Location: National Clonal Germplasm Repository for Citrus
Project Number: 2036-21000-012-000-D
Project Type: In-House Appropriated
Start Date: Mar 6, 2023
End Date: Mar 5, 2028
Objective:
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.
