Conditions required for enzyme activity :

•  Enzymes are pH and temperature dependent, thus, for enzymatic release of protoplast an enzyme showing activity at pH range 4.7-6.0 and temperature range of 25-30°C is used

•  Duration of enzyme pretreatment and condition of light presence required for incubation may also be determined.

•  Enzyme mixture used should essentially consist of cellulose, hemicellulase and pectinase which facilitate the degradation of cellulose, hemicelluloses and pectin, respectively.

•  The concentration of sugar alcohols used as osmoticum (mannitol) must be empirically defined.

 

2.1.  Factors affecting yield and viability of protoplasts

i.  Source of material : Leaves were the most convenient source of the plant protoplasts because it allows the isolation of a large number of relatively uniform cells without killing the plants. Moreover the mesophyll cells are loosely arranged, the enzymes have an easy access to the cell wall. The parent plant age and the conditions in which it is growing have profound effect on the yield of protoplast. Due to the difficulty in isolating culturable protoplast from leaf cells of cereals and some other species their cultured cells can be used as a source material. The yield of protoplasts depends upon the growth rate and growth phase of the cells. Generally embryogenic suspension cultures are used to obtain totipotent protoplasts .

ii.  Pre-enzyme treatments : To facilitate the penetration of enzyme solution into the intercellular spaces of leaf, which is essential for effective digestion, various methods are followed. The most commonly used method is to peel the lower epidermis and float the stripped pieces of leaf on the enzyme solution in a manner that the peeled surface is in a contact with the solution. Most of the time it is not convenient to peel the epidermis, in such cases cutting the leaf or tissue into small strips (1- 2 mm wide) has been found useful. When combined with vacuum infiltration the latter approach has proved very effective. Brushing of leaves with a soft brush or with the cutting edge of a scalpel may also improve the enzymatic action. Large calli are chopped into pieces and can be transferred to enzyme mixture. Agitation of incubation mixture during enzyme treatment improves protoplast yield from cultured cells.

iii.  Enzyme treatment : The release of protoplast is very much dependent on the nature and concentration of the enzymes used. The two major enzymes required for the isolation of protoplast are cellulase and pectinase. The cellulase is required to digest the cellulosic cell walls and the pectinase mainly degrades the middle lamella. Some of the tissues may require other enzymes like, hemicellulase, driselase, macerozyme and pectolyase. The activity of enzyme is pH dependent. The pH of the enzyme solution is adjusted somewhere between 4.7 to 6.0.