Abstract

Transformation of the basidiomycete Coprinopsis cinerea makes use of unicellular haploid asexual spores called oidia. Protoplasts of oidia are generated by a cellulase/chitinase enzyme mix. Protoplasts and DNAs are incubated together in 25 mM Ca2+ and 5% PEG (polyethylene glycol) 4000 on ice and more PEG is added (23% final concentration) after a ‘heat shock’ step at RT (room temperature). Upon regeneration on selective media, transformation rates of several hundreds of clones might be obtained per 1 μg DNA and 107 protoplasts. Although the technique has been invented 25 years ago by Binninger et al. (1), there are reoccurring pitfalls in the method that can cause failure. Successful transformation needs a good amount of skilful knowhow about the fungus and the method. Here we present our experiences with C. cinerea transformations, call attention to potential flaws and to optimal handlings in fungal cultivation, harvesting, protoplasting, transformation, and subsequent regeneration of the fungus.