Long-term, large scale banking of citrus species embryos: comparisons between cryopreservation and other seed banking...

The long-term, large scale application of embryo cryopreservation has been assessed rarely and comparisons of viability loss for partially dried material with conventional seed bank storage conditions infrequently made. Five citrus species were cryopreserved following air drying of embryos (seed minus the testa) and embryonic axes: rough lemon (Citrus jambhiri), pommelo (C. grandis), mandarin (C. reticulata), citron (C. medica) and kagzi lime (C. aurantifolia). Although drying rates to c. 10 percent moisture content (MC) were approximately 10-times faster for isolated axes compared to embryos, the optimum MCs for cryopreservation were generally similar within a species, varying from c. 10 percent (C. jambhiri) to c. 20 percent (C. medica). Nonetheless, the hydration window for cryopreservation of the axis was usually wider than for the embryo. For all species, embryo or axis survival after cryopreservation ranged from 65 to 96 percent (C. medica axes), producing normal healthy seedlings from embryos and plantlets from axes without intervening callus growth in vitro. Whilst partially dried embryos of all five species survived fully liquid nitrogen vapour storage for 120 days, viability loss was rapid at -20 degree C, 5 degree C and ambient temperature, with a maximum interpolated half-life across these temperatures of c. 80 days for C. grandis at 5 degree C. The developed cryopreservation protocols were applied routinely to cryobank 377 accessions of Citrus germplasm from field genebanks, farmer's orchards, semi-wild and wild sources. After an average of 6.3 to 8.4 years cryo-storage, between 69 and 81 percent of accessions per species retained > 70 percent of the viability after desiccation. The results provide irrevocable evidence for the importance of cryopreservation for the banking of seeds of higher plants.