Culture Technique in Plant Tissue Culture
Plant cells are cultured in a suitable nutrient medium composed of inorganic salts, carbon source, vitamins, and growth regulators and organic supplements. In general , plant parts, tissue and cells can grow on media containing only the salts, of nitrogen and other essential elements, sucrose, certain amino acids, vitamins and growth factors. In several plants, the formation of shoot in vitro is promoted by higher levels of cytokinins relative to auxins while the reverses promote the root development. Some commonly employed cytokinins are kinetin and 6- benzylamino- purine and some commonly used and effective auxins are IBA , NAA and 2,4-D. These tend to induce rapid callus proliferation. Higher levels of 2,4-D strongly suppresses the oliogenesis. The most commonly used culture media are Murahige and Skoog medium, Gamborg et. al medium and White’s medium.
The procedure for establishing the culture is as follows. A 2-4 mm3 sterile segment excised from stem or root of the plant is placed on 30 ml of nutrient agar or liquid medium and incubated t 20-30 0C in light. Within few days, cell proliferates and callus culture is obtained. In this, dividing cells form a layer of meristem and build a globular mass of non-dividing parenchyma. Alternaively, they may form small meristematic zones interspersed in non-meristematic regions, yielding a sort of nodulated callus.
After 2-3 subcultures, small bits from soft callus can be cut and incubated into liquid medium where they give rise to suspension culture.
Cell clones can be raised in the same manner as in case of micro-organisms, by plating a suspension of cells on agar plates. Colonies are formed, each representing a clone. They can be picked up individually and inoculated in liquid medium.