Tissue Culture

II B.Sc. GENETICS: PLANT TISSUE CULTURE Plant tissue culture refers to inoculation and growth of plant explants on artificial culture medium under controlled conditions like temperature, light etc. The part of plant used for inoculation and culture is called explant. It may be single cell, isolated protoplast, tissue, excised embryo, anther, pollen grain etc. The explants after incubation under controlled conditions of pH, temp, light etc. may divide and regenerate into complete plants. Plant tissue culture is a non-conventional method exploited for the production of unique plants, rare hybrids and valuable plant products with medicinal or commercial value. The entire technique is based on the unique feature of plant cells called totipotency i.e. the ability of single cell to divide, differentiate and regenerate into complete plant. Totipotency or regeneration capacity is the unique feature of all live plant cells. But in animals totipotency is present in only primitive organisms and fertilized egg cells of higher animals. Under controlled conditions like temperature, light, pH and availability of nutrients, plant cells on artificial medium divide to form an undifferentiated mass called callus which on further divisions regenerate into complete plants. Historical Aspects: > henri-Louis Duhamel du Monceau (1756) first time observed wound healing in plants by spontaneous callus formation. His observations and studies is beginning for the art of in-vitro plant tissue culture. > Further contributions to the PTC can be attributed to the Schleden cell doctrine acoording to which cell is capable of autonomy and potential totipotency. > Trecul (1853) observed callus formation in a no. of decorticated trees. > Vochting (1878) suggested polarity as acharacteristic feature guiding the development of tissue culture. > Weisner 91884) suggested the presence of organ forming substances distributed in polar fashion. Minimal size of explants is an important factor that determines the potential for differentiation. > Haberlandt (1902) developed the concept of in-vitro cell culture. He was the first man to isolate and culture the cells on a nutrient medium containing glucose, peptone etc. > Hanning (1904) initiated culture of embryogenic tissue and later developed embryo culture. Important Applications of in-vitro plant tissue culture: Production of rare and unique plants : In some tree species and ornamental plants, seed dormancy is for longer times. In such plants dormancy is eliminated by resorting to in-vitro tissue culture. Eg: Orchids seeds undergo prolonged dormancy. To avoid dormancy, they are subjected to micro propagation Production of Rare hybrids: Some times it is not possible to produce Interspecific or intervarietal hybrids are due to post zygotic incompatibility barriers. To overcome these barriers, fertilized embryos are excised and placed on nutritional medium to promote normal growth. Production of somatic cell Hybrids: Some times it is not possible to produce viable hybrids between two geneticall different plants. In such case protoplasts are isolated from these

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II B.Sc. GENETICS: STERILIZATION TECHNIQUES IN PLANT TISSUE CULTURE Sterilization is defined as the process of making any material aseptic in nature i.e. making a given environment or substance free from all forms of microorganisms. In in-vitro plant tissue culture, microorganisms like bacteria and fungi often contaminate cultures. Hence it is highly essential to maintain culture media, tissue explants, culture tubes, vessels, inoculating instruments, culture room etc. in aseptic conditions. Sterilization is done for different materials by different methods. Depending on the nature of the material to be sterilized, the methods are categorized into physical and chemical methods.

Physical methods: sterilization by heat : Heat sterilization is the simplest and most reliable method. It can be applied in two forms. a. Dry heat – is used for sterilizing glassware like Petri plates, test tubes, pipettes and inoculation instruments. Dry heat is provided by hot air ovens with thermostat. At high temp. i.e. 160 – 1700c, for one to two hours, eliminates all forms of micro organisms due to oxidation. Some chemicals, which could not be autoclaved, are also sterilized by dry heat. Eg: Oils, powders, and anhydrous fats. b. Moist heat: Heat in the form of saturated steam under pressure is the most practical and reliable agent for sterilization. Steam under pressure provides tremendous heat above the boiling point. Moist heat kills microbes by coagulating and denaturing their enzymes and structural proteins. Moist heat is provided by an apparatus called autoclave in which pressure and temperature can be regulated. Moist heat sterilization is used for sterilizing media components, dissolved chemicals. It is also used for decontamination of contaminated culture tubes. Radiation: Radiations are used to eliminate different microorganisms especially in inoculation chambers and culture rooms. U.V. light is the most suitable sterilization agent. Maximum effect of U.V. can be observed at 260nm wavelength. In plant tissue culture procedures inoculation chambers, instruments and culture media are exposed to U.V. light prior to inoculation. Filtration: Filtration is used to remove microorganisms from solutions, which are heat sensitive. Unique chemicals like enzymes and Phytohormones are sterilized by filtration before adding to culture stocks. Different types of filters are used to remove bacterial endospores and fungal spores. These filters are made from asbestos, diatomaceous earth or biological membranes or cellulose papers. Membrane filters and Millipore filters are most frequently used filters for sterilization. Inoculation chambers and culture rooms are freed from microorganisms by air filters called HEPA filters (High Efficiency Particulate Air filters). Sterilization temperature for filters should be critical and should not exceed 1210c. Chemical Methods: Different chemicals like surface sterilants, disinfectants, antibiotics etc. are used at different steps in in-vitro plant tissue culture. The chemical agents used for sterilization must have certain features like antimicrobial activity, lack of toxicity, ability to penetrate and activity at ordinary temperature. Explants used in tissue culture like seeds, tissue pieces, excised embryos, pollen grains etc. are treated with disinfectants like H2O2, sodium hypochlorite, ethyl alcohol, mercuric chloride etc. to remove different forms of microorganisms (surface sterilization). Inoculation tools like forceps, scalpels, and needles are autoclaved and surface sterilized by dipping in alcohol followed by flame sterilization. Culture rooms are maintained under aseptic conditions through fumigation with potassium permanganate in chloroform. Sometimes the culture stocks and prepared culture media may retain heat resistant endospores of bacteria which subsequently germinate and contaminate culture media. To avoid their germination, antibiotics like streptomycin are added to the medium.

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CULTURE MEDIA FOR PLANT TISSUE CULTURE Selection (or) development of culture medium is very important for successful plant tissue culture. No single medium will support the growth of all explants because growth response to a particular medium varies form cell to cell within a single plant, plant to plant, species to species. So changes in the medium are often necessary for different types of growth response in a single explant. Different types of cultures like embryo culture, anther culture, protoplast culture, culture of somatic tissues require different media. These media are Murashigo-Skoog medium (MS medium), Gamborgs medium, Nitsch medium etc. The formulation of these media shows variation in terms of inorganic and organic salts, Phytohormones and essential chemicals. In general, the medium contains inorganic salts, plant growth regulators, vitamins, pH stabilizers, carbohydrates and gelling agents. In addition, the medium also includes amino acids, antibiotics and natural substances. Such a medium composed of ‘chemically defined compounds is referred to as “ Synthetic Medium” a) Inorganic salts: Growth of explants is supported by a variety of macro and micro nutrients. The macronutrients include nitrogen (N), potassium (K), calcium (Ca), magnesium (Mg), and sulphur (S). Nitrogen is the critical among macronutrients and is provided as different inorganic salts. Micronutrients include iron (Fe), Manganese (Mn), zinc (Zn) boron (B), copper (Cu), cobalt (Co) and molybdenum (Mo). Inorganic salts used in culture media are nitrates, sulphates, halides, along with chelating compounds like Fe-EDTA. MS medium contains high concentrations of nitrate, potassium and ammonium compared to other media. Usually inorganic salts are prepared as stock solutions that can be useful for preparation of several liters of media. Reagent grade chemicals are used for making inorganic stock solutions. b) Plant growth regulators: Phytohormones are incorporated in culture medium for different purposes like callus induction, shooting, rooting etc. The nature and quantity of Phytohormones is according to type of culture being undertaken.

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Auxins: Auxins are required for initiation of cell division and root initiation. High conc. of IAA inhibits morphogenesis and promotes callus induction. Eg: 2,4–D (2,4–Dichloro phenoxy acetic acid) is widely used for callus induction. IA3 (Indole-3-acetic acid) is used for root induction. Other auxins include NAA (1-naphthalene acetic acid), IBA ( Indole-3-butyric acid) etc. Auxins are thermo stable. Hence they are not denatured during medium autoclaving.

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Cytokinins: Cytokinins like Kinetin, Benzyl amine, and Zeatin promote morphogenesis i.e. shoot induction. Usually cytokinin stocks are added to the medium after filter sterilization because they are thermo sensitive and may get denatured, if added before autoclaving.

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Gibberlic acid: also promotes morphogenesis and inhibits callus growth. GA3 is included in the medium for the regeneration of shoots from callus explants. GA3 is also thermo stable like auxins and is added to the medium prior to sterilization.

c) Vitamins : Most of the vitamins act as catalysts in enzymatic reactions and are essential for in-vitro plant tissue culture. They include thiamine (Vit. B1), Nicotinic acid (Vit. B3) and Pyridoxine (Vit. B6) etc. Biotin, Ascorbic acid, and Folic acid are also added to the media as vitamin supplements. Vitamins are common requirement for any type of culture. So they are prepared as a stock and added to the medium ingredients before autoclaving. But to study the effect of specific vitamin, the vitamins are filter sterilized and then added to autoclaved medium. d) Carbohydrates: In-vitro growing cells cannot synthesize carbohydrates by their own because they are not photo synthetically active. The carbohydrate requirement for growing culture is supplemented by the addition of simple sugars like sucrose or glucose approximately 2 – 5%. Sucrose is commonly used sugar. Other carbohydrates sources like fructose and starch are also used for specific cultures. Usually sugar concentration will be kept low for protoplast cultures and high for embryo (or) Anther cultures. e) Haxitoles: Hexitoles mediate important metabolic reactions in in-vitro culture cells. They take part in germination of seeds, sugar transport, mineral nutrition, carbohydrate metabolism, cell wall formation, hormonal homeostasis etc. Myoinositol is an important haxital added to culture medium. It acts as growth enhancer, offers it self as carbohydrate source and sometimes exert vitamin like action. Other hexitols used in in-vitro culture are mannitol and sorbitol.

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f) Gelling agent: Solidifying / gelling agent is used to provide surface and support for in-vitro growing explants. Common gelling agent used is agar. It is a non – nutritive organic substance. g) Amino acids: Amino acids play vital role in morphogenesis. Tyrosine is essential for shoot initiation and Arginine promotes rooting. Glutamine and Aspergine enhance somatic embryogenesis. h) Antibiotics: Sometimes the medium may get contamination even after sterilization due to the presence of heat resistant endospores of bacteria and fungal conidia. In order to avoid their germination in autoclaved or inoculated medium, antibiotics are added to the medium. General antibiotics used in in-vitro culture are streptomycin, penicillin and Ampicillin. i) Natural substances: Based on the nature of explant and specific genus or species, some natural substances are added to the medium. They include coconut liquid endosperm, yeast extract, tomato juice, malt, banana extract, potato extract, casein, hydrolysates, fish emulsion etc. These natural substance supplement several amino acids, vitamins, proteins, enzymes , cofactors to the growing explants. CULTURE STOCKS : 1. Nitrate stock 2.

3. 4. 5.

Ammonium nitrate Potassium nitrate Sulphate stock Magnesium sulphate Manganese sulphate Zinc sulphate Cupric sulphate Halide stock Calcium chloride Potassium Iodide Cobalt chloride PBM stock Potassium phosphate Boric acid Sodium molybdanate Na Fe EDTA stock Ferrous sulphate Na2 EDTA

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Anther Culture Anther culture is meant for the production of haploid plants. Regeneration of haploid plants in-vitro from anthers and excised pollen grains is called androgenesis. Androgenesis is induced by inoculating haploid cells on suitable aseptic culture medium. In-vitro anther culture and regeneration of haploids was first time done by Shimakura (1934). Since then, haploids are being produce by anther culture in several food and commercial crops. Eg: Wheat, Rice, Nicotiana, Chillies, Pepper, Grapes etc. Importance of haploids: 1. Since haploids possess only single set of chromosomes, any recessive mutation can be expressed phenotypically in monozygous condition. Hence haploids are used for mutation breeding. 2. By spontaneous or induced chromosome doubling, dihaploids may be produced from haploids. These dihaploids are completely homozygous and used as pure line varieties. In-vitro culture of haploids: Regeneration of haploids in in-vitro conditions require explants, suitable culture media and inoculation facilities. Explant preparation: For haploid culture, anthers (or) pollen grains are used as explants. Usually early flowers at unopened bud stage are suitable. Anthers from these buds are excised and macerated on slide in acetocaramine stain to determine the state of microspore development. Uni-nucleate pollen grains are most ideal and highly responsive either for callus induction or for regeneration. During inoculation, the flower buds are disinfected by SDS (Sodium Dodecyl sulphate) and anthers are surface sterilized by sodium hypochlorite. Anthers can be directly used as explants (or) they are ruptured to release the pollen grains before inoculation. Culture media for anther culture: Standard media like Murashigo-Skoog and Nitsch media with different proportions of sulphates, nitrates, halides, and EDTA salt are used. The media are supplemented with inositol and sucrose as carbon requirement for ex- plants. Phytohormones like Kinetin and IAA in different proportion are added to the medium and for callus induction and regeneration. The explants on medium are supported by Agar dissolved in the medium. Medium pH always maintained at 5.7 for good in-vitro response. Before inoculation of explants, all medium ingredients are sterilized by autoclaving and dispensed into the culture tubes in aseptic conditions. Factors effecting anther culture: Formation of callus and regeneration of haploids from inoculated explants is influenced by physiological and genetic factors. Generally herbaceous plants are more suitable than tree species. Members of solanaceae family are more responsive and promising for anther culture. Effect of temperature and light: The buds treated with cold temperature at 30C or 50C for 72 hours for inducing pollen embryos in same solanaceous members. Frequency of haploids formed and rich growth of plantlets is generally better in light. Embryo culture Embryo culture refers to growth of excised embryos in-vitro on artificial culture medium. Embryo culture is useful in several plant breeding applications like obtaining rare hybrids, breakdown of dormancy, seed viability testing etc. Most important application of embryo culture is embryo rescue from post zygotic incompatibility barriers. Mature embryos were first time cultured in-vitro from Raphanus sativus by Hanning (1904). In-vitro culture of immature embryos was done for first time by Knutson (1922) in orchids. Excised embryos can be easily grown on culture media with minimum requirement of salts and nutrients. They don’t require hormonal supplement. The medium used for embryo culture is MS medium supplemented with sulphates, Non–sulphates, sucrose along with Agar. Embryos are excised from mature seeds by enzymatic treatment with cellulase and pectinase. These excised embryos are disinfected and sterilized by H2O2 or sodium hypochlorite and inoculated on medium under aseptic conditions. Then the culture tubes with inoculated embryos are kept in culture room with sufficient light and temp. around 18-200C. Applications of in-vitro embryo culture: 1. Embryo rescue: Some hybridization programmes leads to the production of only abortive embryos due to pre zygotic and post zygotic incompatibility barriers. In such cases, a critical stage of embryo abortion is identified and the embryos are excised before reaching that stage. Then the excised embryos are grown by in-vitro on suitable medium until they develop into complete plants. The embryo rescue is used for obtaining rare hybrids, which are not possible, by conventional methods.

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Break down of dormancy: Seeds of some fruit crops undergo long periods of dormancy. Physical and mechanical treatments to breakdown dormancy. Some times leads to embryo damage. If the embryos are excised and get freed from ABA, they may develop into plants without any dormancy. Somatic cell hybridization (Protoplast fusion)

Somatic cell hybridization is a non – conventional method involving fusion between isolated somatic protoplasts under in-vitro condition. The fusion product may be a rare hybrid between two incompatible plants or it may be a cybrid or cytoplasmic hybrid carrying cytoplasmic genes of two parents. Plant protoplasts ( cells devoid of cell wall ) or animal cells can be forced to fuse by physical or chemical methods to form somatic cell hybrids. These hybrid cells are further cultured until the regeneration of complete hybrid plant. Kuster (1909) first reported sponteneous fusion between protoplasts of the same callus. Barski et. al., (1960) demonstrated somatic cell hybridization in mice cells. Harris and Watkins (1965) used inactivated sandai virused to induce fusion between cells. Carlson et. al., (1972) produced somatic cell hybrids ( inter specific) in tobacco.[N glauca with N. langsdorfii] Types of somatic cell hybrids:1. Hetero Karyon (or) Binucleate hybrid:- The nuclei of both protoplasts exist seperately after fusion. 2. Synkaryon (or) uni nucleate hybrid:- Both the nuclei ( of protoplaste) fused to give hybrid cell with single nucleus. 3. Cybrid (or) cytoplasmic hybrid:- One of the parental nuclei in hybrid degenerate to give a hybrid ( In cybridization heterozygosity of extra chr. Material can be obtained) with single nucleus but cytoplasm of both the parental protoplaste. Process of protoplast fusion resulting in the development of cybride is called cybridization. Mechanism of somatic cell hybridization:I Source of Protoplaste:- Protoplaste are generally selected from epidermal and mesophyll cells of leaves because it allows isolation of large no. of relatively uniform cells. Young callus cultures with undifferentiated mass of cells and suspension cells are also best source of protoplaste. Cell suspension cultures also provide excellent source materials for isolating protoplasts. II. Isolation of Protoplasts:A. Mechanical isolation:- Tissues are first plasmolysed by high concentrated salt solution and cut into small pieces. There pieces are again deplasmolysed to get protoplasts. In mechanical isolation protoplasts released are few in number. Isolation of protoplasts mechanically was pioneered by Klerck ( 1892) in higher plants Highly vacuolated cells of storage tissues. B. Chemical isolation:- Explants are cut into small pieces and incubated in different enzymes like cellulase, hemicellulase and pectinase to dissolve cell wall components of cells. a. Sequential emymatre b. mixed emymatre. III. Protoplast fusion:- Protoplasts devoid of cell walls are ready to fuse with other cells under modified physical and physiological conditions. Spontaneous fusion:- During enzymatic degradation to obtain protoplasts, protoplasts of the same tissue may fuse to give homokaryons. More frequent homokary on formation has been observed in protoplasts isolated from dividing cultured cells. Mechanical fusion:- protoplasts from different plants or animals all mixed together subjected to violent shaking or centrifugation result in cell hybrids. The fusion doesn’t depend upon the presence of fusion inducing agent. The most prominent mechanical method is electro fusion. Under low uniform electric fields ( 6 amps) the protoplasts get fused to give somatic cell hybrids. Senda et. al first attempted fusion. Chemical fusion:- chemical agents used for fusion are called fusogens. Several chemical fusogens are employed for protoplast fusion. They are poly Ethylene Glycol (PEG) , NaNo3, CaCl2 , polyvinyl alcohol etc. PЄG is most potential fusogen though it is cytotoxic. PЄG with proper concentration along with calcium chloride added to mixture of protoplasts and incubated. Then the protoplasts mixture is thoroughly washed by centrifugation. ( high frequency of heteokaryon formation). IV. Selection of Somatic cell hybrids:If two different types of protoplasts are treated with suitable fusogen, after incubation the mixture of cells may contain unfused protoplasts, homokaryons (fusion product between similar cells ), Heterokaryans (somatic cell hybrids) and cybrids cytoplasmic hybrids). From this mixture, different cell lines are to be isolated by suitable selection methods.

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Bio – chemical selection:- knowledge on nutritional requirements of protoplasts is necessary for biochemical selection. After fusion, the mixture of cells are grown in a specific medium which selects only fusion products i.e. somatic cell hybrids eg: HAT medium selects only hybridoma cells.

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b. c. d.

Drug sensitivity:- Protoplasts and fusion products exhibit differential sensitivity to drugs like actinomycin – D. In high actinomycin – D content in culture medium, the hybrid cells are able to form callus and differentiate into plants, but normal cells cannot regenerate. Flourescent activated selection:- Parental protoplasts are tagged with flourescent dyes. The fusion products may give different colours and unfused cells give their original colour. visual Selection:- fusion products sometimes produce coloured callus where on unfused cells may form colourless callus.

Applications of somatic cell hybridization:In some vegetatively propogated plants genetic recombination is not possible by conventional methods. Somatic cell hybridization provides an efficient means of bringing genetic recombination. 1. Inter specific hybridization is not possible due to fore and post zygotic incompatibility barriers between different plants. Protoplasts derived from there plants can be fused to get interspecific hybrids. 2. In normal hybridization procedures, some cytoplasmic characters ( characters influenced by DNA of chloroplasts (or) mitochondria ) transferred through materanl cytoplasm only. In somatic all hybridization, it is possible to provide nucleus as well as cytoplasm of both parents to the hybrid. 3. Hybridoma cells – monocloral antibody production. Somatic cell hybrids from B- Lymphocytes and myeloma lymphocytes are called hybridoma cells. these hybridoma cells divide continuously and produce large amounts of antibodies called monoclonal anti – bodies. 4. Through somatic cell hybridizaiton ( between human cells and cells of mice) gene mapping an human chromosomes in possible.

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PLANT TISSUE CULTURE FROM CALLUS INDUCTION TO REGENERATION Plant tissue culture is a non – conventional, in –vitro method for developing rare plants (or) unique hybrids from excised organs (anthers or embryos) or single cells or tissue ex plants. Plant tissue culture is based on the unique feature of plant cells called Totipotency i.e. the ability of single cell to divide and regenerate into complete plant. Plant organs like excised embryos; anthers and small pieces of viable tissue called explants are inoculated on artificial nutrient media under aseptic conditions. These explants divide to form an undifferentiated mass of tissue called callus. When this callus in sub unaltered on to other nutrient media with variation in Phytohormones balance, callus regenerates into plant lets. Plant tissue culture is different types based on specific objective. They are • Anther culture for the production of haploids. • Embryo rescue culture for development of rare plants or hybrids. • Protoplast culture for the production of somatic cell hybrids. • Micro propagation for the bulk production of clones of commercial crops. Callus Initiation : The tissue ex plants are selected from leaves, tubers, root cuttings, stem regions, shoot apex, nodal segments, hypocotyls etc. for immediate response. Explants with actively dividing cells are selected. The explants are treated with surface sterilants and disinfectants like H2O2, ethyl alcohol, sodium hypochlorite, mercuric chloride etc. The medium used for callus culture is Murashigo –Skoog medium or Nitsch medium or whites medium. Medium provides micro and macro elements, vitamins like Thiamine, pyridoxines, phyto hormones, Fe – EDTA, sucrose and gelling agent agar. Prepared medium is poured into test tubes or conical flasks and autoclaved. Sterilized ex plants are placed on culture media of slants and conical flasks in a laminar airflow chamber. After inoculation, the medium slants with ex plants are maintained at 21 – 25°C under sufficient light in culture room. Sub culture of callus for shoot initiation: After 2 or 3 weeks of inoculation, some ex plants show visible growth of callus, which is in pale yellow color. Depending on Phytohormones balance, sometimes ex plants directly give rise to differentiated organs like shoots (or) roots (or) complete plantlets. When callus in accumulated in sufficient quantity the medium gets depleted. The callus from these cultures are taken out on the surface of laminar air flow chamber, cut into small pieces and again inoculated onto fresh callus media or shooting media without much pretreatment with sterilization agents. The sub-cultured callus divides and differentiates into shoots. The media for shoot initiation contains high concentration of cytokinin and low concentration of auxins. After shoot initiation, nutrient medium depletes rapidly. Hence it is necessary to transfer shoots onto rooting media. Root initiation: Callus with in–vitro grown shoots are taken out on laminar airflow chamber and shoots are excised carefully. The shoots are inoculated on to rooting medium, which contains high auxins to cytokinin concentration. With in few days after inoculation, adventitious roots get developed from the bottom of each shoot. Simultaneously shoots increase size. Hardening and Acclimatization: In – vitro cultured plants are highly sensitive and more susceptible to pathogens if brought into field conditions directly from culture room. Before transplanted into field, they are subjected to hardening effect and acclimatization. In–vitro grown plants are taken out from their culture tubes and thoroughly washed to remove the traces of medium to avoid contamination. Each plant let in placed in a small pot containing autoclaved (sterilized) soil and water. These pots with transplants are grown further in green houses, which always maintained under sufficient light, required temperature and humidity. The transplants are grown in these green houses for 15 – 30 days. During this period the plants become robust and hard. Then they can with stand any climatic condition. After 15 – 30 days the plants are transferred to the field. In case of dicot plants, root grafting is done to develop tap root system. Because in–vitro rooting always give adventitious roots irrespective the plant is monocot or dicot. These adventitious roots in dicots must be substituted with tap root system. Before field transfer, the shoots of in–vitro plants are excised and granted onto other dicot shoots.

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STERILIZATION TECHNIQUES IN PLANT TISSUE CULTURE Sterilization is defined as the process of making any material aseptic in nature i.e. making a given environment or substance free from all forms of microorganisms. In in-vitro plant tissue culture, microorganisms like bacteria and fungi often contaminate cultures. Hence it is highly essential to maintain culture media, tissue explants, culture tubes, vessels, inoculating instruments, culture room etc. in aseptic conditions. Sterilization is done for different materials by different methods. Depending on the nature of the material to be sterilized, the methods are categorized into physical and chemical methods.

Physical methods: sterilization by heat : Heat sterilization is the simplest and most reliable method. It can be applied in two forms. c. Dry heat – is used for sterilizing glassware like petriplates, test tubes, pipettes and instruments. Dry heat is provided by hot air ovens with thermostat. At high temp. i.e. 160 – 1700c, for one to two hours, eliminates all forms of micro organisms due to oxidation. Some chemicals, which could not be autoclaved, are also sterilized by dry heat. Eg: Oils, powders, and anhydrous fats. d. Moist heat: Heat in the form of saturated steam under pressure is the most practical and reliable agent for sterilization. Steam under pressure provides tremendous heat above the boiling point. Moist heat kills microbes by coagulating and denaturing their enzymes and structural proteins. Moist heat is provided by an apparatus called autoclave in which pressure and temperature can be regulated. Moist heat sterilization is used for sterilizing media, vitamins and dissolved chemicals. It is also used for decontamination of contaminated culture tubes. Radiation: Radiations are used to eliminate different microorganisms especially in inoculation chambers and culture rooms. U.V. light is the most suitable sterilization agent. Maximum effect of U.V. can be observed at 250nm wavelength. In plant tissue culture procedures inoculation chambers, instruments and culture media are exposed to U.V. light prior to inoculation. Ionising radiations like X – rays and γ- rays are rarely used for sterilization because they are most effective and may cause mutations in explants and also pores some health hazards. Filtration: Filtration is used to remove microorganisms from solutions, which are heat sensitive. Unique chemicals like enzymes and Phytohormones are sterilized by filtration before adding to culture stocks. Different types of filters are used to remove bacterial endospores and fungal spores. These filters are made from asbestos, diatomaceous earth or biological membranes or cellulose papers. Membrane filters are most efficient means of sterilization. Inoculation chambers and culture rooms are freed from microorganisms by air filters called HEPA filters (High Efficiency Particulate Air filters). Sterilization temperature for filters should be critical and should not exceed 1210c. Chemical Methods: Different chemicals like surface sterilants, disinfectants, antibiotics etc. are used at different steps in in-vitro plant tissue culture. The chemical agents used for sterilization must have certain features like antimicrobial activity, lack of toxicity, ability to penetrate and activity at ordinary temperature. Explants used in tissue culture like seeds, tissue pieces, excised embryos, pollen grains etc. are treated with disinfectants like H2O2, sodium hypochlorite, ethyl alcohol, mercuric chloride etc. to remove different forms of microorganisms (surface sterilization). Inoculation tools like forceps, scalpels, needles are autoclaved and surface sterilized by dipping in alcohol followed by flaming. Sometimes the culture stocks and prepared culture media may retain heat resistant endospores of bacteria which subsequently germinate and contaminate culture media. To avoid their germination, antibiotics like streptomycin are added to the medium.

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