Strain Improvement of Pleurotus Species by Protoplast Fusion

Objective: To improve the strain of Pleurotus mushroom by protoplast fusion technique Methods: The white rot fungus Pleurotus species was isolated from fresh fruiting body of Oyster mushroom by spore print technique and was cultured on Potato dextrose agar. The selected strain was identified based on the morphological and cultural characters on Potato dextrose agar. The protoplasts were isolated from both wild and UV mutant strains of Pleurotus species. These were examined microscopically and the isolated protoplasts of wild and mutant strains of Pleurotus were counted by haemocytometer. Protoplast fusion between wild and UV mutant Pleurotus species was carried out by isolating protoplasts from 4-day-old monokaryotic mycelia cultured on malt extract broth. The mycelia were then agitated at 100rpm for 2h in 1ml osmotic stabilizer (0.6 M MgSO4.7 H2O in 0.05 M sodium maleate buffer, pH 5). The freshly prepared protoplasts were then mixed and incubated in 40% PEG (Polyethylne glycol 6000)/0.05 M CaCl2.2H2O for 20 minutes at room temperature. All protoplasts were regenerated on Regeneration Medium for 7-12 days. The regenerated colonies were detected and fusants were selected based on their clamp connections on their mycelia. Results: The fusants were proved to be hybrids of wild and UV mutant strain of Pleurotus species. Their mycelia were significantly faster in growth and larger in size than the parental strains. They showed bands common to their parents when laccase was used for isozyme studies. Conclusion: Fusants possessing clamps was selected and proved to be hybrids of wild and mutant strains of Pleurotus species by comparing their mycelial growth, hyphal size and isozyme patterns. Employing the fungal strains, which show synergistic associations in co culture for the development of protoplast fusants with the desirable characters of both the parents, will be a viable option for various imdustrial and medicinal applications of laccase.