Updates for this quarter:Site management and field trials are progressing well. The site remains available for access to all researchers and all regulatory protocols for the care and disposal of transgenic material are being observed. The trees have been hedged and topped to promote growth, open canopies and access to nutritional sprays. The foliar spray program, applied every two weeks, includes: 435 horticultural oil, Magna-Bon CS2005 copper, 20-10-20 and/or 15-5-15 CA-MG, N-Sure 28-0-0, Keyplex 1400 DP, and Ocean Organics 0.2-0-6. Wedgeworth’s 12-13-15, 2Mg slow release nitrogen and potassium with greening guard was also applied above the root zone in March. Discussions have begun with APHIS-BRS to set conditions for new or expanded transgenic release permits to allow field trials of a novel system using engineered tissue to delivery therapeutic peptides to non-transgenic trees. This effort is in support of NIFA project 2020-70029-33176, Therapeutic Molecule Evaluation and Field Delivery Pipeline for Solutions to HLB, with field trials expected to begin later this year once all regulatory requirements are met. The testing site is also being actively utilized this breeding season. Crosses have been made with transgenic pollen to help elucidate if sexual embryos can be rescued from polyembryonic females, making use of the transgenic markers to determine if sexual hybridization is successful. This could greatly impact both transgenic and conventional breeding efforts by allowing the use of polyembryonic citrus accessions as females. A third year of crossings has also been made with the early flowering (FT) transgenics, continuing the work described below. Recent quarters:A significant USDA-funded infrastructure project has been completed, fully renovating the water management systems and significantly improving storm and flood protection. USDA has also acquired a topper hedger to facilitate canopy management and reflect the best practices of commercial farms. An additional BRS transgenic release permit was approved (AUTH – 0000043620) for material with confidential business information (CBI) for a project led by R. Shatters. The primary BRS permit has also been renewed and amended to include a new construct from UF (Now AUTH – 0000206702). The annual site review from APHIS/BRS has been conducted successfully. Four new plantings from UF expressing resistance genes and two new plantings from USDA-CRADA partners expressing antimicrobial peptides and anti-CLas plantibodies have been made. With recent plantings, the transgenic site is operating at full capacity. Fall 2021 assessments were completed for USDA plantings as described below for trials #8, #9, #10, #11 and #15. Fruit have been harvested from the second year of controlled crosses using pollen from early flowering (FT) transgenics on traditional varieties maintained in the testing site. Seeds from these fruit and those of future crossings will be used to assess inheritability of the phenotype and for CRISPR gene stacking to combine genome editing with accelerated breeding traits. The UCRiverside-led trifoliate and trifoliate hybrid trial has concluded, a manuscript regarding identified HLB-tolerance is in preparation; and these trees can be removed as needed to make space available for future plantings. Dr. Stover analyzed data on canker incidence for this trial and a manuscript detailing these results has been published in HortScience DOI: 10.21273/HORTSCI15684-20. Previously established at the site:A number of trials are underway at the CRDF funded Picos Test Site. A detailed current status is outlined below this paragraph. We continue investigation of potential pollen flow from transgenic trees to assess the possibility of reducing the isolation distances. Availability of the test site for planting continues to be announced to researchers. Supplemental: Full details on trial plantings.1) The UF Grosser, Dutt and Gmitter transgenic effort has a substantial planting of diverse transgenics. These are on an independent permit, while all other transgenics on the site are under the USDA permits.2) Under the Stover permit, a replicated planting of 32 transgenic trees and controls produced by Dr. Jeff Jones at UF were planted. These trees include two very different constructs, each quite specific in attacking the citrus canker pathogen. 3) A broad cross-section of Poncirus derived material is being tested by USDA-ARS-Riverside and UCRiverside, and led by Chandrika Ramadugu. These are seedlings of 82 seed source trees from the Riverside genebank and include pure trifoliate accessions, hybrids of Poncirus with diverse parents, and more advanced accessions with Poncirus in the pedigree. Plants are replicated and each accession includes both graft-inoculated trees and trees uninfected at planting. 4) More than 100 citranges, from a well-characterized mapping population, and other trifoliate hybrids (+ sweet orange standards) were planted in a replicated trial in collaboration with Fred Gmitter of UF and Mikeal Roose of UCRiverside. Plants were monitored for CLas titer development and HLB symptoms. Data from this trial should provide information on markers and perhaps genes associated with HLB resistance, for use in transgenic and conventional breeding. Manuscripts have been published reporting HLB tolerance associated QTLs and differences in ACP colonization. Trees continue to be useful for documenting tolerance in a new NIFA project.5) A replicated Fairchild x Fortune mapping population was planted at the Picos Test Site in an effort led by Mike Roose to identify loci/genes associated with tolerance. This planting also includes a number of related hybrids (including our easy peeling remarkably HLB-tolerant 5-51-2) and released cultivars. Genotyping, HLB phenotyping and growth data have been collected and analysis will continue to be conducted under a new NIFA grant.6) Valencia on UF Grosser tertazyg rootstocks have been at the Picos Test Site for several years, having been CLas-inoculated before planting, and several continue to show excellent growth compared to standard controls (Grosser, personal comm.).7) In a project led by Fred Gmitter, there is a planting of 1132 hybrids of C. reticulata x C. latipes. C. latipes is among the few members of genus Citrus reported to have HLB resistance, and it is expected that there will be segregation for such resistance. The resulting plants may be used in further breeding and may permit mapping for resistance genes. 8) Seedlings with a range of pedigree contributions from Microcitrus are planted in a replicated trial, in a collaboration between Malcolm Smith (Queensland Dept. of Agriculture and Fisheries) and Ed Stover. Microcitrus is reported to have HLB resistance, and it is expected that there will be segregation for such resistance. The resulting plants may be used in further breeding and may permit mapping for resistance genes. 9) Conventional scions on Mthionin-producing transgenic Carrizo are planted from the Stover team and are displaying superior growth to trees on control Carrizo.10) Planting of USDA Mthionin transgenics with 108 transgenic Hamlin grafted on wild type Carrizo (7 events represented), 81 wild type Hamlin grafted on transgenic Carrizo (16 events represented) and 16 non-transgenic controls.11) Multiple plantings with grafted trees of l Hamlin, Valencia and grapefruit scions on transgenic rootstock expressing antimicrobial citrus-thionin and bacterial recognition domain fusion proteins (219 trees with controls) as a collaboration between USDA and the New Mexico Consortium.12) Planting was made of transgenics from Zhonglin Mou of UF under Stover permit, with 19 trees of Duncan, each expressing one of four resistance genes from Arabidopsis, and 30 Hamlin expressing one of the genes, along with ten non-transgenic controls of each scion type.13) Planting from Zhonglin Mou of UF that includes transgenic grapefruit (31 plants) and sweet orange (60 plants) scions expressing two different resistance genes and grafted on WT swingle rootstocks; as well as non-transgenic controls. 14) Transgenic trees expressing FT-ScFv (12 transgenic and 12 control) to target CLas from Tim McNellis of Penn State15)Numerous promising transgenics identified by the Stover lab in the last two years have been propagated and will be planted in the test site.