**AWARDS ISSUED PRIOR TO JANUARY 20, 2025, WERE FUNDED UNDER PREVIOUS ADMINISTRATIONS AND MAY NOT REFLECT THE PRIORITIES AND POLICIES OF THE CURRENT ADMINISTRATION.** 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. TO THIS END, SPECIFIC MEASURES TO INDUCE MALE STERILITY OR TOTAL STERILITY FOR TRANSGENE CONTAINMENT USUALLY NEED TO BE IMPLEMENTED IN PARALLEL WITH THE INTRODUCTION OF GENES OF INTEREST FOR TRAIT MODIFICATIONS IN TRANSGENICS, WHICH NOT ONLY COMPLICATE THE WHOLE PROCESS OF PLANT GENETIC ENGINEERING, BUT ALSO CAUSE ACCUMULATION OF UNWANTED FOREIGN DNAS IN THE HOST GENOME. THE MAJOR OBJECTIVE OF THIS PROJECT IS TO DEVELOP AND EVALUATE A NOVEL APPROACH THAT EXPLORES THE USE OF DIFFERENT MIRNA GENES TAKING ADVANTAGE OF THEIR DUAL ROLES IN PLANT REPRODUCTIVE DEVELOPMENT AND BENEFICIAL AGRONOMIC TRAIT IMPROVEMENT TO PRODUCE SELF-CONTAINED SUPERIOR TRANSGENIC SWITCHGRASS AND TURFGRASS WITH SIGNIFICANTLY IMPROVED MULTIPLE AGRONOMIC TRAITS. SPECIFICALLY, A MICRORNA GENE, MIR396 THAT REGULATES BOTH PLANT STERILITY AND ABIOTIC STRESS RESPONSES WILL BE INTRODUCED INTO SWITCHGRASS AND TURFGRASS TOGETHER WITH THREE ADDITIONAL MIRNA GENES, MIR319, MIR528 AND MIR393 THAT ARE ALL POSITIVE REGULATORS OF PLANT ABIOTIC STRESS RESPONSES. TRANSGENIC LINES WITH THE STACKED MIRNA GENES WILL BE EVALUATED IN THE GREENHOUSE IN POLLEN-CAGE STUDY AND IN THE FIELD TRIAL GENE FLOW STUDY FOR THE EFFICACY OF MIRNA396-MEDIATED STERILITY INDUCTION AND TRANSGENE CONTAINMENT AS WELL AS THE OVERALL IMPACTS OF ALL MIRNA GENES ON BENEFICIAL AGRONOMIC TRAITS. RESULTSFROM THIS RESEARCHWILL ALLOW THE ESTABLISHMENT OF A NOVEL GENE CONTAINMENT SYSTEM IN GRASSES AND PRODUCE ENVIRONMENTALLY SAFE, SELF-CONTAINED SUPERIOR TRANSGENIC PERENNIAL GRASSES WITH IMPROVEMENT OF MULTIPLE BENEFICIAL AGRONOMIC TRAITS. THE SYSTEM DEVELOPED FROM THIS RESEARCH IS ALSO TRANSFERABLE AND COULD EASILY BE ADAPTED TO OTHER PERENNIAL GRASSES AND FOOD CROP SPECIES TO DEVELOP SELF-CONTAINED TRANSGENIC PLANTS WITH SIGNIFICANTLY IMPROVED MULTIPLE TRAITS. THE PROJECT DIRECTLY ADDRESSES USDA BRAG PROGRAM AREA #1 MANAGEMENT PRACTICES TO MINIMIZE THE ENVIRONMENTAL RISK OF GENETICALLY ENGINEERED (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 (1D).