Establishment Of The Blossom System And Obtaining Of Herbicide Resistant Transgenic Variety In Pak Choi

Pak choi (Brassica campestris L.ssp.chinensis Makino) belongs to Cruciferae Brassica and is rich in the nutrition that has many functions to care health. Along with the methods of transgenic technology become mature, breeding new varieties by modern biotechnology has become success. Because traditional agrobactierium-mediated transformation method is complex, the floral dip method was used to transformation in the plants and is successful in Arabidopsis thaliana. Therefore, the research established the blossom system of pak choi with ’Hangzhou Youdonger’ and ’75#’ as the material. The selection antibiotics concentration and related factors to transformation was studied using GV3101(with pCAMBIA3301). The transgenic herbicide resistant variety was obtained. The main results are as follows:1. In order to establish a flowering system of pak choi so as to carry out the related research, with ’Hangzhou Youdonger’,’75#’ and ’78#’ three varieties (lines) as the materials, the germinated seeds were treated at low temperature for10d,20d and30d respectively. Then the initiation time and leaves number at budding, flowering initiation and half flowering time and budding rate at designated time were investigated so as to research the effect of different vernalization days on flowering of different varieties in pak choi. The results showed that the treatment time under low temperature for promoting flowering of ’Hangzhou Youdonger’ and ’75#’ was20d, while the ’78#’ was30d. Comparing the flowering performance of three varieties after cold treatment for20d,’Hangzhou Youdonger’ and ’75#’ were similar, while the flowering time of ’78#’ was later17-21d than the others, so the first two varieties (lines) were more suitable than the latter to the flowering research in pak choi.2. In order to determine the best concentration of selection antibiotics so as to screen the transgenic plants, the concentration of kanamycin and basta were researched. The results showed that the sensitivity of ’Hangzhou Youdonger’ and ’75#’ to basta is almost the same, the optimal concentration of basta solution was0.002%and optimal frequency for spraying was7times. The optimal screening concentration of kanamycin was100mg/L for ’Hangzhou Youdonger’, while that for ’75#’ was75mg/L. 3. Genetic transformation was carried out in pak choi using floral dip method and the factors affecting genetic transformation efficiency were researched. The results showed that floral dipping was carried out when the plant treated by low temperature for20d grew to a few flowers in the plants. Next day the inflorescence need not been watered after dipping and7days later dip again. By this way the transgenic plant in pak choi was obtained. The best time for dipping was2.5min and dip twice with7days interval time.PCR test results to the resistant plants have indicated that the target gene have been integrated into the genome of pak choi. This provides some references for the transformation of pak choi in the future.