Preparation and regeneration of protoplasts and spheroplasts for fusion and transformation of Schizosaccharomyces pombe

Preparation and regeneration of protoplasts is essential for somatic hybridization and transformation of yeasts. We present conditions that were found to be optimal for preparing and regenerating Schizosaccharomyces pombe protoplasts for cell fusion. In contrast to these conditions, genetic transformation of S. pombe requires spheroplasts that are osmotically sensitive, but still have some wall material attached to the cell. The main finding were as follows: (a) For protoplast formation with Novozym SP234, 0.9 M sorbitol was found to be the optimal osmotic milieu and β-mercaptoethanol is not necessary. (b) Embedding in soft agar yields considerably better regeneration frequencies than direct plating. (c) Cell fusion is optimal when both fusion partners are fully protoplasted, although considerable fusion occurs between spheroplasted cells as well. (d)Schizosaccharomyces pombe transformation frequencies are much higher with spheroplasts than with protoplasts. Inclusion of β-mercaptoethanol did not enhance transformation frequency.