Fluorescent antibiotic resistance marker for tracking plastid transformation in higher plants

Plastid transformation in higher plants is accomplished through a gradual p rocess, during which all the 300-10,000 plastid genome copies are uniformly altered. Antibiotic resistance genes incorporated in the plastid genome fa cilitate maintenance of transplastomes during this process. Given the high number of plastid genome copies in a cell, transformation unavoidably yield s chimeric tissues, which requires the identification of transplastomic cel ls in order to regenerate plants. In the chimeric tissue, however, antibiot ic resistance is not cell autonomous: transplastomic and wild-type sectors both have a resistant phenotype because of phenotypic masking by the transg enic 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 i ndividual transformed and wild-type plastids in tobacco and rice plants aft er bombardment with FLARE-S vector DNA and selection for spectinomycin and streptomycin resistance, respectively. This method facilitates the extensio n of plastid transformation to nongreen plastids in embryogenic cells of ce real crops.