Establishment of Agrobacterium-mediated transformation system to Juglans sigillata Dode 'Qianhe-7'.

Abstract

An efficient genetic transformation system is of great significance for verifying gene function and improving plant breeding efficiency by gene engineering. In this study, a stable Agrobacterium mediated genetic transformation system of Juglans sigillata Dode 'Qianhe-7' was investigated using callus and negative pressure-assisted and ultrasonic-assisted transformation selection. The results showed that the axillary shoot leaves were suitable to induce callus and the callus proliferation rate could reach 516.27% when induction calli were cultured on DKW medium containing 0.5 mg L^-1 indole-3-butyric acid, 1.2 mg L^-1 2,4-dichlorophenoxyacetic acid and 0.5 mg L^-1 kinetin for 18 d. In addition, negative pressure infection was the optimal infection method with the lowest browning rate (0.00%), high GFP conversion rate (16.67%), and better growth status. To further prove the feasibility of this genetic transformation system, the flavonol synthetase (JsFLS₅) gene was successfully transformed into the into leaf-derived callus of 'Qianhe-7'. JsFLS₅ expression and the content of total flavonoids in transformed callus were improved significantly compared with the untransformed callus, which proved that we had an efficient and reliable genetic transformation system using leaf-derived callus of Juglans sigillata.