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

Plumbago europaea L. is the main source of plumbagin which is a well-known pharmacological active compound. In this investigation, genetically transformed roots of P. europaea were obtained by improving some factors affecting the efficiency of Agrobacterium rhizoigenes-mediated transformation such as explant type, A. rhizoigenes strain, bacterial infection period, co-cultivation period and acetosyringone concentration. The leaf, hypocotyl and stem explants from in vitro grown plantlets were infected with bacterial strains (A4, ATCC15834, MSU440 and A13). The highest transformation rate of 69.3% was achieved after 7-9 days by inoculating A. rhizogenes MSU440 strain onto the 3-week-old stem explants followed by a co-cultivation period of 2 days on a medium containing 100 mu M acetosyringone. To investigate the existence of the rolB gene, polymerase chain reaction was carried out using specific primers. Effects of growth media (MS, 1/2 MS, MS-B5 and 1/2 MS-B5), different sucrose concentrations and illumination on biomass production and plumbagin biosynthesis in P. europaea hairy root cultures were analyzed using stem explants after infection with MSU440 strain. 1/2 MS-B5 liquid medium containing 30 g L-1 sucrose incubated in the dark resulted in the efficient biomass production of transformed hairy roots (12.5 g fresh weight, 1.8 g dry weight) with 3.2 mg g(-1) DW plumbagin accumulation. This procedure provides a framework for large-scale cultivation of hairy roots for plumbagin production. This is the first report describing the establishment of P. europaea hairy root culture with special emphasis on plumbagin production.