An index [kv: average isolation rate of viable protoplast (number/ml min)] was established to evaluate the optimal conditions for protoplast isolation from cultured plant cells. The optimal conditions for protoplasts isolation from Nicotiana tabacum BY2 cultured cells could be determined on the basis of the kv [31.7 x 10(3) (number/ml min)]. The colony-forming efficiency of the protoplasts was about 46%. The optimal conditions for protoplasts isolation from Catharanthus roseus [kv = 38.1 x 10(3) (number/ml min)] and Wasabia japonica [kv = 14.2 x 10(3) (number/ml min)] cultured cells could also be determined. Furthermore, a method for rapid regenerating cell wall of protoplast in liquid culture using alginate gel containing locust bean gum was developed.