1. Develop new rootstocks that impart HLB-tolerance to scion cultivars. We have produced a list of candidate rootstock seed trees to be tested by DPI for seed transmissible pathogens. Receiving a clean bill of health for these trees will allow us to harvest and distribute seeds to collaborative nurseries and other organizations within Florida, in the US, and globally, to establish advanced trials to compare our best rootstock selections with industry standards. We continue to update and add new rootstock trial files to our website (https://crec.ifas.ufl.edu/citrus-research/rootstock-trials/), currently there is information from 24 locations. Two rootstock candidates selected from the Gauntlet screen for good HLB tolerance and tree performance have been entered into the DPI Parent Tree Program (DPI-PTP) for shoot-tip grafting.2. Develop new, HLB-tolerant scion cultivars from sweet orange germplasm, as well as other important fruit types such as grapefruit, mandarins, and acid fruit. Embryo rescue from 2019 crosses resulted in shoots from >1750 germinating embryos from 36 interploid crosses (including 11 targeting sweet orange-like hybrids, and 8 targeting grapefruit improvement), and nearly all have now been micrografted to rootstocks, and transferred to the greenhouse to grow off. Cybridization experiments were conducted to combine Meiwa kumquat cytoplasm with OLL and EV sweet oranges, to attempt improvements in citrus canker resistance; plant regeneration is underway. Somatic hybridization of Tango and W. Murcott suspension lines with leaf protoplasts of nine CREC and other public cultivars, and advanced selections, was attempted; most combinations already have produced embryos and shoots for propagation and establishment as new breeding parents for scion improvements. Transformation experiments using possible HLB resistance genes have taken place using seven different scion and rootstock selections, and some are nearing size for micrografting and establishment. Five new seedless and easy to peel mandarins have been entered into the DPI-PTP. Additionally, a nearly seedless Valencia orange mutant selected from an irradiation experiment in the 1990s, exhibiting unusual HLB tolerance compared with other oranges in a replicated planting, has also been entered for cleanup at DPI-PTP. 3. Screen our ever-growing germplasm collection for more tolerant types and evaluate fruit quality of candidate selections. We used new approaches to quantify tree responses to HLB, in addition to the previously used subjective approaches; we measured photosynthetic parameters and leaf canopy indexes, to produce repeatable and reliable quantitative data in support of further genetic analyses of tolerant types. Objective quantitative data of tree responses provides more reliable information that improves the precision with which we can associate genome regions with tolerance or sensitivity; see Obj 4. 4. Conduct studies to unravel host responses to CLas and select targets for genetic manipulations leading to consumer-friendly new scion and rootstock cultivars. We selected ~ 450 mandarin hybrids for GWAS studies, using the data referred to in Obj 3 above. DNA samples from these trees were prepared, and each individual was genotyped using the citrus Axiom SNP array. GWAS analysis is in progress now. This work will validate previously identified or identify new genomic regions associated with HLB tolerance or sensitivity. Further, we have contributed budwood of 54 UF-CREC selections to a top-working trial of approximately 200 selections in total, that is a collaboration with USDA-ARS Ft. Pierce and supported by industry. These have been topworked in a commercial grove and are now growing; this smaller but more diverse population will be used for GWAS in the future. Finally, our multi-location transgenic field permit has been renewed by the USDA; this will enable us to continue testing the effects of certain genetic modifications on HLB incidence, disease development, and potential tolerance or resistance, under real world field conditions.