| Chinese cabbage(Brassica rapa L.ssp.pekinensis)belongs to the Brassica genus Brassica of the Brassicaceae family in the botanical classification.Because of its rich nutrients such as crude fiber,protein,various minerals and multivitamins,it had been widely popular in China and even Asia.Therefore,Chinese cabbage has extensive planting area and large markets of supply and demand in China and even Asia,and it is one of the most popular vegetables in my country.But clubroot seriously affects the yield of Chinese cabbage.Although most of clubroot resistance-related genes have been found,their functions have not been verified,which attributed to the limitation of genetic transformation system for clubroot resistance-related genes of Chinese cabbage.There have been some reports on the establishment of the genetic transformation system of Chinese cabbage,however the influence of genotype due to different varieties with different genetic transformation efficiency.In this study,we screened resistant/susceptible Chinese cabbage varieties to Plamodiophora brasssicae Woron,and one of these varieties with high regeneration ability was used to systematically research genetic transformation system.Finally,a stable genetic transformation system with high transformation efficiency was established.This research provided important technical support for subsequent research on functional verification test of clubroot-resistant genes.The detailed results are as following:To screen the genotypes for genetic transformation system,the regenerative abilities of nine Chinese cabbage materials with different clubroot-resistance were investigated.Among them,the Chinese cabbage cultivar ‘SN157’,which is susceptible to P.brassicae,had the highest in vitro regeneration differentiation rate of 89.2 %.To found the best vitro regeneration system,P.brassicae susceptible variety ‘SN157’of Chinese cabbage was used for following research.In this research,cotyledons with petiole and hypocotyls were used for selection of explant type;3-7 d aseptic seedings were used for selection of seedling age;A different combinations of 0、0.25、0.5、0.75、1.0 mg/L NAA or TDZ were used for the selection for plant exogenous hormone combinations.The results show that the 5 d seedling age of Chinese cabbage variety ‘SN157’ with stalked cotyledons is used as explants,and exogenous hormone NAA 0.5 mg/L+TDZ 0.5 mg/L are the better vitro regeneration system,its adventitious bud differentiation rate is more than 45 %.Basing on the above vitro regeneration system of Chinese cabbage,the genetic transformation system of Chinese cabbage was optimized.Agrobacterium solution concentrations of OD600=0.5、0.8、1.2 were used to infect explants of plant materials,and the best concentration was selected,the results showed that OD600=0.5 agrobacterium liquid issuitable to infect plants for its less contamination and high induction percentage(74.23 %)of adventitious shoots.Cotyledons with petiole were infected with OD600=0.5 agrobacterium,and then cultured on the regeneration medium with 0、100、200、300、400 or 500 mg/L Tim,the results showed that 200 mg/L Tim has high resistance to agrobacterium and high induction percentage(71.33 %)of adventitious shoots.To sum up,the optimized genetic transformation system of Chinese cabbage for the research of clubroot-resistant genes should be: ‘SN157’as plant material,5d seedling-age stalked cotyledons as explants,OD600=0.5 as Agrobacterium to infect outside after centrifugal resuspension and then cultured in differentiation medium with 200 mg/L,after subculture,the sufficiently large plant was root cultured.Above optimizing improved the transformation rate and be verified by repeated trials.PCR detection,GFP and q RT-PCR technologies were used to detect these obtained transgenic plants.The results showed that most of them were transgenic positive plants.It is suggested that an efficient and stable Chinese cabbage genetic transformation system was successfully established,and the average transgenic rate is about 2.13 %. |
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