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.

Potential of the genetically transformed root cultures of Plumbago europaea for biomass and plumbagin production

“…These features are actually significant parameters in the transformation systems in order to improve time management strategies. In addition to the bacterial strain, the type of explant is also an essential factor for effective transformation . The stem explants were significantly more suitable for infection by the strains of A. rhizogenes .…”

“…In addition to the bacterial strain, the type of explant is also an essential factor for effective transformation. 22,31 The stem explants were significantly more suitable for infection by the strains of A. rhizogenes. In comparison, hypocotyl explants showed the lowest frequency of transformation.…”

“…In hairy root system, some factors including medium composition, light and temperature have significant impacts on growth and production of natural products, which are frequently species dependent . Previous reports focused mainly on a number of factors such as composition of nutrients medium (eg, salts concentration), temperature and light to achieve maximum growth rates of roots and efficient biosynthesis of natural products . A few studies have been described the stimulation of transgenic hairy root growth and plumbagin synthesis including Plumbago indica and Plumbago zeylanica .…”

“…20 Previous reports focused mainly on a number of factors such as composition of nutrients medium (eg, salts concentration), temperature and light to achieve maximum growth rates of roots and efficient biosynthesis of natural products. 16,21,22 A few studies have been described the stimulation of transgenic hairy root growth and plumbagin synthesis including Plumbago indica 23 and Plumbago zeylanica. 24,25 Surprisingly, hairy root cultures of P. europaea received no attention.…”

Ex situ preservation for some endemic and rare medicinal plants in Taif, KSA

“…Rhazya stricta aqueous extract has been reported to have antimicrobial activity [26,27], central nervous system depressant properties and mutagenic activities of leaf aqueous extract on a wide range of cell types [25]. There are few studies on application of in vitro propagation of R. stricta [28], evaluation of genetic stability of in vitro propagated R. stricta plants [29], establishment of transgenic R. stricta hairy roots modulate terpenoid indole alkaloid production [30].…”