Implementation of Citrus shoot tip cryopreservation in the USDA-ARS National Plant Germplasm System

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. An additional 815 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 h. This large-scale effort has revealed that shoot tip cryopreservation can be successfully scaled-up to secure the NPGS Citrus collection.