Location: National Clonal Germplasm Repository for Citrus
2024 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 progress for project 2036-21000-012-000D, titled, “Citrus and Date Genetic Resource Conservation and Utilization”, which started in March 2023.
In support of Sub-objective 1A, libraries of selected selections were in preparation for re-sequencing. Plants were propagated for field planting for future observation of fruit quality characteristics. Freedom from pathogens was demonstrated via laboratory tests (mostly real time PCR). In support of Sub-objective 1.C, selected accessions were screened for polyembryony and prepared for Kompetitive allele specific PCR (KASP) single nucleotide polymorphism (SNP) analysis. For Sub-objective 1.D, capabilities to efficiently generate large numbers of in vitro micro propagated plantlets were established in FY 2024. Protocols were generated for three rootstock accessions (Carrizo, C. macrophylla, and Schaub rough lemon) and five biological indicators (Mexican lime, Madam Vinous sweet orange, Dweet tangor, Standard sour orange, and C. excelsa). A total of 69 explants (seeds and nodal stems) were planted onto Murashige and Skoog (MS) medium for shoot production. Successful shoots from these explants (and from explants planted last FY) were then excised and transferred onto MS medium supplemented with rooting hormone, which created 858 plantlets. Plantlets that were free from contamination were kept in culture tubes and multiplied or transplanted as needed. In FY 2024, over 60 plantlets were transplanted into soil and acclimated into the greenhouse to be used for rootstocks. Also, in support of Sub-objective 1.D, experimental protocols on difficult to propagate and vulnerable citrus relatives were attempted. A total of 40 nodal explants were excised from Eremocitrus glauca, Glycosmis pentaphylla, and seedless or low seeded sweet oranges such as Parent Washington were planted onto MS medium for shoot production. There were 94 successful shoots excised and placed onto rooting medium. Excessive contamination issues and lack of root growth led to unsuccessful attempts. Alternate disinfestation protocols, media contents, and plant growth regulators need to be optimized. Supporting Sub-objective 1.E, a virtual and a live meeting was held with two CGC’s (citrus and date). Priority areas were identified for plan development. Additional meetings will be held in late FY2024 or early FY2025 to begin plan development.
In support of Sub-objective 2.A, all male date accessions are now cryopreserved at the National Laboratory for Plant Genetic Resource Preservation (NLRGP). Citrus accessions were sent to NLGRP for cryopreservation. Through 2024-07, 15 accessions of budwood were distributed to eight requestors (78 total bud sticks) and 64 accessions of seed were sent to 27 requestors (11,628 total seeds). Draft release materials for 17 accessions of pigmented finger limes were prepared and are currently (2024-07) being revised for finalization towards the end of FY2024. For Sub-objective 2.B, and under agreement 58-3012-2-023F0, a preliminary plant exploration in Vietnam was carried out and a number of seed lots collected. These have not yet been sent to the United States for establishment and sanitation. Also, in support of Sub-objective 2.B, 32 citrus accessions were biologically indexed, and laboratory-based pathogen tests were completed. Quarantine release is anticipated late in FY2024. In addition, a testing protocol for date palm accessions held in quarantine was developed and quarantine release is anticipated late in FY2024. Under Sub-objective 2.C, the Germplasm Resources Information Network (GRIN)Global (GG) accession records were updated with additional information. Discussions were held with GG Database Management Unit (DBMU) and other knowledgeable persons regarding uploading inventory information into GG. The staff is receiving intensive training on the use of GG so that transition to more dependence on GG will proceed smoothly. Pathogen testing records were digitized.
Accomplishments
1. Thirty-two citrus accessions released from quarantine. Increasing the number of date palm accessions involves bringing them in under a Plant Controlled Import Permit (PCIP) issued by the USDA, Animal and Plant Health Inspection Service (APHIS). The PCIP requires therepeutics and testing for specific pathogens before the accessions are released from quarantine. ARS researchers in Riverside, California, completed the quarantine processing of 32 accessions in FY 2024. 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 or even commercial cultivation.
2. Development of a pathogen-testing pipeline for date accessions. Increasing the number of citrus accessions involves bringing them in under a Plant Controlled Import Permit (PCIP) issued by USDA, Animal and Plant Health Inspection Service (APHIS). The PCIP requires testing for specific pathogens before the accessions are released from quarantine. ARS researchers in Riverside, California, developed a pipeline for pathogen detection utilizing next generation sequencing (NGS). This will allow rapid assaying for pathogen detection and increase the speed with which new accessions can be released from quarantine. New date accessions will broaden the scope of date genetic resources maintained at the genebank and potentially aid date research internationally. In addition, the new accessions may be niche cultivars if produced commercially.
Review Publications
Keremane, M.L., Singh, K., Ramadugu, C., Krueger, R., Skaggs, T.H. 2024. Next generation sequencing, and development of a pipeline as a tool for the detection and discovery of citrus pathogens to facilitate safer germplasm exchange. Plants. 13(3). Article 411. https://doi.org/10.3390/plants13030411.
Salomon-Torres, R., Acosta-Perez, J., Gutierrez-Pacheco, M., Krueger, R., Ruisanchez-Ortega, Y., Oihabi, A., Abul-Soad, A., Zaid, A. 2024. Influence of pollination in liquid suspension on the date palm (Phoenix dactylifera L.) Mejhoul cultivar in Mexicali Valley, Mexico. Emirates Journal of Food and Agriculture. 36:1-8. https://doi.org/10.3897/ejfa.2024.120425.
Ahmed, M., Hu, J.S., Strickland, J.A., Krueger, R., Shannon, C., Zhang, A. 2024. Reproductive behavior and development of the global insect pest cotton seed bug. Insects. Insects 2024, 15(1), 65;. https://doi.org/10.3390/insects15010065.
Huang, Y., He, J., Xu, Y., Zheng, W., Wang, S., Chen, P., Zheng, B., Yang, S., Jiang, X., Liu, Z., Wang, L., Wang, X., Liu, S., Lu, Z., Liu, Z., Yu, H., Zhang, Y., Yue, J., Gao, J., Zhou, X., Long, C., Zeng, X., Guo, Y., Zhang, W., Xie, Z., Li, C., Ma, Z., Jiao, W., Zhang, F., Larkin, R.M., Krueger, R., Smith, M., Ming, R., Deng, X., Xu, Q. 2023. Pangenome analysis provides insight into the evolution of the orange subfamily and a key gene for citric acid accumulation in citric fruits. Nature Genetics. 55:1964-1975. https://doi.org/10.1038/s41588-023-01516-6.
Lavagi-Craddock, I., Harper, S., Krueger, R., Vidalakis, G. 2023. Chapter 18 - Policies, regulations, and production of viroid-free propagative plant materials for sustainable agriculture. In: Adkar-Purushothama, C., Sano, T., Perreault, J., Yanjarappa, S., Di Serio, F., Daros, J., editors. Fundamentals of Viroid Biology. Amsterdam, NL: Elsevier. p. 337-361. https://doi.org/10.1016/B978-0-323-99688-4.00023-7.