灵芝原生质体制备与融合条件的研究

Research on preparation and fusion of Ganoderma lucidum protoplasts

以美国大灵芝和信州灵芝为起始菌株,对其原生质体制备和融合条件进行研究。结果表明:美国大灵芝原生质体制备最佳条件:摇瓶培养4d的菌丝球,复合酶液酶解,温度31℃,时间3h,原生质体制备率为5.80×106个/mL,再生率为0.32‰;信州灵芝原生质体制备最佳条件:摇瓶培养5d的菌丝球,复合酶液酶解,温度31℃,时间2.5h,原生质体制备率为4.71×106个/mL,再生率为0.24‰。两种原生质体最佳融合条件:促融剂PEG浓度30%,Ca2+浓度0.03mol/L,时间30min,温度35℃,融合率0.47%。

The time and the temperature of treatment of enzyme were studied to find out the effects on formation and regeneration of protoplasts of big Ganoderma lucidum of America and Ganoderma lucidum of Shinshu.The following conclusions were obtained：The optimum conditions for formation and regeneration of protoplasts of big Ganoderma lucidum of America were obtained by using 4-day-cultured mycelia,the enzyme mixed with 2% lywallzyme,1% snailase and 1% cellulase,temperature of treatment of enzyme was 31℃,time of treatment of enzyme was 3h,regenerated by CYM,the rate of formation was 5.80×106/mL,the rate of regeneration was 0.32‰.The optimum conditions for formation and regeneration of protoplasts of Ganoderma lucidum of Shinshu were obtained by using 5-day-cultured mycelia,the enzyme mixed with 2% lywallzyme,1% snailase and 1% cellulase,temperature of treatment of enzyme was 31℃,time of treatment of enzyme was 2.5h,regenerated by CYM,the rate of formation was 4.71×106/mL,the rate of regeneration was 0.24‰.The suspension of protoplasts were regulated into same concentration and being mixed,using different concentrations of PEG 4000,changing different concentrations of Ca2＋,using different time and temperature on fusion.The optimum conditions on fusion were：concentration of PEG was 30%,concentration of Ca2＋ was 0.03mol/L,time on fusion was 30min,temperature on fusion was 35℃,and the fusion frequency was 0.47%.