Establishment And Optimizationof Soybean Cotyledonary-node Genetic Transformation System Of Jiyu47

Soybean （Glycine max （L.） Merr） is a kind of important economic crop, oil cropwhich is planted worldwide. The applications of transgenic technology can directionaloperate the key genes regulating soybean quality and accelerate the process of highquality soybean cultivar selection. Thus diverse quality soybean varieties can meetconsumer’s needs. Using transgenic technology, exogenous genes are introduced intosoybean to improve its quality traits or endow it herbicide-resistant, pest-resistantcharacteristics. The principle of transgenic technology is connecting the target genewith a suitable carrier, and then transferring it to the plant cell for replication andexpression, until the transformed plants with desired traits was obtain.Since soybean transformation is affected by the factors like soybean genotype,strain, carrier, infection method, tissue differentiation, plant hormones during shootinduction and elongation. It is still difficult to transform soybean plants for the lack ofa high-efficiencies transformed system. Therefore it’s urgent to screen a genotypewhich has high shoot regeneration efficiency with a good sensitivity to Agrobacterium.After the genotype is determined, factors during the infection process should beoptimized in order to establish a soybean genetic transformation system with highregeneration rate and transformation rate.The cotyledon node explants was chosen to establish a regeneration system forthis study. GUS transient expression of ten soybean genotypes was conducted todetect the sensitivity to Agrobacterium and then four sensitive genotypes wereobtained for the measurement of multiple shoots regeneration rate and average shootnumber. Jiyu47had a shoot regeneration frequency of81.2%and the average number3.54, so it was selected as an idea genotype for transformation. After utilization ofsterilization time, germination conditions, plant hormones, C/N ratio and IBA concentration during rooting phase, the most suitable regeneration conditions for theJiyu47soybean cotyledon node was fixed. The cot-node should be sterilized for6-10hours with chlorine and then germinate for5-7days in B5medium added0.5mg·L^-16-BA at16h light+8h dark,25°C.2mg·L^-16-BA0.1mg·L^-1IBA was added tothe medium during shoot induction, after the shoot was induced,0.3mg·L^-1IAA0.5mg·L^-1GA can be added for elongation.Continuous observation of shoot occurrence location and state was carried outwith stereomicroscope, then we found clustered buds located in the meristem ofcotyledon node zone and were visible about7-10days after determination. Themeasurement of GUS fluorescence expression showed that the optimal condition was:OD600=0.7-0.8, infected in vacuum pump for15min or just immerse in the bacterimsolution for30min. The added of0.02%SilwetL-77to the co-cultivated medium canimprove the GUS staining efficiency. With a orthogonal test, We found the browningrate was decreased by amending thiol compounds in liquid and solid co-cultivationmedium. The best combination is400mg·L^-1L-Cys、248mg·L^-1sodium thiosulfate、300mg·L^-1DTT. After studying the effect of GA₃and IAA on shoot elongation, thehighest elongation rate was got when1.0mg·L^-1GA₃and0.5mg·L^-1IAA was added toshoot elongation medium.Jiyu47soybean cotyledonary node was infected with Agrobacterium tumefacienscontaining the objective gene following the method above. Progenies were screenedby selections and the detection including PCR, basta painting and bar gene strips wereconducted for resistant plants.14T₀plants and3T₁plants were detected forcontaining the target gene.