A Protocol for the Selection of Spontaneous Variants from Established Catharanthus roseus Hairy Root Cultures

Proper cell organization in hairy roots plays an important role in defining their capacity to synthetize specialized metabolites. Furthermore, since cell division within complete tissues must occur in an organized manner to ensure the correct cell differentiation and identity, high genetic stability is frequently associated to organ in vitro culture, such as hairy roots. However, this stability could hamper the appearance of cell variants because their integration into the tissue mass. Using in vitro Catharanthus roseus hairy roots, a protocol for the selection of spontaneous variants for alkaloid accumulation was implemented. Cultured hairy roots were exposed to IAA in liquid medium in order to produce the release of cells to the medium. Cell suspensions were recovered by filtration and root regeneration was then resumed by depriving them from IAA. Newly formed roots were isolated, and alkaloid contents, ajmalicine and catharanthine, were analyzed in five regenerated hairy root cultures. Spontaneous variation from the original culture was detected, among the new root lines.