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An Protocol for Genetic Transformation of Catharanthus roseus by Agrobacterium rhizogenes A4

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Abstracts

Catharanthus roseus (L.) G. Don is a plant species known for its production of a variety of terpenoid indole alkaloids, many of which have pharmacological activities. Catharanthine can be chemically coupled to the abundant leaf alkaloid vindoline to form the valuable anticancer drug vinblastine. To study and extract catharanthine and other metabolites from C. roseus, a technique was developed for producing hairy root cultures. In this study, the Agrobacterium rhizogenes A4 was induced in the hairy roots from leaf explants, and the concentration of antibiotics (100 mg/L kanamycin) was elucidated for selection after transformation. The polymerase chain reaction amplification of rol genes results revealed that transgenic hairy roots contained rol genes from the root induced (Ri)-plasmid. Catharanthine from C. roseus hairy roots was separated and analyzed using high-performance liquid chromatography. Over-expression of CrOrca3 (octadecanoid-responsive Catharanthus AP2/ERF domain), and cytohistochemical staining methods were used to validate transgenic hairy roots from C. roseus. Hairy root culture of C. roseus is a valuable approach for future efforts in the metabolic engineering of terpenoid indole alkaloids in plants.

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Acknowledgments

We thank Pang Jun-Feng for technical assistance with RNA extraction and Du Hai for work discussions. We are also grateful to Trillian Ross (Plant Cell Physiology, Institute of Biology, Leiden University, The Netherlands) for giving some useful comments on this manuscript. This work was supported by a grant from the National Transgenic Program (2009ZX08005-004B and 2009ZX08009055B).

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  1. Mei-Liang Zhou

    Present address: Plant Cell Physiology, Institute of Biology, Sylvius Laboratory, Leiden University, Sylviusweg 72, P.O. Box 9505, 2300, RA Leiden, The Netherlands

Authors and Affiliations

  1. Biotechnology Research Institute, Chinese Academy of Agricultural Sciences (CAAS), Room 211, Zhongguancun south, Street No. 12, Haidian District, Beijing, 100081, China

    Mei-Liang Zhou, Yan-Min Wu & Yi-Xiong Tang

  2. School of Life Science, Sichuan Agricultural University, Yaan, Sichuan, 625014, People’s Republic of China

    Mei-Liang Zhou & Ji-Rong Shao

  3. School of Resources and Environment, Sichuan Agricultural University, Yaan, Sichuan, 625014, People’s Republic of China

    Xue-Mei Zhu

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  1. Mei-Liang Zhou
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Correspondence to Ji-Rong Shao, Yan-Min Wu or Yi-Xiong Tang.

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Zhou, ML., Zhu, XM., Shao, JR. et al. An Protocol for Genetic Transformation of Catharanthus roseus by Agrobacterium rhizogenes A4. Appl Biochem Biotechnol 166, 1674–1684 (2012). https://doi.org/10.1007/s12010-012-9568-0

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  • DOI: https://doi.org/10.1007/s12010-012-9568-0

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