Elementary plant Biochemistry and Biotechnology (2+1)

Factors affecting yield and viability of protoplasts

• Source of material: Leaf has been the most favorite source of plant protoplasts because it allows the isolation of a large number of relatively uniform cells without the necessity of killing the plants. Since the mesophyll cell are loosely arranged, the enzymes have an easy access to the cell wall. The leaves from in vitro roots or shoots released twice as many viable protoplasts as the leaves from field grown material. Owing to the difficulty in isolating culturable protoplasts from leaf cells of cereals and other species, their cultured cells have been used as an alternative source material.
• Physiological state of the tissue and cell material: younger the tissue, more the success of protoplasts isolation.
• Pre enzyme treatment: The lower epidermis is peeled and float the stripped pieces of leaf on the enzyme in manner that the peeled surface is in contact with the solution. This will facilitate easy penetration of enzyme in to intercellular spaces of leaf. Mesophyll protoplasts of cereals could be isolated within 2 hr by infiltrating the leaf pieces with enzyme solution under a partial vacuum for 3-5 min. The criterion used to check adequate infiltration is that leaf pieces will sink when the vacuum is removed. Brushing the leaf with a soft brush or with the cutting edge of scalpel may also improve enzymatic action.
• Enzyme treatment: The two enzymes, essential to isolate protoplasts from plant cells are cellulase and pectinase. Pectinase degrades mainly the middle lamella and the cellulase required to digest the cellulosic cell wall. The crude commercial enzymes carry nucleases and proteases as impurities which may be harmful to protoplasts viability. The activity of the enzymes is pH dependant and it is also affected by the temperature. The optimal temperature for the activity of these enzymes is 40-50⁰C which happens to be too high for the cells. Generally 25-30⁰C is found adequate for isolation of protoplasts.
• Osmoticum: A fundamental property of isolated protoplasts is their osmotic fragility and hence, there is a need for a suitable osmotic stabilizer in the enzyme solution, the protoplast medium and the protoplast culture medium. Protoplasts are more stable in a slightly hypertonic rather than isotonic solution. a higher level of the osmoticum may prevent bursting and budding but it may inhibit the division of the protoplast. The most widely used osmotica are sorbitol and mannitol in the range of 450-800 mmol.