The Roles Of The LBD Genes In Vitro Regeneration And The Genetic Transformation Of The Vernerlization Related BrVIN3 Gene In Chinese Cabbage

Chinese cabbage is one of cruciferous Brassica crops,which was considered the regeneration rate is low and difficult to apply in genetic engineering.Improving the regeneration frequency and establishing efficient regeneration system in vitro are necessary foundation for genetic engineering of Chinese cabbage.Callus formation,embryogenesis and adventitious bud development are major steps of plant regeneration,in which callus formation is a key step.In the study,37 inbred lines of Chinese cabbages and 1 commercial cultivar were tested to evaluate their shoot regeneration frequency.Then,selected seven materials with significant different regeneration frequency were used to further study.Here,we focused on the function of Brassica LBD genes in callus formation and plant regeneration by analyzing.The results preliminary suggested that LBD genes are involved in callus formation and may impact shoots regeneration frequency.Main results are as follows:1.Optimization of shoot regeneration system as the basic condition was used for regeneration rate analysis,the results showed that different inbred lines had different regeneration rate,the rate ranged from the lowest 0%to the highest 67.2%.2.21#inbred line which was derived from 'Xin Du You Feng',with a regeneration rate of 2.4%,and 29#inbred line which was derived from 'Xia Bao',with a regeneration rate of 55.7%,were used for function study of LBD.Expressions of LBD genes were recorded by real-time quantitative PCR in the course of 0 d,7 d,14 d,and 21 d after shoot induction.The results showed that LBD impression first increased and then decrease in the process of regeneration.The expresson of LBD genes were also different within different materials of regeneration frequency.3.The expression pattern of LBD genes were further proved by 4 materials with different regeneration rates.Moreover,LBD were induced by exogenous auxin,a hormone which could effectively promote the formation of callus.Based on the cotyledon regeneration system,the genetic transformation system of the Agrobacterium-mediated for the Chinese cabbage was successfully established in vitro.A QTL for flowering time was detected on A02 chromosome based on SLAF sequencing and genome-wide association analysis,which was consistent with the QTL determined by traditional genetic mapping.In order to understand the function of Bra006824,we selected few high regeneration frequency inbred lines.The Agrobacterium-mediated transformation method was taken to transform Bra006824 gene into the plant genome.The results showed that:1.In the screening process of regeneration,the suitable concentration of hygromycin for screening resistant buds was 3 mg/L;At least 5 positive transgenic Chinese cabbage plants were obtained,which was further confirmed by PCR analysis of candidate Bra006824.2.Bra006824 gene closely related to Chinese cabbage bolting was transformed into Arabidopsis thaliana by Agrobacterium floral dip.High ectopic expression of Bra006824 gene promotes early bolting in Arabidopsis.