Cryopreservation of Citrus germplasm: a seed and embryo freezing strategy used at INRAE-Cirad (France)

International audience

Inglés. The INRAE-Cirad citrus collection of San Giuliano is an open field conservatory that includes about 1,000 cultivars of which mandarins (C. reticulata) and their hybrids as well as orange trees (C. × aurantium var. sinensis) are the main species represented with 340 and 160 accessions, respectively. In order to guarantee the long-term sustainability of the conservation of these genetic resources, cryobanking at low temperature (-196°C) was initiated after the construction of a laboratory dedicated to this activity and the purchase of a liquid nitrogen generator. The cryobank began with the freezing of polyembryonic seeds with accessions from lime (C. × aurantiifolia) and lemon (C. × limon) species. Seeds of these species, like all species related to citron (C. medica), are tolerant to desiccation and therefore whole seed drying is performed in an atmosphere with 75% relative humidity until the moisture content reaches equilibrium. High germination rates (70-90%) were observed and SSR molecular markers were used to verify the nucellar origin of regenerated seedlings after thawing and thus ensure clonal multiplication. Orange and mandarin seeds are more sensitive to desiccation because their germination levels after drying at 75% relative humidity decreases by 30 to 70%. The method of drying the embryos (seeds without septa) under air flow offers better germination levels for these two citrus species after drying and freezing in liquid nitrogen compared to that of whole seeds. However, an alternative method will be necessary for recalcitrant species such as Poncirus trifoliata and Fortunella sp.