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Solidifying OR Gelling Agents - Gelling or solidifying agents are commonly used for preparing semisolid or solid tissue culture media. In static liquid cultures the tissue or cells become submerged and die due to lack of oxygen. Gels provide a support to tissues growing in static conditions. Agar, a polysaccharide obtained from seaweed, has several advantages over other gelling agents.
First, agar gels do not react with media constituents. Secondly, they are not digested by plant enzymes and remain stable at all feasible incubation temperatures. Normally, 0.5 to 1 % agar is used in the medium to form a firm gel at the pH typical of plant cell culture media. In nutritional studies the use of agar is avoided because commercially available agar contains impurities in the form of Ca, Mg, K, Na, and trace elements. Consequently, a change in the agar concentration also affects the nutrients present in it as well as the overall nutrient concentration in the experiment.

Impurities can be removed, however, for such critical experiments by washing agar in double distilled water for at least 24 hr, rinsing in ethanol and drying at 600 C for 24 hr.

Gelatin at high concentrations (10%) has been tried as a gelling agent but has limited use because it melts at low temperature (250 C). Other compounds successfully tested include biogel (polyacrylamide pellets) alginate, phytagel and Gelrite.

The FMC Corporation has recently developed a highly purified agarose called Sea Plaque(k), which can be used in the recovery of single protoplasts from cultures. Perforated cellophane, filter paper bridges, filter paper wicks, polyurethane foam and polyester fleece are alternative methods of support used in the medium for cell or tissue growth.

The advantage of working with synthetic gelling compounds is that they form clear gels at relatively low concentrations (1.25-2.5 g 1-1) and are valuable aids for detecting contamination that may develop during the span of cultures. Whether explants grow better on agar or other supporting agents depends on the tissue and the species.
pH: Plant cells and tissues require optimum pH for growth and development in cultures. While preparing a medium, the pH can be adjusted to the requirement of an experiment. The pH affects uptake of ions and for most of the culture media pH 5.0 to 6.0 before sterilisation is considered optimal.
Higher pH is likely to give a hard medium while a low pH results in unsatisfactory solidification of the agar. Most tissue culture media are poorly buffered, resulting in pH fluctuations which could prove detrimental to long term survival and growth of single cells or cell populations at low density.

 
 

 
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