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| Format: | Recurso digital |
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Zenodo
2016
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| Online-Zugang: | https://doi.org/10.5281/zenodo.10530239 |
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- <p><b><i>2.1. Establishment and characterization of V. officinalis hairy roots</i></b></p><p><i>V. officinalis</i> hairy roots were generated using <i>Agrobacterium rhizogenes</i> -mediated transformation of <i>in vitro</i> grown leaf explants. This genetic manipulation relies on conjugative transfer and integration of <i>A. rhizogenes</i> root-inducing plasmid DNA into the host plant nuclear genome. <i>V. officinalis</i> seeds were aseptically germinated and hairy roots were initiated on <i>in vitro</i> 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).</p><p>As a metabolic qualification of the hairy root cultures, chemical profiles of hairy roots transformed with <i>A. rhizogenes</i> harboring Ri plasmid pRi15834 were compared to those of roots from wild type, soil-grown <i>V. officinalis</i> 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 (<b>5</b>) and valerenal (<b>7</b>) were elevated while valerenadiene (<b>6</b>) and valerenic acid (<b>8</b>) were comparable (Fig. 2).</p>