A combined culture system for leaf explants of Lycium ruthenicum with high genetic transformation rates and low seedling vitrification rate

Abstract

[Objective] We aim to establish an efficient and stable genetic transformation system of Lycium ruthenicum and reduce the vitrification rate of the regenerated seedlings in order to promote gene function study and genetic improvement. [Methods] L. ruthenicum leaves were used as explants and Agrobacterium (LBA4404, EHA105) was used to transform L. ruthenicum. By adjusting the types of basic medium and adding plant hormones, we selected the optimal callus-induction medium, differentiation and selection medium, and rooting-induction medium. The transformation rate of L. ruthenicum was increased to over 65%, while the seedling vitrification rate was decreased to below 10%. This combined culture system laid a foundation for the molecular breeding of L. ruthenicum. [Results] (1) The optimal infection concentration of Agrobacterium (OD600) was 0.6 and the infection time was 25 min for the combined culture system of L. ruthenicum. Under this condition, the callus-induction rate was 78.2%-96%; (2) The optimal differentiation and selection medium contained: MS+inositol 50 mg/L+nicotinic acid 0.25 mg/L+vitamin B6 0.25 mg/L+Fe salt storage solution 1 mL/L+glycine 1.0 mg/L+ vitamin B1 0.05 mg/L+6-BA 0.25 mg/L+sucrose 30 g/L+agar 6 g/L+Kanamycin 30 mg/L+Timentin 300 mg/L, pH 6.0. The optimal rooting medium contained: WPM+IBA 0.25 mg/L+sucrose 30 g/L+agar 6 g/L +Kanamycin 30 mg/L+Timentin 300 mg/L, pH 6.0. (3) On the optimal differentiation and selection medium, the seedling vitrification rate infected by Agrobacterium LBA4404-pBI121 was about 65%, while that infected by Agrobacterium EHA105-pBI121 was below 10%. (4) The rooting efficiency of the regenerated seedlings reached 81.2% using a low-salt WPM medium of woody plants. (5) The ratio of the positive callus to the total number of inoculated leaves was used to evaluate the transformation efficiency. Using the optimal transformation system, the transformation rates of Agrobacterium LBA4404-pBI121 and EHA105-pBI121 were 51% and 65.2%, respectively. [Conclusion] The combined culture system of L. ruthenicum leaves can improve transformation rate while reducing seedling vitrification rate.

Keywords

• lycium ruthenicum
• agrobacterium
• combined culture system
• transformation efficiency
• seedling vitrification