Liquid culture enhances protoplast formation from the auxotroph (ser(-)) ofLentinula edodes

The optimal conditions for the production and regeneration of the protoplasts fromLentinula edodes were studied. Protoplast formation from the mycelia ofL. edodes which were cultured in liquid medium showed a significantly high yield compared with that of the mycelia which were cultured on cellophane covered agar media. A mixture of Novozyme 234 (15 mg/ml) and Cellulase Onozuka R10 (10 mg/ml) in 0.6 M mannitol (pH 4) was optimal lytic enzyme for the protoplast release. The optimal incubation time and mycelia age were 3.5-4 hours at 30 degrees C and 6-8 days, respectively. Regeneration frequency was 0.18% plated onto a medium containing 0.6 M sucrose, and 0.08% plated onto a medium containing mannitol. But hardly any regeneration was observed in the media containing NaCl, KCl, or MgSO(4). More than 90% of the protoplasts contianed nuclei and the nucleus number per protoplast was 1.1. The DNA content per nucleus was 5.1 pg. The diameter of the protoplast was 3-5 mum and it had a well defined cell structure.