** AWARDS ISSUED PRIOR TO JANUARY 20, 2025, WERE FUNDED UNDER PREVIOUS ADMINISTRATIONS AND MAY NOT REFLECT THE PRIORITIES AND POLICIES OF THE CURRENT ADMINISTRATION.** AMERICAN CHESTNUTS WERE ONCE AMONG THE MOST PROMINENT HARDWOOD TREES IN EASTERN US FORESTS. THESE LONG-LIVED TREES WERE CULTURALLY, ECOLOGICALLY, AND ECONOMICALLY IMPORTANT: THEY PRODUCED A UNIQUELY CONSISTENT MAST CROP, PROVIDING FOOD TO A VARIETY OF WILDLIFE, AS WELL AS A MARKETABLE FOOD PRODUCT AND PLENTIFUL ROT-RESISTANT LUMBER. AN INVASIVE FUNGUS (CRYPHONECTRIA PARASITICA), INTRODUCED FROM EASTERN ASIA TO THE US IN THE LATE 1800S, CAUSES A DISEASE CALLED CHESTNUT BLIGHT, WHICH KILLED BILLIONS OF AMERICAN CHESTNUT TREES AND EFFECTIVELY REMOVED THEM FROM THEIR ENTIRE NATIVE RANGE BY THE 1950S. SEVERAL EFFORTS HAVE BEEN PREVIOUSLY UNDERTAKEN ATTEMPTING TO RESTORE AMERICAN CHESTNUTS TO THE LANDSCAPE, STARTING WITH USDA-SPONSORED PROGRAMS IN THE EARLY 1900'S TO PLANT NON-NATIVE ASIAN OR HYBRID CHESTNUTS. BY THE 1990S, THE AMERICAN CHESTNUT FOUNDATION (TACF) WAS LEADING EFFORTS TO ENHANCE BLIGHT RESISTANCE THROUGH A BACKCROSS BREEDING PROGRAM, BUT YEARS OF DATA AND NEW GENOMIC ANALYSES HAVE REVEALED THAT BACKCROSSING ALONE IS UNLIKELY TO PRODUCE RESTORATION CHESTNUT TREES WITH OPTIMAL COMBINATIONS OF BLIGHT RESISTANCE AND AMERICAN CHESTNUT TRAITS. WE SUCCESSFULLY PRODUCED A TRANSGENIC TREE THAT EFFECTIVELY TOLERATES CHESTNUT BLIGHT WHILE RETAINING ITS FULL COMPLEMENT OF AMERICAN CHESTNUT TRAITS. THE ENHANCED BLIGHT TOLERANCE COMES FROM A GENE FROM WHEAT CALLED OXALATE OXIDASE (OXO), WHICH DEGRADES A TOXIN (OXALIC ACID) PRODUCED BY THE BLIGHT FUNGUS. THIS MEANS THAT THE TRANSGENIC TREE IS NOT ACTUALLY PREVENTING OR RESISTING BLIGHT INFECTIONS, BUT RATHER TOLERATING THEM WITH LESS DAMAGE. THIS TREE, KNOWN AS DARLING 58, HAS BEEN PLANTED UNDER USDA-APHIS PERMITS AND OUTCROSSED TO MANY WILD AMERICAN CHESTNUTS AND CLOSELY RELATED SPECIES, AND PLANS ARE UNDERWAY TO START RESTORATION EFFORTS PENDING REGULATORY APPROVAL, WHICH IS EXPECTED IN 2023. WITH NUMEROUS SAFETY ASSESSMENTS AND ENVIRONMENTAL COMPARISONS ALREADY COMPLETED (SOME UNDER PREVIOUS BRAG SUPPORT), REGULATORY REVIEWS MOSTLY COMPLETE, AND PUBLIC DISTRIBUTION LIKELY STARTING SOON, THE WORK NOW TRANSITIONS FROM CONFINED RESEARCH PLOTS TO LARGER-SCALE PLANTINGS IN PREPARATION FOR RANGE-WIDE REINTRODUCTION EFFORTS. SOME OF THE SPECIFIC ASSESSMENTS WE WILL CONDUCT UNDER THIS GRANT INCLUDE MONITORING GROWTH, BLIGHT RESISTANCE, FLOWERING DEVELOPMENT, AND PHOTOSYNTHETIC PERFORMANCE OF DARLING 58 CHESTNUT TREES AS THEY START TO MATURE IN REAL-WORLD CONDITIONS. THESE MEASUREMENTS WILL BE FOCUSED ON DARLING 58 CHESTNUTS AND RELATED CONTROLS IN THREE LOCATIONS ACROSS THE CHESTNUT'S RANGE (NY, PA, AND VA), EACH OF WHICH HAS PLOTS IN TWO ENVIRONMENTS (OPEN AND WOODED), FOR A TOTAL OF SIX PLOTS. WE WILL ALSO MEASURE THE DISPERSAL DISTANCE OF DARLING 58 POLLEN IN A ONE-OF-A-KIND PLOT IN NY THAT WAS SPECIFICALLY DESIGNED FOR THIS PURPOSE. THESE TYPES OF OBSERVATIONS WILL BE CRITICALLY IMPORTANT TO UNDERSTAND FITNESS AND LANDSCAPE-SCALE DISPERSAL OF REPRODUCTIVELY MATURE GENETICALLY ENGINEERED TREES,, AND THEY ARE CURRENTLY NOT POSSIBLE IN ALMOST ANY OTHER CONTEXT BESIDES THE ESTABLISHED DARLING 58 CHESTNUT PLOTS IN THIS STUDY. RESULTS FROM THIS WORK WILL BE INFORMATIVE TO DIVERSE AUDIENCES INCLUDING RESTORATION PRACTITIONERS, FOREST LAND MANAGERS, INDIVIDUAL CHESTNUT ENTHUSIASTS, STUDENTS OF VARIOUS AGES, OTHER UNIVERSITY RESEARCHERS, AND FEDERAL BIOTECHNOLOGY REGULATORY AGENCIES AS THEY PREPARE TO EVALUATE OTHER GENETICALLY ENGINEERED TREES IN THE FUTURE. IF SUCCESSFUL AMERICAN CHESTNUT RESTORATION CAN BE INITIATED USING DARLING 58 TREES, THERE WOULD NOT ONLY BE SOCIETAL BENEFITS RESULTING DIRECTLY FROM THE CHESTNUT REINTRODUCTION, BUT THIS WOULD ALSO FACILITATE TREMENDOUS POTENTIAL FOR USING SIMILAR BIOTECHNOLOGIES TO ADDRESS OTHER DIRE THREATS TO NATIVE TREES.