The formation of protoplasts from Mucor species

Abstract

Methods were developed for the reproducible production of high yields of stable protoplasts from mesophilic and thermophilic strains of Mucor. This is a pre-requisite for the genetic analysis and manipulation of Mucor by cell fusion or transformation and is of special importance in these species since conventional genetic analysis by recombination studies is not feasible. The cell wall lytic enzyme used was the concentrated culture fluid of Streptomyces sp no. 6 grown on chitosan and chitin. Using germlings of M. circinelloides f. lusitanicus the optimum conditions for protoplast formation were determined as: 0.5-1.0 mg·ml-1 streptozyme (1.2 units chitosanase·ml-1) plus 2.0 mg·ml-1 novozym 234 or 1%(v/v) helicase in 0.5 m-sorbitol, 0.01 m-sodium phosphate buffer, pH 6.5 for 3-4 hours at 23°C. Treatment of 107·ml-1 germlings gave yields of up to 3×107·ml-1 protoplasts with regeneration and fusion frequencies of up to 40% and 1.7%, respectively. With the thermophilic strains growth at low temperature (25°C) was critical for the subsequent formation of stable protoplasts. Synchronous growth and germination was obtained at this temperature by a short pre-incubation (1-3 hours) of the sporangiospores at 40°C. © 1984 Carlsberg Laboratory.