农杆菌介导的叶用莴苣遗传转化体系的优化与改良

黄妍; 贺淑萍; 许恒; 徐光柳; 周懿雯; 火国涛; 杨涛; 张停林; 李珍珍; 韩颖颖; 魏仕伟

农杆菌介导的叶用莴苣遗传转化体系的优化与改良

Optimization and Improvement of Agrobacterium-Mediated Genetic Transformation System for Leaf Lettuce

摘要

摘要:
目的提高叶用莴苣遗传转化的效率，优化叶用莴苣的遗传转化体系。
方法本试验以‘缤纷3号’为受体材料，构建pKSE401-MYB75 敲除载体，导入农杆菌GV3101，农杆菌介导法进行转化，以MS为基本培养基，对预培养/共培养的培养基设置不同植物生长调节剂配比（6-BA、NAA、KT、2,4-D），分化培养基的抗生素的种类和浓度一致的基础上，设计4种转化体系方案（方案A、B、C、D）。
结果确定叶用莴苣高效诱芽培养基为预培养/共培养培养基：MS + 0.1 mg·L⁻¹ NAA+ 0.05 mg·L⁻¹6-BA；分化培养：MS + 0.1 mg·L⁻¹ NAA+ 0.05 mg·L⁻¹ 6-BA + 300 mg·L⁻¹ Tim+50 mg·L⁻¹ Kan），其愈伤组织诱导率达99%，出芽率为50%，阳性苗转化效率达到6.4%。
结论通过对农杆菌介导的愈伤再生转化过程中植物生长调节剂配比影响因素进行试验，初步优化了‘缤纷3号’的遗传转化体系。

Abstract:
Objective Leaf lettuce genetic transformation was optimized by reformulating the hormones used in culture medium.
Methods BINFEN-3 was used as the receptor to construct the pKSE401-MYB75 knockout vector to be introduced into Agrobacterium GV3101 for the transformation. On the basic MS medium with the types and concentrations of plant growth regulators kept constant, varied ratios of 6-BA, NAA, KT, and 2,4-D were added for pre-culture and co-culture. Schemes A, B, C, and D were designed for the formulation optimization.
Results For efficient lettuce bud induction, a pre-culture/co-culture medium of MS + 0.1mg NAA·L⁻¹ + 0.05mg 6-BA·L⁻¹ and a differentiation/selective medium of MS + 0.1mg NAA·L⁻¹ + 0.05mg 6-BA·L⁻¹ + 300mg Tim·L⁻¹ + 50mg Kan·L⁻¹ were used. A callus rate of 99%, a budding rate of 50%, and a conversion efficiency of positive seedlings reaching 6.4% were obtained on the culture.
Conclusion The genetic transformation of BINFEN-3 was achieved with the optimized plant growth regulators ratio for the Agrobacterium-mediated callus regeneration.

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