PRINCIPLES OF CROP PRODUCTION ABT-320 (3 CREDIT HOURS)) LECTURE 3 LECTURE-WISE COURSE BREAKUP TISSUE CULTURE MAJOR COMPONENTS OF TISSUE CULTURE MEDIA STERILIZATION METHODS THE TECHNIQUE OF TISSUE CULTURE

TISSUE CULTURE Tissue culture is the technique of enhancing vegetative plant formation from in vitro cultured explants. Direct regeneration of plantlets from cultured plant parts (explants) or regeneration through callus are employed for the purpose. For both the purposes, plant parts are grown in culture media under aseptic conditions. Hormonal proportions are altered so as to obtain callus formation or direct regeneration. The callus or plantlets formed are transferred to hardening media for hardening and acclimatization followed by field transfer.

MAJOR COMPONENTS OF TISSUE CULTURE MEDIA AGAR- Agar is a galactan, a complex carbohydrate with galactose molecules . Agar is used to solidify the medium. ORGANIC COMPOUNDS- These are used as the source of carbon and energy. Sucrose is used usually in all standard media. INORGANIC COMPOUNDS- These include micronutrients and macronutrients. N, P, K, Ca, Mg, S are important macronutrients whereas B, Mo, Cu, Zn, Mg, Fe etc are micronutrients. GROWTH HORMONES – Hormones such as cytokinins, auxins and gibberellins are used to regulate growth in tissue culture. Cytokinins promote cell division and regulate growth. The most widely used auxins are adenine, kinetin, zeatin and benzyl adenine. Auxins stimulate shoot elongation.

MAJOR COMPONENTS OF TISSUE CULTURE MEDIA Auxins include indole acetic acid (IAA), naphthalene acetic acid (NAA) and 2,4-dichlorophenoxyacetic acid (2,4-D). Gibberellins are of less importance. However, gibberellic acid (GA3) is used in apical meristem culture. VITAMINS – Vitamins regulate the metabolic activities of cells. They are required in minor quantities. Vitamin B1 (thiamine) is used for all types of tissue culture. Nicotinic acid, riboflavin, pyridoxin, ascorbic acid, biotin and cyanocobalamin are also used in different cases. AMINO ACIDS – Amino acids such as L-aspartic acid, L-asperagin, L-glutamic acid, L-glutamine etc are used in tissue culture as sources of nitrogen.

STERILIZATION METHODS USE OF CHEMICALS: Chemicals such as Chromic acid, Mercuric chloride, Sodium hypochlorite, Calcium hypochlorite and alcohol are used for the sterilization of glassware, work tables and source materials of explants. USE OF OVEN: A dry heat oven is used to sterilize glassware, metallic instruments etc by hot air (200-300C) for 1 hour. USE OF AUTOCLAVES: Autoclaving is done to sterilize nutrient media, distilled water etc with the help of steam (121C for 30 minutes) ULTRA FILTRATION: Vitamins, hormones etc are unstable at high temperatures. They are sterilized using millipore membrane filter etc. USE OF UV LIGHT: UV light is used in the incubation chamber to make it germ-free.

THE TECHNIQUE OF TISSUE CULTURE The major steps involved in the method of in vitro culture of an explant are; 1. Surface Sterilization of the Explant 2. Preparation of Nutrient Medium 3. Inoculation 4. Callus Growth 5. Subculturing 6. Organogenesis 7. Direct Regeneration 8. Acclimatization and transfer to the field

SURFACE STERILIZATION OF THE EXPLANT The plant part which is used for in vitro culture is known as explant. Explants are taken from the most appropriate part of the parent plant. Development of a tissue is the result of cell division, cell elongation and cell differentiation. Therefore, explants from young and healthy plants are usually used. The presence of parenchyma is the first consideration, because parenchyma quickly responds to culture conditions. Explants are usually 2-5mm in size. They are sterilized with 1-2% solution of sodium or calcium hypochlorite or 0.1% solution of mercuric chloride. All the operations in tissue culture are done under aseptic conditions.

PREPARATION OF NUTRIENT MEDIUM An appropriate nutrient medium is prepared in advance. The medium may be solidified by using agar (6gm/l) or may be used as a liquid, depending on the requirement. It is poured to culture vials under aseptic conditions.

INOCULATION Inoculation is the transfer of explants to culture vials. It is done in the inoculation chamber under aseptic conditions.

CALLUS GROWTH The explant, a 2-5 mm sterile segment excised from the appropriate part of the plant, is transferred to the nutrient medium and incubated at 25-28C in an alternate light and dark regime (usually 12 hrs each). Nutrient medium, supplemented with auxins, induces cell division. As a result, the upper surface of the explant gets covered by an amorphous mass of loosely arranged thin-walled cells. This mass of tissue is called callus. It is characterized by abnormal growth and has the potential to produce roots, shoots and embryoids. Callus formation is controlled by the source of the explant, composition of the medium and environmental factors. Explants obtained from meristematic regions develop more rapidly than those of other tissues.

CALLUS GROWTH Usually, callus growth is completed in three stages, namely induction, cell division and cell differentiation. During callus induction, the metabolic rate of the cells increases and cells get packed with metabolic products. This is followed by the rapid division of such cells. In the later stage, cellular differentiation starts.

SUBCULTURING When the callus has grown for some days (say 28 days), it is essential to subculture it on a fresh medium. Otherwise, nutrient depletion, accumulation of toxic metabolites and paucity of water are sure to occur, leading to the death of the callus.

ORGANOGENESIS Organogenesis starts in the callus in response to the stimulation given by the chemicals in the medium. Organogenesis takes place in two stages, namely caulogenesis or shoot initiation and rhizogenesis or root initiation. Both types of organogenesis are controlled by the hormones present in the medium. Skoog and Miller (1957) demonstrated that a high auxin:cytokinin ratio may induce shoot formation. In 1966, Torrey proposed the hypothesis of organogenesis. According to this hypothesis, organogenesis starts with the development of a group of meristematic cells called meristemoids, which initiate the formation of a primordium. Depending on the factors within the system, this primordium develops into either shoot, root or embryoid.

ORGANOGENESIS Generally, in dicots, buds develop when cytokinin:auxin ratio is 1:100. On the other hand, callus production will be favored by an auxin:cytokinin ratio 1:100. The formation of an embryoid from the callus is called somatic embryogenesis. If the hormonal conditions are correctly balanced, an entire plantlet can be induced to grow on the culture medium. This process is called regeneration.

DIRECT REGENERATION In many plants, subculturing of callus results in undesired variations of clones (somaclonal variations). To avoid this, direct regeneration of the explants into plantlets can be tried. This has been achieved in many plant species by altering the hormonal combination of the culture media.

OTHER METHODS OF IN VITRO CULTURING Plant parts like anther, pollen grains, embryo and endosperm are also grown in vitro for different purposes. Pollen culture and anther culture are practiced to produce haploid plants. in vitro culture of embryos is very useful in some plant species in which the embryo gets aborted under in vivo (natural) conditions. In many orchids, embryos are rescued and grown in artificial media to ensure proper development of seeds.

ACCLIMATIZATION & TRANSFER TO THE FIELD In the last stage, the rooted plantlets are subjected to acclimatization, so that they can easily adjust to the field conditions. The plantlets are taken out from the medium, washed thoroughly in running water to remove the agar and are then put in a low mineral salt medium (LMSM) for 24-48 hours. These plantlets are then transferred to pots containing autoclave sterilized mixture of clay, sand and leaf molds in 1:1:1 proportion. The pot is usually covered with transparent polythene to maintain humidity. It is kept undisturbed for 15-30 days. At this stage, the plant becomes fully acclimatized. Finally, these fully acclimatized plantlets can be transferred to the field.

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