Study On Marker-Free Genetic Transformation Of Poplar(Populus×euramericana CV. 'Nanling895')

In this study, Populus×euramericana cv.'Nanlin895'was the experiment material, we used the marker-removable vector PX6-GFP and PX6-CpTI, developed the research on insect-resistant gene transformation of Populus×euramericana cv.'Nanlin895'by Agrobacterium. tumefaciens. We analyzed the efficiency of transformation and marker removing, so that we can provide the evidence for breeding marker-free transgenic poplar. The main conclusion of this experiment was as following:1. Constructing the marker-removing vector system: We used PCR amplification to obtain the insect-resistance gene CpTI of vector pFZY1, after confirming by sequencing and analyzing, we constructed the expressed marker-removing vector PX6-CpTI on the basis of marker-removing vector system PX6.2. Obtaining transgenic poplar plants: Using the optimized Agrobacterium -mediated transformation system and marker-removing vector PX6-GFP and PX6-CpTI, we developed the research on transformation of Populus×euramericana cv.'Nanlin895'..We obtained 51 km resistant plants with vector PX6-GFP and 11 with vector PX6-CpTI. PCR analysis showed that it had 26 positive plants with vector PX6-GFP and 1 positive plant with vector PX6-CpTI.3. Induction of marker excision: We choosed some PCR-positive transgenic plants, cultured on the rooting medium with 5μMβ-estradiol. We also sprayed the inducing medium(1/2MS with 5μMβ-estradiol) towards the plants every 3 days. It lasted 30 days.4. Analyzing Efficiency of marker excision: We analyzed the induced transgenic plants with PCR, DNA sequencing and GFP fluorescence, confirmed that marker gene had been removed from the transgenic plants. In 21 transgenic plants, finally we obtained 1 marker-free transgenic plants, and there's also 1 showed incomplete excision.5. Km sensitivity test of transgenic plant after marker excision: Through the contrast experiment of non-induced and induced leaf disc's sensitivity to Km . We found that after marker excision, the leaf disc of transgenic plants had the similar sensitivity to Km as the control(normal plants), so it is workable to use Km selection system for the multi-transformation of poplar.