Zygotic and nucellar embryo survival following dehydration and cryopreservation of citrus intact seeds

A cryopreservation procedure by dehydration and direct immersion in liquid nitrogen was developed for seeds of four polyembryonic Citrus species, and the sexual or nucellar origin of the recovered seedlings was investigated. Seeds of three species could be desiccated in a sterile air flow to 16% (C. sinensis) or 10% (C. aurantium and C. limon) moisture content with a negligible reduction in germination levels. Differently, the germinability of C. deliciosa seeds dropped to 50% after drying to 15% moisture content. Following dehydration treatments, a reduction in the average number of seedlings per germinated seed was always observed. However, all four species benefited from desiccation in terms of protection during immersion in liquid nitrogen, with C. sinensis and C. aurantium showing the greatest survival (93% germination) after cryopreservation. The Inter-Simple Sequence Repeat analysis of seedlings recovered from cryopreserved seeds showed that the dehydration/cryopreservation procedure promotes the germination of zygotic embryos and reduces the number of apomictic seedlings per seed.