The steps involved in preparing a medium are summarized below:

  Add appropriate quantities of various stock solutions, including growth regulators and other special supplements. Make up the final volume of the medium with distilled water.

  Add and dissolve sucrose.

  After mixing well, adjust the pH of the medium in the range of 5.5-5.8, using 0.1 N NaOH or 0.1 N HCl (above 6.0 pH gives a fairly hard medium and pH below 5.0 does not allow satisfactory gelling of the agar).

  Add agar, stir and heat to dissolve. Alternatively, heat in the autoclave at low pressure, or in a microwave oven.

  Once the agar is dissolved, pour the medium into culture vessels, cap and autoclave at 121°C for 15 to 20 min at 15 pounds per square inch (psi). If using pre-sterilized, non-autoclavable plastic culture vessels, the medium may be autoclaved in flasks or media bottles. After autoclaving, allow the medium to cool to around 60°C before pouring under aseptic conditions.

  Allow the medium to cool to room temperature. Store in dust-free areas or refrigerate at 7°C (temperature lower than 7°C alter the gel structure of the agar).

 

1.2.  Gelling agents

The media listed above are only for liquids, often in plant cell culture a ‘semi-solid' medium is used. To make a semi-solid medium, a gelling agent is added to the liquid medium before autoclaving. Gelling agents are usually polymers that set on cooling after autoclaving.

i.  Agar: Agar is obtained from red algae- Gelidium amansii . It is a mixture of polysaccharides. It is used as a gelling agent due to the reasons: (a) It does not react with the media constituents (b) It is not digested by plant enzymes and is stable at culture temperature.

ii.  Agarose: It is obtained by purifying agar to remove the agaropectins. This is required where high gel strength is needed, such as in single cell or protoplast cultures.

iii.  Gelrite: It is produced by bacterium Pseudomonas elodea . It can be readily prepared in cold solution at room temperature. It sets as a clear gel which assists easy observation of cultures and their possible contamination. Unlike agar, the gel strength of gelrite is unaffected over a wide range of pH. However, few plants show hyperhydricity on gelrite due to freely available water.

iv.  Gelatin: It is used at a high concentration (10%) with a limited success. This is mainly because gelatin melts at low temperature (25°C) and as a result the gelling property is lost.

 

1.3.  Plant growth regulators

In addition to nutrients, four broad classes of growth regulators, such as, auxins, cytokinins, gibberellins and abscisic acid are important in tissue culture. In contrast with animal hormones, the synthesis of a plant growth regulator is often not localized in a specific tissue but may occur in many different tissues. They may be transported and act in distant tissues and often have their action at the site of synthesis. Another property of plant growth regulators is their lack of specificity- each of them influences a wide range of processes.

The growth, differentiation, organogenesis and embryogenesis of tissues become feasible only on the addition of one or more of these classes of growth regulators to a medium. In tissue culture, two classes of plant growth regulators, cytokinins and auxins, are of major importance. Others, in particular, gibberellins, ethylene and abscisic acid have been used occasionally. Auxins are found to influence cell elongation, cell division, induction of primary vascular tissue, adventitious root formation, callus formation and fruit growth. The cytokinins promote cell division and axillary shoot proliferation while auxins inhibit the outgrowth of axillary buds. The auxin favours DNA duplication and cytokinins enable the separation of chromosome. Besides, cytokinin in tissue culture media, promote adventitious shoot formation in callus cultures or directly from the explants and, occasionally, inhibition of excessive root formation and are, therefore, left out from rooting media. The ratio of plant growth regulators required for root or shoot induction varies considerably with the tissue and is directly related to the amount of growth regulators present at endogenous levels within the explants. In general, shoots are formed at high cytokinin and low auxin concentrations in the medium, roots at low cytokinin and high auxin concentrations and callus at intermediate concentrations of both plant growth regulators. Commonly used plant growth regulators are listed in Table 4.