In a variation of this protocol, which was developed primarily to improve and to simplify the handling procedure during culture, the 30 ml glass bottles were replaced with tissue culture plates each with 24 circular wells (Costar Corporation, Cambridge, USA). Each well contained either 1 ml of 0.4% (w/v) Sea Plaque agarose-solidified KPR medium with embedded protoplasts (control) or 1 ml of the same protoplast suspension over 2 ml of oxygenated PFC. The dishes were sealed with Nescofilm. After 7 d culture in the dark, the agarose layers containing dividing protoplasts were transferred to 3.5 cm diameter Petri dishes (1 layer per dish), the dishes each containing 2 ml of liquid KPR medium. Cultures were incubated as in the initial assessments.