The steps involved in plant tissue culture are: Selection of trimmed plant material and surface sterilization:
The selected plant material used for In-vitro culture is called explants. The explants should be washed first in the running water for about 20 minutes in order to remove the dirt and dust. These explants should be trimmed and surface sterilization should be done with 1 -2 % sodium hypochlorite solution or 0.1% mercuric chloride solution for 10 -15 minutes. Then these explants should be rinsed with double distilled water to remove the surface sterilant. Inoculation of the explants: Inside the inoculation chamber the explants are then introduced into the culture tubes containing nutrient medium with a sterilized forceps. This is called inoculation. Regeneration: Within 8 -10 days, the cut end of explants starts to divide to form a mass of undifferentiated cell called callus. The process of formation of callus is called callogenesis. The callus undergoes differentiation to produce a number of shoot buds or embryoids. Since these embryoids are developed from the vegetative cells of the explants, they are called somatic embryoids. The embryoids are cut and separated from the callus and introduced into the separate culture tubes containing nutrient medium. Inside the culture tube, each embryoid or shoot bud develops into plantlet or micro-shoot. The shoot is formed under the effect of high cytokinin to auxin ratio, while to induce root the medium having high auxin to cytokinin ratio is used. The micro-shoots are transferred to the plastic bags containing sterilized soil. These bags are placed in greenhouse where the plantlets grow & get adjusted to the natural environmental conditions. This is called hardening. After several weeks, the hardened plants are transferred to the pots or garden soil.