Agrobacterium-Mediated Transformation Of Isatis Indigotica

Isatis indigotica Fortune that belongs to Cruciferae Isatis herb, is a traditional Chinese medicine. This study discusses the effects of several factors in the process of Isatis Indigotica genetic transformation. Based on this efficient Agrobacterium-mediated transformation, P35-Iap and Ace-Tlp gene was successfully transformed into Isatis indigotica. This study also detect the offspring of the transgenic Bar-Cry IC gene through PCR、RT-PCR、Basta spray、Bar test strip and Southern Blot.The main results are as follows:1. Discuss the different infection time、co-culture time and the concentration of AS for genetic transformation mediated by Agrobacterium transformation in Isatis Indigotica. The results showed that infection 10 min, adding 200 μmol/L AS 60 h in co-culture medium was the highest Basta resistance rate. 2. P35-Iap and Ace-Tlp gene was successfully transformed into Isatis indigotica. 46 strains transplant survival and detect 28 strains were positive, the positive rate reached 60.87% to create a material basis for disease resistance Isatis Indigotica. 3. Bar transgenic Isatis indigotica T1 generation plants were detected with Bar test strip, and compared with the results of PCR, the results show that the coincidence rate could reach about 70%. 4. With RT-PCR test, Bar gene of 7 strains of T0 generation of transformed resistance gene Isatis indigotica expressed. 9 transformed Cry IC gene of Isatis indigotica T1 generation were Bar gene expression, which has 6 Cry IC gene expression. 5. The non-transgenic Isatis indigotica were sprayed with Basta, which made sure that 400mg/L Basta could distinguish between the transgenic and the non transgenic plants. With 400mg/L Basta spraying transgenic plants, 28 strains of resistant plant were sorted out from 60 strains of Bar-transgenic Isatis indigotica. All of them were postive with PCR test.6. The amount of Cry IC protein was tested with ELISA kit, which was up to 204.77 ±0.389ng/g. The Plutella xylostella were feed with the Cry IC-transgenic Isatis indigotica, after 3 days, the mortality of Plutella xylostella up to 83.33%. Bisides, the leaves of transgenic Isatis indigotica didn’t have trace of being eaten. All of theses showed that the transgenic Isatis indigotica has a certain resistance. 7. The T1 generation transgenic Bar-Cry IC gene and T0 generation disease resistance of Isatis indigotica were detected by Southern: two T1 generation transgenic Isatis indigotica was a single copy, two T1 generation transgenic Isatis indigotica Fort is two copies, one T0 disease resistance of Isatis indigotica Fort detect one copy.