Co-transformation With Binary Expression Vectors Containing Insect Resistace Genes Bt-pta And Primary Quality Analysis On Tetraploid Isatis Indigotica

The plant binary expression vectors p3300-CryIA-pta carrying two insect resistance genes CryIA-pta was constructed. The engineered strain was obtained by transferring into Agrobacterium tumefaciens EHA105.MS basal medium supplying with 2mg/L BA and 0.5mg/L NAA was found to be the best for regeneration of Isatis indigotica. To determine a suitable screening pressure for positive transgenic plants, a series concentration of PPT in the regeneration medium for the culture of cotyledon and hopocotyl from Isatis indigotica were tested. The result showed 5mg/L PPT was suitable for screening positive transgenic plants.The cotyledon and hypocotyl of Isatis indigotica were used as explants for transformation and the CryIA-pta genes were transformed into the explants through Agrobacterium-mediated transformation simultaneously. The regeneration rate of cotyledon was 46.7%, while that of hypocotyl was 24.3%. The shoots were then cultured on the medium containing 0.2mg/L NAA for root inducing.In the PCR detection of transformed plants from Isatis indigotica, two bands about 680bp and 804bp were obtained, the same as the positive control Agrobacterium tumefaciens EHA105 with p3300-CryIA-pta while those of untransformed showed no band. The rate of PCR positive was 32.5%. Three of the PCR positive plants were selected for Southern blot. The three transgenic lines all showed hybridization bands, while the control showed no hybridization band. The results of molecular analysis confirmed that the foreign CrylA-pta had been integrated into the genome of Isatis indigotica with low copy number (all single copy).The leaf and petiole from the positive transgenic plants by Southern blot were used as explants for establishing an efficient transgenic plant regeneration system. MS basal medium supplying with 0.2mg/L BA and 0.02mg/L NAA was found to be the best for regeneration of Isatis indigotica. The rate of PCR positive was 86.7%. After one week of acclimation, the transgenic plants can be transferred.The resistance of CryIA-pta transgenic Isatis indigotica to Plutella xylostella L.