Comparison of elicitor-based effects on metabolic responses of Taxus × media hairy roots in perfluorodecalin-supported two-phase culture system

Abstract

Key messageTwo lines of Taxus × media hairy roots harbouring or not the TXS transgene demonstrated diverse gene expression and taxane yield during cultivation in PFD-supported two liquid-phase culture system.AbstractTwo lines of Taxus × media hairy roots were subjected to single or twice-repeated supplementation with methyl jasmonate, sodium nitroprusside, l-phenylalanine, and sucrose feeding. One line harboured transgene of taxadiene synthase (ATMA), while the second (KT) did not. Both hairy root lines were cultured in two-phase culture systems containing perfluorodecalin (PFD) in aerated or degassed form. The relationship between TXS (taxadiene synthase), BAPT (baccatin III: 3-amino, 3-phenylpropanoyltransferase), and DBTNBT (3′-N-debenzoyl-2-deoxytaxol-N-benzoyltransferase) genes and taxane production was analysed. The ATMA and KT lines differed in their potential for taxane accumulation, secretion, and taxane profile. In ATMA biomass, both paclitaxel and baccatin III were detected, while in KT roots only paclitaxel. The most suitable conditions for taxane production for ATMA roots were found in single-elicited supported with PFD-degassed cultures (2 473.29 ± 263.85 µg/g DW), whereas in KT roots in single-elicited cultures with PFD-aerated (470.08 ± 25.15 µg/g DW). The extracellular levels of paclitaxel never exceeded 10% for ATMA roots, while for KT increased up to 76%. The gene expression profile was determined in single-elicited cultures supported with PFD-degassed, where in ATMA roots, the highest taxane yield was obtained, while in KT the lowest one. The gene expression pattern in both investigated root lines differed substantially what resulted in taxane yield characterized particular lines. The highest co-expression of TXS, BAPT and DBTNBT genes noted for ATMA roots harvested 48 h after elicitation corresponded with their higher ability for taxane production in comparison with the effects observed for KT roots.Electronic supplementary materialThe online version of this article (10.1007/s00299-018-2351-0) contains supplementary material, which is available to authorized users.

Production of bioactive plant secondary metabolites through in vitro technologies—status and outlook

“…g −1 DW S S HR Salehi et al ( 2019b ) Farhadi et al ( 2020 ) Jalalipour Parizi et al ( 2020 ) Taxus x media 2.47 mg . g −1 DW HR Sykłowska-Baranek et al ( 2019 ) Steviol glycosides (stevioside + rebaudioside A) Stevia rebaudiana 92.58 mg . g −1 DW AR Ahmad et al ( 2018 ) Ursolic acid Lantana camara 3.87 mg .…”

“…Attempts to increase the in vitro yields of paclitaxel are still ongoing. Sykłowska-Baranek et al ( 2019 ) investigated paclitaxel formation in two hairy root lines of Taxus x media , one of which harbored a gene for taxadiene synthase ( TXS ). In their study they evaluated the effects of various elicitor treatments and a perfluorodecalin-supported two liquid-phase culture system on product formation and expression pattern of three paclitaxel biosynthetic genes.…”

Application of Priming Strategy for Enhanced Paclitaxel Biosynthesis in Taxus × Media Hairy Root Cultures

“…DBTNBT participates in formation of 2 -deoxytaxol through ligation of benzoyl CoA group to 3 -N-debenzoyl-2-deoxytaxol [15,16]. For gene expression analysis, primed BABA24 or BABA1 and elicited culture samples were collected after 1 h, 3 h, 6 h, 12 h, 18 h, 24 h, 48 h, 72 h. The total RNA was isolated from hairy roots frozen in liquid nitrogen according to the method described by Sykłowska-Baranek et al [17]. One µ of total RNA was used to reverse transcription carried out with cDNA RevertAid First Strand cDNA Synthesis Kit and random hexamer primer (Thermofisher Scientific, Waltham, MA, USA) according to the manufacturer's instruction.…”

The Cream of the Crop: Biology, Breeding and Applications of Cannabis sativa

“…In these systems, an aqueous phase is used to support cell growth while a non-aqueous phase, typically consisting of a solvent or resin, is employed to act as a sink for the accumulation of the desired product and in some cases facilitates its subsequent extraction. Two-phase systems have been shown to greatly increase metabolite yield in both plant cell suspension and hairy root culture cultures (Chiang and Abdullah, 2007;Malik et al, 2013;Rudrappa et al, 2004;Syk lowska-Baranek et al, 2019;Wu and Lin, 2003), although to the best of our knowledge such an approach has not yet been attempted in Cannabis.…”