Development of Rhizobium rhizogenes-induced hairy root cultures of Vitex negundo for vitexin production.

Vitex negundo L. is a significant indigenous medicinal plant with diverse therapeutic potentials owing to its rich reservoir of bioactive compounds including sitosterol, vitexoside, agnuside, isovitexin and vitexin which are predominantly present in the root. To induce hairy roots, explants of V. negundo L. were transformed using two strains of Rhizobium rhizogenes: MTCC 532 and MTCC 2364. Among them, the MTCC 2364 strain exhibited the highest transformation efficiency (94.44%). Genetic transformation of the induced hairy roots was confirmed by PCR amplification using primers specific for the rolB and rolC genes and man2 opine primers. Hairy root culture in MS liquid medium at 90 RPM shaking conditions showed an overall higher growth index (9.47 fresh weight in 18-21 months). The vitexin compound was verified and quantified in both hairy roots and wild-type roots by HPLC analysis, revealing a higher concentration in hairy roots (0.49 mg/g Dry Weight) than in the wild-type roots (0.065 mg/g Dry Weight). Furthermore, the multi-elemental analysis using an EDXRF spectrometer revealed the trace elemental analysis in both hairy roots and wild-type roots. To date this is the first report confirming the production of vitexin in hairy root culture of V. negundo which, represents an efficient and economical approach for the large-scale production of such secondary metabolites.