Generation of Stable Catharanthus roseus Hairy Root Lines with Agrobacterium rhizogenes

Agrobacterium rhizogenes is the bacterial agent that causes hairy root disease in dicots and is purposefully engineered for the development of transgenic hairy root cultures. Due to their genetic and metabolic stability, hairy root cultures offer advantages as a tissue culture system for investigating the function of transgenes and as a production platform for specialized metabolites or proteins. The process for generating hairy root cultures involves first infecting the explant with A. rhizogenes, excising and eliminating A. rhizogenes from the emerging hairy roots, selecting for transgenic hairy roots on plates containing the selective agent, confirming genomic integration of transgenes by PCR, and finally adapting the hairy roots in liquid media. Here we provide a detailed protocol for developing and maintaining transgenic hairy root cultures of our medicinal plant of interest, Catharanthus roseus.