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.
Similar content being viewed by others
Abbreviations
- CaMV:
-
Cauliflower mosaic virus
- HPT:
-
Hygromycin phosphotransferase
- MSRB:
-
Methionine sulfoxide reductase B
- MV:
-
Methyl viologen
- PLB:
-
Protocorm-like bodies
References
Chan MT, Chan YL, Sanjaya (2007) Orchids (Cymbidium spp., Oncidium, and Phalaenopsis). In: Wang K (ed) Agrobacterium protocols volume 2, vol 344. Methods in Molecular Biology. Humana Press, pp 331–338
Chang YY, Chiu YF, Wu JW, Yang CH (2009) Four orchid (Oncidium Gower Ramsey) AP1/AGL9-like MADS box genes show novel expression patterns and cause different effects on floral transition and formation in Arabidopsis thaliana. Plant Cell Physiol 50(8):1425–1438
Chang YY, Kao NH, Li JY, Hsu WH, Liang YL, Wu JW, Yang CH (2010) Characterization of the possible roles for B class MADS box genes in regulation of perianth formation in orchid. Plant Physiol 152(2):837–853
Girhepuje PV, Shinde GB (2011) Transgenic tomato plants expressing a wheat endochitinase gene demonstrate enhanced resistance to Fusarium oxysporum f. sp. lycopersici. Plant Cell Tiss Organ Cult 105:243–251
Guo B, Hexige S, Zhang T, Pittman JK, Chen D, Ming F (2007) Cloning and characterization of a PI-like MADS-box gene in Phalaenopsis orchid. J Biochem Mol Biol 40(6):845–852
Hsu HF, Huang CH, Chou LT, Yang CH (2003) Ectopic expression of an orchid (Oncidium Gower Ramsey) AGL6-like gene promotes flowering by activating flowering time genes in Arabidopsis thaliana. Plant Cell Physiol 44(8):783–794
Hsu CT, Liao DC, Wu FH, Liu NT, Shen SC, Chou SJ, Tung SY, Yang CH, Chan MT, Lin CS (2011) Integration of molecular biology tools for identifying promoters and genes abundantly expressed in flowers of Oncidium Gower Ramsey. BMC Plant Biol 11:60
Kerbauy GB (1984) In vitro flowering of Oncidium varicosum mericlones (orchidaceae). Plant Sci Lett 35(1):73–75
Keshamma E, Rohini S, Rao KS, Madhusudhan B, Udaya Kumar M (2008) Tissue culture-independent in planta transformation strategy: an Agrobacterium tumefaciens-mediated gene transfer method to overcome recalcitrance in cotton (Gossypium hirsutum L.). J Cotton Sci 12(3):264–272
Kwon SJ, Kwon SI, Bae MS, Cho EJ, Park OK (2007) Role of the methionine sulfoxide reductase MsrB3 in cold acclimation in Arabidopsis. Plant Cell Physiol 48(12):1713–1723
Lee SH, Li CW, Koh KW, Chuang HY, Chen YR, Lin CS, Chan MT (2014) MSRB7 reverses oxidation of GSTF2/3 to confer tolerance of Arabidopsis thaliana to oxidative stress. J Exp Bot. doi:10.1093/jxb/eru270
Li CW, Su RC, Cheng CP, Sanjaya, You SJ, Hsieh TH, Chao TC, Chan MT (2011) Tomato RAV transcription factor is a pivotal modulator involved in the AP2/EREBP-mediated defense pathway. Plant Physiol 156(1):213–227
Li CW, Lee SH, Chieh PS, Lin CS, Wang YC, Chan MT (2012) Arabidopsis root-abundant cytosolic methionine sulfoxide reductase B genes MsrB7 and MsrB8 are involved in tolerance to oxidative stress. Plant Cell Physiol 53(10):1707–1719
Li CW, Lee SH, Chan MT (2013) Utilization of the plant methionine sulfoxide reductase B genes as selectable markers in Arabidopsis and tomato transformation. Plant Cell Tiss Organ Cult 113(3):555–563
Liao LJ, Pan IC, Chan YL, Hsu YH, Chen WH, Chan MT (2004) Transgene silencing in Phalaenopsis expressing the coat protein of Cymbidium Mosaic Virus is a manifestation of RNA-mediated resistance. Mol Breed 13(3):229–242
Liau CH, Lu JC, Prasad V, Hsiao HH, You SJ, Lee JT, Yang NS, Huang HE, Feng TY, Chen WH, Chan MT (2003a) The sweet pepper ferredoxin-like protein (pflp) conferred resistance against soft rot disease in Oncidium orchid. Trans Res 12(3):329–336
Liau CH, You SJ, Prasad V, Hsiao HH, Lu JC, Yang NS, Chan MT (2003b) Agrobacterium tumefaciens-mediated transformation of an Oncidium orchid. Plant Cell Rep 21(10):993–998
Lin CS, Chen JJ, Huang YT, Hsu CT, Lu HC, Chou ML, Chen LC, Ou CI, Liao DC, Yeh YY, Chang SB, Shen SC, Wu FH, Shih MC, Chan MT (2013) Catalog of Erycina pusilla miRNA and categorization of reproductive phase-related miRNAs and their target gene families. Plant Mol Biol 82(1–2):193–204
Men S, Ming X, Liu R, Wei C, Li Y (2003) Agrobacterium-mediated genetic transformation of a Dendrobium orchid. Plant Cell Tiss Organ Cult 75(1):63–71
Oh SK, Baek KH, Seong ES, Joung YH, Choi GJ, Park JM, Cho HS, Kim EA, Lee S, Choi D (2010) CaMsrB2, pepper methionine sulfoxide reductase B2, is a novel defense regulator against oxidative stress and pathogen attack. Plant Physiol 154(1):245–261
Pan IC, Liao DC, Wu FH, Daniell H, Singh ND, Chang C, Shih MC, Chan MT, Lin CS (2012) Complete chloroplast genome sequence of an orchid model plant candidate: Erycina pusilla apply in tropical Oncidium breeding. PLoS One 7(4):e34738
Porebski S, Bailey LG, Baum BR (1997) Modification of a CTAB DNA extraction protocol for plants containing high polysaccharide and polyphenol components. Plant Mol Biol Rep 15(1):8–15
Sangwan RS, Bourgeois Y, Sangwan-Norreel BS (1991) Genetic transformation of Arabidopsis thaliana zygotic embryos and identification of critical parameters influencing transformation efficiency. Mol Gen Genet 230(3):475–485
Thiruvengadam M, Yang CH (2009) Ectopic expression of two MADS box genes from orchid (Oncidium Gower Ramsey) and lily (Lilium longiflorum) alters flower transition and formation in Eustoma grandiflorum. Plant Cell Rep 28(10):1463–1473
Thiruvengadam M, Hsu W-H, Yang C-H (2011) Phosphomannose-isomerase as a selectable marker to recover transgenic orchid plants (Oncidium Gower Ramsey). Plant Cell Tiss Organ Cult 104(2):239–246
Tokuhara K, Mii M (1993) Micropropagation of Phalaenopsis and Doritaenopsis by culturing shoot tips of flower stalk buds. Plant Cell Rep 13(1):7–11
Tsai TM, Chen YR, Kao TW, Tsay WS, Wu CP, Huang DD, Chen WH, Chang CC, Huang HJ (2007) PaCDPK1, a gene encoding calcium-dependent protein kinase from orchid, Phalaenopsis amabilis, is induced by cold, wounding, and pathogen challenge. Plant Cell Rep 26(10):1899–1908
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.
Author information
Authors and Affiliations
Corresponding author
Additional information
Shu-Hong Lee, Chia-Wen Li, Chia-Hui Liau and Pao-Yi Chang have contributed equally to this work.
Electronic supplementary material
Below is the link to the electronic supplementary material.
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)
Rights and permissions
About this article
Cite this article
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
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11240-014-0596-z