Pannee Dhitaphichit and Chaninun Pornsuriya

pp. 975 - 982

Abstract

Protoplast fusion between Pleurotus ostreatus and P. djamor was carried out by isolating protoplasts
from 4-day-old monokaryotic mycelia cultured on malt extract broth. The mycelia were then agitated at 100
rpm for 2 h with 9 mg Lysing Enzyme (Sigma L-1412) in 1 ml osmotic stabilizer (0.6 M MgSO₄
·7H₂O in 0.05 M sodium maleate buffer,pH 5). The freshly prepared protoplasts were then mixed and incubated in 40%
PEG (polyethylene glycol 6,000)/0.05 M CaCl₂·2H₂O for 20 min at room temperature. All protoplasts were
regenerated on Regeneration Medium for 7-12 days. There were 412 regenerated colonies detected but only
two of them were selected as fusants by posessing clamp connections on their mycelia. The fusants were
proved to be ″hybrids″ of P. ostreatus and P. djamor. Their mycelia were significantly faster in growth and
larger in size than the parental strains. They showed bands common to their parents when esterase was used
for isozyme studies. The fruiting bodies of the fusants also showed recombined characteristics of the parental
strains.