Seed cryopreservation for conservation of ancient Citrus germplasm

The aim of this study was to develop a cryogenic procedure for the conservation of an ancient Citrus collection, maintained at the botanical garden of the “Villa Medicea di Castello” in Florence, Italy. The collection was initiated by Cosimo I de’Medici in the 16th century, and it includes more than 600 accessions, conserved mainly in big earthenware basins. An effective procedure of seed dehydration and direct immersion in liquid nitrogen was developed for a sample of five polyembryonic Citrus accessions, i.e., C. aurantium ‘Foetifera’, C. volkameriana, C. lumia ‘Pyriformis’, C. sinensis and the hybrid C. aurantium x C. paradisi. The initial seed misture content was between 33 and 55%. Seed dehydration was performed in open Petri dishes, exposed to sterile air flow. All the accessions showed adaptability to seed cryopreservation, after the seeds were appropriately dehydrated between 25 (C. lumia) and 15% (C. aurantium x C. paradisi). Maximum post-freezing germinabilità ranged from 27 (C. aurantium) to 100% (C. aurantium x C. paradisi). In general, one seedling per seed was obtained after the dehydration/cryopreservation procedure. Molecular analysis showed the nucellar origin of seedlings. Only in C. volkameriana the occasional development of zygotic embryos was pointed out.