Regeneration of protoplasts from hyphal strands of Volvariella volvacea

A series of experiments on the preparation and regeneration of protoplasts from hyphal strands of Volvariella volvacea (Bull. ex. Fr.) Singer were con ducted with the aim of optimizing the conditions for its efficient regenera tion. One commercial (Vvc1) and two wild (EAAC-0001 and EAAC-0002) strains of V. volvacea from the Philippines were used and subjected to varying cond itions to determine the most efficient means for regeneration of their prot oplasts, The effects of age and type of strain, pH, type and concentration of osmotic stabilizer, enzymatic composition, treatment time, temperature, reciprocal frequency during enzymatic lysis of the cell wall, and centrifug ation conditions were investigated. Results showed that the three strains o f V. volvacea had varying responses in terms of yield, size, and ability of their protoplasts to regenerate into the protoplast regeneration medium. A mong the three strains, EAAC-0002 had the highest rate of regeneration, The 5-day-old culture of V. volvacea, when subjected to a combination of 2% No vozyme 234 and 0.2% chitinase in 0.6 M mannitol (pH 6.0) for 3 h at 30 degr ees C, 90 strokes/min and centrifuged at 1100g for 10 min, produced an effi cient yield of protoplasts with a relatively high regeneration rate.