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

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Abstract

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 μM). 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 °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.

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Abbreviations

AG:

Astragalosides

AG I:

Astragaloside I

AG II:

Astragaloside II

IAG II:

Isoastragaloside II

AG III:

Astragaloside III

AG IV:

Astragaloside IV

AMFGRs:

A. membranaceus field grown roots

AMHRCs:

A. membranaceus hairy root cultures

AMHRLs:

A. membranaceus hairy root lines

ANOVA:

Analyses of variance

CCD:

Central composite design

CY:

Cycloastragenol

DW:

Dry weight

HRCs:

Hairy roots cultures

LC–MS/MS:

Liquid chromatography–tandem mass spectrometry

SRM:

Selected reaction monitoring

TAG:

Total astragalosides

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Acknowledgments

The authors gratefully acknowledge the financial supports by Agricultural Science and Technology Achievements Transformation and Program (2012GB23600641), Fundamental Research Funds for the Central Universities (2572014AA06), Special Fund of National Natural Science Foundation of China (31270618), Key Program of National Natural Science Foundation of China (81274010), and Heilongjiang Province Outstanding Youth Fund (JC201101).

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Authors and Affiliations

  1. State Key Laboratory of Tree Genetics and Breeding, Northeast Forestry University, Harbin, 150040, People’s Republic of China

    Jiao Jiao & Wei Ma

  2. Key Laboratory of Forest Plant Ecology, Ministry of Education, Northeast Forestry University, Harbin, 150040, People’s Republic of China

    Qing-Yan Gai, Yu-Jie Fu, Li-Ping Yao, Chen Feng & Xin-Xin Xia

  3. School of Pharmaceutical, Heilongjiang University of Chinese Medicine, Harbin, 150040, People’s Republic of China

    Wei Ma

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  1. Jiao Jiao
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  2. Qing-Yan Gai
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  3. Yu-Jie Fu
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  4. Wei Ma
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  5. Li-Ping Yao
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  6. Chen Feng
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Correspondence to Yu-Jie Fu or Wei Ma.

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Jiao Jiao and Qing-Yan Gai have contributed equally to this work.

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Jiao, J., Gai, QY., Fu, YJ. et al. Optimization of Astragalus membranaceus hairy roots induction and culture conditions for augmentation production of astragalosides. Plant Cell Tiss Organ Cult 120, 1117–1130 (2015). https://doi.org/10.1007/s11240-014-0668-0

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