Establishment of in vitro culture system for Codonopsis pilosula transgenic hairy roots.

The aim of the study was to establish a reliable system of transgenic hairy roots in Codonopsis pilosula through Agrobacterium-mediated genetic transformation. For this, we optimized several steps in the process of A. rhizogenes strain C58C1 mediated hairy root induction, including the most appropriate medium, explant type, time for infection and co-cultivation. We achieved an induction rate of up to 100% when the roots of C. pilosula seedlings were used as explants, infected with A. rhizogenes C58C1 harboring pCAMBIA1305 for 5 min, followed by induction on 1/2MS supplemented with 0.2 mg/L naphthylacetic acid and 200 mg/L cefotaxime sodium. The co-transformed hairy roots were confirmed by PCR amplification of hygromycin phosphotransferase II gene and histochemical GUS assay, and the efficiency of transformation was 70% and 68.3%, respectively, when no hygromycin selection pressure was exerted. To increase biomass production, we excised and self-propagated the transformed hairy roots, which produce saponins. Our successful establishment of an in vitro culture system of transgenic hairy root for this species lays the foundation not only for assessing gene expression and function but also for obtaining high levels of secondary metabolites through genetic engineering technology.