FLP/FRT-mediated restoration of normal phenotypes and clonal sectors formation in rolC transgenic tobacco.

Abstract

Site-specific recombination systems have been shown to excise transgene DNA sequences positioned between their cognate target sites, and thus be used to generate clonal sectors in transgenic plants. Here we characterized clonal sectors derived from genetic reversion of rolC (A. rhizogenes)--induced vegetative and reproductive phenotypes, mediated by FLP recombinase from S. cerevisiae, in tobacco. The constitutive expression of rolC induces pleiotropic effects including reduced apical dominance and plant height, lanceolate and pale green leaves and small, male-sterile flowers. Two transgenic male-sterile tobacco lines (N. tabacum, Samsun NN) expressing a 35sP-rolC gene construct flanked by two FRT (FLP recombinase target) sites, were cross-pollinated with pollen from a constitutive 35sP-FLP expressing line. Three main phenotypes were generated in result of recombinase-mediated excision of the 35sP-rolC locus in the F1 (FLP x FRT-35sP-rolC-FRT) hybrid progenies: (a) restoration of male fertility, associated with reversion to normal leaf phenotypes prior to flower bud formation, (b) development of normal and fertile lateral shoot sectors on the background of rolC-type plants, (c) restoration of partially fertile flowers, associated with display of peripheral normal leaf sectors surrounding rolC-type inner-leaf tissues, consistent with periclinal chimeras. These results, supported by DNA molecular analysis, indicate that site-specific recombination might be used as a relatively efficient tool for generation of transgenic periclinal chimeric plants.