Plant tissue culture is an essential component of plant biotechnology. Apart from mass multiplication of elites, it also provides the means to multiply and regenerate novel plants from genetically engineered cells. The promising plant thus produced may be readily cloned in cultures under aseptic conditions.
Tissue Culture is widely used in
Obtaining disease free plants.
Rapid propagation of plants those are difficult to propagate.
Genetics improvement of commercial plants.
Obtaining androgenic and gynogenic haploid plants for breeding programmes.
Tissue Culture is becoming as an alternative means to vegetative propagation of plants. In vitro growing plants are usually free from bacterial and fungal diseases. Virus eradication and maintenance of plants in virus free stage can also be rapidly achieved in cultures.
Three main methods generally used in tissue culture are
Micro propagation through the enhanced multiplication of axillary bud.
At present the most successful and commonly used method is enhanced shoot multiplication from axillary bud. Axillary buds are present in the axis of leaves. In tissue culture, by using optimum concentration of cytokinin or combination of cytokinin and Auxin the dormancy of the axillary buds can be broken. Once the dormancy is broken, they develop into shoots.
By using media containing optimum concentrations of plant growth regulators, they can be made to multiply very rapidly. Modern plant tissue culture is performed under aseptic conditions under filtered air. Living plant materials from the environment are naturally contaminated on their surfaces (and sometimes interiors) with microorganisms, so surface sterilization of starting materials (ex plants) in chemical solutions (usually alcohol) is required.
Mercuric chloride is used as a plant sterilizing agent today, as it is dangerous to use, and is difficult to dispose off. Explants are then usually placed on the surface of a solid culture medium, but are sometimes placed directly into a liquid medium, particularly when cell suspension cultures are desired.
Solid and liquid media are generally composed of inorganic salts plus a few organic nutrients, vitamins and plant hormones. Solid media are prepared from liquid media with the addition of a gelling agent, usually purified agar. The composition of the medium, particularly the plant hormones and the nitrogen source (nitrate versus ammonium salts or amino acids) have profound effects on the morphology of the tissues that grow from the initial explant. For example, an excess of auxin will often result in a proliferation of roots, while an excess of cytokinin may yield shoots.
A balance of both auxin and cytokinin will often produce an unorganized growth of cells, or callus, but the morphology of the outgrowth will depend on the plant species as well as the medium composition. As culture grows, pieces are typically sliced off and transferred to new media (subcultures) to allow for growth or to alter the morphology of the culture.
The skill and experience of the tissue culturist are important in judging which pieces to culture and which to discard. As shoots emerge from a culture, they may be sliced off and rooted with auxin to produce plantlets which, when mature, can be transferred to potting soil for further growth in the greenhouse as normal plants.