Preparation and regeneration of protoplasts from mycelia of Tricholoma matsutake

Protoplasts are used in fungal breeding through cell fusion and gene transf er, thus it is very important that the rate of regeneration is high. Theref ore, various conditions for the preparation of protoplasts from mycelia of Tricholoma matsutake (S. Ito et Imai) Sing. were examined. Protoplasts were obtained as follows: mycelia of T. matsutake (strain No. M-03 and M-52) we re cultured separately in semi synthetic liquid medium for 30 days, treated for 3 h in 0.05 M maleic acid NaOH buffer (pH 5.5) containing 0.5 M mannit ol as osmotic stabilizer and previously reported enzyme composition. The pl ating efficiency of protoplasts from mycelia were 0.77% and 0.55% in M-03 a nd M-52 respectively. Futhermore, to obtain protoplasts of higher regeneration rates, protoplasts from mycelia in solid medium for 30 days were prepared under the former co ndition. The plating efficiency of the protoplasts was increased about 2 times. The plating efficiency of protoplasts in suitable enzyme formulation containing 1% Novozym-234, 1% Cellulase "ONOZUKA" R-10, 0.2% Zymolyase-20T, 0.1% Chit inase and 1% Acremoniumcellulase were 2.3% and 1.9%, in M-03 and M-52 respe ctively. From these results, it was shown that Acremoniumcellulase contains enzymes effective for the isolation of active protoplasts from a brush lik e young mycelia.