Developing an efficient protocol for hairy root transformation of Corydalis saxicola

Corydalis saxicola Bunting is a rare perennial species in the Corydalis genus of the Papaveraceae family. It has been traditionally used for treating liver diseases for over a millennium. Research on molecular biology of C. saxicola is just beginning, and there is an urgent need to establish a mature genetic transformation system for conducting functional genomics and germplasm innovation. In this study, we established an Rhizobium rhizogenes-mediated transformation protocol for C. saxicola, exhibiting high efficiency. After screening R. rhizogenes strains, optimizing the root induction medium, evaluating various explants, and standardizing infection time and co-cultivation duration, we found that infecting adventitious buds with the AR1193 strain at an OD600 of 0.6 for 15 min, followed by three days of co-cultivation and sub-culturing on root induction medium supplemented with 1-Naphthaleneacetic acid (NAA), resulted in highly efficient induction of hairy roots containing the reporter gene DsRed. The transformation efficiency reached up to 52.63% in our experiments. This protocol serves as a valuable tool for molecular biology investigations, particularly in the research of secondary metabolic pathways, and will for sure accelerate the development of CRISPR/Cas9-based genome editing in C. saxicola.