Factors affecting the production and regeneration of protoplasts: the case of the mycorrhizal fungus Tulasnella calospora from roots of orchids

Protoplast isolation is relevant for many different applications and has been principally used in procedures involving genetic manipulation. In this study, the age of mycelium, osmotic stabilizers, enzyme, incubation temperature and incubation time were evaluated in terms of their effects on protoplast yield. The young mycelia (3 d) of Tulasnella calospora were digested for 6 h at 30℃ in a mixture of 1.2 mol·L^-1 MgSO₄+10 mmol·L^-1 K₂HPO₄ as the osmotic stabilizer, with a 1.0% lysing enzyme and 1.5% driselase:more than 10⁶ protoplasts mL^-1 were obtained. When collected by density gradient centrifugation, the concentration of protoplasts can reach 10⁷-10⁸ protoplasts mL^-1, an amount suitable enough for experiments of transformation in fungi. For every 10⁵ protoplasts, about 15-25 protoplasts can regenerate after 24-36 h cultivation in a liquid medium and after 8-10 d in an agar medium. This study produced an efficient method for protoplast production, reverting them into a typical mycelia morphology using a Tulasnella calospora isolate.