Kanamycin resistance as a selectable marker for plastid transformation in tobacco |
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Authors: | Helaine Carrer Tish Noel Hockenberry Zora Svab Pal Maliga |
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Affiliation: | (1) Waksman Institute, Rutgers, The State University of New Jersey, 08855-0759 Piscataway, NJ, USA;(2) Present address: Centro de Biotecnologia Agricola, Escola Superior de Agricultura Luiz de Queiroz, Universidade de Sao Paulo, Ave. Padua Dias, 11, 13400 Piracicaba-SP, Brazil |
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Abstract: | We report on a novel chimeric gene that confers kanamycin resistance on tobacco plastids. The kan gene from the bacterial transposon Tn5, encoding neomycin phosphotransferase (NPTII), was placed under control of plastid expression signals and cloned between rbcL and ORF512 plastid gene sequences to target the insertion of the chimeric gene into the plastid genome. Transforming plasmid pTNH32 DNA was introduced into tobacco leaves by the biolistic procedure, and plastid transformants were selected by their resistance to 50 g/ml of kanamycin monosulfate. The regenerated plants uniformly transmitted the transplastome to the maternal progeny. Resistant clones resulting from incorporation of the chimeric gene into the nuclear genome were also obtained. However, most of these could be eliminated by screening for resistance to high levels of kanamycin (500 g/ml). Incorporation of kan into the plastid genome led to its amplification to a high copy number, about 10000 per leaf cell, and accumulation of NPTII to about 1% of total cellular protein. |
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Keywords: | Tobacco Plastid Kanamycin resistance Neomycin phosphotransferase Protein expression |
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