Factors affecting protoplast isolation and regeneration in Microsporum gypseum

ABSTRACT

Factors affecting high yields, regeneration frequencies, and viablity of protoplasts from clonal cultures of Microsporum gypseum were investigated. Maximum yields of protoplasts were obtained after 6 hr digestion of 2-4 days old mycelium with Novozyme 234 using CaCl2 [0.4M] as an osmotic stabilizer and glycine [+] HCL [pH 4.5] as the buffer system. Mercaptoetoethanol + dithi-othreitol [0.01 M] proved to be the best pretreatment of mycelium prior to digestion with enzyme. A regeneration frequency of 94.4% was obtained using the top agar method with complete medium [pH 6.5] containing 0.5% agar and 0.4 M CaCl2 as an osmoticum. Colonies from regenerated protoplasts on medium containing were pigmented and completely powdery with high sporulation