【作者】 牛艳芳；

【导师】 张功；

【作者基本信息】 内蒙古师范大学 ， 植物学， 2004， 硕士

【摘要】 原生质体融合技术是近些年来迅速发展起来的细胞工程育种新技术，它具有许多常规杂交方法所无法比拟的独到之处。本文以蒙古口蘑和双孢蘑菇为出发菌株，研究了酶浓度、酶解时间、菌龄和渗透压稳定剂、再生培养基等对蒙古口蘑和双孢蘑菇原生质体制备和再生的影响，结果表明，蒙古口蘑原生质体制各和再生的最佳条件是：培养5d的菌丝体用2.5％（w／w）的溶壁酶酶解4h，渗透压稳定剂为0.6MKCL，MB为再生培养基，制备率为7.53×10⁷个／ml，再生率为8.4×10^-4；双孢蘑菇原生质体制备和再生的最佳条件是：培养5d的菌丝体用2％（w／w）的溶壁酶酶解3h，渗透压稳定剂为0.6MKCL，PQA为再生培养基，制备率为6.96×10⁷个／ml，再生率为7.4×10^-4。同时对两种菌的原生质体释放过程进行了形态观察，两菌株原生质体均为顶端释放；并采用双亲灭活原生质体融合技术对蒙古口蘑和双孢蘑菇原生质体融合进行研究，蒙古口蘑原生质体采用热灭活，65℃处理30min，灭活率达100％，双孢蘑菇采用紫外灭活，即在15W紫外灯下，距离30 cm，处理20min，灭活率达100％，两菌株按1：1混合，以30％PEG（6000）为促融剂，融合10min，融合率为5.6×10^-5，经遗传稳定性检验，融合菌株的遗传稳定率为80％，并从菌落特征、菌丝形态、菌丝生长量、酯酶、游离全蛋白、乳酸脱氢酶、乙醇脱氢酶和过氧化物酶等同工酶方面对融合子进行筛选，获得一株具有双亲性状的融合株。更多还原

【Abstract】 The protoplast fusion technology has been a new fast developing breeding technology in cell engineering recent years. It has many exceptional advantages that many other normal hybridization methods cant compare with. The concentrations of lywallzyme, the time of lywallzyme treatment and the culture age regeneration medium were studied to find out the effects on preparation and yield of and Tricholoma mongolicam Imai and Agaricits bispopus Sing in this paper. The optimum conditions for regeneration the yield of Tricholoma mongolicam Imai were obtained by using 5-day-cultured mycelia with the treatment of 2.5% (w/w) lywallzyme for 4 hours and the osmotic-pressure stabilizer 0.6MKCL,were regeneration MB. The rate of formation 7.53 l07 /ml, the rate of regeneration 8.4x10 -4.The optimum conditions for regeneration the yield of Agaricus bispopus Sing were obtained by using 5-day-cultured mycelia with the treatment of 2% (w/w) lywallzyme for 3 hours and the osmotic-pressure stabilizer 0.6MKCL, were regeneration PQA. The rate of formation 6.96 107 /ml, the rate of regeneration7.4 10-4. The releasing process of two protoplasts were also observed morphologically. Tricholoma mongolicam Imai and Agaricus bispopus Sing protoplasts fusion were studied by using the inactivated parents protoplasts fusion technique. Used lethality method, Tricholoma mongolicam Imai was handled in 65 C 30min.Lefhality rate of Tricholoma mongolicam Imai were 100%; Used UV method, Agaricus bispopus Sing was handled in 15W 20min. Lethality rate of Agaricus bispopus Sing were 100%. The ratio of Tricholoma mongolicam Imai and Agaricus bispopus Sing 1 to l,they were mixed and fused in 10 min. The fusion rate was 5.6 10-5.The fusion strain heredity stability was tested to find out its stability 80%. The zygote was identified from its colony character, morphological state, growth rate, is dynamic enzyme of EST, soluble free holophote, LDH, ADH, POD and so on. A strain containing the characters of both parents was picked out.更多还原