3.2. Culture medium
The nutritional requirement of protoplast is almost similar to that of the cultured plant cells. Mostly the salts of MS (Murashige and Skoog, 1962) and B5 (Gamborg et al. 1968) media and their modifications have been used. Ammonium salts have been found detrimental to protoplasts survival of many species, and media have been devised that either have a reduced concentration of ammonium or lack it. Concentration of zinc is reduced while the concentration of calcium is increased as it enhances the membrane stability. Osmolarity is maintained by addition of sorbitol, mannitol, glucose or sucrose and mannitol being widely used osmoticum as it is not used by the dividing cells, thus, maintains the osmolarity of the medium.
Glucose is preferred carbon source as sucrose do not satisfy protoplast culture. One or two amino acids are added at low concentration. Growth regulators are required essentially in protoplast culture generally high concentration of auxins (NAA, 2,4-D) along with lower concentration of cytokinins (BAP, Zeatin ) is used.
Environmental conditions: High light intensity inhibits growth of protoplast hence initially protoplast is grown in dim light for few days and then transferred to light of about 2000-5000 lux. However, better results are obtained when cultured in darkness.
3.3. Plating density
Like cell cultures, the initial plating density of protoplasts has profound effect on plating efficiency. Protoplasts are cultured at a density of 1 x 104 to 1 x 105 protoplasts ml-1 of the medium. At high density the cell colonies arising from individual protoplasts tend to grow into each other resulting into chimera tissue if the the protoplast population is genetically heterogeneous. Cloning of individuals cells, which is desirable in somatic hybridization and mutagenic studies, can be achieved if protoplasts or cells derived from them can be cultured at a low density.