Insect-resistance And Genetic Stability Analysis Of Transgenic Cabbage With Pyramided Bt Genes

Cabbage is an important cruciferous vegetable. The cultivation of cabbage is severely challenged by infestation of Lepidopteran pests, including diamondback moth (DBM), Pieris rapae, and etc.. The use of insect-resistant cultivars is the most effective method of controlling the pests. Despite extensive screening, high levels of natural resistance to lepidopteran pests have not been found in cabbage germplasms. Therefore, introducing foreign insect-resistance genes into cabbages through genetic engineering may provide effective germplasm resources for breeding.Both crylla8and cry1Ba3were cloned from the Bt strain. Bioassays showed that Cry1Ia8and Cry1Ba3toxins were highly resistant to DBM and predominant Lepidopteran pests, and there were no cross resistance with the CrylAc. Pyramiding cry1la8and cry1Ba3into cabbage can not only improve the insect resistance of cabage but also dealy the resistant evolution of pests. In addition, the research on the genetic stability, insect-resistance, and risk assessement of transgenic cabbages will make it possible to enable transgenic plants to commercialization.In the study, cry1la8and cry1Ba3were pyramided into cabbage inbred line by Agrobacterium tumefaciens-mediated transformation. Then the genetic stability, insect-resistance, and risk assessement of transgenic cabbages were assessed. The main results were as follows.1. Obtaining of homozygous transgenic cabbage line expressing both cry1Ia8and cry1Ba3.cry1Ia8and cry1Ba3were introduced simultaneously into cabbage inbred line by Agrobacterium tumefaciens-mediated transformation and53kanamycin-resistant plantlets were obtained. There are33Southern blot positive plants. Molecular analyses confirmed that the33transfomed plants were also expressed in RNA and protein level. Bioassays showed that the transgenic plants could effectively control both susceptible and Cry1Ac-resistant DBM.9homozygous transgenic lines were obtained by means of self-pollination and molecular-assisted selection. Then Bt hybrid cabbage were recoveryed by pollinating with other cabbage inbred lines.2. Genetic stability analysis of transgenes in Cryla8-Cry1Ba3cabbage.To plants were selfed and advanced to the third generation. Selfed progeny were harvested from each individual plant for the analysis of genetic stability. Analysis of transgene activities in four sinlge-copy lineages T0-1, T0-9, To-13, T0-17provided evidence that inheritance of the introduced gene was in Mendelian fasion. ELISA analyses indicated that content of Bt toxin was not significantly difference in four successive generations of lineage T0-1. Furthermore, data from field trials indicated that there were no significant differences in most agronomic traits between transgenic plants and the control plants.3. Insect-resistance evaluation of Cryla8-Cry1Ba3cabbage.The results of insect-resistance evaluations in greenhouse and field domonstated that the homozygous transgenic lines were highly resistant against DBM and Pieris rapae as compared to the control while susceptible to Mamestra brassicae and Spodoptera exigua.4. Influences of transgenic cabbage pollen or Bt toxins on survival rates of bees.The bees were fed with the pollens of transgenic cabbages, Crylla8toxin (3000ng/ml), Cry1Ba3toxin (3000ng/ml), mixed pollens, and untransformed cabbages pollens, respectively.20days later, there were no significant differences in mortalities of bees among the5treatments.