Breeding of edible mushroom using cell fusion technique

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Optimum result in obtaining protoplast suitable for an electrofusion technique and having a high viability as well,was observed when hydrolysis was conducted for 3 hours at 30゜C in an enzyme solution on mycelia cultured for 3 to 5 days in soyonion medium. The solution contained 2% of cellulase, 0.6% zymolyase, and 6 units of chitinase, respectively. The yield of protoplast was over 10/ml, the average plating efficiency being approximately 20%. Investigation of the ultrastructure of the protoplast showed that those dikaryon protoplasts with a dense concentration of cytoplasm, had a higher plating efficiency. In terms of fusion conditions,the best result was obtained by adding 0.5mM magnesium chloride and 0.1mM calcium chloride to a 0.5M mannitolsolution, with the pulse length and voltage set at 40 sec, and 4kV/cm, respectively. And this gave a fusion rate of approximately 60%. However, further investigations have yet to be conducted with regard to the decrease in aggregation of protoplasts in a high frequency electric field caused by the addition of electrolyte, and on influences of increased high frequency voltage on the hyphae regeneration, etc. Study of isozyme analysis exhibited that a specific isozyme pattern could be observed for esterase and malate dehydrogenase, thus constituting an efficient selection index for the screening of somatic hybridoma. The development of strains having markers, such as auxotrophic mutants is also under consideration. Screening tests are now being carried out although as yet no new somatic hybridoma have been obtained.