Protoplast fusion between isoleucine-, arginine- and thymidine-requiring auxotroph (lle, Arg, Thy) ofLentinula edodes and arginine-requiring auxotroph (Arg) ofCoriolus versicolor has been achieved using 30% polyethylene glycol (M.W. 4000) in 10 mMCaCl(2)-glycine solution (pH 8.0). Fusion hybrids were selected in the 0.6 M sucrose supplemented minimal media on the basis of nutritional complementation with fusion frequency of 7.4x10(-6). The hybrids included both parental and non-parental types in colony morphology, growth rate and isozyme patterns. We succeeded inter-order protoplast fusion between the auxotrophs ofLentinula edodes and Coriolus versicolor overcoming the natural barriers of incompatibility. We examined the characteristics of the hybrids and clarified the fusion process using electron microscopy.

Heterokaryotic nuclear hybrids overcoming the natural barriers of incompatibility have been studied in basidiomycetes. To produce these nuclear hybrids between incompatible mushrooms, which have several potent pharmacological effects, nuclear transfer was performed between Lentinula edodes and Coriolus versicolor. Nuclei from serine auxotrophs of Lentinula edodes, LE207 (Ser-) were transferred into the protoplasts of arginine auxotrophs of Coriolus versicolor, CV17 (Arg-), using 30% polyethylene glycol 4000 in 10 mM CaCl2-glycine solution (pH 8.0). Nuclear transfer progenies were selected by nutritional complementation on minimal media supplemented with 0.6 M sucrose. The progenies were classified based on colony morphology to L. edodes-like, C. versicolor-like and non-parental type. Most of the progenies grew slower than either parent. The number of nuclei per cell was similar but the DNA content varied between progenies. The isozyme patterns of nuclear hybrids resembled either of the parent profiles or showed a mixed profile.

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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.

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