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Developing an Agrobacterium tumefaciens-mediated genetic transformation for a selenium-hyperaccumulator Astragalus racemosus

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Abstract

Agrobacteriumtumefaciens strain LBA4404 containing the plasmid pBI121, carrying the reporter gene uidA and the kanamycin resistance gene nptII, was used for gene transfer experiments in selenium (Se)-hyperaccumulator Astragalus racemosus. The effects of kanamycin on cell growth and division and acetosyringone on transformation efficiency were evaluated. The optimal concentration of kanamycin that could effectively inhibit cell growth and division in non-transgenic tissues was 50 mg l−1 and thus all putative transgenic plants were obtained on induction medium containing 50 mg l−1 kanamycin. The verification of transformants was achieved by both histochemical GUS assay and PCR amplification of nptII gene. Southern blot analysis was performed to further confirm that transgene nptII was stably integrated into the A. racemosus genome. A transformation frequency of approximately 10% was achieved using this protocol, but no beneficial effect from the addition of acetosyringone (50 μM) was observed. This transformation system will be a useful tool for future studies of genes responsible for Se-accumulation in A. racemosus.

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Abbreviations

CPPU:

N-(2-chloro-4-pyridyl)-N′-phenylurea

GUS:

β-Glucuronidase

nptII:

Neomycin phosphotransferase II

NAA:

α-Naphthaleneacetic acid

MS:

Murashige and Skoog’s medium

PCR:

Polymerase chain reaction

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Acknowledgments

This work was supported by a Biotechnology Research Grant (2007-BRG-1223) from the North Carolina Biotechnology Center, a United States Department of Agriculture—Cooperative State Research, Education, and Extension Service grant (2009-35318-05032) and a startup fund from the Golden LEAF Foundation to the Biomanufacturing Research Institute & Technology Enterprise (BRITE).

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Authors and Affiliations

  1. Department of Pharmaceutical Sciences, Biomanufacturing Research Institute & Technology Enterprise, North Carolina Central University, Durham, NC, 27707, USA

    Diane E. Darlington, Chiu-Yueh Hung & Jiahua Xie

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  1. Diane E. Darlington
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  2. Chiu-Yueh Hung
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  3. Jiahua Xie
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Correspondence to Jiahua Xie.

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Darlington, D.E., Hung, CY. & Xie, J. Developing an Agrobacterium tumefaciens-mediated genetic transformation for a selenium-hyperaccumulator Astragalus racemosus . Plant Cell Tiss Organ Cult 99, 157–165 (2009). https://doi.org/10.1007/s11240-009-9588-9

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