Preservation of Quercus robur Germplasm by Cryostorage of Embryogenic Cultures Derived from Mature Trees and Rapd Analysis of Genetic Stability

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 24°C, 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.