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Culture
Media
Constituents
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The major constituents of a culture medium include
(i) inorganic nutrients,
(ii) organic nutrients,
(iii) growth hormones and
(iv)agar.
Among the inorganic nutrients, besides C (carbon), H (hydrogen), a (oxygen), additional 12 elements (N, P, S, Ca, K, Mg, Fe, Mn, Cu, Zn, B, Mb), which are essential for plant growth, are included in culture media.
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Six of these (N, P, S, Ca, K, Mg ) are major or macroelements (cone. above 0.5 mmol) and the remaining six are minor or microelements. The various culture media differ mainly in quantity rather than in quality of these elements. The original White’s medium (1943) though widely used earlier, was later found inadequate quantitatively for good callus growth, so that the currently used media are richer in mineral salts.
The organic nutrients can be broadly classified into
(i) nitrogen and
(ii) carbon sources, the former mainly including vitamins ( B1, B3, B5, B6) and amino acids and the latter mainly including sucrose, glucose and fructose, or other carbohydrates (including starch).
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Other complex nutrients of undefined composition include casein hydrolysate (CR), coconut milk (CM), corn milk, malt extract (ME), tomato juice (TJ), and yeast extract (YE). It is, however, recommended to avoid their use and replace each by a single amino acid, so that the quantity and quality of constituents may not vary at different times.
The growth hormones included in culture media involve
(i) auxins,
(ii) cytokinins and
iii) gibberellins.
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The auxins ae mainly used to facilitate cell division and root differentiation. Commonly used auxins are IBA (indole-3-butyric acid), NAA (napthaleneacetic acid), NOA (naphthoxyacetic acid), p-CPA (para-chlorophenoxyacetic acid), 2, 4-D (dichlorophenoxy-acetic acid) and 2,4,5-T (trichlorophenoxyacetic acid). IBA and NAA are widely used for rooting and (in combination with cytokinins) for shoot proliferation. 2, 4-D and 2,4,5-T are very effective for the induction and growth of callus.
The cytokinins facilitate the cell division and differentiation. Commonly used cytokinins include BAP (benzylamino purine), 2, ip (isopentenyl adenine) and kinetin (furfurylamino purine). Among 84 known gibberellins, mainly GA3 is used to induce plantlet formation from adventive embryos formed in culture.
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Another important component of any culture medium is agar (a polysaccharide obtained from seaweeds) to provide solid surface for growth (agar is not a nutrient), because in the liquid medium, the tissue will be submerged and die due to lack of availability of oxygen (agar is used at a concentration of 0.8-1.0%).
Cells can also be grown in suspension cultures devoid of agar, but such cultures need to be aerated regularly either by bubbling sterile air or by gentle agitation.
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