Establishment of high-efficiency hairy root and genetic transformation system in Cynanchum stauntonii

Cynanchum stauntonii is an important medicinal plant with antitussive effect, and its roots and rhizomes are the main medicinal part. The saponins of C. stauntonii are generally considered to be the active ingredients. The hairy root (HR) system is an important production system for secondary metabolites. However, the systems for HR induction and transformation of C. stauntonii have not been reported. Using Agrobacterium rhizogenes A4 and 20-day young stems, we induced HRs in C. stauntonii with a 79.5 % success rate. The results showed that the content of steroid alkaloids, such as cortisone, tomatidine, jurubine and 18-hydroxycorticosterone in HRs, were increased compared with normal roots (NRs). We applied three elicitors-chitosan, turpentine, and MeJA to enhance the biosynthesis of active components in HRs. The results showed that 10 mg/L chitosan could increase the content of active ingredients in HRs by over two-fold. Additionally, we successfully established a genetic hairy root transformation system with a 56.36 % success rate, enabling betalain production up to 1.24 mg/g in dry weight of HRs. This research is the first to report HR induction and transformation systems for C. stauntonii, laying a crucial foundation for future functional genomics studies and the production of active components in this valuable medicinal plant.