Efficient Regeneration Transformation of Brassica and Agrobacterium-mediated napus Cultivar Qingza No. 5

摘要

Using the hypocotyl and cotyledon explants of Brassica napus L. cultivar Qingza No. 5 as receptors,hormone combinations in bud differentiation medium,bud growth medium and rooting medium were optimized to establish an efficient plantlet regeneration system of B. napus cultivar Qingza No. 5. The results showed that the highest differentiation efficiency of hypocotyls of B. napus cultivar Qingza No. 5 reached about 90%,which was three times that of cotyledons. The appropriate differentiation medium was MSB + 5 mg/L thidiazuron( TDZ) + 7. 5 mg/L AgNO_3+ 0. 1 mg/L NAA + 2 mg/L proline( L-pro) + 250 mg/L casein acid hydrolysate( CH) + 3% sucrose; the appropriate growth medium was 1 /2 MSB + 1 mg/L IBA + 2 mg/L L-pro + 250 mg/L CH + 1. 5% sucrose; the appropriate rooting medium was 1 /2 MSB + 0. 2 mg/L IAA + 1. 5% sucrose. On this basis,a binary expression vector harboring insect resistance gene B12 was constructed and introduced into B. napus hypocotyls by Agrobacterium-mediated transformation. Positive plants were screened using hygromycin and carbenicillin.Transgenic plants were verified by PCR and GUS histochemical staining. The results showed that insect resistance gene B12 was successfully integrated into the nuclear genome of B. napus plants and could be expressed normally. Leaves of transgenic plants with high expression levels were collected for indoor inoculation test with Plutella xylotella larvae to evaluate insect resistance of transgenic plants.