NIVEDITA SINGH AND U.S. BAGDE
Abstract
Spherical, osmotically sensitive protoplasts were efficiently isolated from young cells of yeast Williopsis saturnus by the 1:,/tic action of cell wall lysing enzyme Lyticase at a concentration of 0.25%. A variety of experimental conditions were investigated in order to achieve optimal conditions for viable protoplast formation and regeneration. Detailed analysis of different factors affecting the synthesis of protoplasts has been reported. The most suitable conditions for protoplasting have been optimized using 20 hour old culture and 0.8M sorbitol as the most suitable osmotic stabilizer. Protoplast formation was spectrophotometrically estimated to be over 98% complete in the end of 90 minutes. Transmission electron micrographs detailed the protoplast ultrastructure and showed the preparations to be completely devoid of cell wall.