| The regeneration system and effects of key factors on transformation mediated by Agrobacterium in Chinese cabbage (Brassica campestris SSp.chinensis L) were studied, and also the effects of irradiation on the regeneration and transformation were explored in this thesis.Some factors showing effects on regeneration were tested for establishment of the regeneration system in Chinese cabbage. In the process of in vitro culture, hormones played an important role. Among 15 combinations of different concentration of 6-BA with NAA, the combination of 5mg/L BA and 0.5mg/L NAA showed the highest rate of shoot regeneration. AgNOs showed obvious effects prompting shoot regeneration and an addition of 5~7.5mg/L in the medium could significantly increase regeneration frequency. Explants from different time of cultured seedlings showed great difference in regeneration rate. The suitable seedlings age varied from 3 to 6 days hi different genotypes, with 3, 4 and 6 days for Aikangqing, Shanghaiqing and Suzhouqing respectively. Four types of explants, cotyledons, cotyledons with l-2mm of petiole, peioles and hypocoltyls, were compared in regeneration rats. Among them cotyledons with l-2mm of petiole showed the highest and could be selected as the suitable explants for establishment of regeneration system. Genotypes also played an important role in the regeneration and varied in regeneration rate. Among the 5 genotypes tested, Xianggangcaixin showed the highest rate of shoot regeneration, 78%, followed orderly by Suzhouqing, Aikangqing, Shanghaiqing and Gaohuabaicai, with frequency of 60%, 52%, 46% and 22% respectively.For effective elimination of Agrobacterium and screen of transformed cells after co-cultivation, sensitivity of cabbage cultured in vitro to 5 antibiotics was testes. Results showed that cultured cotyledons were more sensitive to hygromycin than to knamycin. So the hygromycin could be used for selection of transformats with the suitable concentration of 25mg/L. Among carbenicillin, ampicillin and cefotaxime used for elimination of Agrobacterium, carbenicillin should be preferable based on their effects on shoot regeneration.In order to establish the effective transformation system of Chinese cabbage, key factors on in the process of Agrobacterium-mediated transformation were optimized based on the frequency of transient expression of gus gene. The highest frequency was observed when explants were immersed in bacterium suspension with ODeoo value about 0.5 for 10 to 15 minutes followed by co-cultivated for about 5 days. Transformation frequency varied with different Agrobacterium strains. The frequency of transient expression of gus gene was higher by using EHA105 than that by using LBA4404. Differences in transformation frequency were also observed even using same stain with different plasmids. Among the combination of bacterium strain EHA105 with 3 kinds of plasmids, pKUI^ pKUC and pKSB, the combination of EHA105 with pKUC was better <, Genotypes also showed important effect on transformation. Among the 5 varieties of Chinese cabbage, Gaohuabaicai showed the highest frequency (54%) of transient expression of gus gene, followed by Shanghaiqing, Suzhouqing, Aikangqing, Xianggangcaixin in turn.Effects of irradiation on regeneration and transformation were also probed. In the experiment of regeneration, seedlings and explants were irradiated respectively. It was found that irradiation treatment with lower dose about 1 to 5Gy could increase shoot regeneration rate. In process of transformation, pre-cultured explants and Agrobacterium-infected explants were also treated with y-ray. Results showed that the frequency of transient expression of gus gene was increased hi Agrobacterium-infected explants radiated with the lower dose about 2.5Gy. Therefore, irradiation with lower dose can be used as an assisted method to improve frequency of regeneration and transformation in Chinese cabbag.
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