GENOME EDITING IS A REVOLUTIONARY TECHNOLOGY THAT ALLOW ONE TO MAKE PRECISE CHANGES IN A PLANT'S GENETIC BLUEPRINT FOR AGRICULTURE. HOWEVER, ITS APPLICATION TO MANY MINOR CROPS LIKE ALFALFA, GRAPEVINES, LEMONS, LIMES AND ORANGES, IS LIMITED. THE LIMITATION IN USING GENE-EDITING TECHNIQUES IN THESE CROPS IS MAINLY DUE TO THE INABILITY OF STEM CELLS OF THESE PLANTS TO REFORM WHOLE PLANTS IN TISSUE CULTURE. THERE ARE ONLY A VERY FEW VARIETIES OF ALFALFA, ORANGES, LIMES, LEMONS AND GRAPEVINES FOR WHICH WE ARE CAPABLE OF FORMING WHOLE PLANTS FROM CELLS IN TISSUE CULTURE AND THE FEW VARIETIES THAT WE HAVE BEEN SUCCESSFUL WITH DO SO AT A VERY LOW FREQUENCY.WE RECENTLY DISCOVERED THAT A GENE SEQUENCE ENCODING A CHIMERIC PROTEIN THAT COMPRISES A GROWTH REGULATING FACTOR (GRF) AND ITS COFACTOR GRF INTERACTING FACTOR (GIF) (HENCEFORTH GRF-GIF) DRAMATICALLY INCREASED THE ABILITY OF WHEAT STEM CELLS TO REFORM WHOLE PLANTS IN TISSUE CULTURE. WHEN COMBINED WITH THE GENE EDITING SYSTEM CRISPR-CAS9, WE FOUND THAT THIS TECHNOLOGY ALSO SIGNIFICANTLY INCREASES THE TOTAL NUMBER OF GENE-EDITED PLANTS WE CAN PRODUCE USING TISSUE CULTURE. A NOVEL FEATURE OF THIS TECHNOLOGY IS THAT ALTHOUGH THE CHIMERIC GENE ENHANCES REGENERATION IN YOUNG CELLS, THE CHIMERIC GENE IS TURNED OFF IN OLDER TISSUE SO THAT THE PLANT GROWS NORMALLY TO MATURITY. IN WHEAT, THE TECHNOLOGY ALSO ALLOWS US TO MAKE GENE EDITS IN PLANTS WITHOUT THE USE OF A PLANT SELECTABLE MARKER GENE. THE GRF-GIF SYSTEM STIMULATES THE CELLS THAT CONTAIN IT TO REFORM PLANTS ON TISSUE CULTURE PLATES THAT LACK CERTAIN GROWTH FACTORS NORMALLY ADDED TO THE CULTURE MEDIUM AND WHICH ARE REQUIRED FOR THE CELLS TO REFORM PLANTS IN A PETRI DISH. PLANT SELECTABLE MARKER GENES ARE USED TO ALLOW PLANT CELLS TO GROW ON COMPOUND ON WHICH THEY NORMALLY CANNOT GROW. THESE SELECTIVE AGENTS ALLOWS ONLY CELLS CONTAINING THE NEW GENE EDIT ALONG WITH THE PLANT SELECTABLE MARKER GENE TO REFORM WHOLE PLANTS IN THE PRESENCE OF THE SELECTIVE AGENT. PLANT SELECTABLE MARKER GENES CURRENTLY USED ARE OFTEN THOSE THAT CONFER RESISTANT TO ANTIBIOTICS OR HERBICIDES. ALTHOUGH REGULATORY AGENCIES HAVE APPROVED GENETICALLY MODIFIED PLANTS CONTAINING THESE GENES FOR COMMERCIALIZATION, THE PUBLIC WOULD PREFER NOT TO HAVE THESE GENES IN THEIR FOOD SUPPLY.PROMISING PRELIMINARY RESULTS IN CITRUS SUGGEST THAT THIS TECHNOLOGY MAY BE APPLICABLE TO MULTIPLE PLANT SPECIES. THEREFORE THE OVERALL GOAL OF THIS PROPOSAL IS TO USE THIS TECHNOLOGY TO EXPAND THE BENEFITS OF GENE EDITING TO MINOR CROPS BY INCREASE THE EFFICIENCY WITH WHICH PLANT CELLS REFORM PLANTS ACROSS MULTIPLE VARIETIES OF ALFALFA, CITRUS AND VITIS VINIFERA. IN ADDITION, WE WANT TO USE THE ABILITY OF THE GRF-GIF TECHNOLOGY TO REPLACE ANTIBIOTIC AND HERBICIDE PLANT CELL SELECTION SYSTEM. LASTLY, WE WANT TO COMBINE GRF-GIF TECHNOLOGY WITH GENOME EDITING FOR EACH OF THE CROP TO ENHANCE THE PRODUCTION OF GENE-EDITED PLANTS. THIS PROJECT WILL OPEN THE DOOR TO MULTIPLE MINOR CROPS TO BENEFIT FROM THE POWER OF GENOME EDITING TECHNOLOGIES