Assessment of dynamics of global DNA methylation during the cryopreservation process of Pinus pinaster embryogenic lines

Long-term in vitro maintenance of embryogenic lines of Pinus species has been associated with lower maturation capacity, because of this, cryopreservation protocols for the embryogenic lines are needed to maintain valuable genotypes. Since cryopreservation may induce epigenetic variations, we evaluate changes in DNA methylation levels through the course of the cryopreservation of maritime pine embryogenic lines, as compared to those lines maintained by repeated subcultures. Six maritime pine embryogenic lines were cryopreserved following a protocol that includes pre-treatments inducing osmotic stress in liquid media. The percentage of methylated cytosines (%5-mC) in total DNA was determined by using a colorimetric assay. Reactive oxygen species (ROS) accumulation in cell lines was also determined by quantifying dihydroethidium (DHE) fluorescence under a confocal laser-scanning microscope. In the first experiment, we found that global DNA methylation was significantly reduced during the cryopreservation protocol. Subsequently, we evaluated the methylation status of both cryopreserved and no cryopreserved lines (maintained by subcultures) and found differences among embryogenic lines but overall, cryopreservation did not alter %5-mC of the recovered lines while periodical subcultures increased methylation rates. In addition, maltose pretreatment did not increase significantly ROS production in embryogenic lines. Our results demonstrate that the genetic stability during cryopreservation highly depends on the embryogenic line studied, but the protocol allows maintaining methylation DNA rates in most of the recovered lines.