Abstract
A plastid transformation vector was constructed for dicistronic expression of the aminoglycoside 3′-adenyltransferase (aadA) and green fluorescent protein (gfp) genes under the control of the plastid rrn promoter. Gold particles coated with the vector DNA were bombarded onto tobacco leaf explants using a particle delivery system. Leaf explants produced adventitious shoots when cultured on shoot-inducing medium containing 500 mg l−1 spectinomycin. Shoots that exhibited green fluorescence under UV light were selected. Southern blot analysis detected the presence of the aadA and gfp genes between trnA and trnI in the plastid genome. Northern blot analysis revealed that the aadA and gfp genes were both properly transcribed into a dicistronic transcriptional unit. The expression of the gfp gene in the plastid enabled separation of transformed chloroplasts from wild-type chloroplasts in the protoplast under a fluorescent microscope. The overall results indicate that dicistronic expression of the aadA and gfp genes in the plastid simplifies gene manipulation, facilitating selection and tracking of plastid-transformed cells.
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Abbreviations
- aadA :
-
Aminoglycoside 3′-adenyltransferase
- FITC :
-
Fluorescein isothiocyanate
- gfp :
-
Green fluorescent protein
- MS :
-
Murashige and Skoog
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Acknowledgements
This work was supported by a grant (no. CG2111) to J.R.L. from the Crop Functional Genomics Center of the 21st Century Frontier Research Program funded by the Korea Ministry of Science and Technology, a grant to J.R.L. from KRIBB Research Initiative Program, a grant to J.R.L. from the Korea Science and Engineering Foundation through the Plant Metabolism Research Center of the Kyung Hee University, and a grant to D.-W.C. from the Next-Generation New Technology Development Program funded by the Korean Ministry of Commerce, Industry and Energy.
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Communicated by I.S. Chung
S.-W. Jeong and W.-J. Jeong contributed equally to this work
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Jeong, SW., Jeong, WJ., Woo, JW. et al. Dicistronic expression of the green fluorescent protein and antibiotic resistance genes in the plastid for selection and tracking of plastid-transformed cells in tobacco. Plant Cell Rep 22, 747–751 (2004). https://doi.org/10.1007/s00299-003-0740-4
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DOI: https://doi.org/10.1007/s00299-003-0740-4