Exploration Of Improviug Vacuum Infiltration Transformation Effciency Of Brassica Campastris Ssp.Chinese Var. Parachinensis

As a method of agrobacterium plant transformation In planta, vacuum infiltration is the most frequently used method in Brassica campestris ssp. chinensis by far, which is independent on the tissue culture. Even though plenty of researches have involved vacuum infiltration transformation, there are still some problems unsolved. On one hand, transformation efficiency of Brassica campestris ssp. chinensis by vacuum infiltration is volatile and generally low. On the other hand, factors affecting transformation efficiency such as vacuum degree, infiltration time, plant development stage, agrobacterium cell concentration, surfactant and sucrose content of the inoculation medium and interaction within those factors remain unclear.In this research, Brassica Brassica campastris ssp. chinensis var.parachinensis’Youqing 49’used as recipients for genetically modified material, and agrobacterium tumefaciens GV3101 carrying plasmid pCAMBIA1301 used as vector system, the agrobacterium OD6oo, plant development stage, vacuum degree and infiltration time, concentration of surfactant Silwet L-77 and sucrose were studied through one-way ANOVA and analyzed as single factor affecting transformation efficiency of ’Youqing 49’. After that, orthogonal experimental design of OD600, vacuum degree and infiltration time, concentration of surfactant Silwet L-77 and sucrose was conducted to obtain suitable vacuum infiltration system of ’Youqing 49’. In addition, suggested transformation target and time of the inflorescence treated by in planta methods were analyzed by experiments of hybridization pollination and harvesting seeds from different part of transgenic plants. The main results are as follows:(1) Effect of plant development stage on transformation efficiency was analyzed by applying vacuum infiltration method into three different developmental stages of ’Youqing 49’. Among the experiments, about 0.1% frequency of transgenic seeds was obtained from the treatment of plants with several opening flowers in its main inflorescence, which was higher than the bud stage and the stage of lateral inflorescence with several opening flowers. But transformation efficiency of these different stages showed no significant difference. ’Youqing 49’ at the stage of lateral inflorescence achieved up to 3.36%transformation efficiency, which was 1.8 and 3 times as the main inflorescence stage and bud stage, suggesting that lateral inflorescence with several opening flowers is the most suitable stage for vacuum infiltration.(2) Transformation efficiency of different agrobacterium cell concentration showed that agrobacterium cell concentration had significant effects on transformation event. Among the treatments of OD6oo from 0.0025 to 0.4000, the transgenic seeds rate was up to 0.12% at the absorbency value of bacterial liquid of 0.2.(3) The statistical results of transformation efficiency of different vacuum degree and infiltration time showed that the pressure and infiltration time had a significant influence on transformation effect, among which the highest transformation efficiency was achieved at 10 kPa,5 min.(4) The research on four different concentrations of the surfactant Silwet L-77 showed a significant difference among the Silwet L-77 concentration of 0.0025%,0.005%,0.01% and 0.02%. Moreover,0.01% was the most suitable concentrations of Silwet L-77 for transformation.(5) The variance analysis of the transformation efficiency in different sucrose concentration demonstrated that there was no significant difference on transformation efficiency. While sucrose content at 3% can generate better transformation effect.(6) Orthogonal experimental design of OD600, vacuum degree and infiltration time, concentration of surfactant Silwet L-77 and sucrose was conducted to obtain suitable vacuum infiltration system of ’Youqing 49’, showing that 10 kPa,5.5 min infiltration,0.2 of absorbency value of bacterial liquid,0.0125% surfactant and 3% sucrose in the medium respectively is the suitable transformation system.(7) Statistical results of transformation efficiency by pollinating treated plants and wild type with different hybrid methods showed that, transgenic seed frequency was 0.0039% when plants were treated by vacuum infiltration as male parent while the wild type as the female parent, which was far lower than transgenic seed frequency by the vacuum infiltration-treated plant as female parent. It showed that pollen can generate transformation by transferring agrobacterium tumefaciens into the ovule, but the efficiency is extremely low. Comparisons of transformation efficiency of seeds harvested from the top and bottom of treated plants showed that seeds from upper part obtained as high as 0.133% of transformation efficiency, which was 1.6 times of the seed from lower part, indicating that the upper parts of seeds are more impressionable to agrobacterium tumefaciens and transformation takes place before the late stage of the flower bud development.(8) Several plants were transformed by the flower-dip method, no transformation was observed even though the concentration of bacteria liquid and content of surfactant Silwet L-77 were elevated. It indicated that flower-dip method was not suitable for transformation of ’Youqing 49’.(9) Two different ways of selecting transgenic seeds were studied, one was culture medium containing hygromycin and the other was soaking seeds with hygromycin. Transformation efficiency obtained from culture medium containing hygromycin showed four times as seed soaking method, indicating different ways of selecting transgenic seeds had a significant effect on the transformation efficiency.