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Title: Implementation of citrus shoot tip cryopreservation in the USDA-ARS national plant germplasm system

Author
item Volk, Gayle
item Jenderek, Maria
item Walters, Christina
item Bonnart, Remi
item Shepherd, Ashley
item Skogerboe, Dianne
item Hall, Bradford
item Moreland, Brittany
item Krueger, Robert
item Polek, Marylou

Submitted to: Acta Horticulturae
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/24/2018
Publication Date: 2/28/2019
Citation: Volk, G.M., Jenderek, M.M., Walters, C.T., Bonnart, R.M., Shepherd, A.N., Skogerboe, D.M., Hall, B.D., Moreland, B.L., Krueger, R., Polek, M. 2019. Implementation of citrus shoot tip cryopreservation in the USDA-ARS national plant germplasm system. Acta Horticulturae. 1234:329-334. https://doi.org/10.17660/ActaHortic.2019.1234.43.
DOI: https://doi.org/10.17660/ActaHortic.2019.1234.43

Interpretive Summary: The large citrus collection (1522 cultivars and crop wild relatives) growing at the National Clonal Germplasm Repository for Citrus and Dates (NCGRCD) in Riverside, California is expensive to maintain and vulnerable to extreme environmental conditions and pathogens. The collection can be further safe-guarded by cryopreserving germplasm accessions at the National Laboratory for Genetic Resources Preservation (NLGRP) in Fort Collins, Colorado. This safety back-up requires focused implementation of a citrus cryopreservation procedure that had been previously developed. In a concerted drive, a team of 5 skilled technicians spent 4 full days per week for 7 months cryoprocessing citrus shoot tips. Viability was assessed a few months later by a pair of technicians. Of the 451 high-value, pathogen-tested citrus accessions, 354 accessions had regrowth levels of 40% or greater, 17 had 0% regrowth, 47 had viability levels between 10 and 30%, and 33 had endogenous contaminants. It took about 2 months for technical staff already familiar with shoot-tip cryoprocessing to fully learn specific applications of citrus allowing the team of 5 to place over 60 accessions per month in cryostorage at peak season. This large-scale effort has revealed that shoot tip cryopreservation can be successfully scaled-up to secure the NPGS Citrus collection.

Technical Abstract: The USDA-ARS National Plant Germplasm System (NPGS) maintains a citrus genetic resource collection of 1522 accessions of Citrus cultivars and crop wild relatives. Of these, 540 are maintained as pathogen-free duplicate clones in a screenhouse at the National Clonal Germplasm Repository for Citrus and Dates (NCGRCD) in Riverside, California. Eight-hundred-fifteen accessions of citrus and citrus relatives are maintained in greenhouses or orchards in Riverside until sanitation for pathogens can be completed. This paper describes progress in backing up the 540 pathogen-tested accessions using cryopreservation techniques and methods to accelerate processing and viability testing. Vegetative budwood was harvested from trees in the screenhouse and sent to NLGRP. At least 170 shoot tips from each accession were excised from surface-sterilized budwood, and then cryopreserved using a PVS2 droplet-vitrification technique. Viability was assessed by micrografting 10 thawed shoot tips onto in vitro grown ‘Carrizo’ seedling rootstock. The remaining shoot tips are held in long-term liquid nitrogen storage. A total of 451 of the pathogen-tested citrus accessions were cryoprocessed: 354 accessions had regrowth levels of 40% or greater, 17 had 0% regrowth, 47 had viability levels between 10 and 30%, and 33 had endogenous contaminants. Technical staff familiar with shoot-tip cryoprocessing could be fully trained for specific applications of citrus in about 2 months and thereafter processed a single accession of at least 170 shoot tips in about 16 hrs. This large-scale effort has revealed that shoot tip cryopreservation can be successfully scaled-up to secure the NPGS Citrus collection.