What are the steps for plant tissue culture?


Basic steps involved in plant tissue culture:

Preparation of suitable nutrient medium:
MS medium, White’s medium, B5 medium are few culture media. Auxins, cytokines and Gibberellins are the most commonly used growth hormones in culture medium prepared for tissue culture.

Agar: It solidifies the medium and acts as a substratum. It doesn’t have any nutritive importance. It is obtained from sea weeds and is used to provide solid surface for growth.
All these important nutrients are incorporated in the culture medium and transferred into suitable containers and then sterilized.

Selection of explant:
Explants are any excised part of the plant to be used in tissue culture like axillary buds, leaf and stem segments, root tip, shoot tip, anther, ovary and endosperm.
Always young and healthy parts of plants are selected as explants.

Sterilization of the explant:
Explants are sterilized by using different types of disinfectants like sodium hypochlorite (2%), silver nitrate (1%) calcium hypochlorite (9-10%), bromine water (1-2%), mercuric chloride (0.1-1%), hydrogen peroxide etc. and different types of antibiotics.

Inoculation of explant:
The sterile explant is inoculated on the surface of the solidified nutrient medium under aseptic (free from contamination) condition.

Laminar air flow can be used for this inoculation process.

Incubation:
Incubation is done at 25+/- 2 0C, with a relative humidity of 50-60% with 16 hours of photoperiod.
After defined period of incubation, an unorganized and undifferentiated (no root and shoot) mass of cells called callus is obtained from each explant. In some cases, root and shoot directly develop from the explant after incubation.

Transfer of callus (subculture) and regeneration:
The callus obtained from incubation of explant is transferred into another medium and reincubated.
Plantlet regenerates from the callus with the induction of roots and shoot directly from the callus.

Hardening or acclimatization:
Hardening is the gradual exposure of the plantlets for acclimatization or adjustment to the environmental conditions.

Transfer of plantlets to the field:
After the hardening process, plantlets are transferred to either the green house or the field conditions outside.

These plantlets will grow into independent and healthy plants.



https://larixconferences.com/biotechnology/

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