农杆菌介导的“野大1号”高效遗传转化体系建立

Establishment of Agrobacterium-mediated“Yeda No.1”Efficient Genetic Trans-formation System

栽培大豆在驯化过程中丢失了大量的优异基因,我国野生大豆种质资源丰富,蕴含着许多高蛋白、抗逆方面的基因。大豆新品种“野大1号”作为一份重要的种质资源,保存着宝贵的遗传基因和许多优良的性状,具有较高的营养价值,其中异黄酮的含量高达千分之二,在农业育种上具有重要的价值。本研究以“野大1号”为材料,通过设计不同的消毒方式和时间以及不同的芽诱导6-BA浓度配比,通过GUS染色方法比较摇床和针管真空渗漏两种侵染方式在不同侵染时间下对基因转化效率的影响,分析侵染后外植体最佳抗生素筛选浓度,建立“野大1号”大豆品种的高效遗传转化体系。结果表明:“野大1号”种子消毒最佳方法为75%酒精消毒45 s后再使用15%过氧化氢消毒30 min;最佳侵染方法为针管真空渗漏浸染30 min;最佳不定芽诱导培养基为MS+2 mg·L^(-1)6-BA;抗性筛选标记潮霉素的最适浓度为60 mg·L^(-1)。通过该遗传转化体系成功移栽了14株植株,其中8株为转基因阳性植株。该高效再生及遗传转化体系的建立为大豆的新品种培育奠定坚实基础。

During the domestication process,cultivated soybeans have lost a large number of excellent genes.China′s wild soybean germplasm resources are abundant,containing many genes related to high protein content and stress resistance.The new variety“Yeda No.1”serves as a crucial germplasm resource,preserving valuable genetic traits and numerous desirable characteristics.It possesses high nutritional value,with its isoflavone content reaching as high as two thousandths,rendering it of significant value in agricultural breeding.In this study,“Yeda No.1”was used as the material,and the effects of the two infection methods of shaker and needle vacuum leakage on the gene transformation efficiency under different infection times were compared by GUS staining method by designing different disinfection methods and time,as well as different bud-induced 6-BA concentration ratios,and the optimal antibiotic screening concentration of explants after infection was analyzed,and an efficient genetic transformation system of“Yeda No.1”soybean variety was established.The results indicate that the optimal method for seed sterilization of“Yeda No.1”is to first disinfect with 75%alcohol for 45 seconds followed by 15%hydrogen peroxide for 30 minutes.The best infiltration method is needle vacuum infiltration for 30 minutes.The optimal shoot induction culture medium is MS supplemented with 2 mg·L^(-1)of 6-BA.The most suitable concentration for resistance selection marker hygromycin is 60 mg·L^(-1).After employing this genetic transformation system,we successfully transplanted 14 plantlets,out of which 8 were confirmed as transgenic positive plants.The establishment of this highly efficient regeneration and genetic transformation system lays a solid foundation for the breeding of new soybean varieties.