DEVELOPMENT OF PROTOCOLS FOR THE CRYOPRESERVATION OF ZYGOTIC EMBRYOS OF PINUS RADIATA AND SUBSEQUENT PLANT REGENERATION

Whole zygotic embryos of Pinus radiata D.Don were evaluated for survival after storage in liquid nitrogen (-196°C). Zygotic embryos were cryoprotected with selected levels of sorbitol and dimethylsulphoxide (DMSO) and were then frozen, employing an isopropanol-filled cryovial container and—80°C freezer for 1.5 hours prior to immersion in liquid nitrogen. After removal from liquid nitrogen the condition of embryos was assessed and then cotyledons were excised for shoot induction. Up to 61 % of cotyledons cryopreserved in a cryoprotectant formula of 0.4 M sorbitol and 15 % DMSO regenerated shoots. A sample of the regenerated shoots was successfully rooted. Cryopreserved cotyledons from 84 % of the genotypes were able to regenerate shoots upon return to in vitro culture as compared to 92 % of the unfrozen controls