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• Specific terms are used to describe the sexual expression of individual plants within a population. • Hermaphrodite - A plant that has only bisexual reproductive units (flowers, conifer cones, or functionally equivalent structures). • Monoecious - an individual that has both male and female reproductive units (flowers, conifer cones, or functionally equivalent structures) on the same plant; from Greek for "one household". • Individuals bearing separate flowers of both sexes at the same time are called simultaneously or synchronously monoecious. • Protoandrous describes individuals that function first as males and then change to females; protogynous describes individuals that function first as females and then change to males. • Dioecious - refers to a plant population having separate male and female plants. That is, no individual plant of the population produces both microgametophytes (pollen) and megagametophytes (ovules); individual plants are either male or female. From Greek for "two households". [Individual plants are not called dioecious; they are either gynoecious (female plants) or androecious (male plants).] • In other words, Individual species which have separate male and female plants, and are either one sex or the other, are called dioecious – Androecious - plants producing male flowers only, produce pollen but no seeds, the male plants of a dioecious population. – Gynoecious - plants producing female flowers only, produces seeds but no pollen, the female of a dioecious population. In some plant populations, all individuals are gynoecious with non sexual reproduction used to produce the next generation. • Subdioecious, a tendency in some dioecious populations to produce monoecious plants. • The population produces normally male or female plants but some are hermaphroditic, with female plants producing some male or hermaphroditic flowers or vice versa. • The condition is thought to represent a transition between hermaphroditism and dioecy.. – Gynomonoecious - has both hermaphrodite and female structures. – Andromonoecious - has both hermaphrodite and male structures. – Subandroecious - plant has mostly male flowers, with a few female or hermaphrodite flowers. – Subgynoecious - plant has mostly female flowers, with a few male or hermaphrodite flowers. • Polygamy - Plants with male, female and perfect (hermaphrodite) flowers on the same plant, called trimonoecious or polygamomonoecious plants. • Diclinous , an angiosperm term, includes all species with unisexual flowers, although particularly those with only unisexual flowers, i.e. the monoecious and dioecious species. Ornamental plants or garden plants are typically grown in the flower garden or as house plants. Most commonly they are grown for the display of their flowers. Other common ornamental features include leaves, scent, fruit, stem and bark. In some cases, unusual features may be considered ornamental, such as the prominent and rather vicious thorns of Rosa sericea. In all cases, their purpose is the enjoyment of gardeners and visitors. Ornamental plants may also be used for landscaping, and for cut flowers. The adequate spacing between pots of plants prevents them from competing for sunlight. Similarly trees may be called ornamental trees. This term is used when they are used as part of a garden setting, for instance for their flowers, their shapes or for other attractive characteristics. By comparison, trees used in larger landscape effects such as screening and shading, or in urban and roadside plantings, are called amenity trees. For plants to be considered as ornamental, they may require specific work and activity by a gardener. For instance, many plants cultivated for topiary and bonsai would only be considered as ornamental by virtue of the regular pruning carried out on them by the gardener, and they may rapidly cease to be ornamental if the work was abandoned. Pruning is the process of removing certain above-ground elements from a plant; in landscaping this process usually involves removal of diseased, non-productive, or otherwise unwanted portions from a plant. Ornamental plants and trees are distinguished from utilitarian and crop plants, such as those used for agriculture and vegetable crops, and for forestry or as fruit trees. This does not preclude any particular type of plant being grown both for ornamental qualities in the garden, and for utilitarian purposes in other settings. Thus lavender is typically grown as an ornamental plant in gardens, but may also be grown as a crop plant for the production of lavender oil. Other types of ornamental plants include the lily, rose, morning glory and the pink oak. Simple sterile cultures for micropropagation of ornamentals using direct application of chlorine disinfectants by preparing sterile medium without autoclaving and inoculating explants without the laminar air-flow cabinet were developed. These techniques could be applied to various micropropagation processes of ornamentals. The sterile medium could be prepared without autoclaving by immediately incorporating chlorine disinfectants into the medium. In these cases, all chlorine disinfectants tested were effective for sterile medium preparation. The media could be used for sterile cultures in various micropropagation processes. Spraying the surface of a medium and the whole explants with chlorine disinfectants after inoculating was effective for inoculating explants sterilely and for subsequent sterile cultures under nonsterilized conditions. These techniques could be applied to the following cultures, shoot tips of chrysanthemum and Cymbidium, stem section explants of chrysanthemum. The treated levels of incorporated and sprayed chlorine disinfectants suppressed in vitro contamination and did not appear to be toxic to shoots or plantlets of ornamentals tested. Propagated plantlets which were cultured on the disinfectant incorporated medium and handled with spraying treatments under nonsterile conditions could survive without harming tissues and were raised without in vitro contamination. Horticulture is the industry and science of plant cultivation including the process of preparing soil for the planting of seeds, tubers, or cuttings. Horticulturists work and conduct research in the disciplines of plant propagation and cultivation, crop production, plant breeding and genetic engineering, plant biochemistry, and plant physiology. The work particularly involves fruits, berries, nuts, vegetables, flowers, trees and shrubs. Horticulturists work to improve crop yield, quality, nutritional value, and resistance to insects, diseases, and environmental stresses. Horticulture usually refers to gardening on a smaller scale, while agriculture refers to the large-scale cultivation of crops. The word is composite, from two words, horti, meaning grass, originating in the Greek meaning the same (grass) and the word culture. Propogation is by suckers or off-shoots which spring at the base of a banana-tree from underground rhizomes. Vigorous suckers, with stout base, tapering towards the top and possessing narrow leaves, are selected for plant. Each sucker should have a piece of underground stem with a few roots attached to it. Banana suckers can be planted throughout the year in southern India, except during summer, whereas in the rest of the country, the rainy season is preferred. They are planted in small pits, each just enough to accommodate the base of a sucker. The planting-distance varies from 2m X 2m in the case of dwarf varieties to 4m X 4m in the case of very tall varieties. An application of 20 to 25 kg of farmyard manure, together with about 5 kg of wood-ashes per plant is given at planting time. In southern India, ammonium sulphate is applied one month, five months and nine months after planting 20 kg per hectare each time. In western India, a little over 2 kg of oilcake per stool is applied during the first three months after planting. A complete fertilizer mixture may be applied to supply 100 to 200 kg of N, 100 to 200 kg of P2O5 and 200 to 400 kg of K2O per hectare. A green-manure crop is also considered beneficial. Trials at the Indian Institute of Horticultural Research have shown that for the 'Robusta' variety, a fertilizer mixture comprising 180 g of N + 108 g of P2O5 + 225 g of K2O per plant is ideal. (phospjorous pentoxide+potassium peroxide mixture). The banana and mango-plants require very heavy irrigation. Irrigation is given in most places once in seven to ten days. Stagnation of water in the soils is not very congenial to the proper growth of banana and, hence, the drainage of soil is also essential. Early varieties commence flowering in southern and western India about seven months after planting, and the fruits take about three months more to ripen. In the Andhra Pradesh delta areas, the fruits are ready for harvesting about seven to eight months after planting. The first crop of the 'Poovan' variety matures in 12 to 14 months and the second in 21 to 24 months after planting. In other parts of India, the first crop is usually gathered a year after planting, whereas the succeeding crop may be ready in six to ten months thereafter. No systematic pruning is done. The removal of dead-wood and the thinning of over-crowded and mis-shapen branches after about four years is all that is necessary; flowers that appear during the first three or four years should be removed. The ripening of banana or mango is done in several ways, e.g. exposing the bunches to the sun, placing them over a hearth, wrapping them in closed godowns or smoking them in various ways. One of the common ways is to heap the fruits in a room and cover them with leaves, after which fire is lit in a corner and the room is closed and made as air-tight as possible. Ripening takes place usually in 30 to 48 hours. In a cool store, the bunches ripen well at about 15o to 20oC. The application of vaseline, a layer of clay or coaltar to the cut-ends of the stalks prevents rotting during ripening and storage. Wrapping up the fruits and packing them in crates help to reduce the damage during transport. In animal or plant development, organogenesis (organo-genesis, compound of the Greek words "that with which one works", and "origin, creation, generation") is the process by which the ectoderm, endoderm, and mesoderm develop into the internal organs of the organism. Organogensis refers to that period of time during development when the organs are being formed. After an egg has been fertilized, and has been implanted in the uterus, the developing form is known as the embryo. Organogenesis takes place during this embryonic phase. In fact, most organogenesis has begun as early as week five in humans (remember that a normal human pregnancy lasts an average of 40 weeks). Therefore, damage to any of the organ systems of the body which may ultimately result in some type of birth defect usually strikes during this time frame. By week five, the buds of tissue which will become the limbs are in place. The structures which will become the skeleton, nervous system, and circulatory system of the face, neck, and jaws are in place. A five-week-old embryo has the early developmental structures of the esophagus, stomach, intestine, liver, and pancreas. The heart is already functioning, and continues to develop and change over this period of time. The respiratory system begins developing, as do blood vessels, blood cells, nervous and endocrine organs. Clearly, the most crucial organs of the human form are developing during organogenesis. Essentially, the earlier the injury to these developing buds of tissue, the more severe the ultimate defect. This is because these tiny buds of tissue hold all the primitive cells which should differentiate into the myriad number of cells necessary to create all of the varied organs of the human body. It is an irony that, during this crucial period of development, when toxins from the outside world can have such devastating effects on the ultimate development of the embryo, many women are not even yet aware that they are pregnant, and are therefore not in the mindframe of protecting the developing embryo from exposure to such harmful substances as cigarette smoke, alcohol, certain drugs or medications, or extremes of heat . Embryo Rescue • • • • • • • One of the most infamous agents (teratogens) responsible for widespread deformities during the period of organogenesis is a drug called thalidomide. Thalidomide was administered to women (particularly in Europe in the 1950s) because it was thought to combat the nausea present in early pregnancy. Over time, however, it became evident that babies born of thalidomide-using mothers had very high rates of serious limb deformities. In particular, the long bones of the limbs were either absent or seriously deformed. Furthermore, many of these children had associated defects of the heart and intestine. Thalidomide was ultimately determined to be at fault, causing the most severe defects when given between weeks four and six of pregnancy: the period of organo-genesis. Thalidomide was subsequently withdrawn from the market. Plant embryogenesis is the process that produces a plant embryo from a fertilised ovule by asymmetric cell division and the differentiation of undifferentiated cells into tissues and organs. It occurs during seed development, when the single-celled zygote undergoes a programed pattern of cell division resulting in a mature embryo. A similar process continues during the plant's life within the meristems of the stems and roots. Embryogenesis occurs naturally as a result of sexual fertilization and the formation of the zygotic embryos. The embryo along with other cells from the motherplant develops into the seed or the next generation, which, after germination, grows into a new plant. Embryogenesis may be divided up into two phases, the first involves morphogenetic events which form the basic cellular pattern for the development of the shoot-root body and the primary tissue layers; it also programs the regions of meristematic tissue formation. The second phase, or postembryonic development, involves the maturation of cells, which involves cell growth and the storage of macromolecules (such as oils, starches and proteins) required as a 'food and energy supply' during germination and seedling growth. Embryogenesis involves cell growth and division, cell differentiation and programed cellular death. The zygotic embryo is formed following double fertilisation of the ovule, giving rise to two distinct structures: the plant embryo and the endosperm which together go on to develop into a seed. Seeds may also develop without fertilization, which is referred to as apomixis. Plant cells can also be induced to form embryos in plant tissue culture; such embryos are called somatic embryos. Following fertilization, the zygote undergoes an asymmetrical cell division that gives rise to a small apical cell, which becomes the embryo and a large basal cell (called the suspensor), which functions to provide nutrients from the endosperm to the growing embryo. From the eight cell stage (octant) onwards, the zygotic embryo shows clear embryo patterning, which forms the main axis of polarity, and the linear formation of future structures. These structures include the shoot meristem, cotyledons, hypocotyl, and the root and root meristem: they arise from specific groups of cells as the young embryo divides and their formation has been shown to be positiondependent. In the globular stage, the embryo develops radial patterning through a series of cell divisions, with the outer layer of cells differentiating into the 'protoderm.' Embryonic tissue is made up of actively growing cells and the term is normally used to describe the early formation of tissue in the first stages of growth. It can refer to different stages of the sporophyte and gametophyte plant; including the growth of embryos in seedlings, and to meristematic tissues, which are in a persistently embryonic state, to the growth of new buds on stems. In both gymnosperms and angiosperms, the young plant contained in the seed, begins as a developing egg-cell formed after fertilization (sometimes without fertilization in a process called apomixis) and becomes a plant embryo. This embryonic condition also occurs in the buds that form on stems. The buds have tissue that has differentiated but not grown into complete structures. They can be in a resting state, lying dormant over winter or when conditions are dry, and then commence growth when conditions become suitable. Before they start growing into stem, leaves, or flowers, the buds are said to be in an embryonic state. Somatic embryos are formed from plant cells that are not normally involved in the development of embryos, i.e. ordinary plant tissue. No endosperm or seed coat is formed around a somatic embryo. Applications of this process include: clonal propagation of genetically uniform plant material; elimination of viruses; provision of source tissue for genetic transformation; generation of whole plants from single cells called protoplasts; development of synthetic seed technology. Cells derived from competent source tissue are cultured to form an undifferentiated mass of cells called a callus. PGR’s in the tissue culture medium can be manipulated to induce callus formation and subsequently changed to induce embryos to form from the callus. The ratio of different PGR’s required to induce callus or embryo formation varies with the type of plant. Asymmetrical cell division also seems to be important in the development of somatic embryos, and while failure to form the suspensor cell is lethal to zygotic embryos, it is not lethal for somatic embryos. Somatic embryogenesis refers to the remarkable ability of nonzygotic plant cells (including haploid cells) to develop through characteristic embryological stages into an embryo capable of developing into a mature plant. Somatic embryogenesis is an expression of totipotency and the associated differential gene expression. Somatic embryos may be produced in nature in certain plant species as a form of apomixis known as adventitious embryony. Somatic embryogenesis in plants usually refers to the induction of somatic embryos in vitro, first demonstrated by both Steward and Reinert in 1958. Research into somatic embryogenesis has intensified as plant regeneration in vitro has come to be widely utilized in transformation and somatic hybridization. Artificial Seeds- An artificial seed is a bead of gel containing a somatic embryo (or shoot bud), and the nutrients, growth regulators pesticides, antibiotics, etc. needed for the development of a complete plantlet from the enclosed SE/shoot bud. Synthetic seeds or artificial seeds are the livingseed like structure derived from somatic embryoids (or, somatic embryos) in-vitro culture after encapsulation by a hydrogel. The preserved embryoids are called synthetic seeds. Artificial seeds The use of somatic embryos as artificial seeds for large scale clonal propagation of plants is close to becoming a reality. The quality of the artificial seed depends on the temporal, quantitative and qualitative supply of growth regulator and nutrients along with an optimal physical environment. Desiccation of somatic embryos provides a quiescent phase analogous to true seeds, facilitating the convenience of year round production, storage and distribution. Somatic embryos possess the ability to express desiccation tolerance in response to an external chemical or physical stimuli. The mechanisms of desiccation tolerance involve stabilization of membranes in dry state and prevention of oxidative degradation of biomolecules. Encapsulation of embryo may control the water uptake, release of nutrients and provide mechanical protection required for field planting. Artificial seeds may be produced using one of the following two ways: (1) Desiccated systems and (2) Hydrated systems. In the desiccated system, SE s is first hardened to withstand desiccation and then are encapsulated in a suitable coating material to yield desiccated artificial seeds. SE s may be hardened either by treating/coating mature SE s with a suitable polymer followed by drying, or treating them with ABA during their maturation phase. ABA treatment also improves germination of SE s, and is used even in the hydrated systems. In the hydrated systems, SE s are enclosed in gels, which remain hydrated. Of the many gels evaluated, calcium alginate is the most suitable. Artificial seeds can be easily made as follows. A 2% solution of sodium alginate is filled in a burette and allowed to drip drop by drop into a 100 mM CaCl2 solution. As the sodium alginate bead or drop forms at the tip of the burette, an SE is inserted into it with the help of a spatula before the drop falls into the CaCl solution. The beads become hardened as calcium alginate is formed; after about 20-30 min the artificial seeds are removed, washed with water and used for planting. Hydrated artificial seeds are sticky and difficult to handle on a large scale, and dry rapidly in the open air. These problems can be resolved by providing a waxy coating over the beads. Alternatively, a desiccated system may be used to produce artificial seeds. However, hydrated artificial seeds have to be planted soon after they are produced. Precision machines for a large scale encapsulation of SE s have been devised. General Procedure The general procedure is as followed: 1) Induction of somatic embryogenesis 2) Maturation of somatic embryos 3) Encapsulation of somatic embryos (synthetic seeds) 4) Evaluation of embryoid and plant conversion 5) Planting in fields/green house Advantages 1) It could be preserved for long time at lower temperature 2) Rooting, hardening and conversion steps are waved off as these seeds can directly be sowed in the fields like natural seeds 1) 2) 3) 4) 5) 6) For the development of hybrid plants which have unstable genotypes or show seed sterility. In case that the seeds become sterile, the immature embryo from the seed can be rescued (called as embryo rescue) and then can be encapsulated artificially with appropriate growth medium to allow its maturation and desiccation for its germination. Germplasm conservation. For various research and analysis purposes like studying the role of endosperm, etc. To produce large number of the clones of elite species at the cheaper cost. To supply adjuvant like plant growth regulators, pesticides, etc. Anther Culture • Anter culture is the process of using anthers to culture haploid plantlets. • The technique was discovered in 1964 by Guha and Maheshwari. • This technique can be used in over 200 species, including tomato, rice, tobacco, barley, and geranium. • Some of the advantages which make this a valuable method for obtaining haploid plants are: The technique is fairly simple It is easy to induce cell division in the immature pollen cells in some species A large proportion of the anthers used in culture respond (induction frequency is high) Haploids can be produced in large numbers very quickly. In experiments using Datura innoxia, induction frequencies of almost 100% and a yield of more than one thousand plantlets or calluses have occurred under optimal conditions from one anther. Success can be determined within 24 hours as cells begin to divide. Some disadvantages of using anther culture to obtain haploids are: 1) When working with some species, the majority of plants produced have been non-haploid . 2) In cereals, very few green plants are obtained; many of the plants are albinos or green-albino chimeras. (a chimera is an animal that has two or more different populations of genetically distinct cells that originated in different zygotes; ) 3) It is tedious to remove the anthers without causing damage. 4) Sometimes a particular orientation is necessary to acheive a desired response. This diagram shows the various stages of anther and isolated pollen culture. The stages of anther culture from anther to haploid plantlet can be described as follows: a) an unopened flower bud, 1b) anthers, 1c) the anthers in culture, 1d) and 1e) proliferating anther, 1f) haploid callus, 1g) differentiating callus, h) haploid plantlet. Isolated pollen culture is as follows: a) an unopened flower bud, 3b) isolated pollen from a cultured anther, 3c) pollen culture, 3d) multinucleate pollen, 3e) and 3f) pollen embryo. Homozygous plants can be obtained by treating the haploid plantlets with colchicine. Colchicine is a toxic natural product and secondary metabolite, originally extracted from plants of the genus Colchicum and used originally to treat rheumatic complaints. Colchicine's present medicinal use is in the treatment of gout and familial Mediterranean fever. It is also being investigated for its use as an anti-cancer drug. In neurons, axoplasmic transport is disrupted by colchicine. Protocol for Pollen Culture 1. Flower buds are collected from the plants after all preparations for culturing in the laboratory have been made. 2. Buds are collected in a nonsterile petri dish and the length of each bud measured using a cm scale. Buds of corolla length 21-23 mm are chosen as the pollen in them would have usually completed the first mitosis. 3. The buds are chilled for 12 days (7 to 8°C) in a refrigeration unit. 4. They are then surface sterilized in a petri dish containing a suitable sterilizing agent, for instance 0.01 % solution of mercuric chloride. • 5. Using sterile, double distilled water, the buds are rinsed 3-4 times in a sterile air cabinet. 6. Using forceps and a dissecting needle, the buds are carefully teased open and the anthers removed. 7. The anthers dissected out from each bud are grouped separately on removal. 8. One anther from each group is then removed and squashed in acetocarmine stain in order to determine the stage of pollen development. The pollen should be in the first pollen division. 9. For each culture, anthers from tobacco buds are placed in 5 ml of liquid medium in a petri dish. 10. After regular intervals of 6, 10 and 14 days of culture, the anthers are removed from the culture and discarded. That ensures the dehiscence of all anthers and release of pollen into the culture medium. 11. The dishes are then sealed with parafilm and incubated at 28°C in darkness for the first 14 days of culture. 12. After 14 days, the cultures are transferred to an illuminated growth chamber (500 lux, 12-h daylength, 25°C). 13. The growth of haploid embryos to plantlets developed from released pollen should be observed. 14. Follow steps 13 and 14 given in the protocol for anther culture for growing the plantlets to maturity. These are cultured primarily for the production of Haploid plants which find important application in the field of plant breeding. Pollen culture is also termed as Microspore Culture. Haploids are sporophytes of higher plants with gametophytic chromosome constitution. It is possible to induce haploidy either using anther culture, henceforth reffered to as ‘androgenesis’ or from cultures of individual pollen grains. Pollen culture offers certain advantages over anther culture due to the elimination of anther wall, e.g., i. Studies on differentiation and development are easier and more precise, ii. No callus formation can occur from wall tissue and iii. Products from different pollen grains ordinarily do not get mixed up (this eliminates the risk of chimera). A). Culture Medium Medium requirements may vary with: 1. Species, 2. Genotype, 3. Age of donor plants and anthers, and 4. Conditions under which the donor plants are grown. For example, pollen grains of Datura and tobacco produce embryos on an agar medium containing only 2-4% sucrose. For most plant species, a complete tissue culture medium is required. MS, LS (Linsmaer and Skoog) and some other tissue culture media are generally used. Media with dilute salt solutions, e.g., White’s and Heller’s media, are ordinarily supplemented with coconut milk. B). Growth Regulators C). Stage of Pollen Development etc., the optimum stage is just before or just after the first pollen mitosis, while the early binucleate stage is the most suitable for species Anther cultures are generally maintained in alternating periods of light (12-18 hr; 5,00010,000 lux m2) at 28°C and darkness (12-16 hr) at 22°C, but the optimum conditions vary with the species. The walls of responsive anthers turn brown and after 3-8 weeks they burst open due to the developing callus or embryos. After the seedlings (from embryos) or shoots (from callus) become 3-5 cm long, they are transferred to a medium conducive to good root development. Exposure of excised flower buds to a low temperature for some time e.g., at 3-5°C for 2 days or at 7-8°C for 12 days for tobacco, prior to removal of anthers for culture may markedly enhance the recovery of haploid plants. In some species, however, a brief exposure of anthers to a high temperature is reported to have a promotory effect. Androgenesis in barley is promoted by the use of wheat or barley starch as gelling agent (in place of agar), and by the addition of ficoll (a neutral, highly branched, high-mass, hydrophilic polysaccharide which dissolves readily in aqueous solutions)in liquid medium. In many species, activated charcoal (in agar-gelled media) is promotive. In addition, amino acids like glutamine, proline, serine etc. enhance the frequency of responsive anthers. Anther extracts and media conditioned byculturing anthers for few days improve androgenesis; thus anther wall seems to provide some nutritional factors. Somaclonal variation It is the term used to describe the variation seen in plants that have been produced by plant tissue culture. Chromosomal rearrangements are an important source of this variation. Somaclonal variation is not restricted to, but is particularly common in plants regenerated from callus. The variations can be genotypic or phenotypic, which in the later case can be either genetic or epigenetic in origin. Typical genetic alterations are: changes in chromosome numbers (polyploidy and aneuploidy), chromosome structure (translocations, deletions, insertions and duplications) and DNA sequence (base mutations). Typical epigenetic related events are: gene amplification and gene methylation Historically, plant cell culture has been viewed by most to be a method for rapid cloning. In essence, it was seen as a method of sophisticated asexual propagation, rather than a technique to add new variability to the existing population. ORIGINS AND MECHANISMS OF SOMACLONAL VARIABILITY: Somaclonal variation can be of two sorts: 1)Genetic (i.e. heritable) variability – caused by mutations or other changes in DNA. 2)Epigenetic (i.e. non-heritable) variability – caused by temporary phenotypic changes. Various molecular mechanisms are responsible for genetic variability associated with somaclonal variation. One of the more frequently encountered types of somaclonal variation results from changes in chromosome number, that is, aneuploidy, polyploidy, or mixoploidy. Changes in ploidy originate from abnormalities that occur during mitosis. For example, extra chromosomal duplication during interphase, spindle fusion or lack of spindle formation and cytoplasmic division. A plant cell grows and age, the frequency of changes in ploidy increases. Therefore, changes in ploidy observed in cultures and regenerated plants might have their origins in the source of tissue explants used. Another cause of variability due to changes in ploidy is the in vitro culture regime itself. The longer the cell remains in culture the greater is its chromosomal instability. In addition, the composition of the growth medium can trigger changes in ploidy. For example, both kinetin and 2, 4-D are implicated in ploidy changes and cultures grown under nutrient limitation can develop abnormalities. Selecting a suitable explant and an appropriate culture medium can therefore enhance the chromosomal stability of the culture. However, high variations of ploidy in cultures do not always lead to high frequencies of somaclonal variation in regenerated plants. This is because, in mixed cultures, diploid cells appear to be better fitted than aneuploid or polyploidy cells for regeneration, as they are more likely to form meristems. Structural changes in nuclear DNA appear to be a major cause of somaclonal variation. The changes can modify large regions of a chromosome and so may affect one or several genes at a time. These modifications include the following gross structural rearrangements. Deletion: loss of genes Inversion: alteration in gene order Duplication: duplication of genes Translocation: segments of chromosomes moving to new locations. Epigenetic changes somaclonal variation can be temporary and over time are reversible. However, sometimes they can persist through the life of the regenerated plant. One common phenotypic change seen in plants produced through tissue culture is rejuvenation. Rejuvenation causes changes in morphology such as earlier flowering and enhanced adventitious root formation. Epigenetic changes may be caused by DNA methylation and thus may be one of the important causes of somaclonal variation. As plant tissues are composed of heterogeneous array of cells of various ages, different physiological states and degree of differentiation and cells with different ploidy level exist. By placing cells in tissue culture, the genome at different molecular states is suddenly placed under stress to cope with in vitro conditions. It has also been reported that changes in tissue culture conditions could influenced the frequency of variation. The end effect seems to be an array of genetic engineering changes. Studies concerning different aspects of somaclonal variation are important for the following reasons. Firstly, however successful utilization of SC variation heavily depends upon its systematic evaluation and judicious utilization in breeding programmes. This demands appropriate experimentation. Secondly, SC variation is of interest as a basic genetic process, since it contradicts the concept of clonal uniformity. The cells and tissues which are expected to produce true to type plants through the processes of de-differentiation, division and re-differentiation, possibly perceive the whole process as stress, as a result of which the genome, known for its plasticity, restructures itself to modulate the expression of gene as demanded by the in vitro conditions. Third , SC variation is unwanted when the objective is mricropropagation of elite genotypes or genetic transformation that partly involved tissue culture. Under such circumstances, prevention or at least minimization of variation is of utmost importance. To achieve this, the frequency, nature and magnitude of somaclonal variation in relation to manipulation of media components, explant source, culture conditions etc. should essentially be understood. Majority of studies undertaken on SC variation are confined to early generation of soma clones. Therefore information on the nature, inheritance pattern and stability of morphological and molecular changes expressed in the advanced generation of soma clones is lacking. The different aspects of somaclonal variation investigated so far are as follows 1) Generation of variation 2) Characterization of variant for morphological traits 3) Analysis of biochemical and chromosomal basis of variation 4) Relating the variation to alteration in DNA The mechanism of somaclonal variation According to Bhaskaran (1985) variations in somaclones occur due to the following reasons: 1. The pre-existing genetic variations in the explant tissue, 2. The spontaneous mutations that can accumulate during the many division cycles that cell of the explant go through before differentiating into an in vitro plant. The recessive mutation will naturally require a method by which they can express even diploid cells. Somatic crossing over followed by segregation is a likely mechanism, for the homozygosity and thus phenotypic expression of the recessive (Chopra and Sharma, 1988). 3. Intracellular mutagenic agents produced during in vitro growth. 4. Numerical and structural changed in chromosomes during in vitro growth. 5. Activation of transposable elements or jumping genes, are genetic entities which have the locus at which they get integrated is matured. 6. Agriculturists are very hopeful about practical advantages of somaclonal variation and they are waiting when this technique is fully integrated with the conventional plant breeding procedures. Take an aliquot of suspension and filter off the culture through a wire mesh (300mm). note the volume of the filtrate (F) containing single cells and small clumps and place the drop of this suspension to heamocytometer to determine the number of cells by the equation N = P x 100 x F 0.1 mm where, N = total number of cells and clumps, P = number of cells in the squares of the haemocytometer, f = volume of the filtrate. Forms of somaclonal variation: Many different forms of somaclonal variation arise. The most common forms include point mutation, chromosomal aberrations and increase or decrease in the number of nuclear chromosomes. It is important to realize that not all forms of variability that arise in vitro are heritable. Some morphological and biochemical variants are due to physiological effects and are not exhibited in subsequent generations. There are several different approaches to detecting and isolating somaclaonal variants from cultured plant cell populations. Morphologically distinct cells such as nonphotosynthetic (nongreen) cells or cells that accumulate anthocyanin and other plant pigments are detected visually. To isolate herbicide- and antibiotic-resistant variants, plant cells are simply grown on media containing of the wild type cells in a culture. The surviving cells are then subcultured and retested for growth on herbicide or antibiotic supplemented medium. Through this method, one can eliminate any remaining wild-type cells that may have inadvertently survived the first round of selection. Applications in plant breeding • • • • • • • Somaclonal variation and gemetoclonal variation are the important source of introducing genetic variation that could be of value to plant breeders. Single gene mutation in the nuclear or organelle genome usually provides the best available variety in vitro which has a specific improved character. Somaclonal variations are used to uncover new variant retaining all the favorable characters along with an additional useful trait, e.g., resistance to disease or an herbicide. These variants can then be field tested to ascertain their genetic stability. Gametoclonal variation is induced by meiotic recombination during the sexual cycle of the F1 hybrid results in transgressive segregation to uncover unique gene combinations. Various cell lines selected un vitro and plant regenerated through it prove potentially applicable to agriculture and industry specially resistance to herbicide, pathotoxin, salt or aluminium, useful in the synthesis of secondary metabolites on a commercial scale, etc. The techniques used for development of somaclonal and gametoclonal variation are relatively easier than recombinant DNA technology and is the appropriate technology for genetic manipulation of some crops. The main factors that influence the variation generated from tissue culture are (1) the degree of departure from organised growth, (2) the genotype, (3) growth regulators and (4) tissue source. Despite an increasing understanding of how these factors work it is still not possible to predict the outcome of a somaclonal breeding programme. New varieties have been produced by somaclonal variation, but in a large number of cases improved variants have not been selected because (1) the variation was all negative, (2) positive changes were also altered in negative ways, (3) the changes were not novel, or (4) the changes were not stable after selfing or crossing. Somaclonal variation is cheaper than other methods of genetic manipulation. At the present time, it is also more universally applicable and does not require ‘containment’ procedures. It has been most successful in crops with limited genetic systems and/or narrow genetic bases, where it can provide a rapid source of variability for crop improvement.