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A system for the transformation and regeneration of the recretohalophyte Limonium bicolor

  • Plant Tissue Culture
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Abstract

Limonium bicolor, a typical recretohalophyte, has a specialized salt-secreting structure in the epidermis called the salt gland and plays a significant role in improving saline land. Understanding the molecular mechanisms of salt secretion and salt gland development requires an efficient L. bicolor transformation system, which is described in this report. Leaf explants were incubated with Agrobacterium tumefaciens strain EHA105 harboring the plasmid pTCK303 containing the β-glucuronidase gene (GUS) as the transgene reporter and the hygromycin B resistance gene as a selectable marker. Up to 96.9% of leaves were induced to regenerate shoots on an Murashige and Skoog (MS) medium supplemented with 4.4 μM 6-benzyladenine and 1.1 μM α-naphthaleneacetic acid; roots were induced on the MS medium containing 2.5 μM indole-3-butyric acid. This tissue culture system was suitable for Agrobacterium-mediated transformation of L. bicolor. Pre-cultivated explants (2 d old) were incubated with Agrobacterium (0.6–0.7 at OD600) in a shaking culture for 20 min; the explants and bacterium were co-cultivated for 4 d in the dark before the explants were transferred to a selection medium containing 8 mg/L hygromycin B and 600 mg/L piperacillin sodium (added to prevent continued Agrobacterium growth). Histochemical assays and PCR to detect the GUS gene showed that transformation frequency was 4.43%. Quantitative PCR and Northern blotting further verified the integration and presence of the GUS gene in L. bicolor. This is the first report of an Agrobacterium-based transformation system for L. bicolor. The system will facilitate a research on the identity and function of genes involved in salt gland development and salt secretion.

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Acknowledgments

This work has been supported by the NSFC (National Natural Science Research Foundation of China, project no. 30870138 and no. 31070158), key projects in the National Science & Technology Pillar Program during the 11th 5-yr plan period (2009BADA7B05) and by the Program for Scientific Research Innovation Team in Colleges and Universities of Shandong Province. We thank Prof. Kang Chong (Institute of Botany, Chinese Academy of Sciences) for kindly providing the plasmid pTCK303.

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Authors and Affiliations

  1. Key Lab of Plant Stress Research, College of Life Science, Shandong Normal University, Ji’nan, 250014, Shandong, China

    Fang Yuan, Min Chen, Jianchao Yang, Bingying Leng & Baoshan Wang

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  1. Fang Yuan
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  2. Min Chen
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  3. Jianchao Yang
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  4. Bingying Leng
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  5. Baoshan Wang
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Correspondence to Baoshan Wang.

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Editor: John W. Forster

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Yuan, F., Chen, M., Yang, J. et al. A system for the transformation and regeneration of the recretohalophyte Limonium bicolor . In Vitro Cell.Dev.Biol.-Plant 50, 610–617 (2014). https://doi.org/10.1007/s11627-014-9611-7

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  • DOI: https://doi.org/10.1007/s11627-014-9611-7

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