Genetics And Assay Of Bt Transgenic Cabbage (Brassica Oleracea Var.capitata)

Cabbage (Brassica oleracea var. capitata) is an important vegetable in China with planting area up to several hundred thousands hectares. Pests cause heavy losses in cabbage productions. Among of them cabbageworm larva (pieris rapae L.) and diamondback moth larva (Plutella xylostella L.) are most severe pests.Seldom sources of resistance have been found, which limited the progress of regular breeding on insect resistance. Genetic engineering techniques provide potential approach for transfer such as Bt gene offering protections against insect pests. These techniques are very safe and high efficiency. And it can greatly reduce the cost of production, residues of chemicals in the environment, and killing to non-target insects. Bt genes were derived from Bacillus thuringiensis; its toxin protein could kill lepidopterists specifically but was harmless to higher animals and human beings. Thereby, it was of potential to obtain insect-resistant resources by introducing Bt genes into cabbage.In this research we mainly focus on obtaining transgenic cabbage plants; verifying kanamycin-resistant plants that foreign genes had been integrated into the genome DNA by PCR and Southern blotting hybridization; the bioassays of Bt transgenic cabbage to insect resistance in TI and TO progenies. The main results were as follows:1. The sensitivity screening of cabbage explants to kanamycin. The concentrations of Km from lOmg/L to 15mg/L are effective on inhibiting green shoot regeneration from hypocotyl explants of cabbage.2. Cefotaxime has a side effect to green shoot regeneration from hypocotyl explants of cabbage. With the increase of cefotaxime concentrations the less green shoots could regenerate.3. The transformation frequencies of hypocotyl explants were affected with the co-cultivation periods. We found that the co-cultivation for 48h is the best for transformation in this research.4. Under the optimum conditions, the Bt transgenic cabbage plants were obtained from genotypes of Oxheart and Zhonggan 8.5. The transgenes had been integrated into the cabbage genomes by analysis of Southern blotting hybridization.6. The TI seeds were obtained by the bud pollinations of the TO transgenic plants. The inheritance fashions of the transgenes in TI segregation population were analyzed based on PCR with NPTH primers and Km resistance of leaves. And the results showed that NPJT1 gene inherited as a dominant genetic locus with the segregation ratio 3:1.7. The bioassay against diamondback moth larva in greenhouse on TO plants and TIprogenies respectively showed that all transgenic plants enhanced the resistance to pest, but there were significant variations about insect resistance among transgenic plants.