EFFICIENT AGROBACTERIUM-MEDIATED TRANSFORMATION OF SOYBEAN
DANG Wei1,2 WEI Zhi Ming1 (1Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032; 2Graduate School of the Chinese Academy of Sciences, Beijing 100049)
To improve Agrobacterium tumefaciens mediated transformation of embryonic tips of soybean [Glycine max (L.) Merr],the effect of several factors on transformation efficiency were examined by measuring transient expression levels of β-glucuronidase and the number of resistant explants. The hypervirulent Agrobacterium tumefaciens strain KYRT1 was proved to be a better transformer than EHA105 and LBA4404. Improved transformation efficiencies were obtained when embryonic tips were incubated with an Agrobacterium suspension (A600=0.5) for 20 h. Optimized co-cultivation was performed in acidic medium (pH 5.4) at 22°C in the dark for 5 days. Resting culture and step-by-step selection culture were beneficial to the survial of resistant explants. By combining the best treatments, transgenic soybeans of seven cultivars were obtained that simultaneously express the cryIA(c) and Pinellia ternata agglutinins (pta) genes. Most of the transgenic plants (about 70%) are fertile. The transformation frequency [(the number of PCR-positive regenerated plants/the number of infected explants)×100%] ranged from 4.29% to 18.0%. PCR and Southern analyses confirmed the stable integration of the binary insect resistance genes in the primary transgenic plants.