Studies On Transformation Of Cabbage With Salt Tolerance Gene StP5CS

Cabbage (Brassica oleracea L. var. capitata L.) is one the most of important leafy vegetables in China. Nowdays agricultural production and environment are seriously influenced by soil salinization. Besides, there is a large area of Saline-alkali Soil in China. Thus to develop salt-tolerant cabbage varieties is becoming an urgent and substantial research topic. In this study, four high-generation self-bred lines of cabbage were used to develop an effecient regeneration and transgenic system. The StP5CS gene was introduced into cabbage to improve the ability of salt tolerance by both Agrobacterium-mediated transformation and floral-dip pathway. It is respectively confirmed by PCR, Southern blotting and RT-PCR that StP5CS gene was simultaneously integrated into the genome of plants. Then some of the transgenetic plants were taken as material for salt tolerance test. The main results are as follows:1. G1, G2and G3were used to optimize the regeneration rates of cabbage. Effects of five factors (genetype, type of explants, hormones concentration, seedling age and AgNO3concentration) on the regeneration rate of above were studied. The highest regeneration frequency was obtained with explants from5-day seedings after sowing. Both hypocotyls and cotyledons of G1were the best explants for regeneration. We could obtain high regeneration frequency with hypocotyls by supplementing6-BA1.0mg/L and NAA0.05mg/L on the MS medium. The optimum medium of cotyledons was BA1.0mg/L+NAA0.05mg/L. The higher regeneration rate of adventitious buds could be obtained by adding Silver nitrate. Moreover, the optimal concentration was7.5mg/L.2. Transgenic plants were generated from hypocotyls and cotyledons of G1by Agrobacterium-mediated transformation with StP5CS gene, and the following factors were investigated to establish and optimize explant transgenic system:concentration of Bialaphos sodium, pre-culture period, co-culture period, bacterial concentration, infection time and addition of AS. Non-transgenic green shoots regenerated from hypocotyls or cotyledons of cabbage could be completely inhibited by medium with4mg/L or8mg/L Bialaphos-sodium. The Hypocotyls of G1were pre-cultured for2d, then inoculated with about0.5of OD600Agrobacterium tumefaciens for5min. Inoculated explants were co-culutred in medium with0.1mmol/L AS for2d, then transferred to the SM1medium to screen. The protocal can reach24.47%transformation rate.3. The StP5CS gene was transferred into hypocotyls and the cotyledons of cabbage by Agrobacterium tumefaciens-mediated method.86transformants were obtained and PCR analysis indicated that StP5CS gene was integrated into the genome of cabbage plant. Moreover, Southern blot and RT-PCR analysis showed that StP5CS gene was expressed on both DNA and RNA levels. The resistance to salt of cabbage transgenic plants was significantly increased by measuring SOD, Proline content and REL after treating with different concentration of NaCl.4. To study the transformation factors (the surfactant Silwet L-77, sucrose concentrations and the developmental stage of the plant at the time of inoculation with Agrobacterium) in cabbage by Agrobacterium-mediated floral-dip method, G1, G2and G4were used as the recipients. The results showed that Survival rate was the highest with0.05%Silwet L-77,10%sucrose and less than8mm diameter. PCR, Southern blot and RT-PCR analysis indicated that both StP5CS gene was integrated into the genome of cabbage and was expressed on transcription level.