Study Notes on Culture Media | Biotechnology

The below mentioned article provides a study note on culture media.

Growth and morphogenesis of plant tissues in vitro are largely governed by the com­position of the culture media. Although the basic requirements for culturing plant tissue is same but in practice there are some specific nutritional requirements for promoting optimal growth from different kinds of explants in case of different plant species.

Components of Media:

The principal components of most plant tissue culture media are inorganic nutrients (macronutrients and micronutrients), carbon sources, organic supplements, growth regu­lators, vitamins, amino acids and gelling agent for solid or semisolid media. The various culture media formulated for plant tissue culture are Murashige and Skoog’s medium, Gamborg’s medium, White’s medium, etc. which differ (Table 16.1) mainly in quantity of the components.

(i) Inorganic Nutrients:

A variety of mineral elements (salts) supply the macro- and micronutrients required in the life of a plant. Elements required in concen­tration greater than 0.5 m mol/1 referred to as macronutrients and those less than 0.05 m mol/1 as micronutrients.

The macronutrients include six major elements like Nitrogen (N), Phosphorus (P), Potassium (K), Calcium (Ca), Magnesium (Mg) and Sulphur (S) which are present as salts in various media. The micro­nutrients are iron (Fe), Manganese (Mn), Zinc (Zn), Boron (B), Copper (Cu), Molybdenum (Mo). Among these iron is used in the medium as chelated form with EDTA (ethylene-diamino-tetra-acetic acid).

(ii) Carbon and Energy Source:

Plant cells and tissues in the culture medium lack autotrophic ability and therefore need external carbon for energy. The most pre­ferred carbon source in tissue culture media is sucrose; glucose supports equally for good growth while fructose is less efficient.

(iii) Vitamins:

The plant cells and tissues are capable of synthesizing different vita­mins but in in vitro condition they are being produced at sub-optimal level. Hence it is necessary to supplement the media with required vitamins such as Thiamine (B₁), Nicotinic acid (B₃), Pyridoxine (B₆), Pantothenic acid (B₅) and myoinosi­tol. Except these other vitamins like Biotin, Folic acid, Ascorbic acid are also used in some media.

(iv) Amino Acids:

In vitro grown plant cells or tissues are capable of synthesizing amino acids but the addition of amino acids to media is important for stimu­lating cell growth and protoplast culture and for establishment of cell lines. Glutamine, asparagine, glycine, arginine, cysteine are the commonly used amino acids.

(v) Growth Regulators:

Three broad classes of growth regulators mainly auxins, cytokinins and gibberellins are used in tissue culture. The growth, differentiation and organogenesis of tissues become feasible only on the addition of one or more of these classes of hormones to a medium.

Various kinds of auxins like IAA (Indole-3-Acetic Acid), NAA (Naphthalene Acetic Acid), 2, 4-D (2, 4-dichlorophenoxy acetic acid) are used to induce cell division, elonga­tion of stem, internodes and rooting.

Cytokinins like BAP (Benzyl amino purine), Kinetin (Furfuryl amino purine), Zeatin or 2iP (Isopentynyl adenine) are used which are mainly concerned with cell division, modification of apical dominance and shoot differentiation in tissue culture.

The ratio of auxin and cytokinin is important with respect to morphogenesis in the culture system. For embryogenesis, callus initiation and root initiation the requisite ratio of auxin to cytokinin is high, while the reverse leads to organogenesis and shoot proli­feration. Gibberellins (GA₃) are occasionally used growth regulators to induce plantlet forma­tion from adventive embryos developed in culture.

(vi) Other Organic Supplements:

Culture media are often supplemented with variety of organic extracts which have the constituents of an undefined nature e.g. casein hydrolysate, coconut milk, malt extracts and fruit juice.

(vii) Activated Charcoal:

The addition of activated charcoal to culture media is reported to stimulate growth and differentiation by reducing toxicity by remo­ving toxic substances (e.g. Phenols).

(viii) Antibiotics:

For transformation experiments addition of antibiotics to culture media is required to prevent the growth of Agrobacterium which retards the cell or tissue growth.

(ix) Gelling Agent:

Gelling or solidifying agents are commonly used for preparing semisolid or solid tissue culture media to provide solid surface area for growth.

Agar (a polysaccharide obtained from seaweeds), Gelatin (commercially avai­lable as Phytagel, Alginate or Gelrite) are commonly used solidifying agents at a concentration of 0.8-1.0% (which are not any kind of nutrient), depending on the type of tissue and the species of plant and culture methods.

Media Preparation:

For media preparation it is convenient to use stock solutions of major salts (20X), minor salts (200X), organic supplements (200X), growth regulator (1 m mol or 10 m mol). For final media preparation the stock solutions are mixed together in appropriate quanti­ties. All the stock solutions are stored in proper plastic or glass containers at low tempe­rature.

After preparation of media the pH is adjusted at 5.5-6.0 and agar is mixed when­ever it is required. The media is poured into the desired type of culture vessels (culture tubes, conical flasks, petriplates, etc.), properly plugged with non-absorbent cotton and autoclaved.