493 CryoLetters 29 (6), 493-504 (2008) © CryoLetters, c/o University of Bedfordshire, Luton LU2 8DL PRESERVATION OF Quercus robur GERMPLASM BY CRYOSTORAGE OF EMBRYOGENIC CULTURES DERIVED FROM MATURE TREES AND RAPD ANALYSIS OF GENETIC STABILITY C Sánchez*, M T. Martínez, N. Vidal, M. C. San-José, S. Valladares and A.M. Vieitez Instituto de Investigaciones Agrobiológicas de Galicia, CSIC, Avda de Vigo s/n, Apartado 122, 15080 Santiago de Compostela, Spain. *Correspondence author e-mail: conchi@iiag.csic.es Abstract This study reports on the cryostorage of embryogenic lines derived from selected mature Quercus robur trees, following application of the PVS2-vitrification based procedure. In seven oak genotypes, embryo recovery levels ranging from 57-92% were obtained when 4-6 mg embryo clumps were precultured for 3 days on 0.3 M sucrose basal medium, treated with PVS2 solution for 60 min at 24ºC, and then immersed in liquid nitrogen (LN). Embryos of six out of seven lines were cryostored for one week and one year and used to evaluate cryopreservation tolerance, germination ability and to assess genetic fidelity by random amplified polymorphic DNA (RAPD) markers. There were no significant differences between the recovery frequencies of samples retrieved from LN after 1 week and 1 year of cryostorage. In five out of six lines, RAPD profiles of cryopreserved somatic embryos and regenerated plantlets were identical to those of the controls. Although polymorphisms were detected in only one cryostored embryo of one genotype, no genetic instability was found in the regenerated plantlets. This methodology appears to be suitable for long-term storage of this valuable germplasm, as the recovered plantlets were found to be genetically stable. Keywords: cryopreservation, genetic fidelity, oak, plant regeneration, somatic embryogenesis, vitrification. INTRODUCTION Pedunculate oak (Quercus robur L.) is one of the most important broadleaf tree species in European forests. Oaks have recalcitrant seeds and this, along with difficulties in vegetative propagation (35), makes conservation of the species, especially of high-value genotypes, complicated. For such species, cryopreservation is currently the preferred method for long- term conservation of clonal germplasm (9, 18). During the last decade, cryopreservation of embryogenic cell masses and somatic embryos has become increasingly important in woody plants and there are already examples of large scale applications (10). Cryopreservation is systematically used for storing the thousands of conifer embryogenic cell lines used in large scale clonal planting programmes (16), as well as for storing embryogenic lines of coffee, cacao and oil palm (10). Embryogenic material is cryostored by the traditional slow-cooling method (widely applied with conifer