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Electroporation utilizes high-voltage electric fields for cell permeabilization, This technique has been used for promoting the cellular uptake of exogenous molecules and macromolecules, including nucleotides, dyes, RNA, DNA, and even small proteins (1-7). Electroporation′s useful attributes are its simplicity and its general effectiveness with a wide range of cell types. Because of its general efficacy, electroporation is becoming a valuable technique for the introduction of DNA into cell types that are resistant to transformation by other procedures (6-8). In this chapter, we will outline procedures for introducing foreign DNAs into plant protoplasts by electroporation. DNA uptake is verified by the transient expression of chloramphenicol acetyltransferase (CAT), and by the recovery of plants stably transformed to kanamycin resistance through the introduction of the neomycin phosphotransferase gene. [ABSTRACT FROM AUTHOR]

Reliable methods for obtaining sterile explants (i. e., that part of the parent plant introduced to in vitro conditions) are critical in tissue culture. Normally, explants are soaked in disinfectants in order to eliminate the coating layer of microorganisms ubiquitously found on plants. This chapter is concerned with the use of mechanical and electronic techniques (i. e., an automated Surface Sterilization System [SSS]) to aid in the sterile establishment of explants. The automated SSS reduces labor input and increases the effectiveness and reproducibility of the surface sterilization treatment (1) (seeFig. 1). Numerous disinfectant types and techniques have been developed to surface sterilize plants (2). However, all procedures can be summarized as follows [ABSTRACT FROM AUTHOR]

This chapter is concerned with the use of mechanical and electronic techniques (i. e., the Automated Plant Culture System [APB]) to aid in the sterile cultivation of plants for the purpose of prolonging culture life and/ or increasing yields over that obtained from conventional technology (bd1,bd2). The use of machines to facilitate the culturing of plants in vitro is common (e. g., shakers, roller bottle apparatuses, incubators, magnetic stirrers, and roller drums). However, current techniques employed in plant tissue culture (i. e., agar or liquid culture), developed about 30 yr ago, are highly labor intensive because of the requirement for numerous hand-plant interactions in the culturing of plants in a sterile environment. Culture of any plant in vitro requires periodic transferring to fresh medium. Nutrient medium is either exhausted and/or altered by plant cultures, thus requiring the constant transfers to fresh medium (usually every 4-8 wk or sooner). The system presented in this chapter is meant to reduce dependence on labor through substitution of hand-plant interactions with machine-plant interactions via automatic medium replenishment (Fig. 1). The APCS culture vessel is, therefore, large enough to accommodate the increasing plant culture size associated with long-term growing of plants in a single culture vessel. [ABSTRACT FROM AUTHOR]

There have been numerous reports of enzyme synthesis in cultured plant cells and the presence of certain enzymes in the culture medium. Most of these studies have been performed in the context of the characterization of these enzymes from cell cultures. However, cell cultures-in particular those secreting enzymes into the medium-can be used as a process system for enzyme production. Surprisingly, there is very little information regarding the potential of plant cell cultures as a tool for enzyme productivity. [ABSTRACT FROM AUTHOR]

The diverse group of compounds known as plant secondary products includes many compounds with pharmaceutical activity (e.g., morphine, vincristine), fragrances, pigments, latex, enzymes, and carbohydrates (2). The commercial production of such plant secondary metabolites has, until recently, always been via the harvesting of field-grown plants followed by the extraction and/or direct utilization of the active principle(s). The proportion of the plant that is the active ingredient is invariably only a few percent of the total plant dry weight, (for example, the quinine content of Cinchona bark averages 6-14%) (2). Although annual crops are common for the production of such compounds, it is also often the case that the time required for field growth of a plant may be several years, 7-16 yr in the case of Cinchona, 5-7 yr in the case of the shikonin-yielding root of Lithospermum erythrorhizon (3). Field-grown crops are also subject to the vagaries of the weather, pests, diseases, and nutrient availability. These, with other biotic and abiotic factors (4), have combined to encourage the evaluation of in vitro systems for the production of plant secondary products as an economically sound alternative to conventional growth and processing systems. [ABSTRACT FROM AUTHOR]

Plant cell culture, the growth of plant cells on solid medium or in liquid, was originally used to study the physiology and biochemistry of plants without the complication of the whole plant. However, it was soon found that plant cell cultures were often capable of producing compounds characteristic of the original plant. Of particular interest were those compounds known as secondary products. Secondary products are poorly defined, but in general are products that are not essential for growth, but may confer some advantage. The plant kingdom produces a vast range of secondary products, many of which, such as morphine and diosgenin, are of commercial interest. Since plant cell cultures are capable of producing such compounds (1-3), they have been of interest for the development of a biotechnological process to produce these compounds. Initially, the accumulation of secondary products was fortuitous, but with selection and medium changes, high levels of the products have been achieved that in some cases exceed those found in the whole plant(2-4). [ABSTRACT FROM AUTHOR]

Plant cell culture has great potential as an alternative system for the production of phytochemicals normally extracted from whole plants, many of which have commercial value. Suspension cultures of plant cells have been shown to produce compounds characteristic of the original plant, and in some cases the yield of these phytochemicals has been higher than that from the plant. [ABSTRACT FROM AUTHOR]

Various immobilization strategies have been developed to optimize the biosynthetic potential of cultured plant cells. Immobilization involves the retaining of suspension-cultured plant cells on, or within, a physical barrier that promotes cell aggregation and separates the cells from the surrounding media. The advantages of immobilization include the formation of diffusionary gradients around and between the cells that increase intercellular biochemical communication conducive to the coordinated expression of secondary metabolism. In effect, immobilization simulates the physiological conditions within large aggregates of cells in a manner amenable to manipulation for fermentation purposes. [ABSTRACT FROM AUTHOR]

This chapter ties together ideas introduced in previous articles of this volume in the search for ways to use plant cell cultures as industrial production systems. The economics of a process must always be carefully considered when examining the suitability of plant cell culture for the manufacture of target compounds. The object is to produce the maximum amount of required product in as short a time as possible, using the most cost-effective method. New processes can then be compared with existing systems, and their feasibility assessed. This also requires a knowledge of the market valueof the product under consideration, the projected stability (or growth) of themarket, and the potential market share that your product could hope to attain. [ABSTRACT FROM AUTHOR]

Batch culture involves the inoculation of a known volume or mass of cells into a volume of (normally) defined medium. Growth is allowed to proceed, and the resulting biomass is harvested at some stage during the growth cycle. Throughout the growth period, no additions are made to the media, with the exception of acid and alkali, if needed, for pH control. The scale of operation depends on the requirements. [ABSTRACT FROM AUTHOR]

The large-scale or mass cultivation of plant cells is the growth of plant cell suspensions at volumes above those normally produced in shake flasks, that is, above IL. Attempts to grow plant cells in fermenters or bioreactors started in the early 1960s with converted carboys. The area has developed steadily, such that today bioreactors in excess of 5000 L have been used successfully for large-scale plant cell culture (1,2). Much of the early work was carried out using bioreactors designed for microbial culture. It was soon found, however, that although plant cell suspensions appear to be similar in many ways to microbial cultures, there are, in fact, key differences that can have a significant influence on large-scale cultivation. Plant cells are large, 20-40 µM in diameter, and up to 100 PM in length; further, they rarely occur as single cells, but as aggregates of up to 2 mm in diameter (Fig. 1). The individual plant cell soon after division is typically rounded, containing considerable amounts of cytoplasm; however, as it ages, the cell expands and becomes dominated by a large vacuole. In consequence, the overall metabolic activity is low compared with microbial cells, which in turn gives a very slow growth rate (measured in days, [ABSTRACT FROM AUTHOR]

The chloroplast genome encodes a number of proteins, including thylakoid proteins and the large subunit of ribulose biphosphate carboxylase, associated with the structure and function of the chloroplast (1-2). In addition, many components of the chloroplast translational machinery, such as all of the RNAs and some of the ribosomal proteins, are coded by the chloroplast DNA. Although there have been numerous investigations into the genetics of algal chloroplasts, similar studies with higher plants have been hampered by the uniparental (maternal) pattern of transmission of chloroplasts observed in most species, and the shortage of suitable genetic markers (3,4). [ABSTRACT FROM AUTHOR]

Mutants resistant to chemicals that inhibit growth (antimetabolites) are the most readily selected in plant cell cultures. A number of such mutants have been isolated, with resistance to amino acids and their analogs, base analogs, toxins from pathogenic microorganisms, herbicides, salts, and antibiotics. The mutants can be used for fundamental investigations into cellular metabolism, as markers for plant genetic manipulation, and in efforts to improve crop tolerance to diseases and agrochemicals. Progress in mutant selection has been reviewed extensively in recent years (1-4). Detailed considerations of the technical aspects of in vitro selection are available (1), and some protocols have been published (4-7). [ABSTRACT FROM AUTHOR]

The theory and goals of mutant selection from plant tissue cultures have been reviewed extensively over the past few years (I-5), and will only be dealt with in a cursory manner in this chapter. For more detail, the reader is encouraged to consult these review articles. The ideal plant tissue culture system is useful for mutation selection because it: [ABSTRACT FROM AUTHOR]

Mutants are a valuable tool for solving many problems in physiology, genetics, and molecular biology. Soon after cell suspension and protoplast culture emerged as techniques in plant biology, they were applied to the isolation of selectable markers that were unavailable through classical methods using whole plant systems. This field has been pioneered by Widholm in 1972 1,2, and seeChapter 38 and 40 this vol.) and reviewed several times (3-8). As far as selectable markers are concerned, mostly nuclear-coded mutations have been characterized, with theexceptionof chloroplast-encodedstreptomycinresistance (9,10) and lincomycin resistance (21). No selectable mutations are yet known for the mitochondrial genome in higher plants. [ABSTRACT FROM AUTHOR]

Mutant plant cell cultures can be useful in the study of the physiology and genetics of plants, as well for the improvement of crops. For recent reviews of mutant cell selection from tissue cultures, the reader is referred to Duncan and Widholm (1), Bright et al. (2), Flick (3), and Chapters 39-42, in this vol. Often these mutants have been obtained through selection procedures exploiting spontaneous mutations. However, the use of mutagens in culture systems has been shown to result in higher frequencies of mutant cells. Although mutagens can increase the frequency of the desired mutation in a population of cells, the frequency of undesirable mutations would also be increased. These undesirable mutations can make the produced material unusable because of properties such as slow growth, lack of plant regeneration, and sterility. Thus, one has to weigh the benefits of mutagenesis against the detrimental effects. [ABSTRACT FROM AUTHOR]

Plant tissue cultures are now well-recognized as valuable experimental systems for use in the study of host—pathogen interactions. These techniques have obvious major advantages for the examination of obligately biotrophic fungi and also those with a necrotrophic life style, and it is in these areas that much research effort has been concentrated (1). Success with combined fungal-plant cultures has been variable, especially in terms of establishing cultures that may be maintained in a balanced state for prolonged periods, but there is no doubt that such systems are useful places for the study of cell-cell interactions. [ABSTRACT FROM AUTHOR]

Protoplast fusion provides a nonsexual system for the transfer of genetic information between cell types. This transfer can be between species, genera, families, or kingdoms, thereby allowing unique opportunities to study somatic cell genetics in plants. Individual chromosomes (1) or pieces of chromosomes (2) have been transferred between species in unstable hybrids, but the technology also allows the transfer and recombination of organelles (3). The major obstacle in most protoplast fusion experiments has been developing strategies for the selection of somatic hybrids. This is mainly because of the fact that the fusion process itself is random. The isolation of heterokaryons from homokaryons and unfused protoplasts requires the use of a selection method. There are many selection strategies available (4), but one of the most efficient methods employs the use of resistance complementation (5). [ABSTRACT FROM AUTHOR]

Protoplast fusion produces cells that contain a mixture of the DNA-containing organelles (nuclei, chloroplasts, and mitochondria) from both fusion parents. This chapter describes the production of rapeseed (B. napus) fusion-product cells where one of the nuclei is eliminated. In the absence of selection pressure, the remaining chloroplast/mitochondrial mixture segregates randomly as the cell divides, eventually creating different nuclear/chloroplast/mitochondrial combinations. These novel nucleocytoplasmic combinations are called cytoplasmic hybrids, or "cybrids." [ABSTRACT FROM AUTHOR]

Isolated plant protoplasts can be induced to fuse with protoplasts from different species and, therefore, provide an ideal system for genetic modification and for use in plant breeding. Techniques for electrofusion of plant protoplasts have been developed relatively recently, and specialized apparatus is required, although this is now becoming more widely available. Electrofusion offers definite advantages over more commonly used protocols that make use of chemical stimulation to induce fusion. The most valuable aspects of electrofusion techniques are the high fusion frequencies attained, often tenfold higher than analogous chemical systems (1). The use of potentially toxic chemical stimulants is avoided, and the zones of membrane disturbance are limited to regions of membrane contact alone, all of which tends to preserve protoplast viability. Additionally, fusion events can be monitored microscopically, allowing the precise determination of the effects of various electrical parameters and, therefore, the use of optimum electrical values. Such control, together with manipulation of electrodes, allows more precise definition of parentage (2) than previously was possible with other methods. [ABSTRACT FROM AUTHOR]

The rapid development of tissue culture and recombinant-DNA technology in recent years has enabled plants of many species to be regenerated from cultured cells and their genetic information to be manipulated in various ways. Gene transfer has already resulted in the production of a wide range of transgenic dicotyledons, particularly in the Solanaceue. Although success with monocotyledons is more limited, transgenic rye (1), maize (2), and rice (3) plants have been reported. [ABSTRACT FROM AUTHOR]

The terms "meristem" and "shoot tip" culture have often been indiscriminately interchanged. According to Cutter 1), the apical meristem refers to only that portion of the shoot apex lying distal to the youngest leaf primordium. The shoot apex, or shoot tip, consists of the apical meristem and one to three subjacent leaf primordia. True apical meristem culture of higher plants was first demonstrated by Smith and Murashige 2). The isolated tissues of the apical domes develop directly into plants, demonstrating the developmental automony of the angiosperm shoot apical meristem. [ABSTRACT FROM AUTHOR]

A number of plant species have now been transformed, but there remains considerable uncertainty regarding the optimal procedure to be adopted. Agmbacterium species have been used as vectors for the DNA in some instances, whereas in others direct uptake has been achieved following chemical, physical, or electrical treatment of protoplasts or even of cells (seeChapters 27-30 of this volume). Viruses are another vehicle being considered in this context. One of the most effective techniques of transforming animal cells is that of microinjection, and this, together with the recent claim of very high rates of transformation of plant cells by microinjection (1), has heightened interest in the more general exploitation of this method of introducing DNA to plants. The technique is now sufficiently well developed to enable it to be more generally adopted, and this chapter will describe the method that we have developed. An important feature of the method is the use of low melting point (LMP) agarose, both for holding protoplasts during microinjection and for their subsequent culture. The method will be described in detail for tobacco mesophyll protoplasts, and then variations of the method for rape hypocotyl and sugar beet suspension culture protoplasts will be listed. [ABSTRACT FROM AUTHOR]

Agrobacterium tumefaciens, a soil bacterium that causes crown gall disease on a wide range of dicotyledonous plants, has been used most widely as a vehicle for gene transfer into plants (1,2). The transferred genes are stably integrated into the genomes of the transgenic plants and are transmitted to progeny as dominant mendelian traits (3,4). Since the production of auxins and cytokinins coded by oncogenes on T-DNA is often incompatible with normal plant development, it is often desirable to "disarm" the plasmids by removing the oncogenes and, therefore, allow the transformed plant cells to differentiate in a normal manner, rather than grow in a tumorous fashion. [ABSTRACT FROM AUTHOR]

Agrobacterium rhizogenes causes a disease in susceptible dicotyledonous plants characterized by a proliferation of differentiated root tissue (hairy root) at the site of bacterial injection and following the transfer and integration of the T-DNA from the Ri-plasmid into the host plant genome (1,2). Recently, there has been increased interest in transformation of plants by A. rhizogenes (3,4). The ease with which Ri-TDNA-transformed plant cells of some plant species can be regenerated to whole plants suggests that an Ri-vector system might be a useful alternative to a Ti-vector system. [ABSTRACT FROM AUTHOR]

The bacterial genus Agrobucterium includes two species of considerable interest to plant physiologists and pathologists alike. Infection by virulent strains of A. tumefaciens induces the formation of tumors and infection by A. rhizogenes the proliferation of roots in a wide range of dicotyledenous plants: crown-gall and hairy root diseases are not naturally recorded in monocotyledenous plants (1). Invasion of plant tissues by these free-living soil bacteria usually occurs at a wound site—possibly caused by insect or mechanical damage; nonvirulent strains of the bacteria may invade the plant, but only virulent strains give rise to symptoms of disease. [ABSTRACT FROM AUTHOR]

Plant protoplasts can internalize a variety of particles ranging from molecules (e.g., ferritin) to entire (algal) protoplasts, a size rangeof 0.09-23 µm (1). This capacity to take up foreign bodies provides a powerful tool with which to investigate the functions of cell organelles. Also, protoplasts are the material of choice from which to isolate organelles, because they are easy to rupture, and isolates are not contaminated with cell wall fragments. [ABSTRACT FROM AUTHOR]

A number of different methods have been developed for the enucleation of cells, particularly mammalian cells. Centrifugation of cells adhering to a surface may lead to the nucleus being drawn out from the cytoplasm, resulting in the formation of an enucleated cell or cytoplast and a nucleus surrounded by a thin layer of cytoplasm bounded by a plasma membrane (mini-cell). In those cases where cells do not adhere sufficiently firmly to a surface, enucleation has been achieved by centrifugation in density gradients. This method takes advantage of the differing densities of the nucleus and the cytoplasm. When cells are centrifuged at high speed in a suitable density gradient, the nucleus and the cytoplasm tend to float in those regions of the gradient matching their own densities. If the distance between the regions with densities corresponding to that of the cytoplasm and that of the nucleus is relatively long, considerable stretching forces are generated. Under optimum conditions, this force is usually sufficient to enucleate a high percentage of the cells. The use of density gradients and high-speed centrifugation for generating cytoplasts and mini-cells is much less explored in plant than in mammalian cells. [ABSTRACT FROM AUTHOR]

Plant protoplasts are cells from which the cell wall has been removed enzymatically. Thus, they retain all the normal cell organelles plus the nucleus; the latter is capable of expressing totipotency through the conversion of the protoplast to the regenerated plant using tissue culture technology. The fact that plant regeneration from protoplasts is possible for a large number of species, many of prime agronomic value, means that the protoplast is now seen as an ideal starting point for the gamut of genetic engineering technologies designed to bring about plant improvement. The protoplast is an invaluable tool for a variety of studies, including uptake of exogenously supplied materials, such as bacteria (1), algae (2), organelles (3), viruses (4), and macromolecules like DNA (5), as well as physiological investigations (6), transformation assessments (7), ultrastructural studies (8), and the isolation of subcellular components, including nuclei, chromosomes, and vacuoles (9). [ABSTRACT FROM AUTHOR]

Plantlets cultured in vitro on agar-based media in a water-saturated atmosphere wilt rapidly when transferred to normal greenhouse or field conditions. Water is rapidly lost from the leaves because stomata fail to respond to those stimuli that normally induce closure (1-4), and poor development of epicuticular wax results in loss of water through the cuticle (5-7). Uptake of water by the roots is limited by damage incurred during transplantation and by poor contact with the substrate. Problems of transplantation are accentuated in vitrified plantlets, which grow slowly and wilt rapidly. Reduced deposition of cellulose and lignin in these plantlets causes reduced cell wall pressure, leading to increased water uptake by the cells and a glassy turgescence of leaves and stems (8,9). [ABSTRACT FROM AUTHOR]

Maintenance of collections of succulent plants can be problematic, since many of these species are very susceptible to rots caused by bacteria and fungi. Rooting and establishment of cuttings can also be difficult. At Kew, methods for themicropropagation of cacti and other succulents have been developed over the last 10 yr. These have proved to be very useful for overcoming the problems mentioned above. [ABSTRACT FROM AUTHOR]

Sexual reproduction in higher plants consists of a series of complex processes, including pollination, double fertilization, embryo differentiation, and seed formation. Development of methods for pollination and fertilization under controlled conditions in vitro has greatly facilitated the study of these processes (1). In addition, manipulation of reproductive processes is possible by these methods. Thus, plants have been obtained following self-pollination in vitro with self-incompatible species, and hybrid plants have been obtained from interspecific and intergeneric crosses (2,3). Utilization of in vitro methods for wide hybridization has also provided a method for induction of parthenogenesis and development of haploid plants. [ABSTRACT FROM AUTHOR]

The commercial cultivation of rosaceous fruit trees (e.g., pear, apple, cherry, peach, plum) relies heavily upon the quality and performance of the rootstocks. This is even more the case now that self-rooted scions produce larger trees with a longer juvenile phase (1). It would, therefore, be of special interest for the fruit breeder to have general purpose rootstocks with a wide ecophysiological adaptation and high compatibility coupled with early cropping. In addition, many of the older and highly adapted scion varieties of fruit trees could benefit greatly from the introduction of stable, yet minor changes in their genome. Fruit trees are generally highly heterozygous, outbreeding, and thus are asexually propagated (seeChapter 10, this vol.). Consequently, genetic improvement is likely to be based on protoplast technology, and achieved mainly through somatic methods, such as somaclonal variation or somatic hybridization. [ABSTRACT FROM AUTHOR]

This chapter will deal with methods of clonal propagation for members of the two major morphological groups of orchids. The first group, sympodials, includes such genera as Cymbidium, Cattleya, Dendrobium, and Oncidium. They are characterized by a multi-branchingrhizome that can supply an abundance of axillary shoots for use as explants. They were among the first orchids to be successfully propagated, and techniques for their in vitro initiation (i.e., establishment) and subsequent proliferation are well established (1-8). The second group, monopodials, include Phaluenopsis and Vanda, and are characterized by a single, unbranched axis of growth that possesses few readily available axillary shoots for use as explants. Significantly different in their morphologies, the two groups require different approaches to explant selection and subsequent culturing. The successful large scale micropropagation of monopodials is, in fact, a relatively recent achievement (9): the culmination of a wide variety of studies using different media compositions and supplements (10-18). [ABSTRACT FROM AUTHOR]

In this chapter, a method by which many fern species can be successfully grown from spores in axenic culture will be described. Unlike the conventional method of sowing the spores on compost, this method allows spore populations free from contamination by spores of other species to be sown. The method can be used for the production of mature sporophytes or to provide a controllable system for biosystematic studies of, or experimentation with, fern gametophytes (1,2). [ABSTRACT FROM AUTHOR]

Pioneering research in Brassica microspore culture (1,2,3)rapidly led to the realization that microspores provide a powerful alternative to protoplast culture as a single-celled culture method in plants. These two single-celled systems are fundamentally different, both in tissue origin and in genetic variability. The microspore system improves significantly on the protoplast system by virtually eliminating the large somaclonal variation associated with protoplast selection, by utilizing a true haploid cell system, and by resulting in a more synchronized embryo development that facilitates accurate mutation and selection methods. Most critical, however, are the observations that plant regeneration frequencies in excess of 80% can be readily obtained and that the entire sequence from microspore isolation to plantlet development may take place in as little as 4 wk (4). [ABSTRACT FROM AUTHOR]

The initiation and development of embryos from differentiated tissue (somatic tissue) was first observed by Steward et al. (1) in 1958, but earlier researchers had already observed these formations in culture, without recognizing them as embryos. [ABSTRACT FROM AUTHOR]

The capacity of a cell to produce an embryo is not limited to the development of the fertilized egg. Cells of differentiated tissues (somatic tissues), which are first submitted to the action of an auxin and secondly transferred to a new medium without auxin, are capable of producing embryos (Fig. 1). It seems that the influence of a hormone, such as auxin, rejuvenates the cell so much that it reacquires the capacity of a zygote to produce an embryo. [ABSTRACT FROM AUTHOR]

The culture of embryos has several fundamental purposes. It allows observation of morphogenesis of isolated embryos at regular intervals and direct analysis of the various steps in embryogenesis. In this way, it gives more information than does fixation followed by histological analysis. Also, cultured embryos can undergo surgical operations similar to those performed with great success on animal embryos. In addition, embryo culture is a means of determining the nutritional factors favorable to normal embryogenesis. It is possible, by using appropriate media, to modify the pattern of embryo development. Indeed, if a modification of the experimental conditions of zygotic embryo culture provokes a more rapid growth, a higher survival, and an improved differentiation, it can be assumed that thenew conditions are closer to those that exist in the embryo sac. The culture of zygotic embryos therefore permits us to determine the conditions in which the embryo grows in the ovary. [ABSTRACT FROM AUTHOR]

The culture of fruit tissues as whole organs or isolated tissue sections has been conducted with various species (1). Whole, isolated ovaries have been successfully cultured to give rise to mature fruits (e.g., strawberry). Typically, however, when an isolated portion of the fruit tissue is introduced into a sterile environment, it immediately loses structural integrity and degenerates into a rapidly dividing callus mass (2). Loss of structural integrity is correspondingly associated with an alteration of physiology that is subsequently reflected in the production of an altered metabolism. Therefore, a meaningful study of fruit development using callus derived from fruit tissues is often not possible. Recently, we studied the parameters involved in the maintenance of citrus fruit tissue integrity (2). In this paper, the culture of isolated fruit tissues, as well as half and whole fruit culture, is demonstrated using the lemon fruit (Fig. 1-3). [ABSTRACT FROM AUTHOR]

A variety of factors contribute to flower induction in nature. These same factors are assumed to be responsible for in vitro flowering (I). Flower formation in tissue culture has been observed in several plant species, and arises from a variety of explant sources (2). However, the factors responsible for flowering in culture have not been extensively studied (I). Reasons for studying flower formation in tissue culture can be summarized as follows: [ABSTRACT FROM AUTHOR]

Graft formation in plants involves the severing of the vascular system with consequent loss of water and solute transport throughout the plant. This transport must be restored to prevent death resulting from nutrient starvation or dessication. [ABSTRACT FROM AUTHOR]

Micropropagation techniques are being used by an increasing number of research workers and commercial firms. The main use has been that of mass production of plants ranging from nursery stock species (such as rhododendron or rose), through ornamentals (such as fuchsia or carnation) to fruits (such as apples or raspberries) and vegetables and field crops (such as cauliflower, potato, or lupins) (1-5). As well as enabling the mass production of selected genotypes, micropropagation techniques provide a method whereby viral and bacterial pathogens can be eliminated from infected varieties (1). These techniques are described in Chapter 9, this volume. [ABSTRACT FROM AUTHOR]

The essence of meristem-tip culture is the excision of the organized apex of the shoot from a selected donor plant for subsequent in vitro culture. The conditions of culture are regulated to allow only for organized outgrowth of the apex directly into a shoot, without the intervention of any adventitious organs (1-3). The excised meristem-tip is typically small (often less than 1 mm in length) and removed by sterile dissection under the microscope (Fig. 1). It comprises the apical dome and a limited number of the youngest leaf primordia, and excludes any differentiated provascular or vascular tissues. A major advantage of working with such a small explant is the potential that this holds for excluding pathogenic organisms present in the donor plant from the in vitro culture (see below). A second advantage is the genetic stability inherent in the technique, since plantlet production from adventitious organs can be avoided (3-7). However, if there is no requirement for such benefits, then the related technique of shoot-tip culture (seeChapter 8, this vol.) may be more expedient for plant propagation. In this procedure, the explant is still a dissected shoot apex, but a much larger one, containing a relatively large number of developing leaf primordia. Typically, the explant is between 3-4 mm and 2 cm in length, and development in vitro is still regulated so as to be confined to outgrowth of an organized shoot, without adventitious propagation. [ABSTRACT FROM AUTHOR]

Regeneration of buds, shoots, and roots in cells and explants in vitro has provided useful developmental systems to analyze the processes of cell differentiation and morphogenesis. Interest in these studies was greatly stimulated by the demonstration of chemical regulation of morphogenesis in 1957 by Skoog and Miller (1). They showed that a high ratio of cytokinin to auxin-stimulated formation of buds when these chemicals were provided in the culture medium. Subsequently, their observations have been applied, extended, and found general application with numerous genera and species. [ABSTRACT FROM AUTHOR]

Plant cells can be totipotent, i.e., each cell may be capable of developing into an entire plant when provided with the correct environmental stimuli. Research during the last 30 yr has demonstrated that successful organogenesis in callus cultures can be achieved by the correct choice of medium components, selection of a suitable inoculum, and control of the physical environment (1). The manipulation of plant growth regulator concentration is probably the most widely used technique for the induction of organogenesis, and this methodology has formed the basis of the propagation of commercially important plants via tissue culture in recent years (2). [ABSTRACT FROM AUTHOR]

Plant tissues grown in vitro provide an ideal research tool for the study of a wide range of aspects of plant science. For example, they have been used in the investigation of both primary and secondary metabolism, cytodifferentiation, morphogenesis, plant tumor physiology, and the formation of plant hybrids via protoplast fusion techniques. Plant tissue culture is also being increasingly adopted for the commercial propagation of plants. [ABSTRACT FROM AUTHOR]

Growth and organogenesis in vitro is highly dependent on the interaction between naturally occurring endogenous growth substances and an analogous growth regulator added to the medium. It is often necessary to alter the growth regulator composition and/or concentration for in vitro culture according to the species or variety of plant being grown, the explant origin, the type of culture, and other medium constituents. A balance between auxin and cytokinin growth regulator is most often required for the formation of adventitious shoot and root meristems. The interactions found are often complex, and more than one combination of two substances is likely to produce optimum results. Figure 1 gives an example of the relative amounts of an auxin and a cytokinin that are often required to bring about some kind of morphogenesis. [ABSTRACT FROM AUTHOR]

Cryopreservation, that is, the viable storage of cells at the temperature of liquid nitrogen (-196°C), has wide relevance in many areas of pure and applied biology. Examples of its very successful use can be found in the storage of microbes and of semen (1). More recently, attention has been given to the development of cryopreservation methods for embryos, including those of humans, tissues, and organs for transplantation and blood components. In the context of plant research, realization of the potential applications of cryopreservation has been somewhat latent. However, several clear areas for application can be identified that involve the need to store exact genotypes in a stable state. [ABSTRACT FROM AUTHOR]

Experiments using plant tissue cultures, such as measuring their tolerance to herbicides or metabolite analogs, elucidating biochemical pathways by exposing the tissues to metabolic inhibitors, or cryopreserving the cultures, require at some point the determination of the tissue's viability. Viability is the capacity of a cell or organism to live, and the state of being viable is most commonly recognized by the growth of the studied tissues (1-3). Measurements of growth can include measuring increases in fresh or dry weight of a cultured tissue (2,3) or measuring increases in cell number or size (2-4). [ABSTRACT FROM AUTHOR]