Plant cell Culture: a review Presented by- Pushpendra kumar Tiwari M.Pharm(Pharmacognosy) Gayatri College of Pharmacy, Sambalpur Introduction Most methods of plant transformation applied to GM crops require that a whole plant is regenerated from isolated plant cells or tissue which have been genetically transformed. This regeneration is conducted in vitro so that the environment and growth medium can be manipulated to ensure a high frequency of regeneration. In addition to a high frequency of regeneration, the regenerable cells must be accessible to gene transfer by whatever technique is chosen. The primary aim is therefore to produce, as easily and as quickly as possible, a large number of regenerable cells that are accessible to gene transfer. The subsequent regeneration step is often the most difficult step in plant transformation studies. However, it is important to remember that a high frequency of regeneration does not necessarily correlate with high transformation efficiency. This chapter will consider some basic issues concerned with plant tissue culture in vitro, particularly as applied to plant transformation. It will also look at the basic culture types used for plant transformation and cover some of the techniques that can be used to regenerate whole transformed plants from transformed cells or tissue. Plant tissue culture, the growth of plant cells outside an intact plant, is a technique essential in many areas of the plant sciences. Cultures of individual or groups of plant cells, and whole organs, contribute to understanding both fundamental and applied science. It relies on maintaining plant cells in aseptic conditions on a suitable nutrient medium. The culture can be sustained as a mass of undifferentiated cells for an extended period of time, or regenerated into whole plants. The starting point for all tissue cultures is plant tissue, called an explant. It can be initiated from any part of a plant - root, stem, petiole, leaf or flower - although the success of any one of these varies between species. It is essential that the surface of the explant is sterilised to remove all microbial contamination. Plant cell division is slow compared to the growth of bacteria and fungi, and even minor contaminants will easily over-grow the plant tissue culture. The explant is then incubated on a sterile nutrient medium to initiate the tissue culture. The composition of the growth medium is designed to both sustain the plant cells, encourage cell division, and control development of either an undifferentiated cell mass, or particular plant organs. The concentration of the growth regulators in the medium, namely auxin and cytokinin, seems to be the critical factor for determining whether a tissue culture is initiated, and how it subsequently develops. The explant should initially form a callus, from which it is possible to generate multiple embryos and then shoots, forming the basis for plant regeneration and thus the technology of micropropagation. The first stage of tissue culture initiation is vital for information on what combination of media components will give a friable, fast-growing callus, or a green chlorophyllous callus, or embryo, root or shoot formation. There is at present no way to predict the exact growth medium, and growth protocol, to generate a particular type of callus. These characteristics have to be determined through a carefully designed and observed experiment for each new plant species, and frequently also for each new variety of the species which is taken into tissue culture. The basis of the experiment will be media and protocols that give the desired effect in other plant species, and experience. 2. History History of Tissue Culture Techniques - The in vitro techniques were developed initially to demonstrate the totipotency of plant cells predicted by Haberlandt in 1902. Totipotency is the ability of a plant cell to perform all the functions of development, which are characteristic of zygote, i.e., ability to develop into a complete plant. In 1902, Haberlandt reported culture of isolated single palisade cells from leaves in Knop's salt solution enriched with sucrose. The cells remained alive for up to 1 month, increased in size, accumulated starch but failed to divide. Efforts to demonstrate totipotency led to the development of techniques for cultivation of plant cells under defined conditions. This was made possible by the brilliant contributions from RJ. Gautheret in France and P.R. White in U.S.A. during the third and the fourth decades of 20th century. Most of the modern tissue culture media derive from the work of Skoog and coworkers during 1950s and 1960s. The first embryo culture, although crude, was done by Hanning in 1904; he cultured nearly mature embryos of certain crucifers and grew them to maturity. The technique was utilised by Laibach in 1925 to recover hybrid progeny from an interspecific cross in Linum. Subsequently, contributions from several workers led to the refinement of this technique. Haploid plants from pollen grains were first produced by Maheshwari and Guha in 1964 by culturing anthers of Datura. This marked the beginning of anther culture or pollen culture for the production of haploid plants. The technique was further developed by many workers, more notably by JP. Nitch, C. Nitch and coworkers. These workers showed that isolated microspores of tobacco produce complete plants. Plant protoplasts are naked cells from which cell wall has been removed. In 1960, Cocking produced large quantities of protoplasts by using cell wall degrading enzymes. The techniques of protoplast production have now been considerably refined. It is now possible to regenerate whole plants from protoplasts and also to fuse protoplasts of different plant species. In 1972, Carlson and coworkers produced the first somatic hybrid plant by fusing the protoplasts of Nicotiana glauca and N. langsdorfii. Since then many divergent somatic hybrids have been produced. A successful establishment of callus cultures depended on the discovery during mid-thirties of IAA (idole-3-acetic acid), the endogenous auxin, and of the role of B vitamins in plant growth and in root cultures. The first continuously growing callus cultures were established from cambium tissue in 1939 independently by Gautheret, White and Nobecourt. The subsequent discovery of kinetin by Miller and coworkers in 1955 enabled the initiation of callus cultures from differentiated tissues. Shoot bud differentiation from tobacco pith tissues cultured in vitro was reported by Skoog in 1944, and in 1957 Skoog and Miller proposed that root-shoot differentiation in this system was regulated by auxin-cytokinin ratio. The first plant from a mature plant cell was regenerated by Braun in 1959. Development of somatic embryos was first reported in 1958- 1959 from carrot tissues independently by Reinert and Steward. Thus within a brief period, the tissue culture techniques have made a great progress. From the sole objective of demonstrating the totipotency of differentiated plant -cells, the technique now finds application in both basic and applied researches in a number of-fields of enquiry. 3. Facilities & Requirements Like most other laboratories, tissue culture laboratory needs the sophisticated equipment and facilities. The specific needs of the laboratory can be principally divided into three categories: 1. Essential facilities. 2. Beneficial facilities. 3. Useful facilities. Essential facilities – 1. Incubator 2. Autoclave 3. Refrigerator & Freezer 4. Microscope 5. Washing up Equipments 6. Oven for dry sterilization 7. Water purification 8. Centrifuge 9. Shakers 10. Shelves 11. Culture Room and/or Cabinate 12. Laminar Air Flow cabinet Beneficial Equipment – Desirable additions Useful additions Laminar flow hood Cell counter Vacuum pump CO2 incubator Osmometer Densitometer Phase contrast and fluorescent Microscope Roller racks Magnetic stirrer Separate sterilizing oven and drying oven -70o C freezer Glassware washing machine Colony counter High capacity centrifuge 4. Laboratory: Media storage Transfer room Culture room Office Lockers Lunch Shipping Cold storage There are many textbooks describing very sophisticated laboratory systems for plant cell cultures. However, it is not always necessary to design special laboratories for this technology, but general microbiology laboratories can be used, although aseptic conditions are a prerequisite for incubation of plant cells as well as microbial cultures. The following equipment is required: Laminar air flow cabinets: The cabinets are commercially available in different sizes. They are placed in the laboratory where needed. If there is a sterile room, the cabinets are not always necessary. Autoclave: Autoclaves in different sizes are commercially available. Oven for dry sterilization: Although autoclaves can be used for dry sterilization, an oven is useful for sterilization of scalpels and glass-wares such as petri-dishes, pipets and others. Equipment for sterilization by filtration: The medium containing carbon sources and growth regulators are simultaneously sterilized using a autoclave but sometimes aseptic filtration is favorable to avoid decomposition of unstable chemicals. The equipment is also commercially available. Water distillation apparatus or pure water demineralizer: To prepare media, distilled water or deionized water is generally used although tap water can be used particularly in large- scale cultivation of plant cells in a large fermentor from an economical point of view. Culture rooms and/or Cabinets: To cultivate the plant cells, culture rooms under different temperature or and/or cabinet-type incubators are essential facilities. Temperature and light intensity as well as a duration of lighting in the room and/or in the cabinet are controlled under the optimal conditions. Shelves: Shelves built from rigid wire mesh to allow maximum air movement and minimize shading should be used in the culture room. Shakers: A rotary shaker or a reciprocal shaker is necessary for suspension cultures. Fermentors or bioreactors In order to cultivate plant cells in a large-scale, fermentors with different sizes are useful. Various types of fermentors have been designed by many researchers since the end of 1950's as seen in Fig. 3. The most simple vessel is a carboy system described by Tulecke and Nickell in 1959 (10) which consists of a rubber-stoppered 20 L carboy fitted with four tubes (air-in, air-out, medium-in and sample-out). Filtered compressed air is employed for oxygen supply, aeration and agitation of the medium. A roller-bottled system using a round flask was used by Lamport (31) in 1964. A V-shape fermentor was proposed by Veliky and Martin (32). It is an inverted flask carrying two teflon-coated stirring bars on a glass pin situated at the bottom of the flask. A drain/sample port is also located at the bottom. The top of the flask is fitted with four standard taper penetrations. However, the most common types of system on the bench is a stirred-jar fermentor which is used for microbial cultivation although some minor alteration is made. For example Martin et al. (33) increased the size of each impeller blade to 1 inch with a commercially available 7.5 L New Brunswick Microferm Fermentor. Kato et al. (34) suggested that an agitation speed of 50 to 100 r.p.m. was most appropriate for the growth of tobacco cells in stirred-jar fermentors. It is true that cultured plant cells are more fragile than bacterial cells, however, Martin noted: "it seems obvious that cell lines differ in their resistance to shear effects and that a single optimum agitation speed cannot be designed for all lines" (35). Wagner et al. (36) compared a variety of fermentor types equipped with different agitation and aeration systems with various productivities of cell mass and anthraquinones using Morinda citrofolia cells, and recommended the air-lift type fermentor as the most suitable system (Fig. 4). Tanaka et al. (37) designed a rotary-drum type fermentor having an in-let and an out-let at the side of the fermentor (Fig. 5). The fermentor itself rotates slowly like as a rotary bottle. Recently, Ten Hoopen et al. (38) discussed the problems and profiles of large-scale plant cell culture. However, fermentors installed with agitators which are similar to those of microbial culture have been employed for commercial production of shikonin and ginseng cells although some modification in equipment was made to optimize physical conditions. A company in Germany, DIVERSA, has equipped 5 sophisticated fermentors of up to 75,000 L for plant cell cultures (39). Although detailed modifications to those fermentors has not been disclosed, the photos show that they are similar to ordinary microbial fermentors. The company has cultivated Echinacea purpurea cells for the manufacture immunobiologically active polysaccharides. Figure 3: Various Fermentors for Plant Cell Cultures Located in the heart of the Blue Ridge Mountains of North Carolina, MissVitro Plant Tissue Culture Laboratory specializes in the production of quality Hosta TC Liners. The laboratory, located in our home, has all the equipment, supplies, and technology required to quickly reproduce your favorite Hosta, or hosta liners for your Garden Center or Nursery. Our clients, Hosta Growers, both large and small, as well as individual Hosta enthusiasts, send us dormant Hosta buds or actively growing plants. We propagate them in our laboratory by means of tissue culture. Although some hostas have proven to be difficult to tissue culture due to complex variegation patterns, such as the streaked varieties, most plants "come true" in the TC Laboratory, including many of the medio variegated varieties, and virtually all of the solid colored ones. we ship top quality plants back to our clients, either growing in liners, or as bare root plants. Plants are shipped only after they have become established in a soil-less mix. You receive a healthy vigorous plant ready to grow-on at your location. If the plant you send us for TC is your hybrid or mutation and not yet in the trade you retain all rights to all plants produced, for an agreed upon period of time, or indefinitely if you wish. Although it is necessary for us to require a $50.00 minimum order, there is no limit after that, large or small, on the number of plants you would like for us to produce for you. 5. Methodology: The variety of techniques which can be used to get plant development in vitro (that is, by techniques such as tissue culture) is considerable and completely dependent upon the species in question. Single cells of leaf tissue can regenerate whole plants, as can shoot tips, leaf pieces, root pieces, lateral buds, or stem sections. Not all of these methods are applicable to woody plants, and indeed, not all have been applied to a commercial level to any plants. Shoot tip culture is the method in widest use for the mass propagation of woody species. An actively growing shoot tip is surface sterilized and placed on a defined culture medium under sterile conditions. The culture medium contains inorganic and organic salts (macronutrients, micronutrients and vitamins) as well as an energy source (sucrose or table sugar), growth regulators, and agar )to gel the medium). If the growth regulators are appropriately balanced, the shoot tip elongates, lateral buds break and begin growth, and adventitious shoots are also produced on the stem piece. This rapid proliferation of shoots results in masses of shoots being produced from a single shoot tip. Up to a hundred shoots may be produced in as little as eight to twelve weeks from a single tip. The number of shoots produced and the rapidity of shoot proliferation varies between species, and in some cases, between cultivars of a single species. Shoots are removed from the cultures at regular intervals and a portion of the mass is replaced on fresh media to continue proliferation. The small shoots which are removed are then rooted in a separate medium, either a sterile gelled medium or a peat-perlite medium (in much the same fashion as conventional woody cuttings). Shoots produced through tissue culture are generally easy to root, even though the same cultivar may be difficult to root by cutting propagation. The resulting rooted shoot is referred to as a "plantlet" because of its miniature size, but these can generally be grown in the greenhouse at a rapid rate and with a high degree of survival. Techniques for rooting tissue cultured shoots are currently receiving a great deal of research attention as are methods for establishing these shoots into the greenhouse environment. 6. Culture media: Media - Plants in nature can synthesize their own food material. In contrast, plants growing in vitro are heterotrophic, Le., they cannot synthesize their own food material. Plant tissue culture media therefore require all essential minerals plus a carbohydrate source usually added in the form of sucrose and also other growth hormones (regulators and vitamins). Growth and morphogenesis of plant tissues in vitro are largely governed by the composition of the culture media. Although the basic requirements of cultured plant tissues are similar to those of whole plants, in practice nutritional components promoting optimal growth of a tissue under laboratory conditions may vary with respect to the particular species. Media compositions are thus formulated considering specific requirements of a particular culture system. 'For example, some tissues show better response on a solid medium while others prefer a liquid medium.Considerable progress has been made during the past two decades on the development of media for growing plant cells, tissues and organs aseptically in culture. A significant contribution to formulation of a defined growth medium suitable for a wide range of applications was made by Murashige and Skoog (1962), In their work to adapt tobacco callus cultures for use as a hormone bioassay system, they evaluated many medium constituents to achieve optimal growth of calluses. In so doing, they improved upon existing types of plant tissue culture media to such an extent that their medium (the MS medium) has since proved to be one of the most widely used in plant tissue culture work gives the composition of different media. Composition of Culture Media 1. Inorganic Salts 2. Carbon Sources 3. Vitamins 4. Phytohormones 5. Organic supplements 6. Trace elements Inorganic Salts – o To induce a callus from an explant and to cultivate the callus and cells in suspension, various kinds of media (inorganic salt media) have been designed. o Agar or its substitutes is added into the media to prepare solid medium for callus induction.One of the most commonly used media for plant tissue cultures is that developed by Murashige and Skoog (MS) for tobacco tissue culture (28). o The significant feature of the MS medium is its very high concentration of nitrate, potassium and ammonia. The B5 medium established by Gamborg et al. o (29) is also being used by many researchers. The levels of inorganic nutrients in the B5 medium are lower than in MS medium. o Many other media have been developed and modified and nutrient compositions of some typical media will be described in Table 6. o However, it is not always necessary to test many kinds of basal media when a callus is induced. o It would be better to use only one or two kinds of basal media in combination of different kinds and concentrations of phytohormones. o The most suitable medium composition should be optimized afterwards in order to obtain higher level of products as well as higher growth rate. Carbon Sources – o Sucrose or glucose at 2 to 4% are suitable carbon sources which are added to the basal medium. o Fructose, maltose and other sugars also support the growth of various plant cells. o However, the most suitable carbon source and its optimal concentration should be chosen to establish the efficient production process of useful metabolites. o These factors depend on plant species and products, therefore it is necessary to optimize the medium compositions including carbon sources in each case. o From an economical point of view, the use of more inexpensive carbon sources is appropriate in industry and crude sugars such as molasses have been examined. Vitamins – o The basal media described above such as MS medium include myo-inositol, nicotinic acid, pyridoxine HCl and thiamine HCl. Among these vitamins, thiamine is an essential one for many plant cells and other vitamins stimulate the growth of the cells in some cases. o The level of myo-inositol in the medium is 100 mg/L which is very high although it is not clear whether such a high level of the vitamin is required. Phytohormones – o Phytohormones or growth regulators are required to induce callus tissues and to promote the growth of many cell lines. o As an auxin, 2,4-dichlorophenoxyacetic acid (2,4-D) or naphthaleneaceic acid (NAA) is frequently used. o The concentration of auxins in the medium is generally between 0.1 to 50 µM. Kinetin or benzyladenine as a cytokinin is occasionally required together with auxins for callus induction at concentrations of 0.1 to 10 µM. Other derivatives of auxin and kinetin are also used in some cases. o Since each plant species requires different kinds and levels of phytohormones for callus induction, its growth and metabolites production, it is important to select the most appropriate growth regulators and to determine their optimal concentrations. o Gibberellic acid is also added to the medium if necessary. Organic supplements – o In order to stimulate the growth of the cells, organic supplements are sometimes added to the medium. o These supplements include casamino acid, peptone, yeast extracts, malt extracts and coconut milk. Coconut milk is also known as a supplier of growth regulators. Trace element – Boron (B) BO — Manganese (Mn) Mn++ Zinc (Zn) Zn++ Copper (Cu) Cu+, Cu++ Molybdenum (Mo) MoO4- Chlorine (Cl) Cl- Nickel (Ni) - Aluminium (Al) - Cobalt (Co) - Iodine (I) - Ferrous (Fe) - Sodium (Na) - Most commonly used tissue culture media along with the comparison components Constituents Concentration in culture medium Mg/liter Storage Ms SH B5 A Macronutrients KNO3 NH4NO3 NH4H2PO4 (NH4)2SO4 MgSO4 . 7H2O CaCl2 . 2H2O KH2PO4 NaH2PO4 . 2H2O B Micronutrients MnSO4 . H2O MnSO4 . 4H2O KI H3BO3 ZnSO4 .7H2O CuSO4 . 5H2O Na2MoO4 . 2H2O CoCl2 . 6H2O C Iron Source FeSO4 . 7H2O Na2EDTA D Vitamins Nicotinic acid Pyridoxin-HCI Thyamine-HCI Myo-inositol Glycine E Growth regulators IAA 2,4 –D Kinetin (K) F Sucrose 7. Culture types Cultures are generally initiated from sterile pieces of a whole plant. These pieces are termed ‗explants‘, and may consist of pieces of organs, such as leaves or roots, or may be specific cell types, such as pollen or endosperm. Many features of the explant are known to affect the efficiency of culture initiation. Generally, younger, more rapidly growing tissue (or tissue at an early stage of development) is most effective. Several different culture types most commonly used in plant transformation studies will now be examined in more detail. 1. Callus Explants, when cultured on the appropriate medium, usually with both an auxin and a cytokinin, can give rise to an unorganised, growing and dividing mass of cells. It is thought that any plant tissue can be used as an explant, if the correct conditions are found. In culture, this proliferation can be maintained more or less indefinitely, provided that the callus is subcultured on to fresh medium periodically. During callus formation there is some degree of dedifferentiation (i.e. the changes that occur during development and specialization are, to some extent, reversed), both in morphology (callus is usually composed of unspecialised parenchyma cells) and metabolism. One major consequence of this dedifferentiation is that most plant cultures lose the ability to photosynthesise. This has important consequences for the culture of callus tissue, as the metabolic profile will probably not match that of the donor plant. This necessitates the addition of other components—such as vitamins and, most importantly, a carbon source—to the culture medium, in addition to the usual mineral nutrients. Callus culture is often performed in the dark (the lack of photosynthetic capability being no drawback) as light can encourage differentiation of the callus. During long-term culture, the culture may lose the requirement for auxin and/or cytokinin. This process, known as ‗habituation‘, is common in callus cultures from some plant species (such as sugar beet). Callus cultures are extremely important in plant biotechnology. Manipulation of the auxin to cytokinin ratio in the medium can lead to the development of shoots, roots or somatic embryos from which whole plants can subsequently be produced. Callus cultures can also be used to initiate cell suspensions, which are used in a variety of ways in plant transformation studies. 2. Cell-suspension cultures Callus cultures, broadly speaking, fall into one of two categories: compact or friable. In compact callus the cells are densely aggregated, whereas in friable callus the cells are only loosely associated with each other and the callus becomes soft and breaks apart easily. Friable callus provides the inoculum to form cell-suspension cultures. Explants from some plant species or particular cell types tend not to form friable callus, making cell-suspension initiation a difficult task. The friability of callus can sometimes be improved by manipulating the medium components or by repeated sub-culturing. The friability of the callus can also sometimes be improved by culturing it on ‗semi-solid‘ medium (medium with a low concentration of gelling agent). When friable callus is placed into a liquid medium (usually the same composition as the solid medium used for the callus culture) and then agitated, single cells and/or small clumps of cells are released into the medium. Under the correct conditions, these released cells continue to grow and divide, eventually producing a cell-suspension culture. A relatively large inoculum should be used when initiating cell suspensions so that the released cell numbers build up quickly. The inoculum should not be too large though, as toxic products released from damaged or stressed cells can build up to lethal levels. Large cell clumps can be removed during subculture of the cell suspension. Cell suspensions can be maintained relatively simply as batch cultures in conical flasks. They are continually cultured by repeated subculturing into fresh medium. This results in dilution of the suspension and the initiation of another batch growth cycle. The degree of dilution during subculture should be determined empirically for each culture. Too great a degree of dilution will result in a greatly extended lag period or, in extreme cases, death of the transferred cells. After subculture, the cells divide and the biomass of the culture increases in a characteristic fashion, until nutrients in the medium are exhausted and/or toxic by- products build up to inhibitory levels—this is called the ‗stationary phase‘. If cells are left in the stationary phase for too long, they will die and the culture will be lost. Therefore, cells should be transferred as they enter the stationary phase. It is therefore important that the batch growth-cycle parameters are determined for each cell-suspension culture. 3. Protoplasts Protoplasts are plant cells with the cell wall removed. Protoplasts are most commonly isolated from either leaf mesophyll cells or cell suspensions, although other sources can be used to advantage. Two general approaches to removing the cell wall (a difficult task without damaging the protoplast) can be taken—mechanical or enzymatic isolation. Mechanical isolation, although possible, often results in low yields, poor quality and poor performance in culture due to substances released from damaged cells. Enzymatic isolation is usually carried out in a simple salt solution with a high osmoticum, plus the cell wall degrading enzymes. It is usual to use a mix 44 2 : Plant tissue culture of both cellulase and pectinase enzymes, which must be of high quality and purity. Protoplasts are fragile and easily damaged, and therefore must be cultured carefully. Liquid medium is not agitated and a high osmotic potential is maintained, at least in the initial stages. The liquid medium must be shallow enough to allow aeration in the absence of agitation. Protoplasts can be plated out on to solid medium and callus produced. Whole plants can be regenerated by organogenesis or somatic embryogenesis from this callus. Protoplasts are ideal targets for transformation by a variety of means. 4. Root cultures Root cultures can be established in vitro from explants of the root tip of either primary or lateral roots and can be cultured on fairly simple media. The growth of roots in vitro is potentially unlimited, as roots are indeterminate organs. Although the establishment of root cultures was one of the first achievements of modern plant tissue culture, they are not widely used in plant transformation studies. 5. Shoot tip and meristem culture The tips of shoots (which contain the shoot apical meristem) can be cultured in vitro, producing clumps of shoots from either axillary or adventitious buds. This method can be used for clonal propagation. Shoot meristem cultures are potential alternatives to the more commonly used methods for cereal regeneration (see the Case study below) as they are less genotype-dependent and more efficient (seedlings can be used as donor material). 6. Embryo culture Embryos can be used as explants to generate callus cultures or somatic embryos. Both immature and mature embryos can be used as explants. Immature, embryo-derived embryogenic callus is the most popular method of monocot plant regeneration. 7. Microspore culture Haploid tissue can be cultured in vitro by using pollen or anthers as an explant. Pollen contains the male gametophyte, which is termed the ‗microspore‘. Both callus and embryos can be produced from pollen. Two main approaches can be taken to produce in vitro cultures from haploid tissue. The first method depends on using the anther as the explant. Anthers (somatic tissue that surrounds and contains the pollen) can be cultured on solid medium (agar should not be used to solidify the medium as it contains Culture types 45 inhibitory substances). Pollen-derived embryos are subsequently produced via dehiscence of the mature anthers. The dehiscence of the anther depends both on its isolation at the correct stage and on the correct culture conditions. In some species, the reliance on natural dehiscence can be circumvented by cutting the wall of the anther, although this does, of course, take a considerable amount of time. Anthers can also be cultured in liquid medium, and pollen released from the anthers can be induced to form embryos, although the efficiency of plant regeneration is often very low. Immature pollen can also be extracted from developing anthers and cultured directly, although this is a very time-consuming process. Both methods have advantages and disadvantages. Some beneficial effects to the culture are observed when anthers are used as the explant material. There is, however, the danger that some of the embryos produced from anther culture will originate from the somatic anther tissue rather than the haploid microspore cells. If isolated pollen is used there is no danger of mixed embryo formation, but the efficiency is low and the process is time-consuming. In microspore culture, the condition of the donor plant is of critical importance, as is the timing of isolation. Pretreatments, such as a cold treatment, are often found to increase the efficiency. These pretreatments can be applied before culture, or, in some species, after placing the anthers in culture. Plant species can be divided into two groups, depending on whether they require the addition of plant growth regulators to the medium for pollen/anther culture; those that do also often require organic supplements, e.g. amino acids. Many of the cereals (rice, wheat, barley and maize) require medium supplemented with plant growth regulators for pollen/anther culture. Regeneration from microspore explants can be obtained by direct embryogenesis, or via a callus stage and subsequent embryogenesis. Haploid tissue cultures can also be initiated from the female gametophyte (the ovule). In some cases, this is a more efficient method than using pollen or anthers. The ploidy of the plants obtained from haploid cultures may not be haploid. This can be a consequence of chromosome doubling during the culture period. Chromosome doubling (which often has to be induced by treatment with chem icals such as colchicine) may be an advantage, as in many cases haploid plants are not the desired outcome of regeneration from haploid tissues. Such plants are often referred to as ‗di-haploids‘, because they contain two copies of the same haploid genome. 8. Anther culture Obtain two buds at the appropriate stage. This occurs in tobacco when the sepals and the petals in the bud are the same length. Holding the bud by the pedicel between the thumb and first finger, dip the entire bud in 95% ethanol for 15 seconds Remove bud and allow excess alcohol to drip off. With a pair of sterile forceps, remove the outer layer of tissue, the sepals. Next, remove the inner layer of tissue, the petals, exposing the anthers. Open the petri dish containing the medium for the induction of haploids. Remove each anther from the bud and drop it onto the medium. Do not damage the anther or include any filament tissue. Repeat for another bud. When finished, seal the plates and place in incubator (25°C). In 2–3 weeks examine for somatic embryo initiation. Embryoid-forming cells are characterizedby dense cytoplasmic contents, large starch grains and a relatively large nucleus. Embryoids appear opaque among translucent cells. Embryoids also exhibit high dehydrogenase activity and can be detected by tetrazolium staining (Dodds and Roberts, 1985). Ornamentals, tissue culture and gene transfer Ornamentals In lily. tulip and rose, interspecific crosses are made in order to generate new combinations of specific growth characteristics, colors, and resistances against diseases. Male and female fertility are monitored and pre- and post-fertilization barriers can be identified. Those barriers are studied and in many cases can be overcome technically, resulting in hybrids. Differences in ploidy level can hamper breeding, and can lead to infertility in the progeny. Ploidy manipulations are being applied as a solution to these problems. Backcrossing will sometimes result in recombination between chromosomes leading to introgression of desired traits into the recipient parent. This can be studied by Fluorescent or Genomic In Situ Hybridization (GISH). The genetics behind traits that are of interest for ornamental crops are studied by a thorough monitoring and describing of the traits (phenotyping) including e.g. disease resistance in disease assays. The heritability is determined. Molecular markers are generated and used for mapping. Linkage studies in progenies or association mapping in collections of cultivars can identify molecular markers or QTLs linked to specific traits. Tissue Culture Plant tissue culture is an essential component of many present-day breeding techniques, such as embryo rescue and microsporogenesis. Optimal conditions are to be determined for each new species. Another major application of plant tissue culture is micropropagation: vegetative propagation in vitro. Micropropagation may produce very fast large numbers of vigorous plants with high quality and without endogenous pathogens. Micropropagation can be achieved by inducing outgrowth of axillary buds and suppressing apical dominance, by de novo synthesis of adventitious shoots or by somatic embryogenesis. Parameters involved in those processes aimed at understanding the mechanisms and improving the efficiencies are studied. A major drawback of micropropagation is that it is labor-intensive leading to a high price of micropropagated plantlets. Our research is aimed at cost-reduction and improvement of quality. Cost- reduction is dealt with by developing new technologies, such as micropropagation in the dark via root culture or using anti-giberellins. Because stress is a major cause of poor quality, quality improvement is tackled in a research project on stress related to tissue culture at the physiological, biochemical and molecular level. We have developed various procedures to reduce the detrimental effects of stress. Other research in micropropagation concerns development of protocols for selected crops, e.g. Alstroemeria. In this research, the mechanisms underlying apical dominance are a major item. Gene Transfer Protocols for genetic modification of numerous crops are being developed based on the knowledge of processes involved such as regeneration, gene transfer, and DNA integration. Research is aimed at constantly updating that knowledge. The public acceptance of GM crops can be improved by being able to better control the process of integration, and by targeting the insert DNA to specific locations in the genome of the plant. Controlling expression of the inserted genes by using well-characterized plant promoters is also considered helpful. Antibiotic resistance genes as selectable markers are often still necessary but their presence in the final plant product is no longer required. In order to remove those undesired gene sequences a marker-gene removal system has been developed based on recombination generating so-called marker-free plants. Other new technologies making use of GM in the process of cultivar development but resulting in products with no added foreign DNA are the subject of studies related to the potential application of EU rules and regulations to these crops. Long-term stability of introduced genes and traits is monitored in field trials with genetically modified crops, e.g. apples. Traits studied encompass disease resistance and quality, e.g. starch composition in cassava or color in ornamentals. 8. Application of plant tissue culture Micropropagation is widely used in forestry and in floriculture. Micropropagation can also be used to conserve rare or endangered plant species. A plant breeder may use tissue culture to screen cells rather than plants for advantageous characters, e.g. herbicide resistance/tolerance. Large-scale growth of plant cells in liquid culture inside bioreactors as a source of secondary products, like recombinant proteins used as biopharmaceuticals. To cross distantly related species by protoplast fusion and regeneration of the novel hybrid. To cross-pollinate distantly related species and then tissue culture the resulting embryo which would otherwise normally die (Embryo Rescue). For production of doubled monoploid plants from haploid cultures to achieve homozygous lines more rapidly in breeding programmes, usually by treatment with colchicine which causes doubling of the chromosome number. As a tissue for transformation, followed by either short-term testing of genetic constructs or regeneration of transgenic plants. Certain techniques such as meristem tip culture may be employed that can be used to produce clean plant material from virused stock, such as potatoes and many species of soft fruit. Micropropagation using meristem and shoot culture to produce large numbers of identical individuals. Screening programmes of cells, rather than plants for advantageous characters. Large-scale growth of plant cells in liquid culture as a source of secondary products. Crossing distantly related species by protoplast fusion and regeneration of the novel hybrid. Production of dihaploid plants from haploid cultures to achieve homozygous lines more rapidly in breeding programmes. As a tissue for transformation, followed by either short-term testing of genetic constructs or regeneration of transgenic plants. Removal of viruses by propagation from meristematic tissues. Various urea-derived herbicides and different cytokinin analogues were used to determine their effects on callusing response and shoot regenerating capacity of alfalfa (Medicago sativa L.) and Coleus (Coleus forskohlii Briq.). The herbicides monuron and diuron evoked profuse callusing response from Coleus leaf segments and alfalfa petiole explants on Murashige and Skoog medium. Shoot regeneration by monuron (2.0 mg/l) showed a maximum of 3 multiple shoots both in alfalfa and Coleus with a frequency of 92% and 75%, respectively. Whereas diuron (0.5 mg/l) showed a high frequency of shoot regeneration (89%) with a mean number of 5 shoots in alfalfa, in C. forskohlii, the frequency of regeneration was 90% with a mean number of 6 shoots. Diuron with two chloride groups in the phenyl ring showed signifi cantly higher cytokinin- like activity than single chloride substitution monuron. This study demonstrates the potential use of monuron and diuron as cytokinins in plant tissue culture. 1. Production of lily Bulbetsvia by plant tissue culture: Step I - Disinfection and Initiation It is recommended to use Lilly Bulbs that are in good condition (high turgo) as starting material. If the bulbs cannot be used immediately, they should be kept at 4ºC in conditions of high humidity. Wash the bulb thoroughly with running water. After separating the scales from the basal part, wash scales again with distilled water. The scales are surface sterilized using Alcohol 70%, Hydrogen Peroxide Captan, Benomyl, Cuprum Sulfate or Chlorhexidine Blycolate. Disinfection is performed in flasks, which should be shaken for 10 minutes with the disinfecting agent. In the final step of disinfection, the scales are washed three times with sterile distilled water. Step II - Bulblet Production Each scale is sliced transversal into pieces 1 - 2 mm wide. Place each piece with the basal side on solid medium in a tube. The medium, 700c - Dark Initiating Bulblets is comprised of: MS Salts (Mumashige & Skoog) IBA 1 mg/1 Thiamin HCL 0.4 mg/l Sucrose 30 gr/l Agar 8 gr/l pH 5.7 Place tubes in dark at 25ºC for a period of one week. At the end of this period, first inspection is done. Contaminated tubes are discarded. Inspection for contamination is continued throughout the introduction period which lasts for an additional 5 to 8 weeks. By the end of this period several bulblets, 5mm in diameter, will have been produced on each piece. It is possible to recycle the Dark Procedure and get a second wave of bulblets from mother scales. Each bulblet produced can be separated from the mother scale, and then separated into scalets, and transferred to another tube (5 pieces/tube), containing the same medium (700c). The propagation factor of this stage is 1:2-3. Step III - Production of Initials Scalets from Bulblets produced in the dark are transferred to different solid media (700 T or 700 T1 ), in tubes or in boxes and incubated under 16 hr light at 25ºC. Two to three scalets can be placed in each tube, or about 30 in a box of 10 x 10 cm. Depending upon the variety, after 6 - 9 weeks, initials of the bulblets can be seen. It is possible to synchronize the size of the initials by transferring the earlier produced initials to a lower temperature (18 - 20ºC), thereby slowing down their growth. Light Initiating Media - 700 T and 700 T1 Media Contents: Both media are based on Ms Salts: Sucrose - 30 gr/l Agar - 8 gr/l pH - 5.7 The only difference between the two media are the hormones: 700 T - Kinetine 10.0 mg/l NAA 0.1 mg/l 700 T1 - Kinetine 1.0 mg/l NAA 0.1 mg/l Step IVa - Beginning of Initial Elongation When the Initials reach 5 - 10 mm in size, they can be transferred to Step VIII for bulb fattening (Initials of a size less than 2 mm will not continue to fatten.) or to Step IVb (Continuation of Elongation) for further multiplication. Step IVb Continuation of Initial Elongation The 5 - 10 mm length scalets with initials from the previous stage are transferred to flasks containing elongation medium (Elongation and Fattening media are presented below). The flasks, each containing 20 to 30 units, are incubated in the dark at 25º C. After 3 to 4 days sterility tests are carried out through liquid samples. The contents of the flasks that have been found to be clean can be transferred to a fermenter for the completion of elongation (about 3 weeks total incubation). In a fermenter there are approximately 300 initiation units per liter of medium. Step V Mechanical Cutting The elongated scalets are mechanically chopped to approximately 10mm lengths for production of initials, using the VitroCut mechanical cutter and a 10 mm x 10mm grid. Step VI - Production of Initials in a Light Fermenter The chopped scalets are mixed with sodium alginate beads to maintain the scalets immobile and spaced from one another. The beads can be dissolved by the addition of a 1M phosphate buffer (pH 6,5), The procedure for the production of sodium alginate beads is attached. The alginate beads and scalets are mixed with 700T medium (MS salts, 0.1 mg/l NAA, 10mg/l Kinetin and 30g/l sucrose) and transferred to a LifeReactor with about a 5 liter capacity . The plant material is incubated under continuous cool white fluorescent illumination (100 120 E) at 25 C The medium to scalet ratio is 3ml per scalet, and the medium is changed twice during a 5- week incubation period. After incubation the medium is discharged and the units either transferred to a fermenter for elongation of initials (Step VII) or to a fermenter for fattening (Step VIII). The fermenter for elongation of initials is a four-liter fermenter holding about 1,200 initiation units. The biological process is the same as Step IVb, but takes place entirely in a fermenter. After chopping, the material is transferred, as noted above, to light fermenters. Between 5% and 10% of the chopped material is transferred to a 1-liter LifeReactor that is recycled to a five-liter elongation fermenter. The rest of the chopped material is transferred to a 5-liter LifeReactor which is transferred to Step VIII bulblet production or fattening. Step VIII - Bulblet Production (Bulblet Fattening) Scalets with initials from the light stage (5 10 mm length) are transferred to an aerated vessel containing fattening medium and incubated in the dark at 25 C for about 10 weeks. After one month the medium is replaced. In the initial stages of multiplication 20 30 scalets are first transferred to flasks containing fattening medium and tested for sterility before being transferred to a fermenter. Medium Composition for Elongation and Fattening All media are based on MS salts and differ only in the sucrose concentration and hormones employed. Longiflorum has been found to be responsive to D and C' Medium % Sucrose IAA(mg/l) Kinetin(mg/l) D 5 0.1 1.0 D' 3 - 1.0 D" 8 0.1 1.0 M 3 - 0.1 E 8 1.0 3.0 C 5 1.0 - Medium Components Per Liter For Lilium Micropropagation Preparation of alginate beads 1. Take enough Sodium Alginate powder (Alginic Acid Sodium Salt) High Viscosity Sigma A 7128 for a 0.75% solution. 2. Pour the powder into deionized water (not the reverse). Stir with a stirring rod (not a magnetic stirrer). 3. Heat until dissolved while stirring with a magnetic stirrer. 4. Autoclave at 121C for 20 minutes. 5. Prepare a 50mm solution of Calcium Chloride (CaCl 2.2H2O). Prepare five times the amount of Calcium Chloride as Sodium Alginate. 6. Cool to room temperature under laminar flow (sterile) conditions. 7. Dispense the Sodium Alginate solution drop by drop into the Calcium Chloride solution from a flask with rounded lip, while gently shaking the receiving vessel. You will get Sodium Alginate beads about 2 - 3 mm in diameter. 8. Leave the beads in the Sodium Chloride solution for one hour under sterile conditions at room temperature. 9. Pour off the excess sodium Chloride solution, and wash the beads twice with sterile distilled water maintaining sterile conditions. 10. Mix a 1:1 ratio of beads and chopped plant material. The mix can be stored for up to four hours. 2. Microtubers Produced on LifeRafts Serve Switzerland‘s Potato Market: Introduction The Orchidarium & In Vitro Propagation Laboratory of Dr. Daniel Page is an excellent example of how only a two man, very compact laboratory using LifeRafts in a stock of 2000 LifeGuard vented vessels can produce 200,000 virus free certified microtubers a year. After multiplication in isolated greenhouse conditions and tunnels in the field, they produce enough certified Class A plants to supply almost all of Switzerlandâ€™s 1035 potato producers. In 1995, Daniel Page began his research on potato microtuber culture at the RAC, Station Federale de Recherches en Production Vegetale de Changins, in Nyon Switzerland. Using LifeRafts and LifeGuard vented vessels, he developed specialized microtuber culture techniques, to earn the opportunity to put his research into commercial operation. With Delley Semences et Plantes SA, he established a microtuber production facility that is at the head of the production chain which today supplies almost all of Switzerlandâ€™s certified seed potato growing needs. 3. Production of Microtubers on LifeRafts Mother plant material The process, shown in Figure 1, starts (F0 in Figure 1) with highly indexed, virus free plant stock varieties conserved at the RAC lab. The Erntestolz, Hermes, Agria, Eba, Desiree, Nicola, Urgenta, Ditta and Ostara varieties have all been produced as potato microtubers. Both Dr. Page and the RAC lab use Osmotekâ€™s tall, 700536 vessel bottoms, with the 750547 vented lid to eliminate the effects of ethylene and achieve healthy plant growth on agar medium (Figure 2). Figure 1. Microtuber Production of Potato Seed Class F0 Mother plant PTC Indexing Conservation & Storage Class F1 Propagate Plants on Rafts in Vented LifeGuard Vessels Change to tuberization medium & collect microtubers Class F2 & 3 Grow Microtubers in special soil in boxes under tunnels Grow Microtubers in soil in Tunnels Class F4 Growth in open field isolated 300m from other plants. S Class SE 1-3 E Growth in open field isolated 50m from other plants Class A Certified Plants Growth in open field at a distance of 20 m from other plants This material is then cut and placed in 25 micron membrane, 600250 rafts on 602010 floats (Figure 3), using a novel, hormone free growth medium which Dr. Page developed with the RAC. These vessels are then placed in the growth room and within 3-4 weeks grow up into the healthy plants shown in Figure4. At this point the plants must be induced to form microtubers. One of the big advantages of working with liquid medium and LifeRafts is the ease with which the plant medium can be changed. Unlike work on agar, the plants do not require any handling. Instead, the old growth medium is simply suctioned out under aseptic conditions and replaced with a unique presterilized tuberization medium. By using two peristaltic pumps to create an inexpensive medium replacement system, Dr. Page and an assistant are able to work together and change over the medium of each box at the rate of about 1 box per minute. The plants on tuberization medium are placed in a refrigerated growth chamber (Figures 5 and 6). By carefully controlling the medium replacement frequency, light cycle, and other growth parameters, after about two months the plants produce an average of 15 to 25 microtubers per box with a diameter of 12 â€― 15mm. Dr. Page emphasizes the importance of controlling the size and uniformity of the microtubers produced, which are important elements in achieving success in the conventional multiplication stages to follow. His advice is that uniform size and vigor of the microtubers is much more important than the number of microtubers obtained. A typical package of microtubers ready for planting in the next stage is shown in Generation of Virus-free Minitubers To maintain the plants in a disease free condition during the multiplication stage and generation of minitubers, the microtubers are planted in plastic boxes filled with a special artificial soil mixtures (Figures 8 & 9). These boxes are then placed under net tunnels in the field with controlled irrigation. Each microtuber will rapidly sprout and generate approximately 5 normal potato plants, each of which on average produces 5 large minitubers within about 2 Â½ months. These minitubers are replanted in the soil, under the netting for the second multiplication. From F4 onward, the minitubers are propagated in the traditional method to create certified potato seed. Cost Factors Dr. Page is quick to point out that while the LifeGuard vessels are relatively expensive compared to many other alternatives. However, their height, optical clarity and most importantly, their vented lids, create a contamination free atmosphere for plants with great leaf and strong root systems. His vessels are almost eight years old, and yet, as the pictures show, they do not look the worse for wear, even though they have gone through an average of 32 autoclave cycles. This type of vessel life enhancement is at least partly due to the use of a liquid medium growth system, which is much easier in its cleaning cycle on the polycarbonate plastic. Agar is much more difficult to remove, and the chemicals and higher cleaning temperature slowly attack the plastic, causing it to become cloudy. The cost of cleaning the vessels is also significantly reduced with liquid medium, since it is simply poured out and the vessel is washed in an ordinary dishwasher, with mild detergent and a low temperature cycle. The liquid medium based growth system uses 25 micron membrane rafts, which are part of the cost of a plant growth cycle and microtuber harvest, since the roots grow into the membrane. Since he purchases a 4 year supply of membrane rafts at one time (about 20,000 units), he benefits from a significant volume discount, and lowered cost of shipping and handling. These contribute about US$0.03 to the cost of a microtuber, which is about 2% of the total cost of production. Despite the relatively high costs of microtuber production, the vigor and growth they display in the conventional multiplication steps more than make up for this cost. Each microtuber creates 4-6 plants, and about 25 excellent quality minitubers. This leverage is what makes the entire process so competitive and worthwhile. Thereâ€™s also no question that the technique can be used to greater advantage in lower labor cost countries, for whom food production is a major issue. About 60% of the cost of microtuber production is labor, which is the largest single contributing factor. Since Switzerland is a relatively high labor cost country (both in terms of direct salary and indirect social benefits) it is clear that the technology could be much more economically instituted in lower labor cost environments such as Asia, Africa and Latin America. 4. Production of banana by Plant tissue culture: Introduction Biotechnology is an area with a tremendous potential in solving basic problems of food, fibre, fuel and medicine, particularly in developing Asian countries. It can become versatile tool for Mass multiplication of elite clones, Elimination of disease in planting material, Creation of Super genotypes of agricultural crops, Which hither to it was not possible through conventional plant breeding methods. Tissue culture propagation can greatly enhance our ability to produce consistently uniform superior planting material for export and domestic market. Advantages of tissue culture raised plants against the conventionally propagated (by suckers) plants were clearly demonstrated in banana Field trials. Superiority of Tissue Cultured quality plants in terms of developed root system, greater functional leaf area leading to high photosynthetic rate and higher yields in now well established. Technology The technology can improve continuously, the productivity, profitability, stability and sustainability of the farming system. Each plant cell has the potential to generate into a single plant. This is called Totipotency and when this character is involved into rapid and mass multiplication of propagules at optimum levels is called Micropropagation. This is an alternate to slow vegetative plant propagation. In Tissue Culture when a group of undifferentiated and meristamatically active cell called Tissue is aseptically disserted out and put into a medium containing nutrient and incubated under conducive controlled conditions of light and temperature, it establish it self and starts growth. This is called Culture and the concept of 'tissue culture' was thus conceived. Tissue culture involved following stages Preparation of Stock plant The elite plants are selected and maintained under hygenic conditions (by spraying fungicide, bactericide and insecticide) and then the plant parts are taken for initiation. Initiation (Stage-1) : The innermost tissue of surface sterilised plant in dissected aseptically and put an to the medium of growth, Medium contains major and miner elements, same vitamins. Amino acids and growth promoting hormones, solidified by agar. Multiplication (Stage II) : When the tissue starts growth in stage I and forms a shoot it is transferred to another medium containing growth promoting hormones (enhancing cell division). The growing shoot multiplies and forms a dump of 3-4 shoots. Those are transferred to another medium for shooting and rooting after optimum growth. Shoot Organogenesis Shooting and Rooting (Stage III) : After multiplication, the single shoots are separated and placed into a shooting are rooting medium. At this stage the hormones may or may not be required. The shoot elongates and new root came up. Rooting takes place within 3-4 weeks. Root Organogenesis Hardening (Stage IV) : It involves acclimatisation of bottle grown plants to the natural environment in Green House. The plants are taken out of the bottle and the media adhering to the root system in washed fully. Complete Plantlet After wards the plants are graded as per their size and then transferred singly to wells of portrays containing sterile medium (a mixture of peat moss and perlite). The whole portray with plants is maintained under high humidity conditions for a couple of weeks and there after the portrays are kept in open in the Green House under controlled temperature and humidity. This hardening taken 6 weeks and is called primary hardening - Regular sprays of plant protection chemicals are sprayed to achieve good hygenic condition of the plants. Established Plantlet Established Plantlet in field Marketing Potential (Both Domestic And International) There is a good export market for banana particularly to the countries / places like. UAE, Dubai, Sharjah and AbuDhabi, Saudi Arabia, Qatar, Countries, Singapore, Nepal, Sri Lanka and Malaysia. Small portions of banana are exported to the above Countries from the merchant exporters. No one has registered so far with the APEDA, Bangalore as exclusive growing / manufacturer exporters. Lot of incentives are available from APEDA for fruit exporters like Air freight subsidy financial assistance for inter national publicity to identify suitable irrigator, for the development of package for international standards. To utilise that benefit the farm is to be registered with APEDA as export oriented unit. There is also assured domestic market for banana for high quality banana in Indian cities. 5. Controlling The Performance Of An Interfacial Membrane Raft In Plant Micropropagation Introduction The use of microporous membrane rafts for improved culture of plant cells and protoplasts has been reported several times (1) The reasons cited for their use include: i. significantly enhanced growth rates compared to agar solidified medium, ii. easy removal from inhibitory substances in the medium, iii. ease of preparation and change or modification of media during growth, iv. possibility of removing secondary metabolites with minimum disturbance to the culture, v. simplified aseptic weighing, and vi. facilitated mechanization. Nonetheless, the use of conventional design membrane rafts has remained very limited. This is primarily due to the need to constantly monitor the level of the media in the growth vessel, as the rafts required legs to prevent them from sinking after some time. Improved rafts with a novel bouyant design (2~3) have recently been described and commercially introduced. Termed "Interfacial Membrane Rafts" (IFMR), the membrane bottom of the raft floats in contact with the liquid interface, but slightly above the level of the surrounding liquid. This assures that liquid cannot enter the raft as the result of a hydrostatic pressure forcing liquid through the membrane. Despite this fact, after several days the accumulation of liquid around the periphery of melon explants was observed, a phenomenon which has been termed "water pickup". Further investigation indicated that the amount of water pickup was related to the concentration of the surface active agent used to render the polypropylene membrane hydrophilic. A detailed study was therefore undertaken to quantify the phenomenon, and examine its influence on in vitro micropropagation of melons which are especially sensitive to water. Methods Interfacial Membrane Rafts. LifeRaft™ interfacial membrane rafts, with a nominal membrane pore size of 0.3 micron were obtained from Osmotek Ltd, POB 550 Rehovot, Israel. Two sizes of rafts were used. For water pickup trials, 9x9 cm (model #600030) with O-lOq floats (model 9600005) were used with LifeGuard Tm on the membrane. Water pickup tests were therefore performed at the highest level of wetting agent treatment (4.5% Osmowet S dispersion) with empty rafts and rafts containing several stainless steel washers. The results were followed over a 72 hour period and are shown in Figure I. Each point represents the average weight gain of 3 rafts. in empty rafts, water pickup quickly reaches a very small plateau value. Careful inspection of the raft revealed a tiny amount of liquid accumulating at the point of contact between the edge of the plastic frame and the membrane. As may be seen from Figure 1, 4.5% treated rafts with inert washers as "dummy explants" show significant water pickup which also approaches a plateau value after about 24 hours. In this case the liquid was easily visible, and gathered around the perimeter of the washer. This behaviour appeared identical to that observed with melon explants. Given this behaviour, the effect of the wetting agent treatment was then studied on rafts treated with different concentrations of Osmowet S. A set of five rafts was prepared at each concentration, and water pickup was measured after 24 hours. The average results and standard deviation are shown in Figure 2. It is clear that membrane rafts treated with low concentrations of wetting agent show a very reduced water pickup level, in addition, the average water pickup increases sharply as the treatment concentration approaches 3-4%. These results were used as a guide to prepare a set of rafts with different water pickup for in vitro micropropagation trials with melons. Biological Results: Plants were examined at regular weekly intervals and scored for vitrification. Results for the various treatments are shown in Table 1. Only after three weeks was vitrification apparant, and the same percentage of vitrification was also noted after four weeks. As may be seen from Table I, while all explants vitrified on rafts showing elevated water pickup (surfactant treatments of 2-4.5%), the treatment with only l% surfactant demonstrates a significant improvement in performance (although still less than the control). Vitrified and nonvitrified explants which are 4 weeks old are shown in Figure 3. Discussion The basis for the phenomenon of water pickup in a LifeRaft™ requires explanation. Because the raft floats above the level of the surrounding liquid, any liquid entering the raft must be pulled by a "driving force" against the dual forces of gravity and the hydraulic resistance of the membrane. It is our hypothesis that the reduction of the interfacial tension at the point of contact of an object resting on the membrane provides the force to create the flow. Once the interface is wet by liquid, water ceases to enter the raft, and no further weight gain over time is observed. This is consistent with the behaviour shown in Figure 1. The connection between the level of water pickup to the percent surfactant used in the hydrophilic treatment (Figure 2) is less obvious. One explanation could be that the hydraulic resistance of the membrane is influenced by the amount of surfactant coating the internal surface of the pores. In any case, this provides an additional tool for optimizing the raft performance with the biological requirements. Since melons are known to be highly prone to vitrification, the fact that all explants were vitrified with 2-4.5% surfactant treated rafts is not surprising. The sharp improvement achieved at the lowest surfactant treatment is consistent with the idea that a drier growth environment should reduce vitrification. As in the case of' melons grown on conventional rafts(4), the results are not as favorable as those achieved on agar medium. However, they clearly demonstrate how control of water pickup may be used to improve micropropagation on membrane rafts. Culture of Orchids and Azelea on Membrane Rafts Azelea (Rhodendron ―Sunlight‖) and orchid (Cattleya intermedia xC amethystoglossa) cultures were grown on membrane rafts in liquid medium and on agar (8 g/L) as a reference. The azelea cultures were initiated from 1.6 mg subcultured shoot tips and grown on a medium consisting of Lloyd and McCown's woody plant basal salts, MS modified Orchid cultures were initiated from 12 mg subcultured protocorms and grown on a medium consisting of MS modified basal salts, MS modified vitamin mixture , 20 g/L sucrose, 0.5 mg/L NAA, and 2.0 mg/L BA. Both cultures were grown on 60 ml of medium in Magenta GA-7 vessels with 10 mm vented lids, at 25 C, under cool white fluorescent bulbs at a distance of 25 cm above the bottom of the cultures. Although the experiments were conducted for 9 weeks, most of the growth was observed to take place in the first 3 weeks for the rafts, and 4-6 weeks for the agar cultures. In all cases, whether examined from the point of view of # of shoots or the overall fresh weight, plants grown on rafts showed more than a 100% improvement over the same material grown on agar. In the case of the azelea cultures, the difference was as large as 1000% in favor of the rafts. 6. "Improvement of the Establishment of Transgenic Plants Using Membrane- Based Liquid Culture" The Use of Interfacial Membrane Rafts in Micropropagation of Syngonium White butterfly LifeRaft Supports vs Agar Solidified Medium Fig1,2 :On the left membrane Raft ,on the right solid medium Experimental conditions : The liquid growth media composition was MS Basal salts and vitamin mixture (as per Sigma M5519), 30g/liter sucrose , 2mg/liter kinetin. The solid medium was identical, except that 8g/liter of agar (difcobacto agar ) was added. The volume of the growth medium was 150ml in a 700 534 growth vessel. Plant material 5 propagules per vessel of initial fresh average weight of 1.7g/propagule , were obtained from tissue culture tubes, and culture on either membrane rafts (LifeRaft) floating on liquid medium, or on agar as solid medium. The material was a mixture of adventitious and axilliary buds, in which the main shoot was cut and the roots were removed. Explants were cut with a large base to ensure good contact with the membrane surface. Culture growth conditions The cultures were kept at 25 deg Celcius in a growth room under cool white fluroescent light bulbs (60 micromoles PAR/square meter/sec ). The photoperiod regime was 16 hrs light, and 8 hours dark. The experiments were conducted for 25 days, after which the material was harvested. Results: The experiment was repeated and at least ten replicates were used in each trial. As may be seen from Figures 1 & 2, the growth on the membrane rafts was faster then on the solid medium, with the same resulting quality of plant material. Rafts (LifeRaft) Solid medium Final fresh weight 3.2 0.3 g/propagule 2.3 0.2 g/propagule Number of developed shoots per propagule 6.8 0.6 5 0.3 Length of the developed main shoots 13.8 0.8cm 6.7 0.5 cm Regeneration of both tobacco and potato plants was significantly improved, in fresh weight as well as the number of regenerated shoots, using membrane based liquid cultures with MS complete 50X concentrate medium. Toxic effects of antibiotics such as kanamycin, carbenicillin and hygromycin were shown when the plant tissues were grown in the membrane based liquid cultures or in agar based semi-solid cultures. Transgenic plants were achieved by using Agrobacterium-mediated transformation in tobacco as well as potato grown in the membrane based cultures. The transformation efficiency was improved 6 fold in the number of regenerated tobacco shoots and 60% to 80% of fresh weight of regenerated shoots by growing in the membrane based liquid culture. Transgenic plants obtained from membrane based liquid culture were further analyzed by GUS activity and PCR. From 30 kanamycin resistant plants, 25 plants showed GUS activity, and all 30 plants contained the kanamycin resistance gene by PCR analysis. These results demonstrate that membrane based liquid culture is able to improve not only the regeneration of non-transgenic plants but also the transgenic plants. 7. Cloning Plants By Tissue Culture Many plants are cloned by tisssue culture techniques and sold commercially. Some of the ferns such as Boston fern and staghorn fern are propagated through tissue culture. Also, many varieties of African violet are propagated asexually by tissue culture. We can take a leaf from a plant like the plant below. The leaf is then cleaned of contaminating microorganisms, fungal spores, small insects or whoever might be on board. Part 2 The leaf is then cut into small pieces in a laminar flow hood that provides a clean working surface. The small pieces of plant tissue that are cut out of the leaf are called explants. Below you can see what they look like. The explants are then placed on a chemical medium that provides nutrients for the plant tissues to grow and usually some plant hormones to encourage development of new organs from the plant tissue. Below is an explant that has been placed on a chemical medium inside a test tube. If you look at an explant with a scanning electron microscope, it would look like this. From this explant, new shoots would start to develop. Before they were obvious to you, as they just started to develop, they would look like this with the scanning electron microscope. Part 3 After six to eight weeks, the explant will develop new shoots, as below. These shoots may be cut free from the explant, and placed in a larger container on a new medium that will help roots to develop. The rooted plant can then be transferred to soil. At this stage, the humidity must be kept high until the plant can adjust to the new surroundings. This process of adjustment is called acclimatization, and involves the growth of new leaves that will function in the less humid room air. The cover is slowly opened more and more over a two week period so that the plant can gradually adjust. Then the cover can be removed completely and you have a new African violet plant. From one original violet, you may produce hundreds of genetically identical plants. Because the plants are genetically identical, and are of similar developmental age, they tend to produce flowers at the same time. This is very important to someone who is growing the plants and wants to get them to market just as they start to flower. 9. Role of plant tissue culture in biodiversity conservation and economic development: A national seminar on the ‗Role of Plant Tissue Culture in Biodiversity Conservation and Economic Development‘, and the XXII meeting of the Plant Tissue Culture Association (India) was organized by G. B. Pant Institute of Himalayan Environment and Development, Kosi-Katarmal, Almora during 7–9 June 1999. The Symposium attracted over 150 participants including members of the Plant Tissue Culture Association (India), distinguished scientists, members of corporate bodies and research students from all over the country. The 3-day deliberations were centered around 6 themes, namely (1) Micropropagation and somatic embryogenesis, (2) Growth, differentiation and production of secondary metabolites, (3) Tissue culture studies in medicinal plants, (4) Genetic transformation and transgenics, (5) Cryopreservation, conservation strategies and crop improvement, and (6) Synthetic seeds, hardening, acclimatization and miscellaneous issues. In view of the large number of speakers, oral presentations had to be arranged in two concurrent sessions and sufficient time was allotted for poster presentations. Keynote address, special and invited lectures, however, were presented in a common platform for the benefit of all the participants. In the inaugural session L. M. S. Palni (G. B. Pant Institute of Himalayan Environment and Development) highlighted the main objectives of the meeting; A. N. Purohit (High Altitude Plant Physiology Research Centre, Srinagar-Garhwal) expressed the importance of modern technologies like plant tissue culture and stressed the need for its judicious application for the development of mankind. B. S. Rajput (Kumaun University, Nainital) described the close links between culture and science that have existed in India from ancient times. Manju Sharma (Department of Biotechnology, New Delhi) inaugurated the greenhouse facility, and in her keynote address on ‗Biodiversity Conservation and Socio-economic Development: Role and Relevance of Biotechnology‘, she mentioned that the country‘s rich biodiversity has been sadly and seriously affected with the increasing human population and reckless use of natural resources; however, public awareness towards the value of conserving biodiversity for sustained development has increased considerably, also at all levels of government and private sectors. She emphasized the need of using modern biotechnology, which can greatly facilitate conservation and sustainable utilization of genetic diversity. Plant tissue culture has, therefore, largely been adopted for mass production of selected elite varieties and to conserve endangered and threatened species. She concluded by stressing that concerned and focussed R&D efforts need to be directed for commercialization of products and processes utilizing the existing rich natural resources of the country; a judicious application of biotechnology to convert biological wealth into economic wealth on a sustainable basis should be one of the major aims in the future. Besides visiting the greenhouse facility, Rural Technology Park and laboratories of the Institute, she also attended the scientific and poster sessions. In his special lecture A. R. Mehta (formerly from the Department of Botany, M. S. University) highlighted the environmental impact of crops developed through biotechnology, and indicated that the new approaches for genetically altering plants are time-saving and precise as compared to conventional plant breeding methods. L. M. S. Palni (GBPIHED) enumerated the work on micropropagation of some economi cally important plants of the Indian Himalayan region and specified that conventional methods of propagation need to be supplemented with modern techniques not only for use in afforestation and plantation programmes, but also for plant improvement, conservation and in vitro production of metabolites. Highlighting the success in developing in vitro protocols for tea, ‗maggar bamboo‘, ‗dev- ringal‘ and selected alpine medicinal herbs, he emphasized the underlying problems associated with hardening and field establishment, and discussed cost-effective approaches for hardening, successful field establishment and subsequent growth performance. He specified that proper and long term monitoring of tissue culture-raised plants is essential. N. S. Shekhawat (J. N. Vyas University) elaborated micropropagation work on plants of arid and semi-arid zones, and listed a wide range of species for which tissue culture protocols have been developed and plants subsequently transferred to field. U. Dhar (GBPIHED) presented the progress made thus far in in vitro regeneration of some multipurpose tree species of the Himalayan region focussing on the efficiency and reproducibility of protocols. He pointed out that proper selection procedures need to be developed for initiating future in vitro studies in multipurpose trees of the region. Reports on micropropagation studies and successful field establishment of bamboos were also presented in the Symposium. While I. D. Arya (Forest Research Institute) reported a complete protocol for mass propagation of Dendrocalamus asper and Bambusa multiplex through axillary bud and/or seed culture, Niladri Bag (GBPIHED) described efficient plant regeneration via somatic embryogenesis and use of nodal explants taken from mature as well as physiologically young but field tested plants of Dendrocalamus hamiltonii. The application of tissue culture techniques in horticultural crops was also presented; T. S. Lokeswari (SPIC Science Foundation) reported micropropagation in 3 commercially important S. Indian varieties of mango and R. K. Gupta (Sher-e-Kashmir University of Agriculture & Technology) described micropropagation using nodal explants of selected Citrus aurantifolia (Kagzi lime) trees. In view of the economic importance and endangered status, orchids deserve special attention. S. P. Vij (Panjab University) enumerated the pivotal role of tissue culture in orchid industry. He indicated that since seed-raised orchid plants require several years to reach maturity, the recently developed technique of inducing precocious flowering in the protocorms provides the breeder with an opportunity to select suitable clones from a hybrid progeny for commercial purposes within a short time. Despite the fact that micropropagation protocols have been developed for several Indian orchid species/varieties, development of meaningful orchid based cut-flower and pot plant industry in the country still awaits motivated entrepreneurs. S. K. Sinha (State Forest Research Institute of Arunachal Pradesh) mentioned how biotechnological approaches could be gainfully adopted in conserving some rare and endangered vandaceous orchids, namely, Renanthera imschootiana Rolfe, Vanda coerulea Griff., ex Lindl. and V. coerulescence Griff. which are listed in the IUCN Red Data Book of India. K. V. Krishnamurthy (Bharatidasan University) described a convenient system to manipulate cambial activity and to control the cambial zone diameter as well as the quality of wood produced by in vitro cultivation of cambial meristem, and highlighted the success in in vitro culture of cambium of Eucalyptus tereticornis. Papers on secondary metabolite production were also discussed by B. Ghosh, S. Mukherjee (Calcutta University) and V. Malathy (Mumbai University). P. S. Ahuja (Institute of Himalayan Bioresource Technology) summarized the work carried out on tissue culture of tea, and studies on medicinal plants. While describing the tissue culture protocols developed for certain endangered/threatened alpine and sub-alpine species, he also defined the difference in domestication and cultivation, and cited the example of Valeriana wallichii for which a complete cultivation package has been established. Various achievements in tea tissue culture including micrografting and subsequent ease in field establishment were also indicated. Several speakers dwelt on the potential and use of recombinant DNA technology. K. V. Krishnamurthy (National Chemical Laboratory) while reviewing the status of research on plant transformation in the past and indicating possible future developments, also mentioned that over the years natural genetic variability is getting gradually exhausted and highlighted that recombinant DNA technology involving the manipulation of DNA, transfer, integration and expression of designed genes from microbes, incompatible plant species, animals can greatly benefit crop production. S. K. Sen (IIT-BREF-Biotek) enumerated the development of transgenic Indian cotton cultivar tolerant against bollworm damage which accounts for around three-fourth loss of productivity. He highlighted successful genetic transformation of Indian cultivars by transferring Bacillus thuringiensis (Bt) gene, responsible for insecticidal crystal protein, to elite cultivars; the transgenic plants were shown to possess varied degrees of resistance/tolerance against bollworm damage. U. Mohapatra (Utkal University) elaborated the use of Agrobacterium-mediated transformation of lettuce for bialophos (a herbicide) resistance. Plant regeneration and Agrobacterium-mediated transformation of Leucaena leucocephala, a multipurpose nitrogen fixing tree legume, was presented by U. N. Dwivedi (Lucknow University) who has raised transgenic Leucaena with reduced lignin and minosine contents. M. V. Rajam (University of Delhi, South Campus) recounted studies on genetic engineering of polyamine metabolism with a view to studying plant growth and development as well as to inducing stress tolerance in rice, eggplant and tobacco. Polyamines are known to be essential for normal growth and development, and also for the regulation of various other cellular processes, including stress response. A. K. Kukreja (Central Institute of Medicinal and Aromatic Plants) reported scaling up of Agrobacterium rhizogenes transformed hairy root culture of Hyoscyamus muticus using a plant bioreactor. G. K. Garg (G. B. Pant University of Agriculture and Technology) explained molecular signaling in phytohormone-mediated plant growth and development in tissue cultures. He pointed out that since the plant cell differentiation is a regulated developmental process influenced by various phytohormones, understanding of molecular mechanisms of hormone action can lead to rationalizing its use; the differences in biological efficacies of these regulators in calli indicate that hormone interactions are stereo-specific and different tissues from the same crop may possess different receptors which may lead to diverse responses; phytohormone-binding proteins may be involved in signal transduction. H. C. Chaturvedi (National Botanical Research Institute) mentioned that unscrupulous human indulgence tops all other factors responsible for the present state of deforestation in the country. He explained how phytodiversity could be conserved through in vitro morphogenesis which plays a pivotal role in developing strategies of germplasm preservation, particularly in case of heterozygous hybrids, plants where seeds are recalcitrant or not produced, or if the plant is diseased and/or plant material is very limited. Using similar in vitro approach, A. K. Sharma, from the same Institute, attained success in conserving phytodiversity of Azadirachta indica which has acquired immense significance in pharmaceutical and agroindustries. Somaclonal variation as an important alternative for crop improvement was presented by P. S. Negi (Defence Agriculture Research Laboratory). In vitro raised plants often experience high mortality during or following lab to field transfer; accordingly some papers highlighted the steps required to be taken during hardening of tissue culture raised plants. A. Pandey (GBPIHED) mentioned that apart from various abiotic factors, one major factor was the sudden exposure of delicate root system to soil microbial communities. It was shown that the major cause of mortality in tea plants during lab to land transfer was fungal attack, and bacterial inoculation (isolated from tea soils) resulted in near 100% survival against 45–55% in control plants. At the end of the 3-day deliberations, a concluding session chaired by H. C. Arya (Rajasthan University) was held. The following recommendations were made: (1) Major efforts need to be concentrated on transgenic research and secondary metabolite production, (2) Principles of basic physiology of plants be kept in mind in conducting tissue culture studies, (3) Since physiological levels of plant growth substances are important, their concentrations should be reported as micromoles in place of mg/l or ppm, (4) Emphasis must be laid on mass propagation of economically important and threatened species, (5) Tissue culture being a clonal method of propagation, explants taken from each (seed raised) plant be clearly identified and kept separately, (6) While progress has been made in the field establishment and survival of in vitro raised plants, greater emphasis is necessary for understanding the mechanisms(s) underlying the process of hardening, (7) Cost of production should be kept in mind, and concerted efforts are required to reduce expenditure. ―Response of jojoba shoots to ventilation in vitro‖ Fig 3. Effect of ventilation on epicuticular wax deposition on leaves in clone 879-154 as observed by SEM. A -- closed vessel ; B-- 5mm membrane diameter ; C -- 8 mm membrane diameter ; D -- 10 mm membrane diameter. Scale bar is 10 um. David Mills, Ruth Friedman and Aliza Benzion of Ben Gurion University in Israel have recently published a study of jojoba shoots cultured in Magenta vessels using Osmotek's vented lids with different sized membrane vents to achieve different levels of ventilation. The study was performed with a statistically significant number of shoots per treatment and per clone, and the results were analyzed by the Fisher Anova test at a probability of 5%. After eight weeks of growth, the absolute values of dry weight of shoots and total area differ by a factor of two for different clones, but the effect of ventilation is identical. Both the leaf area and dry weight were 60 – 100% higher for shoots grown in vessels with 10mm diameter vents compared to those grown in vessels with solid lids. Most interestingly, the shoot length was approximately 25% less, and the shoots grew fewer leaves. Ventilation had a positive effect on leaf size and color. In addition, the percentage of water in the most ventilated plants (10mm vent diameter) was 12 –15% lower than the non- ventilated control. This clearly showed the effect of ventilation on reducing the hyperhydric tendency of these plants in tissue culture. Such plants also showed greater wax deposition on the leaf surface, greater lignification and a lower rate of water loss. Propagation of lilies In Liquid Medium with LifeReactor Dr. M. Horita of the Hokkaido Green-Bio Institute and his research team have been developing a propagation system for lily using Osmotek's LifeReactor. They have established the basic culture conditions, and have sufficient production to allow field trails of the products with small lily bulbs. The trials are currently being carrried out. They plan to apply this system to several varieties, including lilies for cut flowers and for consumption. In the above pictures, on the left are lily clusters in the LifeReactor, and on the right the flowers from the field . Interested parties are invited to contact Dr. Horita at the Green Bio Institute. 10. Bibliography 1. 1. Introduction to Plant tissue culture – Second edition, M.K.Razdan ISBN 978-1-57808 – 237-7, 2003 ; 330 pages 2. Barcelo, P. , Rasco-Gaunt, S. Thorpe, C and Lazzeri, P. (2001) Transformation and gene expression In advances in botanical research, Volume 34. Biotechnology of cereals ed. P.R. Shewary, P.A. Lazzeri and K.I. Edwards. PP 59-126 Academic Press, London. 3. Dahleen, L.S. (1999) Donor Plant Environment effects on regeneration from barley embryo 2. 1. Adam S. R. P. and Adams, J.E. 1992, Conservation of Plant genes, DNA Banking and In Vitro Technology Academic Press, New York. 2. Bhojwani, S.S. 1990, Plant tissue culture, Applications and Limitations. Elsevier, Amsterdam. 3. Singh B.D. 1998. Biotechnology, Kalyani Publishers, New Delhi. 4. Lewin B. 2004. Genes VIII Oxford University Pres, New York. 3. 1. Donnelly, D.J. and W.E. Vidaver 1988, Glossary of Plant tissue culture, Portland, Timber Press. 2. Smith, Robert, 1992, Plant tissue culture ; Techniques and Experiments Academic Press. 4. 1. Plant and tissue culture Laboratory exercise; 2nd edition. R.N. Trigiano, D.J.Gray 454 PP. CRC Press 1999. 5. 1. Coffin, R. C.D. Japer and C. Chang 1976, Can I. Bot 54. Page No. 547-551. 2. Nitsch, S.J. and E.G. Sutter 1990; Horticulture Science 25, Page no. 800-802. 3. Nitsch J.P. and C. Nitsch 1969. Haploid plants from Pollen Grains Science 63 Page no. 85-87. 6. 1. Vidyasagar K, National conference on plant biotechnology, Lady Doak College, Madurai April-2006. 2. Chapman 1994 ―An effective biocide for invitro diagnostic reagents and other products.‖ Amclin Laboratory August 1994 Page nos. 13-14 7. 1. Fowler, M.R. (2000) Plant cell culture, laboratory techniques. In encyclopedia of cell technology by R.E. Spier, Page no. 994-1004 Wiley, New York. 2. Gamborg, O.L. (2002), Plant tissue culture ; Biotechnology Milestones Page no. 84-92 3. Sugiyama, M. (1999) Organogenesis in vitro current opinion in Plant biology -2; 61-4 8. 1. www.potato.com www.cropdevelopment.org 2. www.bananagarden.com Yield potential of tissue Banana; V.L. Sheela and S. Ramachandran Nair ; College of Agriculture, Vellayni Thirvananthapuram, India 3. Bhattacharya, N.M and Sen, S.K. (1990) Production of Jojoha; Page no.357-365 4. David Mills, Institute for Applied Research, Ben-Gurian University of Negev, Beer- Sheva 84105, Israel. 5. G.B. Tanny, J.Greenwal D., S. Greenwala D, A.A. Watad and V. Gaba (a) Osmotek Ltd., POB 550, Rehovat, Israel; (b) Dept. of Ornamental Horticulture (c) Dept. of Virology, Agricultural Research Organization, Volcani Center. 6. J.J. LIN, N. Garcia – Assad, Agricultural Biotechnology R & D Life Technologies Inc. 7. Clonging Plants by tissue culture by Michael H. Renfroe, Ph.D.
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