Hairy root cultures of Rehmannia glutinosa and production of iridoid and phenylethanoid glycosides

Abstract

Hairy root lines through the infection of Agrobacterium rhizogenes strain (A4) were established from shoot tips and leaves of Rehmannia glutinosa Libosch. Ten lines of hairy roots were selected on the basis of biomass increase in half-strength Gamborg medium (1/2 B5). Transgenic status of the roots was confirmed by polymerase chain reaction using rolB and rolC specific primers. Iridoid glycosides (catalposide, loganin, aucubin and catalpol) and phenylethanoid glycosides (verbascoside and isoverbascoside) identified using HPLC–ESI–MS, and their contents were compared with untransformed root culture and roots of 1-year-old field-grown plants of R. glutinosa by RP-HPLC. The growth and production of secondary metabolites in ten hairy root lines varied considerably as to the media. Woody plant (WP) medium displayed higher growth in terms of fresh (FW) and dry weights (DW) compared to 1/2 B5 medium. High-yielding hairy root lines produced higher amounts of loganin, catalposide, verbascoside and isoverbascoside in comparison to the untransformed root culture and roots of 1-year-old field-grown plants. The highest amounts of catalposide and loganin in transformed roots were 4.45 mg g−1 DW (RS-2 hairy root line) and 4.66 mg g−1 DW (RS-1 hairy root line), respectively. Aucubin and catalpol were detected in some lines in trace amounts. The highest amounts of verbascoside (16.9 mg g−1 DW) and isoverbascoside (3.46 mg g−1 DW) were achieved in RS-2 root line. The contents of catalposide, verbascoside and isoverbascoside in high-producing lines were several times higher than in untransformed root culture and roots of R. glutinosa plants grown in soil. Loganin and aucubin could not be detected in roots of field-grown plants. However, the levels of catalpol were much lower in the in vitro roots.