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J Integr Plant Biol, 1994, 36 (9): -, Research Article
Transformation of Brassica napus Using Agrobacterium tumefaciens and Regeneration of Transgenic Plants
Cheng Zhen dong, Wei Zhi ming and Xu Zhi hong
Seeds of Brassica napus L. cv. "Yunbei 2" were surface-sterilized and germinated on hormone-free MS medium. After 4—5 days the cotyledons were excised in such a way that each has a 1—2 mm petiole was remained at its base. These cotyledons were used as the explants for tissue culture and genetic transformation. This paper first deals with the improvement of the medium for shoot regeneration. Of the elements tested, AgNO3 and carbenicillin enhanced shoot regeneration. The highest frequency (52 %) was obtained on MS medium containing 4.5 mg/L BAP, 20 μmol/L AgNOa and 500 mg/L earbenicillin. An efficient gene transfer system based on the regeneration procedure was established. After 2 days of cocultivation with Agrobacterium tumefaciens strain A208SE (pTi T37-SE, pROA93), the explants were transferred onto selection medium containing 25 mg/L kanamycin. After 1.5 months shoots emerged from 27% of the explants inoculated. They were excised and transferred onto rooting medium containing 25 mg/L kanamycin and 200 mg/L cefotaxime which is better than carbenicillin for root induction. Whole plants were transplanted into pots, and grew well in the phytotron. Transformation was confirmed by β-glueuronidase assay and Southern blotting analysis.
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