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Establishment of an Agrobacterium-mediated genetic transformation procedure for the experimental model orchid Erycina pusilla

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Abstract

The establishment of a genetic transformation system for Erycina pusilla (E. pusilla) would be a major step for orchid research and help establish the species as a potential experimental model. Here we describe the development of an efficient selection and plant regeneration protocol for Agrobacterium-mediated transformation of E. pusilla protocorms. Plants were transformed with a binary plasmid containing the Arabidopsis thaliana gene Methionine sulfoxide reductase B7 (MSRB7) using the A. tumefaciens strain EHA105. Explants were selected on hygromycin-containing New Dogashima medium (Tokuhara and Mii in Plant Cell Rep 13(1):7–11, 1993) with 6-benzylaminopurine and α-naphthaleneacetic acid for 5 months to diminish the ratio of false positive explants, and it was determined that 3 month-old E. pusilla protocorms represented the optimal stage for transformation. Positive transformants were confirmed by Southern blot and PCR analyses, and by methyl viologen tolerance assays. Since E. pusilla has a shorter life cycle than most other orchids, self-pollinated T1 progeny was obtained within only 18 months. In addition, the results indicated that overexpression of MSRB genes can be used to avoid the effects of abiotic stress in economically important plants such as orchids.

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Abbreviations

CaMV:

Cauliflower mosaic virus

HPT:

Hygromycin phosphotransferase

MSRB:

Methionine sulfoxide reductase B

MV:

Methyl viologen

PLB:

Protocorm-like bodies

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Acknowledgments

This work was supported by a grant from Academia Sinica, Grant (98-2324-B-001-003-CC1) from the Development Program of Industrialization for Agricultural Biotechnology of the Republic of China and Grant (99AS-1.1.1-FD-Z1) from the Agriculture and Food Agency, Council of Agriculture, Executive Yuan of the Republic of China. We thank Dr. Kah Wee Koh for discussion and manuscript editing. We thank PlantScribe (www.plantscribe.om) for editing this manuscript.

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Authors and Affiliations

  1. Institute of Biotechnology, National Cheng Kung University, Tainan, 701, Taiwan

    Shu-Hong Lee & Ming-Tsair Chan

  2. Academia Sinica Biotechnology Center in Southern Taiwan, Tainan, 741, Taiwan

    Shu-Hong Lee, Chia-Hui Liau, Pao-Yi Chang & Ming-Tsair Chan

  3. Department of Biotechnology, TransWorld University, Douliu City, Yunlin County, 640, Taiwan

    Chia-Wen Li

  4. Institute of Life Science, National Kaohsiung Normal University, Kaohsiung, 802, Taiwan

    Li-Jen Liao

  5. Agricultural Biotechnology Research Center, Academia Sinica, Taipei, 115, Taiwan

    Choun-Sea Lin & Ming-Tsair Chan

Authors
  1. Shu-Hong Lee
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  2. Chia-Wen Li
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  3. Chia-Hui Liau
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  4. Pao-Yi Chang
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  5. Li-Jen Liao
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  7. Ming-Tsair Chan
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Corresponding author

Correspondence to Ming-Tsair Chan.

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Shu-Hong Lee, Chia-Wen Li, Chia-Hui Liau and Pao-Yi Chang have contributed equally to this work.

Electronic supplementary material

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11240_2014_596_MOESM1_ESM.ppt

Schematic map of the T-DNA regions of binary vector, pMSRB7 used for plant transformation. P35S, CaMV 35S promoter; T35S, CaMV 35S terminator; MsrB7, MsrB7 cDNA coding sequence; ATG start site as +1 site; Tnos, nopaline synthase terminator; Hpt, hygromycin phosphotransferase; LB, left border; RB, right border. (PPT 105 kb)

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Lee, SH., Li, CW., Liau, CH. et al. Establishment of an Agrobacterium-mediated genetic transformation procedure for the experimental model orchid Erycina pusilla . Plant Cell Tiss Organ Cult 120, 211–220 (2015). https://doi.org/10.1007/s11240-014-0596-z

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  • DOI: https://doi.org/10.1007/s11240-014-0596-z

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