摘要
以花生(Arachis hypogaeaL.)品种花育20为材料,探讨不同的酶液浓度、酶解时间及渗透压对花生原生质体分离的影响。结果表明:原生质体分离的适宜酶液配比是2%纤维素酶(Cellulase Onozuka RS)、0.2%果胶酶(Pectolyase Y-23),甘露醇渗透压调节剂的浓度为0.7mol/L,暗处理10h,叶片原生质体产量为4.86×105个/ml,存活率75.6%,愈伤组织原生质体产量为4.97×105个/ml,存活率75.4%。将分离的原生质体培养在添加1mg/LNAA和4mg/LBAP的改良MS液体培养基中。培养约2～3天后,细胞开始分裂。然后部分细胞继续分裂,并形成细胞团。培养5～6周后,将培养物转移到添加2mg/L2,4-D和3mg/LBAP的MS液体培养基(pH5.8)中进行培养。7～8周后,将形成的直径为1～2mm的小愈伤组织转移到添加1mg/LNAA和5mg/LBAP的MS固体培养基上培养,小愈伤组织迅速生长。转移3～4周后,愈伤组织长至7～9mm。
The effect of different enzyme combination, time of enzymolysis, osmtic pressure on protoplast isolation of peanut （Arachis hypogaea L.） were studied. Young leaves and somatic embryiogenic calli of peanut cuhivar Huayu20 were used as materials. The results showed that the appropriate enzyme solution was contained 2% cellulase, 0.2% pectinase and 0.7 mol/L mannitol. When cultured for 10 hours in dark, the protoplast yield was 4.86× 10^5 protoplasts, and the activity was 75.6%. After purification, the protoplast was cultured in the modified liquid MS medium containing 1mg/L NAA and 4 mg/L BAP. The first cell division occurred within 2 to 3 days. Then some of the cells divided and developed into colonies. After 5 to 6 weeks, colonies were transferred in the liquid MS medium supplemented with 2 mg/L 2,4-D and 3 mg/L BAP. After 7 to 8 weeks of incubation, protoplast-derived calli up to 1-2 mm in diameter were transferred onto the solid MS medium supplemented with 1 mg/L NAA and 5 mg/L BAP, for callus proliferation. After 3 to 4 weeks of transferring, the calli grew to 7-9 mm in diameter.
出处
《中国农学通报》
CSCD
北大核心
2009年第14期47-50,共4页
Chinese Agricultural Science Bulletin
基金
国家自然基金"利用细胞工程技术创造花生种质资源的研究"(30871544)
关键词
花生
原生质体分离
产量
活力
培养
peanut, protoplast isolation, yield, activity, culture
