An improved procedure for the isolation and purification of protoplasts from carrot suspension culture

A procedure is reported for the rapid and highly reproducible isolation of protoplasts from carrot suspension culture. The method utilizes Onozuka R 10 cellulase which has been purified by chromatography on Sephadex G75. Protoplast isolation, using this procedure, is quantitative and complete within 1 to 1.5 h. Intact protoplasts were separated from broken ones and other cellular debris by application of a polyethylene glycol/dextran two-phase system. The protoplasts isolated in this manner lack any detectable cell wall and are greater than 95% viable when assayed using fluorescein diacetate. It is concluded that such protoplasts are highly suitable for biochemical studies.Abbreviation PCM protoplast culture medium