Location: National Clonal Germplasm Repository for Citrus
2025 Annual Report
Objectives
The primary goals of this project plan are to acquire, sanitize, maintain, preserve, characterize, evaluate, distribute, and documentgermplasm of Citrus and other members of the Rutaceae, and date palms (Phoenix dactylifera) and related Phoenix spp. and to conduct research in support of these objectives.
Objective 1: Conduct research to develop genetic resource maintenance, evaluation, or characterization methods and, in alignment with the overall NPGS Plan, then apply them to priority citrus and date genetic resources to avoid backlogs in plant genetic resource and information management.
Sub-objective 1.A: Carry out quality assurance assessment of cryopreservation on Citrus genotypes and phenotypes and verify pathogen status of cryopreserved accessions.
Sub-objective 1.B: Conduct transcriptomic and phenomic analyses of date fruit development. [Deleted with the concurrence of the Office of National Programs.]
Sub-objective 1.C: Screen accessions of citrus and date palm for ploidy level and screen Citrus spp. for frequency of polyembryony and percent zygotic embryos in polyembryonic accessions.
Sub-objective 1.D: Optimize micropropagation techniques to allow effective propagation, conservation, and distribution of Citrus spp. and other Rutaceous accessions. Develop tissue culture-based propagation methods and utilize to conserve priority citrus accessions.
Sub-objective 1.E: Based on data documented in the NPGS National Strategic Germplasm and Cultivar Collection Assessment and Utilization Plan (“NPGS Plan” henceforth), collaborate with the citrus and date Crop Germplasm Committees (CGC), NLGRP colleagues, and other stakeholders and subject matter experts (SME’s) to develop a mid- to long-term plan to address backlogs and other deficiencies in collection composition, pathogen testing and clean up, maintenance, long-term preservation, genetic and phenotypic characterization and evaluation, and documentation in GRINGlobal and other readily accessible digital repositories.
Objective 2: Acquire, distribute, and maintain the safety, genetic integrity, health, and viability of priority citrus and date genetic resources and associated descriptive information.
Sub-objective 2.A: Efficiently and effectively conserve germplasm of citrus, date palms, and related taxa. Ensure all current and future accessions are preserved by multiple methods such as field plantings, potted plants in protective structures, and cryopreserved seeds, pollen, and clonal tissue. Distribute germplasm to qualified requestors worldwide.
Sub-objective 2.B: Expand collection coverage of citrus and date genetic resources, emphasizing crop wild relatives and under-represented accession areas. Rescue potentially valuable citrus and date germplasm in areas under threat of genetic erosion. Sanitize priority citrus accessions.
Sub-objective 2.C: Expand the range of accession-associated information available in the GRINGlobal (GG) Public Web (PW) and other digital repositories. Expand the use of the GG Curator Tool (CT) within NCGRCD for routine use in managing germplasm resources and fulfilling web orders.
Approach
Sub-objective 1.A: Cryopreserved meristems will be regenerated into trees and planted in the field. Phenotypic traits will be compared to the source trees to demonstrate that cryopresrvation has not caused economic changes. Pathogen-testing will be carried out to demonstrate that the phytosanitary status of the cryopreserved materials has not changed. Whole-genome re-sequencing of the regenerated accessions and the source trees will identify any genomic changes that may have occurred.
Sub-objective 1.B: Deleted.
Sub-objective 1.C: Ploidy analysis will be assessed using nuclei isolated from of young leaf tissue and analyzed using flow cytometry. In order to assess the embryony level, 100 seeds will be germinated from each accession studied. The number of seedlings produced will indicated whether the accession is mono- or poly-embryonic. The percentage zygotic seedlings will be determined using KASP (Kompetitive Allele Specific PCR) SNP markers that had been shown to indicate percentage zygotic seedlings in polyembryonic genotypes.
Sub-objective 1.D: We have modified established citrus micropropagation techniques a pilot project to micropropagate a specific accession. During this project cycle, we will apply this propagation method to other plant materials to generate the remaining 6 indicators and 4 rootstocks that we routinely use. Tissue culture protocols for a limited number of related genera that cannot be readily propagated by conventional means will be developed.
Sub-objective 1.E: NCGRCD leadership will consult with stakeholders and subject matter experts to devise a detailed but flexible plan that identifies programmatic areas needing increased resource allocation. Within these areas, accessions or accession groups will be prioritized for needed attention. The first areas to be analyzed in this manner are those indicated above to have the most significant backlogs. Following this, other programmatic areas will be analyzed. The analysis will include recommendations for activities at existing staffing levels but also document the needs for increases in funding and additions in staffing and to facilities and physical resources.
Sub-objective 2.A: Pathogen-tested germplasm is maintained in an APHIS approved screenhouse, un-sanitized material is separated in other protective structures, and most accessions are also maintained in a field planting. Cryopreservation as a long-term backup will continue as accessions are released from quarantine and for wild relatives, seeds and pollen.
Sub-objective 2.B: Gaps in the citrus and date palm germplasm collection are identified by taxonomy, geographic origin, characterization data, and stakeholder input. New germplasm is acquired by exchange with cooperating scientists or industry personnel and by plant exploration for wild germplasm. Therapy and pathogen testing will continue for new citrus accessions a. A pathogen-testing program for date palms will be initiated.
Sub-objective 2.C: Information will be updated and expanded through the GRINGlobal (GG) system. The GG Curator tool will be deployed for germplasm distribution to replace use of the current local database.
Progress Report
This report documents the FY 2025 progress of project 2036-21000-012-000D, titled, “Citrus and Date Genetic Resource Conservation and Utilization”, which began in March 2023.
In support of Sub-objective 1.A, trees propagated for field planting to verify maintenance of trueness-to-type after cryopreservation were maintained in the greenhouse as they are still too small for field planting. Genetic analysis by sequencing has preliminarily demonstrated that there were no significant mutations such as large deletion and structural variants were observed during the process of cryopreservation of any of the genotypes studied so far. For Sub-objective 1.C, seeds of additional genotypes were planted for embryony analysis and determination of zygotic embryo percentage. Preparations are being made to continue with the ploidy level analysis. In support of Sub-objective 1.D, additional routine micropropagations were made of routinely used rootstocks and indicators. Biological indexing of quarantine items was carried out with both micropropagated and conventionally propagated indicators and preliminary observation appear to confirm that there is no difference in sensitivity using either propagation method. Progress on Sub-objective 1.E, included a report that was provided to the Crop Germplasm Committees on input solicited regarding improving the efficiency of the program. However, some recommended actions could not be carried out due to the hiring freeze. In support of Sub-objective 2.A, regeneration of germplasm maintained in planta was carried out as needed. Due to capacity limitations at the National Laboratory for Genetic Resource Preservation (NLGRP), no additional citrus access was sent for cryopreservation. All male (staminate) date accessions are backed up as cryopreserved pollen at NLGRP. Clean source citrus accessions were re-tested for HLB and CTV. In support of Sub-objective 2.B, 32 citrus accessions were released from quarantine and made available to the user community. An additional six citrus accessions were acquired, three of which need quarantine processing. Quarantine processing of additional date palm accessions was completed and a release request is being prepared. Seventeen accessions of Australian finger limes as enhanced germplasm were released by USDA and are being sanitized by our University of California cooperator. Citrus distributions included: 98 accessions to 11 requestors as budwood; 62 accessions to 27 requestors as seed; and 17 accessions to 8 requestors as leaf tissue. There was one distribution of date inflorescence. In support of Sub-objective 2.C, additional information was added to the GRINGlobal (GG) database. The staff received training in GG and distribution requests and documentation using GG were implemented.
Accomplishments
1. Thirty-two citrus accessions released from quarantine. Increasing the number of citrus accessions involves obtaining and/or processing them under a Plant Controlled Import Permit (PCIP) issued by USDA-APHIS. The PCIP requires therapeutics and testing for specific pathogens before accessions can be released from quarantine. An ARS scientist in Riverside, California, released 32 citrus germplasm accessions from quarantine in FY2025. These accessions will be incorporated into the citrus genebank and become available to citrus researchers worldwide. These new accessions may have traits that are useful in research of even commercial cultivation.
2. Development of diagnostic assays for Candidatus Phytoplasma brasiliense. Phytoplasmas are widespread pathogens in many crop and non-crop plants. In some cases, they are economically damaging, and appropriate diagnostics and control measures must be implemented. An ARS researcher in Riverside, California, in cooperation with the University of Florida, developed diagnostic assays for C. Phytoplasma brasiliense (CPb). Previously existing assays used nested PCR, which were not suitable for screening large numbers of samples. Two quantitative PCR diagnostic assays were developed using (a) high-resolution melt analysis and (b) TaqMan. The former allows distinguishing between CPb and other common Phytoplasma groups, whereas the latter is specific to CPb. These assays will be useful for the monitoring of CPb in host species by government agencies and stakeholders, and for use in research aimed at better understanding CPb biology.
Review Publications
Krueger, R., Chen, A.Y., Zhou, J.S., Liu, S., Xu, H.K., Ng, J.C. 2024. An engineered citrus tristeza virus (T36CA)-based vector induces gene-specific silencing and is graft transmissible to commercial citrus varieties. Phytopathology. 114(11):2453-2462. https://doi.org/10.1094/PHYTO-05-24-0167-R.
Volk, G.M., Shepherd, A.N., Araújo De Oliveira, A.C., Krueger, R. 2025. Date palm pollen cryopreservation. In: Volk, G.M., Chen, K., editors. Pollen Preservation. Fort Collins, Colorado: Colorado State University. Available: https://colostate.pressbooks.pub/pollenpreservation/chapter/date-palm-pollen-cryopreservation/
Lane, J., Bloch, M., Helmick, E.E., Krueger, R., Bahder, B.W. 2024. Development of a TaqMan assay for the detection of ‘Candidatus Phytoplasma brasiliense’ and assessment by high-resolution melt-curve analysis (HRMA). Plant Disease. 108(10):3108-3114. https://doi.org/10.1094/PDIS-04-24-0742-RE.
Smith, M.W., Gultzow, D.L., Newman, T.K., Reid, M., Krueger, R., Roose, M.L., Federici, C.T., Bowman, K.D. US-Gallasch: A new citrus rootstock 60 years in the making. HortScience. 60(5): 777–781. 2025. https://doi.org/10.21273/HORTSCI18444-24.
Liu, J., Singh, K., Huff, M., Gottschalk, C.C., Do, M.S., Staton, M., Keremane, M.L., Krueger, R., Ramadugu, C., Dardick, C.D. 2025. Deep R-gene discovery in HLB resistant wild Australian limes uncovers evolutionary features and potentially important loci for hybrid breeding. Frontiers in Plant Science. 15. Article 1503030. https://doi.org/10.3389/fpls.2024.1503030.
Singh, K., Huff, M., Liu, J., Park, J., Rickman, T., Keremane, M.L., Krueger, R., Kunta, M., Roose, M., Dardick, C.D., Staton, M., Ramadugu, C. 2024. Chromosome-scale, de novo, phased genome assemblies of three Australian limes: Citrus australasica, C. inodora, and C. glauca. Plants. 13(11). Article 1460. https://doi.org/10.3390/plants13111460.