IMPROVED CONDITIONS FOR PROTOPLAST FORMATION AND TRANSFORMATION OF PLEUROTUS-OSTREATUS

Conditions suitable for the production and regeneration of Pleurotus o streatus protoplasts from dikaryotic mycelia were examined. Three comm ercially available muralytic enzymes, including Sigma lysing enzyme, N ovozym 234 and Novozym 234 LP, were used for production of protoplasts . Over 2 x 10(7) protoplasts per gram fresh weight mycelia were obtain ed within 1.5 h by using each of these three enzymes. The colony regen eration rate was up to 12-13% on potato-dextrose-agar medium containin g 0.8 m mannitol. Genetic transformation was based on positive selecti on for resistance to hygromycin B (HmB) using the plasmid vector pAN7- 1 and accomplished by either electroporation or a polyethylene glycol (PEG)-divalent cation method. P. ostreatus strains used in this study have innate sensitivity to HmB at a critical inhibitory concentration of between 40-50 mug/ml. Selection for HmB resistance of this fungus, indicative of transformation, resulted in 3-48 HmB-resistant colonies per microgram of pAN7-1 per 10(7) viable protoplasts. No significant d ifferences were apparent when either transformation protocol or either P. ostreatus strain was used. The best electrical condition found for the electrotransformation of P. ostreatus is at a field strength of 2 .6-2.8 kV/cm with a capacitance of 25muF and a parallel resistance of 800 ohms, corresponding to a time constant range of 10-14 ms.