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Plant Cell, Tissue and Organ Culture (PCTOC)

, Volume 120, Issue 1, pp 211–220 | Cite as

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

  • Shu-Hong Lee
  • Chia-Wen Li
  • Chia-Hui Liau
  • Pao-Yi Chang
  • Li-Jen Liao
  • Choun-Sea Lin
  • Ming-Tsair Chan
Original Paper

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.

Keywords

Methionine sulfoxide reductase Methyl viologen Orchid 

Abbreviations

CaMV

Cauliflower mosaic virus

HPT

Hygromycin phosphotransferase

MSRB

Methionine sulfoxide reductase B

MV

Methyl viologen

PLB

Protocorm-like bodies

Notes

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.

Supplementary material

11240_2014_596_MOESM1_ESM.ppt (106 kb)
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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Copyright information

© Springer Science+Business Media Dordrecht 2014

Authors and Affiliations

  • Shu-Hong Lee
    • 1
    • 2
  • Chia-Wen Li
    • 3
  • Chia-Hui Liau
    • 2
  • Pao-Yi Chang
    • 2
  • Li-Jen Liao
    • 4
  • Choun-Sea Lin
    • 5
  • Ming-Tsair Chan
    • 1
    • 2
    • 5
  1. 1.Institute of BiotechnologyNational Cheng Kung UniversityTainanTaiwan
  2. 2.Academia Sinica Biotechnology Center in Southern TaiwanTainanTaiwan
  3. 3.Department of BiotechnologyTransWorld UniversityDouliu CityTaiwan
  4. 4.Institute of Life ScienceNational Kaohsiung Normal UniversityKaohsiungTaiwan
  5. 5.Agricultural Biotechnology Research CenterAcademia SinicaTaipeiTaiwan

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