Study on the Separation and Fusion of Protoplasts of Main Auricularia auricula Cultivars in Qinba Mountains

Abstract:

Single variable method was utilized to determine the optimum conditions of Auricularia auricula protoplasts preparation, regeneration and fusion in Shenlong A8 and Qindan 4#. After cultured for 7 d, the Auricularia auricula mycelium was treated with 0.5 mol/L mannitol as osmotic stabilizer, digested by 2.0% lywallzyme, then incubated in 30 ℃ water for 3.5 h, and the total amount of released protoplasts reached 0.94×107/mL, 1.44×107/mL in the two varieties, respectively.RM5 regeneration medium was selected and the highest regeneration rates were observed in Shenlong A8 and Qindan 4# protoplasts, which attained 1.91% and 1.53%, respectively.The optimum condition for protoplasts fusion: 30% PEG4000 and 0.05 mol/L CaCl2(pH 10)were added as fusion-aided agent, and incubated in 35 ℃ for 30 min. The resulting fusant was obtained by fluorescence microscopy and antagonistic reaction, which served as the theoretical basis for Auricularia auricular breeding.