Biological Characteristics Of Hohenbuehelia Serotina And Protoplast Fusion With Tricholoma Matsutake

Hohenbuehelia serotina is one of the well-known wild edible fungi in northeast of China which is delicious and rich in nutrition. The mycelia were isolated from the wild and the biological characteristics and the fermentation culture conditions with the method of shake flask were studied. The results showed that the optimal carbon source was glucose and the optimal nitrogen source was yeast extract powder and peptone. The range of C/N value for mycelial growth was 10/1-60/1 and 20/1-40/1 was most suitable. The optimal temperature and pH were found to be 23-25℃and 5.4-5.8 respectively. The optimal strain age of spawn and inoculum quantity were 7d and 15% with rotation speed of 180rpm and the maximium biomass was 1.117g/mL.The optimal medium composition of primary spawn and culture spawn of Hohenbuehelia serotina were determined respectively. Among 6 mediums of primary spawn, A₃ was found to be the best for mycelial growth which was composed by 58% sawdust, 30% rice straw, 10% wheat bran, 1% sucrose, 1% CaCO₃ and growth rate was 1.23cm/d. Among 10 mediums of culture spawn, the optimal mediums were A₅ and A₄ and the transformation efficiency were 63.81% and 60.92% respectively. A₅ was composed by 78% sawdust, 10% corn stalk, 10% wheat bran, 1% sucrose, 1% gypsum and A₄ was composed by 58% sawdust, 30% rice straw, 10% wheat bran, 1%sucrose, 1% gypsum respectively.The optimal conditions of protoplasts separation and regeneration of Hohenbuehelia serotina were studied. The results showed that the 8-day-mycelia incubated in lywallzyrne at the consistence of 2% at 32℃constant temperature water-bath for three hours were the most effective for the protoplast yield. The suitable osmotic stabilizers and pH with the lywallzyrne for the maximum release of protoplasts were NaCl at the concentration of 0.6 M and pH 6.5 respectively. In addition, the highest regeneration rate of the protoplast of Hohenbuehelia serotina was 6% when 0.6mol/L sucrose was used for osmotic stabilizer of reproducible medium.At the basis of the protoplasts separation of Tricholoma matsutake studied in our laboratory, the conditions of electrofusion between Hohenbuehelia serotina protoplasts and Tricholoma matsutake protoplasts were studied. The results showed that the optimal alternating current (AC) and duration of aglignment were 150V/cm and 30s respectively. The optimal direct current (DC) was 1000V/cm and duration of pulse was 20μs and the optimal DC pulse was 2 times. The fusion rate were 19.7% and 18.9% respectively.