Poor storability of recalcitrant seeds is due to their inability to tolerate low moisture content. Understanding the processes underlying their recalcitrance is a prerequisite to developing a maintenance strategy and prolonging their lifespan. Multiple studies have investigated the differences between orthodox (desiccation-tolerant) and recalcitrant (desiccation-sensitive) seeds. Information on epigenetic regulation, however, is lacking and thus limits our understanding of the processes defining the physiology of seeds. In the present comparative study, changes in the global levels of 5-methylcytosine (m5C) in orthodox and recalcitrant seeds of Acer platanoides L. and Acer pseudoplatanus L. were characterized during progressive stages of severe drying. Concomitant with their differential sensitivity to desiccation stress, we demonstrate variation in the response of embryonic axes and cotyledons to water deficit at the level of DNA methylation. Results indicate that desiccation-induced changes in m5C are both tissue- and seed category-specific and are highly correlated with recalcitrant seed viability. Moreover, we demonstrate that m5C global changes in response to desiccation are not retained in DNA isolated from seedlings, except in seedlings that are derived from strongly desiccated orthodox seeds (moisture content of 3.5%). Finally, the potential utilization of m5C status as a universal seed viability marker is discussed.

Ageing and deterioration of seeds is a major problem for the maintenance of seed quality and viability during long-term storage. Prediction of early stages of seed deterioration in order to point out the plantlets’ regeneration time is a major challenge of successful storage. In preserved seeds, damages accumulate within cells at the rate mainly related to their moisture content and temperature of storage. Current research reveals global alterations in DNA methylation in lipid-rich intermediate seeds during desiccation and storage at various regimes covering nonoptimal and optimal conditions. We show for the first time that monitoring of 5-methylcytosine (m5C) level in seeds can be used as a truly universal viability marker regardless of postharvest category of seeds and their composition. For seeds stored up to three years, in varied conditions, moisture content, temperature, and time of storage had significant influence on seedling emergence and DNA methylation (p < 0.05). Similarities among lipid-rich intermediate and orthodox seeds regarding different reactions of embryonic axes and cotyledons to desiccation are newly revealed. Along with previous studies on seeds dramatically different in desiccation tolerance (recalcitrant vs. orthodox), results regarding lipid-rich seeds positioned in-between (intermediate) prove that maintaining global DNA methylation status is crucial for maintaining seed viability.

The conservation of the genetic resources of old trees is crucial to their ecological role but is extremely difficult, especially for oak species (Quercus spp.) displaying recalcitrance in seed and vegetative propagation methods. Our study aimed to assess the regenerative potential of Quercus robur trees of different ages (up to 800 years) during micropropagation. We also aimed to determine how in vitro conditions can influence in vitro regeneration responses. Lignified branches collected from 67 selected trees were cultivated ex vitro in culture pots at 25 °C to obtain epicormic shoots (explant sources). The explants were cultivated on an agar medium supplemented with 0.8 mg L−1 6-benzylaminopurine (BAP) for at least 21 months. In a second experiment, two different shoot multiplication conditions (temporary immersion—RITA® bioreactor and agar medium) and two culture medium formulations (Woody Plant Medium and modified Quoirin and Lepoivre medium) were tested. The results showed that the mean length of the epicormic shoots obtained in a pot culture was a function of donor age and was similar among the group of younger trees (ca. 20–200 years), and varied between older trees (ca. 300–800 years). The efficiency of in vitro shoot multiplication strictly depended on the genotype. A sustainable in vitro culture (defined as survival after 6 months) was only possible for half of the tested old donor trees, even when they survived the first month of in vitro growth. A continuous monthly increase in the number of in vitro cultured shoots was reported in younger oaks and in some old oaks. We found a significant effect of the culture system and the macro- and micronutrient composition on in vitro shoot growth. This is the first report demonstrating that the in vitro culture can be successfully applied to the propagation of even 800-year-old pedunculate oak trees.