Cryopreservation of citrus shoot tips using micrografting for recovery

The USDA-ARS National Plant Germplasm System (NPGS) and the University of California Citrus Variety Collection maintain more than 888 unique accessions representing 132 taxa of Citrus, Fortunella, and citrus wild species within field, screenhouse, and greenhouse collections. We have identified a cryopreservation method by which Citrus genetic resources that are not maintained in vitro can be successfully conserved. Shoot tips were excised from actively growing vegetative flushes of protected trees. Surface-disinfected shoot tips were precultured overnight in 0.3 M sucrose, loaded with a loading solution for 20 min and treated with PVS2 for 30 or 60 min at 0 degree C, prior to direct immersion in liquid nitrogen. Rewarmed shoot tips post-cultured overnight on survival medium were then micrografted on 'Carrizo' seedling rootstocks to produce whole plants. Micrografted shoot tips recovered quickly and rooted plants could be transferred to the greenhouse within months. Regrowth of whole plants after micrografting averaged 53 percent for cryopreserved shoot tips of cultivars representing eight Citrus and Fortunella species. This method has several advantages: it uses screenhouse or greenhouse plants as source materials, it is not dependent upon cultivar-specific recovery media, and it avoids seedling juvenility.