Development of a seed cryobank to preserve the citrus genetic resources of INRAE-Cirad germplasm: a complementary strategy to the tree field conservation

In genebanks, fruit trees are often maintained as grafted scions on rootstocks in field or greenhouse settings and are therefore exposed to various risks of decay. To guarantee a secure long-term conservation, cryopreservation in liquid nitrogen is currently developed for many plant species. The success of seed freezing depends on their ability to tolerate cold and desiccation. Citrus seeds are described as semi-orthodox because they are partially tolerant to desiccation. Seeds of many citrus are polyembryonic and thus offer the possibility of clonal propagation by seedling. A seed cryopreservation program for polyembryonic cultivars of the INRAE-Cirad citrus collection has been initiated. We present here the protocol and the adaptations for cultivars sensitive to desiccation. The control of the water content of the seeds is ensured by incubation in atmospheres with controlled relative humidity to eliminate intracellular free water lethal to the cells during freezing. The temperature and duration of incubation during this desiccation step are factors that have a role in maintaining germination capacity. Genetics also has a strong influence on the success of cryopreservation operations. Citrus related to citron (Citrus medica) such as lemons and limes are much more tolerant of low water content than cultivars related to mandarin (C. reticulata) such as sweet and sour oranges. Seeds of Fortunella and Poncirus trifoliata and some of their hybrids are unsuitable for cryopreservation because they are too sensitive to moisture content decrease. Apex cryopreservation could be an alternative for these non-orthodox citrus trees. However, regeneration by sowing of frozen seeds is simpler than regeneration of apexes that need to be micrografted to develop a plant. Although improvements to the protocol are still needed to increase germination rates of some varieties, we estimate that we are able to cryopreserve in seed form about half of the citrus collection (~500 accessions).