The effect of co-cultivation and selection parameters on Agrobacterium-mediated transformation of Chinese soybean varieties.
In the present study, an efficient Agrobacterium-mediated gene transformation system was developed for soybean [Glycine max (L.) Merrill] based on the examinations of several factors affecting plant transformation efficiency. Increased transformation efficiencies were obtained when the soybean cotyledonary node were inoculated with the Agrobacterium inoculum added with 0.02% (v/v) surfactant (Silwet L-77). The applications of Silwet L-77 (0.02%) during infection and L-cysteine (600 mg l(-1)) during co-cultivation resulted in more significantly improved transformation efficiency than each of the two factors alone. The optimized temperature for infected explant co-cultivation was 22 degrees C. Regenerated transgenic shoots were selected and produced more efficiently with the modified selection scheme (initiation on shoot induction medium without hygromycin for 7 days, with 3 mg l(-1) hygromycin for 10 days, 5 mg l(-1) hygromycin for another 10 days, and elongation on shoot elongation medium with 8 mg l(-1) hygromycin). Using the optimized system, we obtained 145 morphologically normal and fertile independent transgenic plants in five important Chinese soybean varieties. The transformation efficacies ranged from 3.8 to 11.7%. Stable integration, expression and inheritance of the transgenes were confirmed by molecular and genetic analysis. T(1) plants were analyzed and transmission of transgenes to the T(1 )generation in a Mendelian fashion was verified. This optimized transformation system should be employed for efficient Agrobacterium-mediated soybean gene transformation.
Liu
Sheng-Jun
SJ
Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China.
Wei
Zhi-Ming
ZM
Huang
Jian-Qiu
JQ
D015139:Blotting, Southern; D002934:Cinnamates / Q000494:pharmacology; D003545:Cysteine / Q000494:pharmacology; D006921:Hygromycin B / Q000031:analogs & derivatives / Q000494:pharmacology; D030821:Plants, Genetically Modified; D016133:Polymerase Chain Reaction; D012231:Rhizobium / Q000235:genetics*; D013025:Glycine max / Q000187:drug effects / Q000235:genetics* / Q000254:growth & development; D013696:Temperature; D014170:Transformation, Genetic / Q000187:drug effects / Q000235:genetics*
D002934:Cinnamates; D006921:Hygromycin B; C026273:hygromycin A; D003545:Cysteine