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Overexpression of farnesyl pyrophosphate synthase (FPS) gene affected artemisinin content and growth of Artemisia annua L.

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Abstract

Transgenic plants of Artemisia annua L., a medicinal plant that produces the compound artemisinin which has an anti-malarial activity, were developed following Agrobacterium tumefaciens-mediated transformation of leaf explants. A. tumefaciens strain EHA105 carrying either pCAMBIA1301 or pCAMBIAFPS was used. Both plasmids harbored the hygromycin phosphotransferase II (hptII) gene as a selectable gene, but the latter plasmid also harbored the gene encoding for farnesyl pyrophosphate synthase (FPS), a key enzyme for artemisinin biosynthesis. Shoot regeneration was observed either directly from leaf sections or via intervening callus when explants were incubated on solidified Murashige and Skoog (MS) (1962) medium containing 0.1 mg l−1 α-naphthaleneacetic acid (NAA), 1 mg l−1 N6-benzyladenine (BA), 30 mg l−1 meropenem and 10 mg l−1 hygromycin. Applying vacuum infiltration dramatically increased transformation efficiency up to 7.3 and 19.7% when plasmids with and without FPS gene were used, respectively. All putative transgenic regenerants showed positive bands of hptII gene following Southern blot analysis. Expression of FPS was observed in all transgenic lines, and FPS over-expressed lines exhibited higher artemisinin content and yield, of 2.5- and 3.6-fold, respectively, than that detected in wild-type plants. A relatively high correlation (R 2 = 0.78) was observed between level of expression of FPS and artemisinin content. However, gene silencing was detected in some transgenic lines, especially for those lines containing two copies of the FPS transgene, and with some lines exhibiting reduced growth.

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Abbreviations

FPS :

Farnesyl pyrophosphate synthase gene

GUS:

β-glucuronidase

hptII:

Hygromycin phosphotransferase gene

NAA:

α-naphthaleneacetic acid

BA:

N6-benzyladenine

PGR:

Plant growth regulators

FW:

Fresh weight

DW:

Dry weight

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Acknowledgments

This research work was supported by a Mahidol University grant, Thailand. The authors would like to thanks the scholarship from The Royal Golden Jubilee PhD Program (RGJ) of the Thailand Research Fund and partial support from Graduate Institute of Science and Technology (TGIST). This work is under the collaboration of Mahidol University and Chiba University for double degree program.

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Authors and Affiliations

  1. Department of Biotechnology, Faculty of Science, Mahidol University, Bangkok, 10400, Thailand

    Waleerat Banyai & Kanyaratt Supaibulwatana

  2. National Center For Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Thailand Science Park, Pathumthani, 12120, Thailand

    Chalermpol Kirdmanee

  3. Laboratory of Plant Cell Technology, Faculty of Horticulture, Chiba University, Chiba, 271-8510, Japan

    Waleerat Banyai & Masahiro Mii

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  1. Waleerat Banyai
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  2. Chalermpol Kirdmanee
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  3. Masahiro Mii
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  4. Kanyaratt Supaibulwatana
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Correspondence to Kanyaratt Supaibulwatana.

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Banyai, W., Kirdmanee, C., Mii, M. et al. Overexpression of farnesyl pyrophosphate synthase (FPS) gene affected artemisinin content and growth of Artemisia annua L.. Plant Cell Tiss Organ Cult 103, 255–265 (2010). https://doi.org/10.1007/s11240-010-9775-8

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  • DOI: https://doi.org/10.1007/s11240-010-9775-8

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