| Dioscorea zingiberensis C.H.Wright, as a native species in China, is one of the most important medicinal resource plants, this plants has the highest content of diosgenin among all the species in the world. Since 1970s, the research on variety improvement, sapogenin extraction and micropropagation of D. zingiberensis has a significant development, and formed a very uniform system of rapid regeneration. An easy culture genotype of D. zingiberensis 4-73 was used as explant in this study. Base on the existent Agrobacterium-mediated transformation system, we optimize the regeneration and transformation system and successfully establish a stable and efficiency Agrobacterium-mediated transformation system. Use this system, an production of transgenic D. zingiberensis plants using the BAR gene and EPSPs gene for herbicide resistance was achieved. This study provides a simple and efficient transformation system of D. zingiberensis based on the use of BAR or EPSPs gene as a selectable marker gene, which can be combined with other agronomical important genes for improvement of D. zingiberensis. It also can use to research the metabolic mechanism of sapogenin.The main results are as follows:一、Optimize of regeneration system1. To optimize the condition of calli, calli were subcultured on MS medium supply with 2,4-D 2.0 mg/L,6-BA 0.5 mg/L and different concentration of NAA (0 mg/L、0.1 mg/L、0.2 mg/L、0.3 mg/L、0.4 mg/L、0.5 mg/L). The results showed that the condition of calli is much better with NAA 0.4 mg/L2. Subculture calli on MS medium supply with different concentration of 2,4-D (2.0 mg/L、2.5 mg/L、3.0mg/L、4.0mg/L、5.0 mg/L). The results showed that, with the increase of concentration of 2,4-D, the condition of calli are getting worse. Proliferation of calli is paused when the concentration of 2,4-D is 4.0 mg/L and 5.0 mg/L. It is indicate that high concentration of 2,4-D has no positive effect on the condition of calli. 3. Replaced agar with phytagel as coagulant of subculture medium. As the result shown, the conditions of calli much better than before, the quantity of embryogenic calli was significantly increased and calli are more drier and loose, the color of calli are white but yellow. Surface between calli and medium is no longer significant browning.二、Optimize of transformation system1. The introduction of desiccation. The results showed that desiccation for 2 h after infection and placed a filter paper on co-culture medium can significantly increase the transient expression of GFP.2. Effects of different bacteria preparation methods on the transient expression of GFP: a. Monoclonal Agrobacterium EHA105 were inoculated on a fresh LB solid medium, then cultivate for 30 h. Scraped adequate bacteria to the submerge medium until OD600 is 0.5; b. Monoclonal Agrobacterium EHA105 were inoculated to a fresh LB liquid medium. After 30 h shaking cultivation under 200 rpm, Agrobacterium were accumulated under 3000 rpm centrifugation, and resuspension at 50 mL submerge medium until OD600 is 0.5; c. Inoculate monoclonal Agrobacterium to 50 mL submerge medium, shaking culture until the OD600 is 0.5. After 3 days cocultivation at 20℃, the observed and statistics of transient expression of GFP were performed. The results showed that three methods have little effect on the transient expression of GFP, the rates are maintained at 40%.3. In order to increase the efficiency of transformation, leaves as explant were treated by same concentration of cellulase and pectinase for 0 minutes,10 minutes and 30 minutes, and then submerged with Agrobacterium EHA105 (containing GUS gene). The transient expression of GUS was detected after 3 d coculturation. As the result shown enzyme treatment for 0 min, the leaves have no GUS blue spots, enzyme treatment for 10 minutes, GUS blue spots increased significantly, while enzyme for 30 minutes, there either have no GUS blue spots appeared.三、production of transgenic plants with herbicide-resistance geneGenetic transformation of D zingiberensis mediated by Agrobacterium has been achieved. Embryogenic callus initiated from seeds of genotye 4-73 was infected with an Agrobacterium strain (LBA4404) harboring a vector that contained a herbicide-resistant BAR gene driven by the 35S-P promoter, a NPTⅡgene driven by nas-P promoter and a basnare gene driven by TA29-P promoter, and an Agrobacterium strain (EHA105) harboring a vector that contained a herbicide-resistant EPSPs gene driven by the CaMV 35S promoter. Sensitive of screening agent analysis show that 5.0 mg/L PPT and 800 mg/L glyphosate are useful for selection. The stable transformation efficiency of LBA4404 is 5.4%, and the stable transformation efficiency of EHA105 is 1.3%. PCR analysis and Southern blot confirmed transgene integration in the D zingiberensis genome. All independent transformation events which were analysised are carried one to two copies of the transgene. We report here the successful use of Agrobacterium for the large-scale production of transgenic D zingiberensis can be consummated in 7 months. |
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