| Indigoblue woad (Isatis indigotica Fort.) is a kind of extensively used Chinese medicinal plant and it is cultivated almost all over China. At present, insect pest is one of the main influence factors on the production and quality of Indigoblue woad. CrylA gene is a sort of insecticidal crystal proteins performing toxicity on the insects of Lepidoptera. In order to obtain the transgene plants with the resistance to Lepidoptera insects for the solution of the insect pests problem in the cultivation of Indigoblue woad. We used Agrobacterium tumefaciens as the carrier, and introduced CrylA gene into the nucleus genome of Indigoblue woad.I . Anatomical studies on the mode of regeneration in the explant systems of indigoblue woad.The regeneration of callus and buds, of the explants cotyledons and hypocotyls of the autertraploid indigoblue woad, was investigated using the conventional paraffin-section method. The results indicated that the differentiation (regeneration) capacity of cotyledons is higher than that of hypocotyls. In bud-induced medium, cotyledons differentiated into a few calluses firstly, and then a great deal of adventitious buds. Whereas, hypocotyls differentiated directly intoadventitious buds from cut surface without the stage of callus. The results from anatomical studies indicated that both cotyledon and hypocotyls come from callus. At the same time, the outset-layer of callus differentiated to be buds and restrained the development of callus. The results suggested the original manners of the budof the two explants are all belonging to external origin. The period from 2 to 5 days after cultivation, with the actively splitting of the divided cells, is the optimum period for explants to accept external DNA.II. Optimization of in vitro genetic transformation systemThe effect of the species and quantity of the antibiotic screening agents and degermingagent on the growth of Indigoblue woad explants was examined. The results revealed that the differentiation of adventitious buds was restrained obviously when the concentration of Kan was over 50mg/L. Therefore, the concentration 50mg/L of Kan was used as final seletion tress to obtain the kanamycin-resistant(Kanr) buds, and kan 30mg/L was used in the root-induced medium. Furthermore, the 500mg/L cef was used in the bud-induced stage and the concentration of cb 250mg/L was used in the root-induced medium to restrain the growth of Agrobacterium tumefaciens.The producing rates of Kanr buds in cotyledons and hycopotyls after 30d were 13.33% and 11.42% respectively which were the highest when the concentration (OD600) of Agrobacterium tumefaciens was fed at about 0.6. The same results were obtained when the infect time of cotyledons lasted for 5~8 minutes and hypocotyls for 3~5minutes. The physiological condition of seedling sprouted about 4-5 d is the most suitable one for transformation. The highest producing rates of Kanr buds were achieved when cotyledons was pre-cultured for 2 days and cocultured with Agrobacterium tumefaciens for 3 days, and when hypocotyls was pre-cultured for 2days and co-cultured with Agrobacterium tumefaciens for 2 days. During the pre-culture and co-culture stages, the addition of 4mg/L AgNO3 could improve the inducing rate. It was also found that the inducing rate of Kanr buds could be improved when cotyledon was delayed to put in the selected medium for three days and hypocotyl for two days after co-cultivation. Cutting off the Kanr buds cultured in the bud-induced medium, the root-inducing rate is about 80%.III. Molecular detection, insect resistance and phenotypic character analysis of transformed plantsThe results from PCR detection indicated that the CrylA gene has been successfully transformed into the genome of Indigoblue woad. And the experimental results of leaf damaging showed that insect resistance of transgenic plants were significantly higher than that of non-transformed plants. The non-transformed leaves were damaged seriously. However, the transgenic leaves restrained the damage from t... |
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