1. Develop new rootstocks that impart HLB-tolerance to scion cultivars. Seed from a first group of rootstock crosses was harvested and planted in the calcareous/Phytophthora soil as the first step in the gauntlet screen; parents included several previously selected but unreleased HLB-tolerant rootstocks, as well as some of the UFRs, HLB-tolerant pummelos, and US-897 and US-942. Fifty seedlings exhibiting tolerance of the poor soil challenge were selected, potted up, and rooted cuttings were produced for further gauntlet steps. Seed from a second group of crosses, using LB8-9 Sugar Belle® and other mandarins as eed parents with pollen from various hybrids of Poncirus trifoliata with citrus accessions, Citrus ichangensis (Ci), different Cleopatra mandarin x Ci hybrids, a Palestine sweet lime x Ci hybrid, and two C. latipes hybrids, were planted in a second round of gauntlet screening, to be completed spring 2022. In collaboration with researchers at IFAPA in Spain, new information has been generated regarding performance of selected UFRs and other unreleased rootstock hybrids from our program in response to drought and flooding, Phytophthora, boron and salinity; UFR-6 has demonstrated good tolerance to all conditions except flooding and salinity, and 9 unreleased rootstock hybrids exhibiting tree size control and HLB tolerance in Florida likewise showed good tolerance of Phytophthora, with one of these showing best performance under all stress conditions. HLB ratings, fruit quality measures, or yield data were collected from 8 different rootstock field trials by our team during the reporting period.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. Following extensive phenotyping of a replicated planting of hybrids between Monreal Clementine and an accession of Citrus latipes (perhaps the most HLB-tolerant citrus), we have found at least two hybrids that remain PCR-negative after 6 years under high pressure in the field, produced large fruit somewhat resembling sweet orange. Hundreds of seeds were collected from crosses made using their pollen onto low-acid breeding parents in our program and planted. Seed from new early-maturing (first week of December) Vernia sweet orange clones, which had higher soluble solids than the other selected early-maturing Vernia clones, were collected and planted. Thirty new grapefruit hybrids generated using HLB and canker tolerant breeding parents were moved from embryo rescue into pots for subsequent field planting. One hundred twenty new EV protoclones, grafted on UFR-15 and US-802, were planted, in efforts to select a more robust early maturing Valencia clone. Three hundred ten new grapefruit hybrids or cybrids were planted at the CREC and in the IR area. Protoplast fusion experiments using W. Murcott suspension protoplasts with various leaf parents were caried out to create new tetraploid breeding parents that can be used for orange and mandarin improvement. 3. Screen our ever-growing germplasm collection for more tolerant types and evaluate fruit quality of candidate selections. We have more than 70 5-year-old-trees of `Marathon’ mandarin on sour orange, that set large crops this last season. Although all trees have HLB, there are few to no obvious disease symptoms in fruit, leaves, or canopy, demonstrating a high degree of tolerance thus far. We have followed closely their performance, and individual trees yielded more than 300 pounds of fruit in 2 harevsts in September and October. Fruit size distributions were determined, and post-harvest behavior and fruit quality data were collected and are now under analysis. We made several selections of apparently HLB-tolerant seedlings from breeding populations of oranges and orange-like hybrids, grapefruit and mandarins, and presented fruit and juice samples at displays in the CREC. 4. Conduct studies to unravel host responses to CLas and select targets for genetic manipulations leading to consumer-friendly new scion and rootstock cultivars. Several new genetic constructs have been developed using newly identified citrus specific promoters (phloem and root tissue), and new putative disease resistance genes, or downstream genes. Transgenic plants have been produced with some of these constructs, and additional transformation experiments are underway with several sweet oranges, grapefruit, and rootstocks. We assessed a diverse population of 459 hybrids from 30 crosses and 53 accessions for HLB tolerance using different morphophysiological traits and compared the relationship of these traits with a visual HLB severity score. We found a significant genetic effect on HLB tolerance which indicates opportunities for genetic improvement of HLB tolerance. Leaf area index (LAI) was the trait most highly correlated with HLB score. suggesting that LAI is a rapid, cost-effective, and reliable method in comparison to other existing HLB phenotyping measurements, and can avoid cognitive bias in phenotyping trees for HLB tolerance.