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Optimization of the conditions for Casuarina cunninghamiana Miq. genetic transformation mediated by Agrobacterium tumefaciens

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Abstract

Using epicotyl fragments of the actinorhizal tree Casuarina cunninghamiana and the disarmed strain of Agrobacterium tumefaciens C58C1 (pGV2260) containing the pBIN19-35S-GUSINT binary vector, a method for the genetic transformation of C. cunninghamiana was established. Transformed cells were initially selected for 2 weeks on nutrient medium supplemented with kanamycin 20 mg L−1 during callus induction, and then subcultured with 50 mg L−1 until adventitious bud and shoot differentiation. Different factors involved in the early stages of the T-DNA transfer process were studied. Agrobacterium-mediated DNA delivery was most successful when epicotyl fragments excised from 45-day-old seedlings were co-cultivated with an exponentially growing culture of A. tumefaciens at an OD600nm of 0.3, for 5 days in the presence of 50 μM of acetosyringone. Kanamycin resistant calli were observed on 88.89 % of the explants and transgenic rooted C. cunninghamiana plants were obtained in 6 months. Evidence of genetic transformation was demonstrated by ß-glucuronidase histochemical assays and polymerase chain reaction analyses. The possibility to obtain transgenic nitrogen-fixing nodules after inoculation by the soil actinomycete Frankia was established.

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Acknowledgments

This study was financially supported by the 12th National Five-Year Plan for Forestry Project (2012BAD01B0603), the Ministry of Finance of China through a Specific Program for National Non-profit Scientific Institutions (RITFYWZX201203) and Guangdong Province Forestry Science & Technology Innovation Project (2014KJCX017).

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The authors declare that they have no conflict of interest.

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Authors and Affiliations

  1. Research Institute of Tropical Forestry, Chinese Academy of Forestry, Guangzhou, 510520, People’s Republic of China

    Qingbin Jiang, Chonglu Zhong, Bingshan Zeng & Yong Zhang

  2. College of Life Science and Technology, Central South University of Forestry and Technology, Changsha, 410004, People’s Republic of China

    Yingzi Ma

  3. Black Mountain Laborartories, CSIRO National Research Collections Australia, Canberra, ACT, 2600, Australia

    Khongsak Pinyopusarerk

  4. Equipe Rhizogenèse, UMR DIADE, Institut de Recherche pour le Développement, 911 avenue Agropolis, BP64501, 34394, Montpellier Cedex 5, France

    Didier Bogusz & Claudine Franche

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  1. Qingbin Jiang
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  3. Chonglu Zhong
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Correspondence to Chonglu Zhong.

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Qingbin Jiang and Yingzi Ma equally contributed to this work.

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Jiang, Q., Ma, Y., Zhong, C. et al. Optimization of the conditions for Casuarina cunninghamiana Miq. genetic transformation mediated by Agrobacterium tumefaciens . Plant Cell Tiss Organ Cult 121, 195–204 (2015). https://doi.org/10.1007/s11240-014-0694-y

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