Cryopreservation changes the DNA methylation of embryonic axes of Quercus robur and Fagus sylvatica seeds during in vitro culture

Abstract

Dehydration and cryo-stress (−196 °C) caused a greater increase in the DNA methylation of embryonic axes (EAs) of beech seeds (tolerant to both stresses) than in EAs of oak (sensitive to both stresses). Simultaneously, in vitro survival of cryopreserved beech EAs was also significantly higher than in those of oak. DNA methylation plays a key role in the regulation of growth and differentiation in plants. The present study focused on global DNA methylation of embryonic axes (EAs) isolated from the seeds of pedunculate oak (Quercus robur L.) and European beech (Fagus sylvatica L.) after cryopreservation. Oak seeds can be labeled recalcitrant, i.e., sensitive to dehydration, while beech seeds fall under the suborthodox category, i.e., tolerant to dehydration. DNA methylation was determined using a specific antibody directed at the methylated cytosine in CpG dinucleotides. The analysis was carried out on DNA isolated from EA tissues and subjected to (1) dehydration (control), (2) a combination of dehydration and plant vitrification solution (PVS3) and (3) dehydration with vitrification PVS3, and rapid cooling in liquid nitrogen (LN2, −196 °C) at a cooling rate of 32 °C s−1. Such treatments induced global DNA methylation primarily in tolerant EAs from F. sylvatica seeds. The level of global DNA methylation in this material was 30 % higher than in the control, i.e., dehydrated (not frozen) beech EAs. The same treatment in oak EAs did not evoke such changes. After 120 days of tissue growth, we observed substantially more DNA demethylation in the beech tissues after cryopreservation in LN2 than in the controls or in tissues subjected to vitrification but not cryopreservation. Information on the global level of DNA methylation can be useful in monitoring procedures aimed at increasing the rate of recovery of EAs following cryopreservation.