The Transgenic Study On Marker-free, Flavonoids-enriched And Late Blight Resistance Potato

Potato(Solanum tuberosum) is the fourth crop in the world. Late blight, caused by Phytophthora infestans de Bary, is the most destructive disease. Flavonoids, a class of natural plant secondary metabolites, have antioxidant, anti-virus and other important functions, but only trace amounts can be detected on current widely planting potato varieties. In this study, a plant flavonoids regulating gene AtMYB12 and three new cloned late blight resistance genes (Rpi-vnt1.1, Rpi-mcq1.1 and Rpi-mcq1.2 ) were differently combinated and transformed into a cultivated potato species Desiree in order to obtain a range of flavonol-rich ( such as rutin, etc.) and potato late blight resistant trangenic lines. It is expected to improve both traits of the late blight resistance and nutritional quality.By using the Cre/lox site-specific recombination system PX6-GFP, three transgenic constructs were made with target genes from four donors through a two-step strategy with high-fidelity PCR, which included pX6-patatin:: AtMYB12, pX6-Rpi-mcq1.1-Rpi-mcq1.2 and pX6-patatin::AtMYB12-Rpi-mcq1.1-Rpi-vnt1.1.Three recombinant vectors were mobilised into Agrobacterium strain AGL I, and then were introduced into the susceptible cultivar Desiree via Agrobacterium-mediated stem segment transformation. In total, 28, 20, 20 positive transgenic plant individuals were obtained with 68%, 40% and 45% of transformation efficiency respectively.Rpi gene potato were tested with six P.infestans isoltates with detached leaf method. The potato carried Rpi-mcq1.1 and Rpi-mcq1.2 were resistant only for two isolates, EC1 and 90128 . Potato carried pX6-patatin::AtMYB12-Rpi-mcq1.1-Rpi-vnt1.1 were resistant to all six isolates fallen into the former design.Utilizing HPLC method, We detected the content of flavonoids in potato tubes with different lines carrying AtMYB12 gene. In potato carried pX6-patatin::AtMYB12, the content of rutin is rang from 0.051 mg g-1 to 3.091 mg g-1, and the kaempferol-rutinoside is ranged from 0.041 mg g-1 to 0.951 mg g-1. In addition, flavonoid content in pX6-patatin::AtMYB12 -Rpi-mcq1.1-Rpi-vnt1.1 potato is ranged from 0.08 to 0.2 mg g-1. The result indicated the overall flavonoid content is increased significantly.Based on resistance phenotype, flavonoid content analysis and the stability of mor- phological traits, 2 lines from each selected transgenic combination were treated withβ-estradiol, and were tested by PCR to obtain NPTII marker-free plants, whose resistance were analysed again. The result indicated that the percentage of marker-free in 3 transgenic combinations is 10%, 8%, 8% respectively. In summary, we have obtained new flavonoid-rich or (and) late blight resistance varieties by transgenic strategy, which is expected to be further screened in the field and to pass the safety assessment, and to address the key problem in production.