Question

Arrange the given steps of the tissue culture process in the correct order.
I. Sterilisation of culture media and explant
II. Transfer of explants to culture medium
III. Selection of explant
IV. Organogenesis in callus and formation of plantlets
V. Establishment of plantlets in the field

Answer 1

Tissue culture is a technique used in modern plant breeding programmes for faster multiplication of plants using the vegetative parts of plants. It is the technique of growing plant cells, tissues or organs (explants) in a sterile culture medium under controlled environmental conditions.

The steps involved in tissue culture are-
A. Selection of the explant : Explant is the plant part/cell/tissue used for tissue culture. The explant can be an excised root tip, shoot bud, leaf, petiole, anther, ovule, etc.

B. Preparation of the culture media : It is made using appropriate concentrations of all necessary nutrients such as inorganic salts, sugars, amino acids, vitamins, growth hormones. Agar agar is commonly used as a solidifying agent in callus cultures.

C. Sterilisation of the culture media and explant : Culture vessels and the instruments are sterilised to eliminate any microbial presence. The vessels, culture media and instruments are completely sterilised in autoclaves at 120°C and 12-15lbs of pressure for 15-20 mins. The high temperature kills microbial cells and spores.

D. Transfer for explants to the culture medium (inoculation) : The sterile explant is transferred/inoculated into the sterile culture vessels containing the culture media under aseptic (germ-free) conditions.

E. Incubation (Formation of callus) : The inoculated plant tissues are incubated for 3-4 weeks in the growth room under optimum conditions of light, humidity and temperature. The cells of the explant start dividing actively and form an undifferentiated mass of cells known as the callus. These cells are totipotent (can grow into an entire plant).

F. Organogenesis in callus and formation of plantlets : Under the influence of various concentrations of different growth hormones in the culture medium, the cells of the callus start differentiating into different types of plant tissues. This gradually leads to the formation of plant organs like roots and shoots (organogenesis) and ultimately a small miniature plant (plantlet) is formed.

G. Establishment of plantlets in the field: The plantles are then hardened (adapted to environmental conditions) and then transferred to fields where they grow into whole plants.