A method for the preparation of plant protoplasts.

A simple procedure for the preparation of plant cell protoplasts is described. The leaf is first treated with pectinase under conditions that do not liberate cells; cellulase is then used to release protoplasts. The yields are comparable to those obtained by methods that entail the prior preparation of free cells. The method is of general application but is particularly useful with tissues that cannot tolerate the shaking needed to liberate intact cells. The preparation of protoplasts from leaf mesophyll usually requires the use of several enzymes, typically pectinase and cellulase. Only in a few favourable cases is a single enzyme preparation sufficient. The enzymes are used either simultaneously1 or sequen­ tially2. The simultaneous application of several crude enzymes has the great advantage of simplicity, but for several reasons is not as satisfactory as the sequential procedure. Both cells and protoplasts are exposed throughout to all the toxic substances which may be present in the mixture; the efficiencies of the enzymes, particularly cellulase, are often reduced under these conditions so that higher concentrations or much longer periods of treatment are required1-2. When large numbers of protoplasts are required, for example in work with viruses, long periods of digestion are undesirable because of the difficulties in avoiding infections by microorganisms; the rich enzyme solu­ tions encourage any stray organisms to proliferate rapidly. The sequential procedure2 has been used with great success for preparing protoplasts from tobacco leaf mesophyll. It consists of two steps. 1 . The leaf material is shaken with pectinase solu­ tion at 25 °C to release intact mesophyll cells. 2 . The free cells are collected and shaken gently with cellulase solution at 35 °C to convert them to protoplasts. When this method was applied to leaves of pea (Pisum sativum, cv. Chemin Long), the initial shaking with pectinase to release cells caused so much damage that the subsequent treatment with cellulase produced almost no live protoplasts. Reduction of the violence of shaking to prevent damage resulted in negliglible yields of free cells. An extremely simple modification of the procedure was therefore devised which has proved very successful in many cases where the ori­ ginal method was too traumatic. It has also been used