Marker‐free site‐specific gene integration in rice based on the use of two recombination systems

Summary Transgene integration mediated by heterologous site‐specific recombination (SSR) systems into the dedicated genomic sites has been demonstrated in a few different plant species. This approach of plant transformation generates a precise site‐specific integration (SSI) structure consisting of a single copy of the transgene construct. As a result, stable transgene expression correlated with promoter strength and gene copy number is observed among independent transgenic lines and faithfully transmitted through subsequent generations. Site‐specific integration approaches use selectable marker genes, removal of which is necessary for the implementation of this approach as a biotechnology application. As SSR systems are also excellent tools for excising marker genes from transgene locus, a molecular strategy involving gene integration followed by marker excision, each mediated by a distinct recombination system, was earlier proposed. Experimental validation of this approach is the focus of this work. Using FLPe‐ FRT system for site‐specific gene integration and heat‐inducible Cre‐ lox for marker gene excision, marker‐free SSI lines were developed in the first generation itself. More importantly, progeny derived from these lines inherited the marker‐free locus, indicating efficient germinal transmission. Finally, as the transgene expression from SSI locus was not altered upon marker excision, this method is suitable for streamlining the production of marker‐free SSI lines.