• Open Access

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

Authors

  • Soumen Nandy,

    1. Department of Crop, Soil & Environmental Sciences, University of Arkansas, Fayetteville, AR, USA
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  • Vibha Srivastava

    Corresponding author
    1. Department of Crop, Soil & Environmental Sciences, University of Arkansas, Fayetteville, AR, USA
    2. Department of Horticulture, University of Arkansas, Fayetteville, AR, USA
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(Tel 479 575 4872; fax 479-575-7465; email vibhas@uark.edu)

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.

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