Cryopreservation of Quercus suber somatic embryos by encapsulation-dehydration and evaluation of genetic stability

We describe an encapsulation and dehydration procedure for the cryopreservation of cork oak (Quercus suber L.) somatic embryos that resulted in at least 90% survival. Genetic stability of the regenerated material was assessed by flow cytometry (FCM), amplified fragment length polymorphisms (AFLP) and simple sequence repeats (SSR). Cryopreservation of embryogenic clusters involved encapsulation of each cluster in an alginate bead, followed by a 3-day culture in 0.7 M sucrose and subsequent desiccation to 25 or 35% water content (WC), followed by freezing in liquid nitrogen. Thawed, cryopreserved somatic embryos had high viability and exhibited long-term survival. No morphological differences were observed between somatic embryos desiccated to 25 and 35% WC. Analysis of DNA ploidy stability of control (i.e., encapsulated and dehydrated but not frozen) and cryopreserved material by flow cytometry showed no significant differences. Similarly, DNA-marker analyses (AFLPs and SSR) revealed no significant differences between control and cryopreserved samples at the DNA-sequence level. Nonetheless, because polymorphisms were found between control material and samples cryopreserved and desiccated to 25% WC, the 35% WC method is recommended for cryopreservation of this tissue type. Cryopreservation of Q. suber somatic embryos by this encapsulation-dehydration procedure has potential for use in long-term conservation programs.