Fluorescent antibiotic resistance marker for tracking plastid transformation
in higher plants
Muhammad Sarwar Khan
& Pal Maliga
Waksman Institute, Rutgers, The State University of
New Jersey, 190 Frelinghuysen Rd., Piscataway,
NJ 08854-8020.
Correspondence should be addressed to Pal Maliga maliga@waksman.rutgers.eduaminoglycoside 3"-adenylyl transferasegreen fluorescent proteinplastid transformationricespectinomycin and streptomycin resistancetobacco
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.
