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.
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