1. Develop new rootstocks that impart HLB-tolerance to scion cultivars. Seeds were extracted from 27 candidate rootstock seed source trees, including several UFRs and other experimental rootstocks, that were fund to be free of seed transmissible pathogens by DPI testing. We are authorized to distribute seeds to licensed nurseries for the UFRs, and to collaborative nurseries and other organizations within Florida, in the US, and globally. New trials will be established with these rootstocks in Australia (New South Wales and Queensland) and South Africa in the next year. A rootstock trial with previously exported rootstocks was propagated in Sicily this spring. These trials will provide information relating to performance under diverse soil and environmental conditions and can help development of recommendations for Florida conditions. More than 3000 flowers were pollinated in twenty-one new rootstock cross combinations of 13 different parents, to recombine multiple useful traits for rootstock improvement. We updated and added new data to existing rootstock trial files and added new files to our website (https://crec.ifas.ufl.edu/citrus-research/rootstock-trials/); currently there is information from 26 locations. Seedlings are being selected from 2020 diploid and tetraploid rootstock crosses in calcareous, high pH soil inoculated with two species of Phytophthora, the first step of the `gauntlet’ screening. More than 50 gauntlet rootstock candidates in citripots are growing off for grafting of replicate trees as necessary to meet the new CRDF guidelines for Stage 1 rootstock evaluations (approximately 500 liners). 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. Shoots generated from 37 interploid crosses made using selected HLB tolerant plants in 2020, including 10 crosses for red grapefruit improvement, 10 for sweet orange improvement, and 10 for mandarin improvement are in the process of micrografting on to 3 different rootstocks, and some have been already moved to the greenhouse. Seedlings from somaclone seedling-derived populations of early maturing (January), high soluble solids OLL selections were planted, to screen for even earlier maturing OLL types; seedling populations of a nearly seedless HLB-tolerant mandarin were also planted to get a completely seedless selection, and to extend the maturity season. A September-maturing pink grapefruit hybrid C2-53, with canker and HLB tolerance exceeding standard grapefruit, was approved for release by the IFAS cultivar release committee. 3. Screen our ever-growing germplasm collection for more tolerant types and evaluate fruit quality of candidate selections. We have continued monitoring a unique hybrid family of more than 400 individuals (with many of these planted as 3 tree replicates) from the cross of Clementine mandarin with a wild species reported to be nearly resistant to CaLas attacks, by collecting detailed HLB phenotypic data, including Ct values and other tree health measures as described previously. The frequency of CaLas-negative trees remains unexpectedly high. We have revisited a replicated planting of sweet orange-Poncirus hybrids that was mapped for HLB tolerance QTLs (Huang et al. 2018) and are collecting new phenotypic data to study long term performance of these trees.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 were prepared, each individual was genotyped using the citrus Axiom SNP array, and GWAS analysis is proceeding. This work will validate previously identified, or identify new genomic regions, associated with HLB tolerance or sensitivity. We continued seedling and callus line transformation experiments using the BG gene, and have plants coming from 3 orange, 1 grapefruit, and 2 rootstock lines.