Tissue Culture in Crop Improvement

Haberlandt?

Tissue culture is a technique of growing plant tissues on synthetic medium under controlled and aseptic conditions. G. Haberlandt, a German plant physiologist is considered to be the father of plant tissue culture who conceived the idea of totipotency in 1902 which refers to the capability of a cell to give rise to a complete plant under suitable cultural conditions. Such a property of cell has far reaching implications to manipulate plant cells for rapid multiplication of plants, to cross plants at the level of somatic cells by overcoming limits of crossability, and also to regenerate adult plants after modifying the DNA molecule at cellular level. Suitable explants i.e. organs excised from plants such as roots, hypocotyls, cotyledons, leaves, shoot apices, nodal segments, anthers, embryos, and seeds are surface sterilizedwith a disinfectant like sodium hypochlorite (10-50% w/v; 10-30 minutes) or with mercuric chloride (0.1%, w/v; 5-10 minutes), thoroughly washed with sterile (autoclaved) water and then aseptically cultured on a synthetic medium in the culture vessels like test tubes, jars and petridishes. The cultures incubated at 25±1℃ exhibit growth in 1-3 weeks depending upon the plant species, nature of explant, type of culture medium, kind and concentration of the growth regulators (hormones) used in the medium and the light intensity in the incubation room.A large number of media have been developed for plant tissue culture but the most commonly used include: Murashige and Skoog, 1962 (MS-1962), White (1963), Gamborg et al. (1968) (B5) and Chu (1978) (N6) medium. 

Tissue culture medium contains major (macro) elements, micro elements, vitamins and amino acids, carbohydrattes (sucrose) and growth regulators (auxins, cytokinins). Auxins such as Indole acetic acid (IAA), Indole butyric acid (IBA), Naphthalene acetic acid (NAA) at concentrations ranging from 0.1-5.0 mg/l favour cell elongation and rooting, whereas 2,4-Dichloro-phenoxyacetic acid (2,4-D) at concentrations of 0.5-4.0 mg/l usually induces callus i.e. homogenous mass of undifferentiated cells. Likewise, cytokinins such as 6-furfuryl amino purine (kinetin) and benzyl amino purine (BAP) at concentrations (0.1-2.0 mg/l) cause rapid cell divisions and development of shoot buds/shoots. The solidification of the medium is achieved by adding chemically inert powdered gelling agents such as agar before autoclaving. The medium is poured into culture vessels such as agar before autoclaving. The medium is poured into culture vessels and sterilized using autoclave at temperature 121 ℃, 15 lbs/sq. inch pressure for 20-25 minutes. Inculation of explants in the culture vessels is done in Laminar Air Flow Cabinet fitted with HEPA filters (pore size 0.2-0.3 µm).