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Fluorescent antibiotic resistance marker for tracking plastid transformation in higher plants

Nature Biotechnology volume 17, pages 910–915 (1999)Cite this article

Abstract

Plastid transformation in higher plants is accomplished through a gradual process, during which all the 300–10,000 plastid genome copies are uniformly altered. Antibiotic resistance genes incorporated in the plastid genome facilitate maintenance of transplastomes during this process. Given the high number of plastid genome copies in a cell, transformation unavoidably yields chimeric tissues, which requires the identification of transplastomic cells in order to regenerate plants. In the chimeric tissue, however, antibiotic resistance is not cell autonomous: transplastomic and wild-type sectors both have a resistant phenotype because of phenotypic masking by the transgenic cells. We report a system of marker genes for plastid transformation, termed FLARE-S, which is obtained by translationally fusing aminoglycoside 3"-adenyltransferase with the Aequorea victoria green fluorescent protein. 3"-adenyltransferase (FLARE-S) confers resistance to both spectinomycin and streptomycin. The utility of FLARE-S is shown by tracking segregation of individual transformed and wild-type plastids in tobacco and rice plants after bombardment with FLARE-S vector DNA and selection for spectinomycin and streptomycin resistance, respectively. This method facilitates the extension of plastid transformation to nongreen plastids in embryogenic cells of cereal crops.

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Figure 1: Plastid transformation vector with FLARE-S as selectable marker.
Figure 2: luorescence microscopy to identify transplastomic leaf sectors by FLARE16-S expression.
Figure 3: Localization of FLARE16-S to tobacco plastids by laser scanning confocal microscopy in heteroplastomic tissue.
Figure 4: Immunoblot analysis of FLARE16-S accumulation in chloroplasts.
Figure 5: Amplification of border fragments confirms integration of FLARE-S genes into the plastid genome.
Figure 6: Localization of FLARE11-S3 to rice chloroplasts in the Os-pMSK49-5 line by laser scanning confocal microscopy.

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Acknowledgements

We thank Hiroshi Kuroda for providing plasmids pHK10 and pHK34, Peter Hajdukiewicz for plasmids containing TpsbA and engineered gfp, and Millie Georgiadis for discussions concerning the design of fusion proteins. This research was supported by the Rockefeller Foundation Rice Biotechnology Program through a Postdoctoral Fellowship Award to M.S.K. and a Research Grant to P.M.

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  1. Waksman Institute, Rutgers, The State University of New Jersey, Piscataway, 08854-8020, 190 Frelinghuysen Rd., NJ

    Muhammad Sarwar Khan & Pal Maliga

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Correspondence to Pal Maliga.

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Khan, M., Maliga, P. Fluorescent antibiotic resistance marker for tracking plastid transformation in higher plants. Nat Biotechnol 17, 910–915 (1999). https://doi.org/10.1038/12907

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