The project aims to the duplication in liquid nitrogen of the ancient citrus collection located in the Villa Medicea of Castello in Florence. The collection was initiated by Cosimo I° de’ Medici in the XVI° century and comprehends over 600 accessions of high historical value, some of which present in just one specimen. The project (named “CRIOGERM, Innovative technologies for the safeguard of plant biodiversity by cryopreservation and establishment of a CRYOBANK for the Citrus germplasm of the Villa Medicea of Castello in Florence”) started in the 2007, thanks to the collaboraion of the “Soprintendenza Speciale per il Patrimonio Storico, Artistico ed Etnoantropologico e per il Polo Museale della Città di Firenze” and to the financial support of the bank fundation “Ente Cassa di Risparmio di Firenze”. An effective procedure of seed dehydration and cryopreservation has been developed in the frame of the project. In fact, the cryopreservation of seeds is a good approach not only for the conservation of species reproduced by gamic propagation, but also of cultivars from species characterized by the presence in the seed of a zygotic embryo together with nucellar embryos. This is the case of the genus Citrus, including many polyembryonic species. Up to now, seeds from 14 ancient accessions, coming from 9 Citrus species and one hybrid, have been cryopreserved by dehydration and direct immersion in liquid nitrogen, obtaining for 11 of them a germinability over 75% after the recovery of seeds from liquid nitrogen. The seeds from the polyembryonic species are then conserved in the citrus cryobank of IVALSA, first example in Italy of practical application of seed cryopreservation.