TURFGRASS AND SWITCHGRASS ARE AMONG THE MOST IMPORTANT PERENNIAL GRASSES SIGNIFICANTLY IMPACTING AGRICULTURE PRODUCTION, AGRICULTURE ECONOMY, SUSTAINABLE ENERGY AND ENVIRONMENT. LIKE IN MANY ROW CROPS, GENETIC ENGINEERING OF BOTH SWITCHGRASS AND TURFGRASSES USING TRANSGENIC TECHNOLOGIES OFFERS THE OPPORTUNITY TO INCORPORATE MANY ECONOMIC AND AGRONOMIC BENEFITS THAT ARE DIFFICULT OR IMPOSSIBLE TO ACHIEVE THROUGH TRADITIONAL BREEDING TECHNIQUES. HOWEVER, THE RISK OF TRANSGENE ESCAPE AND THE UNFORESEEN ENVIRONMENTAL CONSEQUENCE BY THE USE OF TRANSGENIC TECHNOLOGY IN PERENNIAL GRASSES REQUIRE DEVELOPMENT OF STRATEGIES FOR TRANSGENE CONTAINMENT. THE MAJOR OBJECTIVE OF THIS PROJECT IS TO DEVELOP AND EVALUATE AN EFFECTIVE MOLECULAR STRATEGY COMBINING AN INTEGRATED DUAL SITE-SPECIFIC RECOMBINATION SYSTEM WITH RELIABLE TOTAL STERILITY INDUCTION MECHANISMS TO PRODUCE TRANSGENIC SWITCHGRASS AND TURFGRASS SELF-CONTAINED FOR DESIRABLE TRANSGENE, BUT FREE OF UNDESIRABLE FOREIGN DNAS. SPECIFICALLY,WE WILL IMPLEMENT A DUAL SITE-SPECIFIC RECOMBINATION PLATFORM THAT WE HAVE PREVIOUSLY TESTED FOR ELIMINATING ALL TRANSGENES NO LONGER DESIRED IN THE FINAL TRANSGENIC PRODUCT. WE WILL ALSO CREATE TOTAL STERILITY FOR TRANSGENE CONTAINMENT BY RNAI- AND CRISPR/CAS9-MEDIATED TARGET GENE KNOCKOUT. THE DUAL SITE-SPECIFIC RECOMBINATION PLATFORM AND THE STERILITY-INDUCING MECHANISMS AS WELL AS THE GENE OF INTEREST (GOI) FOR TRAIT MODIFICATION WILL ALL BE COMBINED INTO AN INTEGRATED SYSTEM IN THE FINAL PRODUCT, GENERATING ENVIRONMENTALLY SAFE, CLEAN TRANSGENIC PERENNIAL GRASSES WITH GENETICALLY IMPROVED TRAITS FOR COMMERCIALIZATION. THE KEY COMPONENTS OF THIS INTEGRATED SYSTEM AND THE GOI WILL BE BUILT INTO TWO CHIMERIC GENE CONSTRUCTS, WHICH WILL THEN BE TRANSFORMED INTO SWITCHGRASS AND TURFGRASS, PRODUCING TWO GROUPS OF DISTINCT PARENTAL TRANSGENIC LINES. NEITHER THE DUAL SITE-SPECIFIC RECOMBINATION PLATFORM NOR THE STERILITY-INDUCING MECHANISM IS FUNCTIONAL IN THE TWO PARENTAL LINES AND THE GOI WILL NOT BE EXPRESSED IN THE PARENTAL LINES EITHER. THIS ALLOWS TWO PARENTAL LINES TO BE SEXUALLY PROPAGATED UNDER CONTAINED CONDITIONS. UPON CROSS-POLLINATION OF THE TWO PARENTAL LINES, THE DUAL SITE-SPECIFIC RECOMBINATION PLATFORM, THE MECHANISMS FOR STERILITY AND GOI EXPRESSION INDUCTION ARE ALL COMBINED INTO AN INTEGRATED, FULLY FUNCTIONAL SYSTEM IN THE HYBRIDS, RESULTING IN TOTAL STERILITY, THE EXPRESSION OF THE GOI AND THE REMOVAL OF ALL TRANSGENES NO LONGER NEEDED. ADDITIONALLY, THE HYBRID VIGOR OF BENEFICIAL TRAITS IN THE FINAL TRANSGENICS IS ALSO ADVANTAGEOUS FOR PRODUCT COMMERCIALIZATION. THE SYSTEM DEVELOPED IS ALSO TRANSFERABLE AND COULD EASILY BE ADAPTED TO OTHER PERENNIAL GRASSES AND FOOD CROP SPECIES FOR UNDESIRABLE TRANSGENE CLEANING AND TRANSGENE CONTAINMENT. OUR PROJECT DIRECTLY ADDRESSES USDA BRAG PROGRAM AREA #1 MANAGEMENT PRACTICES TO MINIMIZE THE ENVIRONMENTAL RISK OF GE ORGANISMS AND SPECIFICALLY, DEVELOPMENT OR EVALUATION OF EFFECTIVE STRATEGIES, INCLUDING MOLECULAR AND/OR GENETIC, TO LIMIT GENE TRANSFER (GENE FLOW) OR OUTCROSSING TO SEXUALLY COMPATIBLE ORGANISMS OR TRANSFER OF GENETIC MATERIAL BETWEEN VIRUSES, INSECTS, OR MICROORGANISMS.