Optimization of Astragalus membranaceus hairy roots induction and culture conditions for augmentation production of astragalosides

摘要

In this study, an efficient and reliable Agrobacterium rhizogenes-mediated hairy root system in A. membranaceus was developed. An optimal transformation rate of 66.84 % was obtained after 13 days by inoculating A. rhizogenes LBA9402 onto 3-week-old leaf explants followed by a co-cultivation period of 2 days with acetosyringone (100 mu M). Among eight representative A. membranaceus hairy root lines (AMHRL), AMHRL VI was found to be the lead line and confirmed by PCR amplification of rolB, rolC and aux1 genes. Culture parameters of AMHRL VI were systematically optimized by central composite design and slogistic kinetic models, and six main astragalosides (AG) were quail-quantitatively determined by liquid chromatography-tandem mass spectrometry. Under optimal conditions (inoculum size 1.54 %, culture temperature 27.8 degrees C, sucrose concentration 3.24 % and harvest time 36 days), A. membranaceus hairy root cultures (AMHRCs) in MS medium gave the optimal biomass production of 15.79 g/L dry weight (DW) with total AG accumulation of 2.657 mg/g DW. This yield was significantly superior to that of 3-year-old field grown roots (2.435 mg/g DW). Overall, this investigation provided the generation of a high-productive AMHRCs that is capable of augmentation production of AG, offering a promising and continuous product platform for naturally derived, high quality and valuable phytochemicals.