Citrus cryopreservation: viability of diverse taxa and histological observations

Diverse citrus cultivars maintained clonally within gene banks serve as valuable resources for research and breeding programs worldwide. These critical collections are kept as trees within field, screenhouse, or greenhouse collections. Ex situ collections are at risk of being lost due to unforeseen environmental or biological disasters. Cryopreservation provides a secure method to back-up these important collections. Herein, we assessed the applicability of a vitrification-based cryopreservation method to conserve citrus collection cultivars. Shoot tips were excised from screenhouse-grown trees from the USDA-ARS National Clonal Germplasm Repository for Citrus and Dates. Shoot tips were then treated with cryoprotectants, plunged into liquid nitrogen (LN), warmed and then recovered by micrografting onto 'Carrizo' citrange seedling rootstocks. Of 150 cryopreserved Citrus accessions representing 32 taxa, 24 taxa had mean regrowth levels that were at least 40 %. The 36 navel orange (Citrus sinensis) accessions had an average regrowth level of 64 %. There was no decrease in viability after 3 years of LN storage for the three accessions that are part of a long-term storage experiment. Histological observations revealed high levels of cell survival after LN exposure and that cellular regrowth occurred between rootstock and shoot tips within 2 days of micrografting. We demonstrate that diverse citrus cultivars can be successfully cryopreserved within gene banks for long-term conservation.