紫花苜蓿与百脉根原生质体培养及不对称体细胞杂交  

Protoplasts Culture and Asymmetric Somatic Hybridization Between Medicago sativa and Lotus corniculatus

作　　者：李玉珠[1] 师尚礼[1] 

机构地区：[1]甘肃农业大学草业学院/草业生态系统教育部重点实验室/甘肃省草业工程实验室/中美草地畜牧业可持续发展研究中心,甘肃兰州730070

出　　处：《核农学报》2015年第1期40-48,共9页Journal of Nuclear Agricultural Sciences

基　　金：国家牧草产业技术体系专项(CARS-35);牧草种质资源保护与利用(NB2130135);青藏高原甘肃甘南社区生态畜牧业关键技术集成与模式示范(201203010);甘肃牧区优质饲草生产技术研究与示范(201003023)

摘　　要：本研究拟通过原生体培养和不对称细胞杂交实现白脉根与苜蓿种间基因重组,为改良苜蓿饲用品质提供新的技术手段,用酶解法分离清水紫花苜蓿（Medicago sativa L.cv.‘Qingshui’）及里奥百脉根（Lotus corniculatus L.cv.‘Leon’）愈伤组织来源的原生质体,研究了酶解时间、酶液组合、甘露醇浓度、预处理条件、继代时间及培养方法等对原生质体分离和培养效果的影响。采用改良的PEG-高Ca2＋-高p H法进行了不对称体细胞杂交,通过荧光染色鉴别异核体并测定融合率。结果表明,采用继代培养第8~12天的苜蓿和百脉根愈伤组织,0.55 mol·L-1CPW溶液中预处理1h或预暗培养24 h,甘露醇浓度为0.55~0.6 mol·L-1时,清水紫花苜蓿在2%纤维素酶＋0.5%果胶酶＋0.3%崩溃酶的酶液中酶解10 h,里奥百脉根在2%纤维素酶＋0.5%果胶酶＋0.3%半纤维素酶的酶液中酶解12 h,经2~3个月的固液＋固体培养模式可分别获得较好的原生质体分离效果及愈伤组织再生植株。清水紫花苜蓿原生质体经3 mmol·L-1IOA处理10 min,里奥百脉根经50μg·m L-1R-6G处理10 min后融合,PEG（6000）最适浓度为35%,异源融合率为3.1%,最终获得了苜蓿与百脉根的杂种愈伤组织,为进一步培育抗臌胀病型苜蓿种质提供了新的材料。The calli of Medicago sativa L. cv. Qingshui and Lotus corniculatus L. cv. Leon were used to study the effect of enzymolysis time,enzyme combination,osmotic pressure, subculture time, pretreatment measures and culture methods so as to isolate protoplasts through enzyme digeston,then the asymmetric somatic hybridization was conducted by modified PEG-high Ca2 ＋- high p H methods,and tested the fusion rate by fluorescent staining. The results showed the optimum protoplasts enzymolysis effects of alfalfa and birdsfoot trefoil can be obtained with callus of pre-culture for 8~ 12 d,preplasmolyzing in CPW-10 for 1 h or maintenance in darkness for 24 h and isolating with 0. 55 to 0. 6 mol·L- 1mannitol. With 2% cellulase onozuka R-10 ＋ 0. 5% pectinase Y- 23 ＋ 0. 3 % driselase harvested at 10 h for Qingshui and with 2% cellulase onozuka R-10 ＋ 0. 5% pectinase Y- 23 ＋ 0. 3% hemicellulose harvested at 12 h can get regenerated calli and plants through solid-liquid ＋ solid culture pattern after 2 ~ 3 months,respectively. ‘Qingshui＇protoplasts was irradiated in 3 mmol·L- 1IOA and ‘Leon＇protoplasts was inactivated in 50 μg·m L- 1R-6G,and the heterologous somatic hybrid calli were obtained between them with 3. 1% fusion rate,and the optimal PEG concentration was 35% in it. The hybrid calli of alfalfa and Lotus corniculatus will become new germplasm resources for cultivating anti-bloat alfalfa.

关 键 词：紫花苜蓿 百脉根 原生质体 培养 不对称体细胞杂交 

分 类 号：S541.9[农业科学—作物学]