DEDIKARYOTIZATION OF THE SHIITAKE MUSHROOM, LENTINULA EDODES BY THE PROTOPLAST REGENERATION METHOD

The present study was carried out to investigate the applicability and usefulness of the protoplast isolation and regeneration method as a new technique for artificially dedikaryotizing Lentinula edodes dikaryons. When protoplasts derived from dikaryans were incubated in a regeneration agar medium at 25 degrees C, about 11% of them individually started regeneration into hyphae within 3 days and formed visible colonies, varying in size, after 7 days of incubation. By isolating preferentially smaller colonies out of these visible colonies, it was found that neohaplonts could be obtained at the high frequencies of 40-92% in every dikaryon sampled and that the two-component nuclear types appeared. These neohaplonts showed considerable variation of mycelial growth rates. However, robust neohaplonts exhibited no apparent change in biological properties such as colony morphology, and electophoretic zymograms of esterase and malate dehydrogenase, suggesting that they might retain the original genetic traits. From these results, it is concluded that the protoplast regeneration method for dedikaryotizing L. edodes dikaryons is more useful than previous methods such as the physical procedure and chemical treatment.