Optimization of cryopreservation protocols for zygotic embryos of Citrus reticulata

Citrus seeds are known to have short life spans in conventional seed banks. Seed of most Citrus species show a typical intermediate storage behaviour where they survive desiccation to c. 7-10%. However, longevity is compromised at this low moisture content at sub-zero temperatures. Cryopreservation of zygotic embryos is an option for the long-term storage of germplasm of this species and we investigated its feasibility for Citrus reticulata var. Mandarin. The experimental strategies evaluated included: seed and embryo desiccation sensitivity assessment, naked embryo cryopreservation, encapsulation-dehydration cryopreservation, and cryopreservation of embryos following the PVS2 vitrification technique. Differential scanning calorimetry was used at every desiccation step to determine the critical water content for cryopreservation. Desiccated and cryopreserved naked embryos with a moisture content of 13% (fresh weight basis) showed the highest regrowth (60%). Maximum regrowth of about 58 and 52% was recorded for encapsulated embryos with a moisture content of ~22% and for embryos treated with PVS2 vitrification for 120 min, respectively. Although encapsulated, PVS2 treated and naked embryo cryopreservation had almost similar regrowth percentages, the seedlings obtained from naked embryo cryopreservation appeared more vigorous, probably because of the lack of mechanical contraints imposed by the alginate encapsulation and no exposure to cryo-protectant, a known plant stressor. We conclude that naked embryo desiccation is the optimum technique for cryopreservation of C. reticulata zygotic embryos.