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

doi:10.19303/j.issn.1008-0384.2025.09.010

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 Lactuca sativa L.

摘要

摘要:
目的提高叶用莴苣（Lactuca sativa L.）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，通过农杆菌介导法进行转化。以MS为基本培养基，对预培养/共培养的培养基的植物生长调节剂，包括萘乙酸(1-naphthale-neacetic acid，NAA)、6-苄氨基腺嘌呤(6-benzylaminopurine, 6-BA)、激动素（kinetin, KT）、2,4-二氯苯氧乙酸（2,4-Dichlorophenoxyacetic acid, 2,4-D）设计不同种类与浓度配比，具体设计如下：0.05 mg·L⁻¹ NAA+ 0.5 mg·L⁻¹ KT、0.1 mg·L⁻¹ NAA+ 0.05 mg·L⁻¹ 6-BA、0.1 mg·L⁻¹ NAA+ 0.05 mg·L⁻¹ 6-BA + 0.5 mg·L⁻¹ 2,4-D、0.1 mg·L⁻¹ NAA+ 0.1 mg·L⁻¹ 6-BA +0.5 mg·L⁻¹ NAA。在分化培养基以300 mg·L⁻¹特美汀（Timentin, Tim）和50 mg·L⁻¹ 卡那霉素（Kanamycin, Kan）抗生素浓度不变的条件下，分别对应上述四种植物生长调节剂配比组合，建立4种转化体系，分别标记为方案A(0.05 mg·L⁻¹ NAA+0.5 mg·L⁻¹ KT+300 mg·L⁻¹ Tim+50 mg·L⁻¹ Kan)、B(0.1 mg·L⁻¹ NAA+0.05 mg·L⁻¹ 6-BA+300 mg·L⁻¹ Tim+50 mg·L⁻¹ Kan)、C(0.1 mg·L⁻¹ NAA+0.05 mg·L⁻¹ 6-BA+0.5 mg·L⁻¹ 2,4-D+300 mg·L⁻¹ Tim+50 mg·L⁻¹ Kan)与D(0.1 mg·L⁻¹ NAA+0.1 mg·L⁻¹ 6-BA+0.5 mg·L⁻¹ NAA+300 mg·L⁻¹ Tim+50mg·L⁻¹ Kan)。
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⁻¹ 6-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 Lactuca sativa L. 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. Using MS as the basic medium, different types and concentration ratios of plant growth regulators, including naphthylacetic acid (NAA), 6-benzylaminopurine (6-BA), kinetin (KT), and 2,4-dichlorophenoxyacetic acid (2,4-D), were designed for pre-culture/co-culture media.The specific designs are as follows: 0.05 mg·L⁻¹ NAA + 0.5 mg·L⁻¹ KT, 0.1 mg·L⁻¹ NAA + 0.05 mg·L⁻¹ 6-BA, 0.1 mg·L⁻¹ NAA + 0.05 mg·L⁻¹ 6-BA + 0.5 mg·L⁻¹ 2,4-D, and 0.1 mg·L⁻¹ NAA + 0.1 mg·L⁻¹ 6-BA + 0.5 mg·L⁻¹ NAA. Under the constant antibiotic concentrations of 300 mg·L-1 timentin (Tim) and 50 mg·L-1 kanamycin (Kan) in the differentiation and selective medium, four transformation systems were established corresponding to the four plant growth regulator combinations mentioned above, labeled as Scheme A (0.05 mg·L⁻¹ NAA + 0.5 mg·L⁻¹ KT + 300 mg·L⁻¹ Tim + 50 mg·L⁻¹ Kan), B (0.1 mg·L⁻¹ NAA + 0.05 mg·L⁻¹ 6-BA + 300 mg·L⁻¹ Tim + 50 mg·L⁻¹ Kan), C (0.1 mg·L⁻¹ NAA + 0.05 mg·L⁻¹ 6-BA + 0.5 mg·L⁻¹ 2,4-D + 300 mg·L⁻¹ Tim + 50 mg·L⁻¹ Kan), and D (0.1 mg·L⁻¹ NAA + 0.1 mg·L⁻¹ 6-BA + 0.5 mg·L⁻¹ NAA + 300 mg·L⁻¹ Tim + 50 mg·L⁻¹ Kan).
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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