Establishment of transgenic Rhazya stricta hairy roots to modulate terpenoid indole alkaloid production.

Abstract

Transgenic hairy roots of R. stricta were developed for investigation of alkaloid accumulations. The contents of five identified alkaloids, including serpentine as a new compound, increased compared to non-transformed roots. Rhazya stricta Decne. is a rich source of pharmacologically active terpenoid indole alkaloids (TIAs). In order to study TIA production and enable metabolic engineering, we established hairy root cultures of R. stricta by co-cultivating cotyledon, hypocotyl, leaf, and shoot explants with wild-type Agrobacterium rhizogenes strain LBA 9402 and A. rhizogenes carrying the pK2WG7-gusA binary vector. Hairy roots initiated from the leaf explants 2 to 8 weeks. Transformation was confirmed by polymerase chain reaction and in case of GUS clones with GUS staining assay. Transformation efficiency was 74 and 83% for wild-type and GUS hairy root clones, respectively. Alkaloid accumulation was monitored by HPLC, and identification was achieved by UPLC-MS analysis. The influence of light (16 h photoperiod versus total darkness) and media composition (modified Gamborg B5 medium versus Woody Plant Medium) on the production of TIAs were investigated. Compared to non-transformed roots, wild-type hairy roots accumulated significantly higher amounts of five alkaloids. GUS hairy roots contained higher amounts two of alkaloids compared to non-transformed roots. Light conditions had a marked effect on the accumulation of five alkaloids whereas the composition of media only affected the accumulation of two alkaloids. By successfully establishing R. stricta hairy root clones, the potential of transgenic hairy root systems in modulating TIA production was confirmed.