Valeriana officinalis subsp. hairy, L.

Ricigliano, Vincent, Kumar, Santosh, Kinison, Scott, Brooks, Christopher, Nybo, S. Eric, Chappell, Joe & Howarth, Dianella G., 2016, Regulation of sesquiterpenoid metabolism in recombinant and elicited Valeriana officinalis hairy roots, Phytochemistry 125, pp. 43-53 : 45

2.1. Establishment and characterization of V. officinalis hairy roots

V. officinalis hairy roots were generated using Agrobacterium rhizogenes -mediated transformation of in vitro grown leaf explants. This genetic manipulation relies on conjugative transfer and integration of A. rhizogenes root-inducing plasmid DNA into the host plant nuclear genome. V. officinalis seeds were aseptically germinated and hairy roots were initiated on in vitro leaf explants. Single root segments were removed from different explants to afford independent transgenic events, which were maintained on hormone-free solid medium. Isogenic lines were used to initiate shake flask cultures and generate clonal biomass for molecular and biochemical analyses ( Fig. S1 ).

As a metabolic qualification of the hairy root cultures, chemical profiles of hairy roots transformed with A. rhizogenes harboring Ri plasmid pRi15834 were compared to those of roots from wild type, soil-grown V. officinalis plants. Root biomass was powdered in liquid nitrogen, the materials fully lysed by mixing with an equal volume of acetone, followed by the addition of an equal volume of water, then partitioned into hexane. Aliquots of the hexane extracts were analyzed directly by GC–MS, as well as dried under nitrogen prior to trimethylsilyl diazomethane derivatization for GC–MS ( Fig. 2 , Fig. S7 ). The chemical profile of extracts from roots of wild type plants exhibited qualitative and quantitative differences relative to the hairy root cultures. The pRi15834 lines tended to accumulate fewer constituents than the roots from soil grown plants, but the levels of β- caryophyllene (5) and valerenal (7) were elevated while valerenadiene (6) and valerenic acid (8) were comparable ( Fig. 2 ).

Figures

Fig. 1. An outline for hypothetical sesquiterpenoid biosynthetic pathways operating in V. officinalis. Not all steps are shown.

Fig. 2. GC–MS profiles comparison of sesquiterpenoid chemistry within V. officinalis hairy root to wild type, soil grown plant root. Extracts prepared from intact, wild type plant root (panels C, D) are compared to a hairy root line harboring the A. rhizogenes pRi15834 T-DNA insertion (panels A, B). The extracts were examined directly (panels A, C) or after derivatization (panels B, D) by GC analysis. The peaks for β-caryophyllene (a), valerenadiene (b), valerenal (c) and valerenic acid (d) are annotated to facilitate comparisons.

Fig. 7. Establishment of binary vector co-transformation conditions and heterologous gene expression. (a) Kanamycin sensitivity assay of pRi15834 control hairy roots. Root tips were inoculated to MS medium containing a range of kanamycin concentrations (0, 20, 40, 60, 80 mg/L) and qualified after 30 days. (b) GUS histochemical staining of cotransformed hairy roots. The lines harbored the pVo257 T-DNA, which contained a GUS-intron overexpression cassette, or the empty vector control (pVo257 EV). The three independent GUS lines exhibited varying degrees of staining, whereas no staining occurred in the empty vector line (C) Magnified visualization of GUS expression along the root axis including individual root hairs.