Cryopreservation of sessile oak (Quercus petraea (Matt.) Liebl.) plumules using aluminium cryo-plates: influence of cryoprotection and drying.

BACKGROUND QUERCUS: seeds that are recalcitrant to desiccation and freezing temperatures cannot be stored in gene banks under conventional conditions. However, the germplasm of some recalcitrant seeded species can be stored in liquid nitrogen (-196 °C). Unfortunately, for many species, among them for almost the whole genus Quercus, an effective cryostorage method is still unknown. In this study, we propose a successful cryostorage protocol for Quercus petraea (Matt.) Liebl. germplasm using plumules (a shoot apical meristem of an embryo) frozen on aluminium cryo-plates. RESULTS: The plumules isolated from the acorns of ten provenances were prestored in 0.5 M sucrose solution (for 18 h). To form alginate beads (one plumule per bead), the plumules were placed in the wells of a cryo-plate and embedded in calcium alginate gel. For cryoprotection, the encapsulated plumules were immersed in cryoprotectant solution containing 2.0 M glycerol and different concentrations of sucrose (0.8-1.2 M) for 40 min at 25 °C and desiccated under a laminar flow cabinet for 1.0-4.0 h. Cryo-plates with plumules were directly immersed in liquid nitrogen and then cryostored for 30 min. For rewarming, cryo-plates with plumules were immersed in 1.0 M sucrose solution and rehydrated for 15 min at 25 °C. Survival rates varied from 25.8 to 83.4 were achieved after cryoprotection in 1.0 M sucrose solution and the drying of plumules for 2 h. The in vitro regrowth rate of cryopreserved plumules varied among provenances and was 26-77%. CONCLUSIONS: This study presents, for the first time, a successful, simple and effective protocol for the cryopreservation of Q. petraea germplasm that could be used in gene banks. The experiment was successfully repeated on seeds from various provenances, each yielding similar, good results. However, seed quality and storage time after harvesting are important factors in plumule regrowth after cryopreservation.

Controlled Polymer Synthesis Toward Green Chemistry: Deep Insights into Atom Transfer Radical Polymerization in Biobased Substitutes for Polar Aprotic Solvents.

Cyrene (dihydrolevoglucosenone) and its derivative Cygnet 0.0, recognized as eco-friendly alternatives to polar aprotic solvents, were utilized in atom transfer radical polymerization (ATRP) of a wide range of both hydrophobic and hydrophilic (meth)acrylates. The detailed kinetics study and electrochemical experiments of the catalytic complex in these solvents reveal the opportunities and limitations of their use in controlled radical polymerization. Both solvents produce precisely controlled polymers using supplemental activator and reducing agent (SARA) ATRP. They offer an efficient reaction medium for crafting well-defined branched architectures from naturally derived cores such as riboflavin, ß-cyclodextrin, and troxerutin, thereby significantly expanding the application scope of these solvents. Notably, Cygnet 0.0 significantly reduces side reactions between the solvent and the catalyst compared to Cyrene, allowing the catalyst complex to be used at a reduced concentration down to 75 ppm. The effective mass yield values achieved in Cyrene and Cygnet 0.0 underscore a substantial advantage of these solvents over DMF in generating processes that adhere to the principles of green chemistry. Furthermore, the copper residue in the final polymers was several hundred times lower than the permissible daily exposure to orally administered copper in pharmaceuticals. As a result, the resulting polymeric materials hold immense potential for various applications, including the pharmaceutical industry.

Successful In Vitro Shoot Multiplication of Quercus robur L. Trees Aged up to 800 Years.

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.

Bio-Inspired Iron Pentadentate Complexes as Dioxygen Activators in the Oxidation of Cyclohexene and Limonene.

The use of dioxygen as an oxidant in fine chemicals production is an emerging problem in chemistry for environmental and economical reasons. In acetonitrile, the [(N4Py)FeII]2+ complex, [N4Py-N,N-bis(2-pyridylmethyl)-N-(bis-2-pyridylmethyl)amine] in the presence of the substrate activates dioxygen for the oxygenation of cyclohexene and limonene. Cyclohexane is oxidized mainly to 2-cyclohexen-1-one, and 2-cyclohexen-1-ol, cyclohexene oxide is formed in much smaller amounts. Limonene gives as the main products limonene oxide, carvone, and carveol. Perillaldehyde and perillyl alcohol are also present in the products but to a lesser extent. The investigated system is twice as efficient as the [(bpy)2FeII]2+/O2/cyclohexene system and comparable to the [(bpy)2MnII]2+/O2/limonene system. Using cyclic voltammetry, it has been shown that, when the catalyst, dioxgen, and substrate are present simultaneously in the reaction mixture, the iron(IV) oxo adduct [(N4Py)FeIV=O]2+ is formed, which is the oxidative species. This observation is supported by DFT calculations.

Identification of DNA Methylation Changes in European Beech Seeds during Desiccation and Storage.

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.

Deterioration in the Quality of Recalcitrant Quercus robur Seeds during Six Months of Storage at Subzero Temperatures: Ineffective Activation of Prosurvival Mechanisms and Evidence of Freezing Stress from an Untargeted Metabolomic Study.

Pedunculate oak (Quercus robur L.) is an economically important forest-forming species in Poland that produces seeds that are sensitive to desiccation; therefore, short-lived seeds are classified as recalcitrant. Such seeds display active metabolism throughout storage. Acorns stored under controlled conditions (moisture content of 40%, temperature -3 °C) maintain viability for up to 1.5-2 years. Meanwhile, oaks only produce large numbers of seeds every few years during so-called mast years. This results in a scarcity of good-quality seeds for continuous nursery production and restoration. The recalcitrant storage behavior and the requirements of foresters make it necessary to develop a new protocol for longer acorn storage at lower temperatures. Two storage temperatures were tested: -3 °C (currently used in forest practice) and -7 °C. Our results showed that acorns stored for six months exhibited deterioration and reduced germination capacity, as well as reduced seedling performance, particularly when acorns were stored at -7 °C. To elucidate the decrease in quality during storage, an untargeted metabolomics study was performed for the first time and supported with the analysis of carbohydrates and percentages of carbon (C) and nitrogen (N). Embryonic axes were characterized by a lower C:N ratio and higher hydration. A total of 1985 metabolites were detected, and 303 were successfully identified and quantified, revealing 44 known metabolites that displayed significantly up- or downregulated abundance. We demonstrated for the first time that the significant deterioration of seed germination potential, particularly in seeds stored at -7 °C, was accompanied by an increased abundance of phenolic compounds and carbohydrates but also amino acids and phosphorylated monosaccharides, particularly in the embryonic axes. The increased abundance of defense-related metabolites (1,2,4-Benzenetriol; BTO), products of ascorbic acid degradation (threonic and isothreonic acid), as well as antifreezing compounds (sugar alcohols, predominantly threitol), was reported in seed stored at -7 °C. We hypothesize that seed deterioration was caused by freezing stress experienced during six months of storage at -7 °C, a decline in antioxidative potential and the unsuccessful rerouting of the energy-production pathways. Additionally, our data are a good example of the application of high-throughput metabolomic tools in forest management.

DNA Methylation as an Early Indicator of Aging in Stored Seeds of "Exceptional" Species Populus nigra L.

Ex situ preservation of genetic resources is an essential strategy for the conservation of plant biodiversity. In this regard, seed storage is the most convenient and efficient way of preserving germplasm for future plant breeding efforts. A better understanding of the molecular changes that occur during seed desiccation and aging is necessary to improve conservation protocols, as well as real-time methods for monitoring seed quality. In the present study, we assessed changes in the level of genomic 5-methylcytosine (5mC) in seeds of Populus nigra L. by 2D-TLC. Epigenetic changes were characterized in response to several seed storage regimes. Our results demonstrate that P. nigra seeds represent an intermediate type of post-harvest behavior, falling between recalcitrant and orthodox seeds. This was also true for the epigenetic response of P. nigra seeds to external factors. A crucial question is whether aging in seeds is initiated by a decline in the level of 5mC, or if epigenetic changes induce a process that leads to deterioration. In our study, we demonstrate for the first time that 5mC levels decrease during storage and that the decline can be detected before any changes in seed germination are evident. Once P. nigra seeds reached an 8-10% reduction in the level of 5mC, a substantial decrease in germination occurred. The decline in the level of 5mC appears to be a critical parameter underlying the rapid deterioration of intermediate seeds. Thus, the measurement of 5mC can be a fast, real-time method for assessing asymptomatic aging in stored seeds.

Coffee Beverage: A New Strategy for the Synthesis of Polymethacrylates via ATRP.

Coffee, the most popular beverage in the 21st century society, was tested as a reaction environment for activators regenerated by electron transfer atom transfer radical polymerization (ARGET ATRP) without an additional reducing agent. Two blends were selected: pure Arabica beans and a proportional blend of Arabica and Robusta beans. The use of the solution received from the mixture with Robusta obtained a high molecular weight polymer product in a short time while maintaining a controlled structure of the synthesized product. Various monomers with hydrophilic characteristics, i.e., 2-(dimethylamino)ethyl methacrylate (DMAEMA), oligo(ethylene glycol) methyl ether methacrylate (OEGMA500), and glycidyl methacrylate (GMA), were polymerized. The proposed concept was carried out at different concentrations of coffee grounds, followed by the determination of the molar concentration of caffeine in applied beverages using DPV and HPLC techniques.

A New Protocol for Ash Wood Modification: Synthesis of Hydrophobic and Antibacterial Brushes from the Wood Surface.

The article presents the modification of ash wood via surface initiated activators regenerated by electron transfer atom transfer radical polymerization mediated by elemental silver (Ag0 SI-ARGET ATRP) at a diminished catalyst concentration. Ash wood is functionalized with poly(methyl methacrylate) (PMMA) and poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA) to yield wood grafted with PMMA-b-PDMAEMA-Br copolymers with hydrophobic and antibacterial properties. Fourier transform infrared (FT-IR) spectroscopy confirmed the covalent incorporation of functional ATRP initiation sites and polymer chains into the wood structure. The polymerization kinetics was followed by the analysis of the polymer grown in solution from the sacrificial initiator by proton nuclear magnetic resonance (1H NMR) and gel permeation chromatography (GPC). The polymer layer covalently attached to the wood surface was observed by scanning electron microscopy (SEM). The hydrophobic properties of hybrid materials were confirmed by water contact angle measurements. Water and sodium chloride salt aqueous solution uptake tests confirmed a significant improvement in resistance to the absorption of wood samples after modification with polymers. Antibacterial tests revealed that wood-QPDMAEMA-Br, as well as wood-PMMA-b-QPDMAEMA-Br, exhibited higher antibacterial activity against Gram-positive bacteria (Staphylococcus aureus) in comparison with Gram-negative bacteria (Escherichia coli). The paper presents an economic concept with ecological aspects of improving wood properties, which gives great opportunities to use the proposed approach in the production of functional hybrid materials for industry and high quality sports equipment, and in furniture production.

Induction of Oxidative Stress in SH-SY5Y Cells by Overexpression of hTau40 and Its Mitigation by Redox-Active Nanoparticles.

Abnormally phosphorylated tau protein is the principal component of neurofibrillary tangles, accumulating in the brain in many neurodegenerative diseases, including Alzheimer's disease. The aim of this study was to examine whether overexpression of tau protein leads to changes in the redox status of human neuroblastoma SH-SY5Y cells. The level of reactive oxygen species (ROS) was elevated in tau-overexpressing cells (TAU cells) as compared with cells transfected with the empty vector (EP cells). The level of glutathione was increased in TAU cells, apparently due to overproduction as an adaptation to oxidative stress. The TAU cells had elevated mitochondrial mass. They were more sensitive to 6-hydroxydopamine, delphinidin, 4-amino-TEMPO, and nitroxide-containing nanoparticles (NPs) compared to EP controls. These results indicate that overexpression of the tau protein imposes oxidative stress on the cells. The nitroxide 4-amino-TEMPO and nitroxide-containing nanoparticles (NPs) mitigated oxidative stress in TAU cells, decreasing the level of ROS. Nitroxide-containing nanoparticles lowered the level of lipid peroxidation in both TAU and EP cells, suggesting that nitroxides and NPs may mitigate tau-protein-induced oxidative stress.

Smart, Naturally-Derived Macromolecules for Controlled Drug Release.

A series of troxerutin-based macromolecules with ten poly(acrylic acid) (PAA) or poly(2-dimethylaminoethyl methacrylate) (PDMAEMA) homopolymer side chains were synthesized by a supplemental activator and reducing agent atom transfer radical polymerization (SARA ATRP) approach. The prepared precisely-defined structures with low dispersity (Mw/Mn < 1.09 for PAA-based, and Mw/Mn < 1.71 for PDMAEMA-based macromolecules) exhibited pH-responsive behavior depending on the length of the polymer grafts. The properties of the received polyelectrolytes were investigated by dynamic light scattering (DLS) measurement to determine the hydrodynamic diameter and zeta potential upon pH changes. Additionally, PDMAEMA-based polymers showed thermoresponsive properties and exhibited phase transfer at a lower critical solution temperature (LCST). Thanks to polyelectrolyte characteristics, the prepared polymers were investigated as smart materials for controlled release of quercetin. The influence of the length of the polymer grafts for the quercetin release profile was examined by UV-VIS spectroscopy. The results suggest the strong correlation between the length of the polymer chains and the efficiency of active substance release, thus, the adjustment of the composition of the macromolecules characterized by branched architecture can precisely control the properties of smart delivery systems.

Stimuli-Responsive Rifampicin-Based Macromolecules.

This paper presents the modification of the antibiotic rifampicin by an anionic polyelectrolyte using a simplified electrochemically mediated atom transfer radical polymerization (seATRP) technique to receive stimuli-responsive polymer materials. Initially, a supramolecular ATRP initiator was prepared by an esterification reaction of rifampicin hydroxyl groups with α-bromoisobutyryl bromide (BriBBr). The structure of the initiator was successfully proved by nuclear magnetic resonance (1H and 13C NMR), Fourier-transform infrared (FT-IR) and ultraviolet-visible (UV-vis) spectroscopy. The prepared rifampicin-based macroinitiator was electrochemically investigated among various ATRP catalytic complexes, by a series of cyclic voltammetry (CV) measurements, determining the rate constants of electrochemical catalytic (EC') process. Macromolecules with rifampicin core and hydrophobic poly (n-butyl acrylate) (PnBA) and poly(tert-butyl acrylate) (PtBA) side chains were synthesized in a controlled manner, receiving polymers with narrow molecular weight distribution (Mw/Mn = 1.29 and 1.58, respectively). "Smart" polymer materials sensitive to pH changes were provided by transformation of tBA into acrylic acid (AA) moieties in a facile route by acidic hydrolysis. The pH-dependent behavior of prepared macromolecules was investigated by dynamic light scattering (DLS) determining a hydrodynamic radius of polymers upon pH changes, followed by a control release of quercetin as a model active substance upon pH changes.

Temperature Regulation of Primary and Secondary Seed Dormancy in Rosa canina L.: Findings from Proteomic Analysis.

Temperature is a key environmental factor restricting seed germination. Rose (Rosa canina L.) seeds are characterized by physical/physiological dormancy, which is broken during warm, followed by cold stratification. Exposing pretreated seeds to 20 °C resulted in the induction of secondary dormancy. The aim of this study was to identify and functionally characterize the proteins associated with dormancy control of rose seeds. Proteins from primary dormant, after warm and cold stratification (nondormant), and secondary dormant seeds were analyzed using 2-D electrophoresis. Proteins that varied in abundance were identified by mass spectrometry. Results showed that cold stratifications affected the variability of the highest number of spots, and there were more common spots with secondary dormancy than with warm stratification. The increase of mitochondrial proteins and actin during dormancy breaking suggests changes in cell functioning and seed preparation to germination. Secondary dormant seeds were characterized by low levels of legumin, metabolic enzymes, and actin, suggesting the consumption of storage materials, a decrease in metabolic activity, and cell elongation. Breaking the dormancy of rose seeds increased the abundance of cellular and metabolic proteins that promote germination. Induction of secondary dormancy caused a decrease in these proteins and germination arrest.

Polymer Brushes via Surface-Initiated Electrochemically Mediated ATRP: Role of a Sacrificial Initiator in Polymerization of Acrylates on Silicon Substrates.

Silicon wafers as semiconductors are essential components of integrated circuits in electronic devices. For this reason, modification of the silicon surface is an important factor in the manufacturing of new hybrid materials applied in micro- and nanoelectronics. Herein, copolymer brushes of hydrophilic poly(2-hydroxyethyl acrylate) (PHEA) and hydrophobic poly(tert-butyl acrylate) (PtBA) were grafted from silicon wafers via simplified electrochemically mediated atom transfer radical polymerization (seATRP) according to a surface-initiated approach. The syntheses of PHEA-b-PtBA copolymers were carried out with diminished catalytic complex concentration (successively 25 and 6 ppm of Cu). In order to optimize the reaction condition, the effect of the addition of a supporting electrolyte was investigated. A controlled increase in PHEA brush thickness was confirmed by atomic force microscopy (AFM). Various other parameters including contact angles and free surface energy (FSE) for the modified silicon wafer were presented. Furthermore, the effect of the presence of a sacrificial initiator in solution on the thickness of the grafted brushes was reported. Successfully fabricated inorganic-organic hybrid nanomaterials show potential application in biomedicine and microelectronics devices, e.g., biosensors.

Oxidation processes related to seed storage and seedling growth of Malus sylvestris, Prunus avium and Prunus padus.

Seeds stored in controlled conditions in gene banks, faster or slower lose their viability. The effects of seed moisture content levels (ca. 5, 8, 11%) combined with storage temperatures (-3°, -18°, -196°C) were investigated in terms of the description of seeds defined as orthodox under oxidative stress after seed storage, during germination, and initial seedling growth. Hydrogen peroxide (H2O2), thiobarbituric acid reactive substances (TBARS) and ascorbate (Asc) were analyzed in relation to seed germinability and seedlings emergence in three species: Malus sylvestris L., Prunus avium L. and Prunus padus L. The effect of seed storage conditions on H2O2 levels appeared in germinated seeds after the third year of storage in each species. The H2O2 levels were negatively correlated with the germination and seedling emergence of P. avium seeds after three years of storage under all examined combinations. The emergence of P. padus seedlings was not linked to any of the stress markers tested. The P. padus seed biochemical traits were least altered by storage conditions, and the seeds produced tolerant seedlings of relatively high levels of H2O2 and TBARS. To cope with different H2O2 levels, TBARS levels, and Asc levels in seeds of three species varying storage conditions different molecular responses, i.e. repairing mechanisms, were applied during stratification to compensate for the storage conditions and, as a result, seeds remained viable and seedlings were successfully established.

Low Ppm Atom Transfer Radical Polymerization in (Mini)Emulsion Systems.

In the last decade, unceasing interest in atom transfer radical polymerization (ATRP) has been noted, especially in aqueous dispersion systems. Emulsion or miniemulsion is a preferred environment for industrial polymerization due to easier heat dissipation and lower production costs associated with the use of water as a dispersant. The main purpose of this review is to summarize ATRP methods used in emulsion media with different variants of initiating systems. A comparison of a dual over single catalytic approache by interfacial and ion pair catalysis is presented. In addition, future development directions for these methods are suggested for better use in biomedical and electronics industries.

Surface Modifications of Poly(Ether Ether Ketone) via Polymerization Methods-Current Status and Future Prospects.

Surface modification of poly(ether ether ketone) (PEEK) aimed at applying it as a bone implant material aroused the unflagging interest of the research community. In view of the development of implantology and the growing demand for new biomaterials, increasing biocompatibility and improving osseointegration are becoming the primary goals of PEEK surface modifications. The main aim of this review is to summarize the use of polymerization methods and various monomers applied for surface modification of PEEK to increase its bioactivity, which is a critical factor for successful applications of biomedical materials. In addition, the future directions of PEEK surface modifications are suggested, pointing to low-ppm surface-initiated atom transfer radical polymerization (SI-ATRP) as a method with unexplored capacity for flat surface modifications.

Ultrasound-Mediated Atom Transfer Radical Polymerization (ATRP).

Ultrasonic agitation is an external stimulus, rapidly developed in recent years in the atom transfer radical polymerization (ATRP) approach. This review presents the current state-of-the-art in the application of ultrasound in ATRP, including an initially-developed, mechanically-initiated solution with the use of piezoelectric nanoparticles, that next goes to the ultrasonication-mediated method utilizing ultrasound as a factor for producing radicals through the homolytic cleavage of polymer chains, or the sonolysis of solvent or other small molecules. Future perspectives in the field of ultrasound in ATRP are presented, focusing on the preparation of more complex architectures with highly predictable molecular weights and versatile properties. The challenges also include biohybrid materials. Recent advances in the ultrasound-mediated ATRP point out this approach as an excellent tool for the synthesis of advanced materials with a wide range of potential industrial applications.

Surface-Initiated Atom Transfer Radical Polymerization for the Preparation of Well-Defined Organic-Inorganic Hybrid Nanomaterials.

Surface-initiated atom transfer radical polymerization (SI-ATRP) is a powerful tool that allows for the synthesis of organic-inorganic hybrid nanomaterials with high potential applications in many disciplines. This review presents synthetic achievements and modifications of nanoparticles via SI-ATRP described in literature last decade. The work mainly focuses on the research development of silica, gold and iron polymer-grafted nanoparticles as well as nature-based materials like nanocellulose. Moreover, typical single examples of nanoparticles modification, i.e., ZnO, are presented. The organic-inorganic hybrid systems received according to the reversible deactivation radical polymerization (RDRP) approach with drastically reduced catalyst complex concentration indicate a wide range of applications of materials including biomedicine and microelectronic devices.

Changes in genomic 5-methylcytosine level mirror the response of orthodox (Acer platanoides L.) and recalcitrant (Acer pseudoplatanus L.) seeds to severe desiccation.

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.