Plant tissue culture refers to growing and multiplication of cells, tissues and organs of plants on defined solid or liquid media under aseptic and controlled environment. The commercial technology is primarily based on micro propagation, in which rapid proliferation is achieved from tiny stem cuttings, axillary buds, and to a limited extent from somatic embryos, cell clumps in suspension cultures and bioreactors. The cultured cells and tissue can take several pathways. The pathways that lead to the production of true-to-type plants in large numbers are the preferred ones for commercial multiplication. The process of micro propagation is usually divided into several stages i.e., pre-propagation, initiation of explants, subculture of explants for proliferation, shooting and rooting, and hardening. These stages are universally applicable in large-scale multiplication of plants. The delivery of hardened small micro propagated plants to growers and market also requires extra care.
1. Preparation of nutrient medium:
A semi-solid medium is prepared in double distilled water containing macro elements, micro elements, amino acids, vitamins, iron source, carbon source like
sucrose and phyto-hormones. The medium is heated for dissolving the agar and 25 to 50 ml is dispensed into each wide mouth bottles. The vessels containing culture media are then sealed and sterilized by auto claving [6].
2. Establishment of aseptic culture:
The starting material for the process is normally an actively growing shoot tip of axiliary or terminal bud or shoot tip of a plant. The process of tissue culture starts from the selection of mother plants having the desired characteristics.Ex-plant preferably the meristematic tissue of the selected mother plant is isolated. The excised tissue/explant is washed with water and then rinsed with a disinfectant such as savlon or detol solution followed by a sterile-water wash. The tissue is then dipped in 10% bleach solution for ten minutes for disinfecting the plant tissue material, killing most of the fungal and bacterial organisms. Sterilization process of explants depends on the plant species and types of explants [7].
3. Inoculation:
Inoculation is carried out under aseptic conditions. In this process explants or micro shoots are transferred on to the sterilized nutrient medium.
4. Development of plants in growth room:
After the inoculation of the plant tissue, the bottles are sealed and transferred into growth room to trigger developmental process under diffused light (fluorescent light of 1000-2000 lux) at 25 ± 2 oC and 50 to 60% relative humidity. Light and temperature requirements vary from species to species and sometimes during the various stages of developments.The cultures are observed daily for growth and any signs of infection/contamination. Cultures, that do not show good growth or infected, are discarded. The healthy cultures grow into small shoot buds. These are sub-cultured on the fresh medium after 4 weeks. The number of subcultures required is specific to the plant species, which are standardized. The shoots generally develop after 4 weeks. After enough number of shoots is developed in each container (10 to 15), to a minimum height of 2 cm they are transferred to another medium for initiating the process of rooting. The constituent of rooting medium for each plant species are specific. Roots are generally formed within 2 to 4 weeks. Plants at this stage are delicate and require careful handling [8].
5. Hardening of micro plants:
Due to very high humidity inside the culture vessel and artificial conditions of development, the plantlets are tender and are therefore are not ready for coping up with the filed conditions. The plants removed from the sterile medium are washed and are maintained under intermittent mist or are covered with clean transparent plastic. After 10 to 15 days under high humidity, the plants are transferred to green house and maintained for another 4 to 6 weeks. They are then ready to be transferred to net house or the field. Normally, the tissue culture plants are sold either as ex-agar plants or hardened plants from the greenhouse.
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