictions. Plant cell culture operates on all levels within agricultural development, from the very basic to the more complex, and either as a system in its own right, or as a tool to facilitate some other system. As such, it has a great role to play in agricultural development and productivity.

12 Bibliography Ashburner G. R., Faure M. G., Tomlinson D. R., and Thompson W. K. (1995). A Guide to the Zygotic Embryo Culture of Coconut Palms (Cocos nucifera L). in ACIAR Technical Reports. No 36. p. 16. ACIAR, Canberra, Australia, [A good technical report on the culture of coconut embryos.] Debergh P. C. and Maine L. (1983). Dracaena and Cordyline. Handbook of Plant Cell Culture. Vol. 5 (eds. Ammirato, P.V, D.A. Sharp, W.R. Sharp and Y.P.S. Bajaj., eds), pp New York: McGraw Hill Publishers. [A report on the culture of two ornamental species.] George E. F. (1993). Plant Propagation by Tissue Culture. Part 1. The Technology. 2 nd Edition. UK: Exegetics Ltd. [A comprehensive book on the theory of plant tissue culture.] Gray D. J. and Purohit A. (1991). Somatic embryogenesis and development of synthetic seed technology. Critical. Review. Plant. Science. 10, [An interesting paper on somatic embryos and the development of synthetic seeds.] Israeli Y., Reuveni O., and Lahav E. (1991). Qualitative aspects of somaclonal variations in banana propagated in vitro techniques. Scientia. Horticulture 48, [This paper discusses the problems of somaclonal variation in bananas.] Lindsey K. and Jones M. G. K. (1992). The biology of cultured cells. Plant Biotechnology in Agriculture. UK: Wiley and Sons Ltd. [A good chapter on cell culture looking at growth characteristics and aspects of structural organization.] Pierik R. L. M. (1997). In Vitro Culture of Higher Plants, 348 pp. The Netherlands: Kluwer Academic Publishers. [This provides a comprehensive introduction to tissue culture of plants.] Proc. Int. Symp. Biotechnology Tropical and Subtropical Species., ed. R.A. Drew (1998). Acta Horticulture 461 ISHS. [This provides information on the application of the techniques discussed in this paper to tropical and sub-tropical species.] Thinh Tien N., Takagi H., and Sakai A. (2000). Cryopreservation of in vitro grown apical meristems of some vegetatively propagated tropical monocotyledons by vitrification. Cryopreservation of Tropical Plant Germplasm. Current Research and Application. (eds Engelmann, F., and H. Takagi). Italy: IPGRI. [Paper discusses recent research into cryopreservation of crops such as taro. The book is an excellent review of recent cryopreservation research and current activities in this field.] Biographical Sketch Mary Taylor obtained her Masters in Horticulture and PhD in Plant Tissue Culture from the University of Bath in UK. The past 8 years she spent as the Regional Tissue Culture Adviser with an EU-funded regional agricultural programme [Pacific Regional Agricultural Programme - PRAP]. Most of these years she was based in Western Samoa at the University of the South Pacific Campus, but worked also with other countries in the region, such as Fiji, Tonga, Vanuatu, Soloon Islands, Papua New Guinea, Tuvalu and Kiribati. Her present position is as Tissue Culture Specialist for an AusAID funded Taro Genetic Resources Project which again has a regional impact. The TaroGen office is based at the Secretariat of the Pacific Community (SPC) in Suva, Fiji. In the early stages of her project, a Regional Germplasm Centre [RGC] was established at SPC, where she also acted as an adviser. The RGC is being developed to house collections of all the important crops in the Pacific, in vitro storage (slow growth and cryopreservation), field conservation, in situ conservation, intellectual property rights issues, and plant genetic resources issues.