Generation of fimgal protoplast is essential for fusion and transformation systems.
Protoplast fusion offers great potential for the improvement of industrially important
microorganisms. To establish conditions for the protoplast isolation and regeneration
of the mycelia of Lentinus lepideus, various enzymes and osmotic stabilizers were
examined. To investigate suitable medium for the culture of L. Zepideus, the mycelia
were grown in ten different media at 28 “C for 10 days. Among them potato dextrose
agar (PDA) medium was found to be the best for colony growth. When Novozym 234,
cellulase and j3-glucuronidase were added to the mycelia in combination or alone,
Novozym 234 alone at the concentration of 10 mg/ml was the most effective for the
protoplast yield. Purified spherical protoplasts of the mycelia were osmotically
hypersensitive and further incubation of the mycelia with the lytic enzyme resulted in
the older parts of the hyphae swollen. When we applied various osmotic stabilizers at
the fixed concentration of 0.6 M on the protoplasts, the yields of protoplasts were
increased until 4-hr incubation. However application of sucrose or MgSO, led to
further protection of protoplasts after that time and reached a plateau on S- and 7-hr
incubations, respectively. The suitable incubation time and optimal pH with the lytic
enzyme for the maximum release of protoplasts were 6 hrs of incubation and pH 5,
respectively. When we examined various osmotic stabilizers for the regeneration of
the protoplast, the complete medium containing 0.6 M sucrose induced highest
hyphal growth with regeneration frequency of 3.28 %.