Formation,regeneration, and fusion of protoplasts in aCellulomonas strain

The effect of different conditions on protoplast formation was studied in the streptomycin-resistant strainCellulomonas sp.M32Bo. The greatest efficiency (75% protoplasts) was achieved by use of 0.5M sodium succinate as osmotic stabilizer, supplemented with 20 mM MgCl₂, 200 µg/ml of lysozyme, and 0.01M EDTA at pH 7.4. Cells harvested at the midexponential growth phase were more suitable for protoplast formation than those of the stationary phase. Electron microscopy observations showed the presence of both protoplasts and spheroplasts in the treated samples, some of them still showing a rod shape. Two regeneration media were developed that showed similar regeneration frequencies (52%). StrainM32Bo was fused with a tetracycline-resistant strain (Cellulomonas sp. Sz). Segregation analysis of fusant colonies suggested the existence of a temporary diploid stage in which both parental genotypes were expressed.