VISUAL CHARACTERIZATION OF RECOMBINATION AT FRT-GUSA LOCI IN TRANSGENIC TOBACCO MEDIATED BY CONSTITUTIVE EXPRESSION OF THE NATIVE FLP RECOMBINASE

FLP/FRT-mediated site-specific recombination was studied with a recomb ination-reporter gene system which allows visualization of P-glucuroni dase (GUS) expression after site-specific excisional activation of a s ilent gusA gene. This system was used for characterization of the func tional activity of the Saccharomyces cerevisiae native FLP recombinase driven by the cauliflower mosaic virus (CaMV) 35s promoter [linked to the tobacco mosaic virus (TMV) omega translational leader] in mediati ng site-specific recombination of chromosomal FRT sites in tobacco FLP x FRT-reporter hybrids. Six hybrids were generated from crosses of li nes containing either a stably integrated recombination-reporter or a FLP-expression construct. The activated gusA phenotype was specific to hybrid progenies and was not observed in either parental plants or th eir selfed progenies. Recombination efficiency in whole seedlings was estimated by the percent of radioactivity on a Southern blot which was incorporated into the recombined DNA product. Estimated efficiency me an values for the six crosses ranged from 5.2 to 52.0%. Histochemical analysis in hybrid plants visualized GUS activity with variable chimer ic patterns and intensities. Recombination efficiency and GUS expressi on varied both among and within crosses, while higher recombination ef ficiency coincided with larger and more intense patterns of GUS activi ty. These data suggest that recombination is induced randomly during s omatic developmental stages and that the pattern and intensity generat ed in a given plant are affected by factors imposing varibility not on ly between but also within crosses. Additionally, while 1-recombinatio n in a population of FLP/FRT hybrids may occur in all plants, recombin ation efficiency may still be low in any given plant. The activity of the native, as compared to a modified, FLP (Kilby et al. 1995) in the activation of transgenic traits in tobacco is discussed.