Study On Transformation Of Cabbage With Pyramided Bt Genes CrylIa8 And CrylBa3

Cabbage(Brassica oleracea L. var. capitata L.)is one of the most important vegetables belonging to the Cruciferae. It plays an important role in supply of the vegetable all year-around. Along with the expansion of cabbage cultivation in recent years, cabbage is damaged by pests more and more seriously. Among them diamondback moth(Plutella xylostella)and cabbage worm(Pieris rapae)are the primary pests. Chemical prevention is one of the most important methods of pest contol at the present stage. But chemical insecticides cause severely environmental pollution and bring about adverse effects on people and animals. Moreover, pests have evolved resistance to many insecticides. The best way to control pests is to breed insect-resistant cultivars. However, insect-resistant germplasm resource available for cabbage breeding is limited. Bt cabbage can be obtained through genetic engineering. The transgenic plants may provide germplasm resources for cabbage breeding.Both cry1Ia8 and cry1Ba3 gene were cloned by the Institute of Plant Protection, Chinese Academy of Agriculture Sciences. Cry1Ia8 toxin is toxic to diamondback, corn borer, and soybean moth. Cry1Ba3 toxin is toxic to diamondback moth, potato beetle, and housefly. Bioactivity results of Cry1Ia8 and Cry1Ba3 toxins showed that there was no cross resistance with Cry1Ac which is widely used for pest control in crop production. Pyramiding two Bt genes in cabbage is promising way to inhibit the evolution of insect resistance.In this study, we optimized the systems of regeneration and genetic transformation of cabbage. cry1Ia8 and cry1Ba3 were transferred into cabbage line by Agrobacterium tumefaciens-mediated transformation method and transgenic plants with pyramided cry1Ia8 and cry1Ba3 were obtained. The main results were as follows:1. Optimization of the regeneration system in cabbageThe system of regeneration was optimized with four cabbage lines. Hypocotyl from seedlings after 5 days from sowing was optimal. MS medium supplemented with 3 mg/L AgNO₃,150 mg/L Timentin, and 2 mg/L 6-BA in combination with 0.1 mg/L NAA could lead to generation of more adventitious buds . The highest regeneration frequency is 96.87%.2. Optimization of the genetic transformation system in cabbageThe binary plasmid vector pCSIaN harboring cry1Ia8 gene was transferred into cabbage to optimize the genetic transformation system. The regeneration of kanamycin-resistant shoots has been obviously increased by using the selective medium supplemented with 2 mg/L 6-BA in combination with 0.1 mg/L NAA, 150 mg/L Timentin, and 3 mg/L AgNO₃ and the transformation frequency reached to 10.42%.3. Transformation of cabbage with pyramided Bt genes cry1Ia8 and cry1Ba3A total of 41 kanamycin-resistant plants with pyramided cry1Ia8 and cry1Ba3 transformation were obtained by Agrobacterium tumefaciens-mediated method. Molecular detections confirmed that cry1Ia8 and cry1Ba3 were successfully inserted into the genome of cabbage and also expressed in RNA and protein level. There are 33 PCR positive and 27 Southern blot positive plants. 24 RT-PCR and Western blot positive plants were obtained. Insect bioassay showed that the transgenic cabbage may control diamondback moth strains both susceptible to Bt toxin and resistant to Cry1Ac toxin.