Development of Site-Specific Recombinase Technology for Targeted Integration with Marker Removal from the Plant Genome

Plant biotechnology has a role in addressing global needs for food, fiber and fuel, by developing new crop varieties with increased pest resistance, biofortification, and abiotic stress tolerance. Publicly acceptable forms of biotechnology offer an avenue for meeting these demands. Recombinase-mediated genetic engineering provides a favorable direction for enhancing the precision of biotechnological approaches. Concerns over the presence of antibiotic resistance genes in the food supply and their escape into the environment can be relieved through the use of recombinase technology to excise unwanted DNA from the genome of genetically engineered (GE) crops prior to marketing or release. Previous studies have documented how use of site-specific recombination can produce transgenics with stable gene expression over multiple generations and also resolve multicopy transgene inserts, initially silenced for expression, to a single functional genomic copy.  Research in this lab addresses the need of novel publicly available recombinases. Our team has developed a series of novel recombinases for use as molecular tools. We have identified and characterized the recombinase Bxb1, CinH, and phiC31 for practical use towards genomic engineering of crop plants. Here we present data in tobacco for a practical combinatorial approach where transgene integration occurs with ~26% efficiency in the T0 generation with subsequent male specific excision of the selectable marker genes at ~90% efficiency in the T1 generation.