SUMMARY: A method for the production of protoplasts of strain N1-4080 utilizing lysozyme and penicillin G was optimized to yield about 100% protoplasts. Treatment of 10 cells left fewer than 10 that were viable and osmo-resistant. With a mutant strain deficient in autolytic activity, about 1% of the protoplasts regenerated walled cells, whereas most gave rise to stable L-form colonies. Addition of sodium polyanethole sulphonate to the medium improved the regeneration frequency by at least a factor of 10, probably by somehow inhibiting the residual autolytic activity of the strain.


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