Improving The Resistance Of Some Important Vegetables To Insects By Genetic Engineering

Chinese cabbage(Brassica campestris ssp. pekinensis}, cabbage(Brassica oleracea var. capitatd) and pepper(Caps/cum annuum L.) are three of the most important vegetables in China. It is estimated that large amount of these vegetables are lost to pests annually, such is the case despite an estimated 5.0 billion annual lost to combat the problem. One limitation hi addressing crop loss due to pests is the almost exclusive reliance on chemical compounds. Prolonged and intensive selection pressure in large monocultures has brought about the evolution of insect resistance against of commonly used insecticides. In the meanwhile, spray of chemical insecticide has led to the destroy of natural balance and brought a matter of universal concern for sustainability of food production, environment and biodiversity. Common cabbage worm(Pzm's rapae), diamondback mo\h(Plutella maculipennis) and sphid(Myzus persicae) are major pests of Brassica vegetables, budvform(Heliothis armigera) and oriental tobacco budwoTm(Helicoverpa assultd) are important pests of pepper. No sources of resistance have been found in the cultivars grown worldwide, which limited the progress of regular breeding on insect resistance. Gene-engineering techniques provide potential for transfer of trait such as engineered genes offering protection against insect pests. These techniques can greatly reduce dependence on chemical control, reduce the cost of production, chemical contamination of the environment, and damage to non-target insects, which have many merits such as strong selection, safe application and high efficiency.Obtaining transgenic plants of Chinese cabbage, cabbage and pepper greatly depends on the establishment of efficient genetic transformation system. Several factors affecting transformation of the three kinds of vegetables were studied, and a quick and effective protocol with optimized condition for transformation of Chinese cabbage, cabbage and pepper was developed, respectively. Different type of hormones and additions of doses, different genetypes, seedling age, explants, preculture or not, bacterial concentration, infection time, and the pH of co-cultivation media, no micro-element CoClj in co-cultivation media, the concentration of kanamycin and cefotaxime, the add of acetosyringone and the appropriate adding time had some effects on transformation frequency. The highest transformation frequency of Chinese cabbage was 5.74%, and the one for cotyledonary petioles and hypocotyls of cabbage was 3.17% and 5.21% respectively, pepper was averagely 16.5%.Several insect-resistant genes, such as Bt, sck( modified cpti gene), gna, Bt-cpri fusionprotein gene and Bt-cpti-gna gene, were successfully transferred to the explants of Chinese cabbage, cabbage and pepper mediated by Agrobacterium tumefaciens. Bt-cpti fusion protein gene was transferred to the shoot apex of Chinese cabbage and cabbage via particle bombardment Obtained kanamycin-resistant plants were verified that foreign genes had been integrated into the genome DNA of Chinese cabbage, cabbage and pepper by PCR, PCR-Southern and Southern blotting hybridization; Northern blotting hybridization showed that Bt, sck and gna had been normally transcribed in transgenic plants of Chinese cabbage, cabbage and pepper, respectively; the bioassays analysis of CpTI inhibitory activity and lection cruor activity indicated that sck and gna had been expressed in transgenic plants. In the meanwhile, no mRNA or little mRNA could be detected in parts of transgenic sck, Bt and gna plants, and all of transgenic plants had strong differences in mRNA signal; there were also significant variance in CpTI relative capacity and cruor activity among transgenic sck and gna plants in Chinese cabbage, cabbage and pepper. All of these suggested that gene silencing happened in parts of the transgenic plants. Except lower self fruitfulness rate which was brought by tissues culture, there were no significant variance in other characters, such as flowering date, plant shape and seed thousand-kemel-weight etc, be...