Genetic Transformation Of Orychophragmus Violaceus And Isatis Indigotica With NPR1by Agrobacterium Tumefaciens

As an ornamental flower, Orychophragmus violaceus (L) O. E. Schulz, contains high linoleic acid and low erucic acid, which is also a novel resource of oil plants. Isatis indigotica Fort. is an important Chinese herbal medicinal plant, and the leaves can also be used as industrial raw materials to make indigo dye, possessing enormous application value. While both of them are vulnerable to damages caused by pathogen in the field, leading to the harm of the plants and the decrease of the yield. In this research, NPR1gene(Nonexpressor of Pathogenesis-related Genes1), which plays a key role in regulating the systemic acquired resistance was transferred to Orychophragmus violaceus and Isatis indigotica by Agrobacterium tumefaciens in order to enhance their resistance of plant disease. Main results of the research are listed as follows.1. In the tissue regenerating test, regenerating frequencies of cotyledon and hypocotyl in the same crop were compared to each other. It is found that, as to Orychophragmus violaceus, average regenerating frequency of hypocotyl is much higher than that of cotyledon. On the contrary, average regenerating frequency of cotyledon in Isatis indigotica is higher. Therefore, hypocotyl of Orychophragmus violaceus and cotyledon of Isatis indigotica are selected as explants to conduct transformation of the plants.2. Hypocotyls of Orychophragmus violaceus and cotyledons of Isatis indigotica are put on differentiation mediums containing gradient concentrations of kanamycin to choose a suitable concentration for regenerating. Then12mg/L kanamycin was selected as the primary sifting concentration for hypocotyls of Orychophragmus violaceus, and16mg/L as the secondary concentration. Similarly,6mg/L kanamycin was the primary sifting concentration and9mg/L was the secondary concentration for cotyledons of Isatis indigotica.3. Transformations of Orychophragmus violaceus and Isatis indigotica were conducted by Agrobacterium tumefaciens EHA105which carries the vector. The hormones contained in regenerating medium of Orychophragmus violaceus are3mg/L6-BA,8mg/L TDZ,0.2mg/L NAA,2mg/L ZT, which is different from3mg/L6-BA,0.2mg/L NAA,2mg/L ZT in Isatis indigotica. In the regenerating progress, kanamycin was used to sift green buds as transgenic tissue.4. After PCR test,17positive transgenic plants of Orychophragmus violaceus were obtained. After PCR test,12positive transgenic plants of Isatis indigotica were obtained5. After RT-PCR test and bacterial inoculate test on the leaves,3Isatis indigotica plants expressed NPR1, and6Isatis indigotica plants showed different resistance to Pst DC3000.Conclusion:Results in this research show that it is feasible to transfer NPR1gene to Isatis indigotica, which makes the plant obtain broad-spectrum resistance and improve the disease resistance.