Plant tissue culture: Plant tissue culture is a collection of techniques used to maintain or grow plant cells, tissues or organs under sterile conditions on a nutrient culture medium of known composition. Callus: Plant callus (plural calluses or calli) an unorganised, growing and dividing mass of cells. Callus is usually composed of unspecialised parenchyma cells. 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. Callus formation takes place under the influence of exogenously supplied growth regulators present in the nutrient medium. 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.History of tissue culture: 1838- Cell theory, indicating towards totipotentiality of cells by Schleiden and Schwann. 1902- First but unsuccessful attempt of tissue culture using monocots by Haberlandt. He also explained the concept of cell totipotency. 1904- First attempt in embryo culture of s d Crucifers by Hannig. 1922- A symbiotic germination of orchid seeds by Knudson. 1922- In vitro culture of root tips by Robbins. 1924- Callus formation on carrot root explants by use of lactic acid by Meyer. 1934- In vitro culture of cambial tissues of different trees and shrubs failed by Guatheret. 1934- Identification of the first plant hormone, IAA, leading to cell enlargement by Kogl. 1941- Coconut Milk used for growth and development of very young Datura embryos by Overbeek. 1942- Observation of secondary metabolites in plant callus cultures by Gautheret. 1943- Tumor-inducing principle of crown gall tumors identified by Braun, 1944- First In vitro culture of tobacco used to study adventitious shoot formation by Skoog. 1946- First whole plants of Lupines and Tropacolum from shoot tips by Ball. 1948- Formation of adventitious shoots and roots in tobacco by Skoog. 1957- Discovery that root or shoot formation in culture depends on auxin: cytokinins ratio by Skoog and Miller. 1958- In vitro culture of excised ovules of Papaver somniferum by Maheshwari.1958- Regeneration of somatic embryos from nucleus of Citrus ovules by Maheshwari and Rangaswamy. 1962- Development of MS medium by Murashige and Skoog. 1964- First haploid plants from Datura androgenesis by Guha and Maheshwari. 1973- Cytokinins found to be capable of breaking dormancy in Gerberas by Pierik 1978- Somatic hybridization of tomato and potato resulting pomato by Melchers. 1978- Industrial scale fermentation of plant cells for production of shikonin. (Selection of cell lines with higher yield of secondary products) by Tabata. 1981- Introduction of the term somaclonal variation by Larkin. 1981- Isolation of auxotroph by cell colony screening in haploid protoplasts of Nicotiana plumbaginifolia treated with mutagens by Sidorov. 1985- Infection and transformation of leaf discs with Agrobacterium tumefaciens and regeneration of transformed plants by Horsch. 1985- Development of disarmed Ti-plasmid vector system for plant transformation by Fraley. 1985- Development of binary vector system for plant transformation. 1985- Gene transfer in protoplasts of Dicot and Monocot plants by electroporation. 1993- In vitro fertilization with isolated single gametes resulting in zygotic embryogenesis and recovery of fertile maize plants by Kranz. 1993- "Green Hairy roots" showing photoautotrophy due to development of photosynthetic ability by Flores. 1996- Development of ‘agrolistic’ method of plant transformation by Hansen.Importance or Advantages of Plant Tissue Culture clones: Plant Tissue Culture allows the production 1, Mass multiplication of of large numbers of plants from small pieces of the mother plant. The production requires relatively short periods of time to grow plants. Depending on the species under production, a single ex-plant can be multiplied into several thousand plants in less than one year. Another purpose for which plant 2. Elimination of diseases in planting materi tissue culture is uniquely suited is in the obtaining, maintaining, and mass propagating of specific disease-free plants. The concept behind indexing plants free of pests is closely allied to the concept of using tissue culture as a selection system. Plant tissues known to be free of the disease under consideration (viral, bacterial or fungal) are physically selected as the explants for tissue culture. Tissue culture could be a useful way of circumventing or eliminating disease, which can accrue in stock plants. 3. Plant improvement through tissue culture: Creation of superior varieties of agricultural crops is possible through tissue culture method, which otherwise is not possible through conventional plant breeding methods. 4. True to Type production: Large number of true to the type plants could be propagated within a short time and space and that too throughout the year. For example, it may be possible to propagate Two to Four lakhs of tissue cultured plants from a single bush or rose against 10 to 15 plants by conventional means. Also, it may take about Two to Four months to produce a healthy planting material by tissue culture means, whereas a minimum of Six to Eight months is required for most species by the latest method of plant propagation.5. Higher Yields: Tissue Culture Plants may have increased branching and flowering, greater vigour and higher yield, mainly due to possibility of elimination of diseases. 6. Beneficial when conventional propagation is difficult: The method may succeed to propagate plants where seeds or conventional propagation is not possible or difficuk or undesirable. 7. Efficient method in saving space and energy: The method saves space and energy of the farmer. For example, in a conventional method the plants are grown in the open farm requiring an area of about 25,000 m , same 10 number of plants would require only 10 m* space, if they are grown in the tissue culture laboratory. 8. Flexible method: The flexibility of nurseries can be improved. As the capital investment on mother plant is reduced to almost zero, it may be easier to adapt to changing conditions. Additionally, a better programme of production is possible, because of the greater plant uniformity and the availability in the mass at any time. 9. Innovation of new varieties: Tissue culture can be utilized for breeding new varieties. Applications of Plant Tissue Culture: 1. The plant tissue culture technique provides a way for rapid multiplication of —————desirable.nlants____4. Large-scale growth of plant cells in liquid culture in bioreactors for production of valuable compounds, like plant-derived secondary metabolites and recombinant proteins used as biopharmaccuticals. 5. To cross distantly related species by protoplast fusion and regeneration of the novel hybrid. 6. To rapidly study the molecular basis for physiological, biochemical, and reproductive mechanisms in plants, for example in vitro selection for stress tolerant plants. 7. To cross-pollinate distantly related species and then tissue culture the resulting embryo which would otherwise normally die (Embryo Rescue). 8. For chromosome doubling and induction of polyploidy, for example doubled haploids, tetraploids, and other forms of polyploids. This is usually achieved by application of antimitotic agents such as colchicine or oryzalin. 9. As a tissue for transformation, followed by either short-term testing of genetic constructs or regeneration of transgenic plants. 10. Certain techniques such as meristem tip culture can be used to produce clean plant material from virused stock, such as sugarcane, potatoes and many species of soft fruit. 11. Production of identical sterile hybrid species can be obtained. 12, Large scale production of artificial seeds through somatic embryogenesisTotipotency: Totipotency is the ability of a single cell to produce all cell types and to organize them into an entire organism when cultured in a suitable culture medium at an appropriate temperature and aeration conditions. Spores and Zygote are examples of totipotent cells. Totipotency in Plant: In plants, even highly mature and differentiated cells retain the ability to regress to a meristematic state as long as they have an intact membrane system and a viable nucleus, Cellular totipotency is the inherent potentiality of a plant cell to give rise to a whole plant, a capacity that is retained even after a cell has undergone final differentiation in the plant body. For a differentiated cell to express its totipotency, it first undergoes “dedifferentiation” followed by “redifferentiation.” Mostly, “dedifferentiation” involves embryonization of cells leading to callus formation. However, embryonic explants often exhibit differentiation of roots, shoots or embryos without an intervening callus phase. Tissue culture techniques offer an excellent opportunity to study the factors that elicit the totipotentiality of cells, and allow the investigation of factors controlling cytological and_ histological differentiation. Two substances that have a profound effect on vascular tissue differentiation are auxin and sucrose. They affect vascular differentiation qualitatively as well as quantitatively.Types of tissue culture: Callus culture: Callus culture may be defined as production and maintenance of an unorganized mass of proliferative cell from isolated plant cell, tissue or organ by growing them on artificial nutrient medium in glass vials under controlled aseptic conditions. Organ culture: That may allow differentiation and preservation of the architecture. The organ culture refers to the in vitro culture and maintenance of an excised organ primordial or whole or part of an organ in way and function. Single cell culture: Single cell culture is a method of growing isolated single cell aseptically on nutrient medium under controlled condition. Suspension culture: Suspension culture is a type of culture in which single cell or small aggregates of cell multiply while suspended in agitated liquid medium. Suspension cultures are used in induction of somatic embryos and shoots, production of secondary metabolites, in vitro mutagenesis, selection of mutants and genetic transformation studies. Embryo culture: Embryo culture may be defined as aseptic isolation of embryo (of different developmental stages) from the bulk of maternal tissue of mature seed or capsule and in vitro culture under aseptic and controlled physical condition in glass vials containing nutrient semisolid or liquid medium to grow directly into plantlet. Anther culture: Androgenesis is the in vitro development of haploid plants originating from potent pollen grains through a series of cell division and differentiation. Pollen culture: Pollen culture is the in vitro technique by which the pollengrains (preferably at the microscope stages) are squeezed from the intact anther and then cultured on nutrient medium where the microspores without producing male gametes.Somatic Embryogenesis: Somatic embryogenesis is the process of a single or group of cells initiating the development pathway that leads to reproducible regeneration of non-zygotic embryos capable of germinating to form complete plants. Protoplast Culture: It is the culture of isolated protoplasts which are naked plant cells surrounded by plasma membrane which is potentially capable of cell wall regeneration, cell division, growth and plant regeneration on suitable medium under aseptic condition 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. Explant Culture: There are variety of forms of seed plants viz., trees, herbs, grasses, which exhibit the basic morphological units i.e. root, stem and leaves. Parenchyma is the most versatile of all types of tissues. They are capable of division and growth.Factors Affecting Tissue Culture Efficiency: Plant regeneration from tissue culture varies with the following parameters: 1) Plant species 2) Genotype within the species 3) Source of the cultured tissue 4) Age and health of the donor plant 5) Nutrient medium, other factorsChoice of explant: The tissue obtained from a plant to be cultured is called an explant. Explants can be taken from many different parts of a plant, including portions of shoots, leaves, stems, flowers, roots, single undifferentiated cells and from many types of mature cells provided are they still contain living cytoplasm and nuclei and are able de- differentiate and resume cell division. In many species explants of various organs vary in their rates of growth and regeneration, while some do not grow at all. The choice of explant material also determines if the plantlets developed via tissue culture are haploid or diploid. Also the risk of microbial contamination is increased with inappropriate explants. Some explants, like the root tip, are hard to isolate and are contaminated with soil microflora that become problematic during the tissue culture process. These associated microflora will generally overgrow the tissue culture medium before there is significant growth of plant tissue. Some cultured tissues are slow in their growth. For them there would be two options: (i) Optimizing the culture medium; (i) Culturing highly responsive tissues or varieties. Necrosis can spoil cultured tissues. Generally, plant varieties differ in susceptibility to tissue culture necrosis. Thus, by culturing highly responsive varieties (or tissues) it can be managed. Aerial (above soil) explants are also rich in undesirable microflora. However, they are more easily removed from the explant by gentle rinsing, and the remainder usually can be killed by surface sterili n. Most of the surface microflora do not form tight associations with the plant tissue. Such associations can usually be found by visual inspection as a mosaic, de-colorization or localized necrosis on the surface of the explant. ‘An alternative for obtaining uncontaminated explants is to take explants from seedlings which are aseptically grown from surface-sterilized seeds. The hard surface of the seed is less permeable to penetration of harsh surface sterilizing agents, such as hypochlorite, so the acceptable conditions of sterilization used for seeds can be much more stringent than for vegetative tissues.Flow chart of plant propagation by tissue culture metho PREPARATION OF MEDIUM ‘SELECTION OF A MOTHER PLANT q Sah STERLISATION IN 1 AUTOCLAVE POURING IN BOTTLES ‘CUTTING OUT THE EX-PLANT PART en WASHING EX-PLANT IN WATER, SOAP AND ANTISEPTIC SOLUTION y a ‘SEALING AND RESTERILISATION ‘COOLING AND SETTING REWASHING AND SURFACE STERILISATION OF EXPLANTS IN CHEMICAL SOLUTION “SHIFTING OF EXPLANT TO LAMINAR AIR, FLOW STATION READY FOR INOCULATION to ‘SUBCULTURING (____,] sreriuiseo MEDIUM oF KNOWN. “SURFACE STERILISATION IN SODIUM HYPOCHLORITE FOLLOWED BY WASING IN DISTILLED WATER FOR 3-4 TIMES. SEER Ee INOCULATION OF EXPLANT ON COMPOSITION — —_ ‘SHIFTING OF CULTURE TO GROWTH ROOM (15-25°C, 3000 LUX) 3-6 WEEKS y_ BUNCH OF IN-VITRO SHOOTS (GROWTH) ‘SEPARATION OF IN-VITRO SHOOTS, TRANSFER TO ROOTING SOLUTION IN GROWTH ROOM TRANSFER TO GREEN HOUSE FOR HARDENING.