(i) Various Sterilization Technique :
Maintenance of Aseptic Environment: Dining in vitro culture maintenance of aseptic environment is the most difficult task, because the cultures are easily contaminated by fungi and bacteria present in the air. The contaminants produce toxic metabolites which inhibit growth of cultures plant tissues. Therefore, each step must be handled aseptically and with great care. Following are some of the sterilization methods for aseptic manipulation of plant tissues.
(a) Sterilization of glassware : Glassware (Petri plates, vials, culture tubes, flasks, pipettes, etc.), metallic instruments are sterilized in a hot air oven at 160-180° C for 2-4 hours.
(b) Sterilisation of instruments : The metallic instruments (e.g., forceps, scalpels, needles, spatulas, etc.) are flame sterilised i.e., dipping them in 25% ethanol followed by flaming and cooling. It is called incineration.
(c) Sterilisation of culture room and transfer area: Floor and walls of culture room are washed first with detergent then 2% sodium hypochlorite or 95% ethanol. Larger surface area is sterilised by exposure to UV light. The cabinet of laminar airflow is also sterilised by exposing UV light for 30 minutes and 95% ethanol 15 minutes before beginning of work inside the cabinet of laminar airflow.
(d) Sterilization of nutrient media : Culture media are properly dispensed in glass container, plugged with cotton or sealed with plastic closures and sterilized by autoclaving (steam sterilization) at 15 psi (that gives 121°C) for 30 minutes. During autoclaving vitamins, plant extracts, amino acids and hormones are denatured. Therefore, the solution of these compounds are sterilized by using millipore filter paper which has 0.2 pm pore diameter.
(e) Sterilization of plant materials : Surface of all plant materials has microbial contaminants. Therefore, disinfectants (e.g., sodium hypochlorite, hydrogen peroxide, mercuric chloride, or ethanol) should be used to make plant materials sterile. Then the chemicals must be washed 6-8 times using sterile distilled water. Then ex plants are transferred aseptically on nutrient medium inside the cabinet of laminar airflow.
(ii) Composition of Nutrient Media: Composition of nutrient media governs the growth and morphogenesis of plant tissue in vitro. Depending on the type of plant cells or tissue used for culture the composition of nutrient media vary. The principal constituents of tissue culture media are inorganic nutrients, carbon sources, organic supplements, growth regulators and gelling agent.
(a) Inorganic nutrients: Several minerals (i.e., macro-and micro-nutrients) are required by the plants. Minerals dissolved in water are dissociated and ionized. For example, in MS medium NH4N03 contributes NO-3 and KNO3 contributes K+ ions. There are six major macro-nutrients such as nitrogen, phosphorus, potassium, calcium, magnesium and sulfur. But the essential or micro-nutrients are required in low amount. These are boron, molybdenum, cobalt, zinc, manganese, iron and copper.
(b) Carbon and energy sources: Mostly sucrose is required as carbon source followed by glucose. These carbon sources enhance cell proliferation and tissue regeneration.
(c) Organic supplements : There is a large number of organic supplements used for best growth of tissues. Vitamins (riboflavin, folic acid ) Amino acids (casein hydrolysate, L-glutamine, L-asperagine, L-glycine, L-arginine, L-cysteine) are commonly used as nitrogen source and enhancers of cell growth. Besides, culture
media are also supplemented with casein, coconut milk, yeast and malt extracts, ground banana, orange juice and tomato juice. Activated charcoal added to culture media is known to stimulate plant growth. If required antibiotics (streptomycine or kanamycin) may be added to culture medium to avoid systemic infection of micro-organisms.
(d) Growth regulators : For proliferation of cultured tissues four classes of growth regulators (e.g., auxins, cytokinins, gibberellins and abscisic acid) are used. For induction of root or shoot the ratio of hormones varies considerably. For example, auxins (e.g., indole acetic acid, 1-naphthaleneacetic acid) induce cell division and cause elongation of stem, and intemodes. Cytokinins (e.g., 6-benzylaminopurine, 6-benzyladenine, zeatin) induce cell division and shoot differentiation of cultured tissues. Different ratios of auxins and cytokinins are important in morphogenesis of callus. High ratio of auxin to cytokinin promotes embiyogenesis, callus and root initiation. But high ratio of cytokinin to auxin leads to axillary and shoot promotion.
(e) Solidifying agents : Most commonly agar is used as solidifying or gelling agent. Agar gels do not react with constituents of media and not digested by plant enzymes. Generally 0.5 -1% agar is used to form gel.
