Conditions for isolation of and colony formation by mycelial protoplasts of Coprinus macrorhizus

Conditions suitable for the release of mycelial protoplasts of Coprinus macrorhizus and for colony formation by the isolated protoplasts were examined. The study culminated in the development of a method using commercially available preparations of chitinase, cellulase and β-glucanase, by which over 2x 10 8 /ml protoplasts could be obtained within 2hr. The protoplasts from the wild type and mutant Fis c of C. macrorhizus regenerated into hyphae in a complex medium containing malt and yeast extract, and subsequently formed colonies with frequencies up to 50%, which paralleled the percentage of nucleated protoplasts. The plating efficiency in a minimal medium was about 20% on average. Sucrose was found to be superior to mannitol as an osmoticum for colony formation. Protoplasts from auxotrophic mutants formed colonies at much lower frequencies even in the complex medium, but addition of N-acetyl glucosamine significantly improved their plating efficiency. Colonies derived from the protoplasts of C. macrorhizus Fis c consistently developed fruiting bodies, when grown on a hypotonic medium.