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.

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.

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.

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.

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.

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.

Manganese(II) complexes with Bn-tpen as powerful catalysts of cyclohexene oxidation.

Manganese(II) complex [(Bn-tpen)MnII]2+ activated dioxygen for oxidation of cyclohexene in acetonitrile (MeCN) and methanol (MeOH). In MeCN, ketone (2-cyclohexen-1-one), alcohol (2-cyclohexen-1-ol) and small amounts of epoxide (cyclohexene oxide) were produced in this reaction, while in MeOH only ketone was formed. In the most efficient experiment, the combination of 2.5 × 10-4 mol% [(Bn-tpen)MnII]2+ and 4 M cyclohexene under dioxygen atmosphere (pO2 = 1 atm) in MeCN after 24 h of reaction, gave the TON equal to 716, and the main oxidation products were ketone (196 mM) and alcohol (147 mM), whereas epoxide was formed in insignificant amounts (15 mM). The formation of [(Bn-tpen)MnIV=O]2+ and [(Bn-tpen)MnIII-OH]2+ species was confirmed. The novelty of this work is the observation, that in both solvents, [(Bn-tpen)MnII]2+ complex is initially oxidized by t-BuOOH to produce Mn(III)-complex, which is reduced back by cyclohexene to [(Bn-tpen)MnII]2+, and the latter species is an active catalyst of c-C6H10 oxidation. Knowledge of the electrochemical properties of the system components may contribute to understanding the mechanisms involving participation of the active agents created in the system.

Synthesis of naturally-derived macromolecules through simplified electrochemically mediated ATRP.

The flavonoid-based macroinitiator was received for the first time by the transesterification reaction of quercetin with 2-bromoisobutyryl bromide. In accordance with the "grafting from" strategy, a naturally-occurring star-like polymer with a polar 3,3',4',5,6-pentahydroxyflavone core and hydrophobic poly(tert-butyl acrylate) (PtBA) side arms was synthesized via a simplified electrochemically mediated ATRP (seATRP), utilizing only 78 ppm by weight (wt) of a catalytic CuII complex. To demonstrate the possibility of temporal control, seATRP was carried out utilizing a multiple-step potential electrolysis. The rate of the polymerizations was well-controlled by applying optimal potential values during preparative electrolysis to prevent the possibility of intermolecular coupling of the growing polymer arms. This appears to be the first report using on-demand seATRP for the synthesis of QC-(PtBA-Br)5pseudo-star polymers. The naturally-derived macromolecules showed narrow MWDs (D = 1.08-1.11). 1H NMR spectral results confirm the formation of quercetin-based polymers. These new flavonoid-based polymer materials may find applications as antifouling coatings and drug delivery systems.

A silver bullet: elemental silver as an efficient reducing agent for atom transfer radical polymerization of acrylates.

Elemental silver was used as a reducing agent in the atom transfer radical polymerization (ATRP) of acrylates. Silver wire, in conjunction with a CuBr(2)/TPMA catalyst, enabled the controlled, rapid preparation of polyacrylates with dispersity values down to D = 1.03. The silver wire in these reactions was reused several times in sequential reactions without a decline in performance, and the amount of copper catalyst used was reduced to 10 ppm without a large decrease in control. A poly(n-butyl acrylate)-block-poly(tert-butyl acrylate) diblock copolymer was synthesized with a molecular weight of 91 400 and D = 1.04, demonstrating good retention of chain-end functionality and a high degree of livingness in this ATRP system.

Global 5-methylcytosine alterations in DNA during ageing of Quercus robur seeds.

BACKGROUND AND AIMS: Epigenetic regulation plays an important role in the management of plant growth, development and response to stress factors, and several reports have indicated that DNA methylation plays a critical role in seed development and viability. This study examines changes in 5-methylcytosine (m(5)C) levels in the DNA of seeds during ageing, a process that has important implications for plant conservation and agriculture. METHODS: Changes in the global level of m(5)C were measured in mature seeds of oak, Quercus robur. The extent of DNA methylation was measured using a protocol based on two-dimensional thin-layer chromatography. Viability of seeds was determined by germination and seedling emergence tests. KEY RESULTS: An ageing-related decrease in total m(5)C during storage of recalcitrant seeds was highly and significantly correlated with a decrease in seed viability, as reflected by a reduction in germination (r = 0·8880) and seedling emergence (r = 0·8269). CONCLUSIONS: The decrease in viability during ageing of Q. robur seeds is highly correlated with a global decline in the amount of m(5)C in genomic DNA, and it is possible that this may represent a typical response to ageing and senescence in recalcitrant seeds. Potential mechanisms that drive changes in genomic DNA methylation during ageing are discussed, together with their implications for seed viability.

Simplified electrochemically mediated atom transfer radical polymerization using a sacrificial anode.

Simplification of electrochemically mediated atom transfer radical polymerization was achieved efficiently under either potentiostatic or galvanostatic conditions using an aluminum wire sacrificial anode (seATRP) immersed directly into the reaction flask without separating the counter electrode. seATRP polymerizations were carried out under different applied potentials, Eapps = E1/2, Epc, Epc -40 mV, and Epc -80 mV. As the rate of polymerization (Rp) can be modulated by applying different Eapp potentials, more reducing conditions resulted in faster Rp. The polymerization results showed similar narrow molecular-weight distribution throughout the reactions, similar to results observed for n-butyl acrylate (BA) polymerization under conventional eATRP. High-molecular-weight PBA and diblock copolymers were synthesized by seATRP with more than 90% monomer conversion. Furthermore, galvanostatic conditions were developed for synthesizing PBA with the two-electrode system.

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.

Climate legacy in seed and seedling traits of European beech populations.

Tree species' ability to persist within their current distribution ranges is determined by seed germination and seedling growth. Exploring variation in these traits in relation to climatic conditions helps to understand and predict tree population dynamics, and to support species management and conservation under future climate. We analyzed seeds and seedlings of 26 European beech populations from the northeastern boundary of the species range to test whether: 1) adaptation to climatic conditions is reflected in depth of dormancy and germination of seeds; 2) climatic characteristics of origin predictably affect seedling traits. The variation in seed dormancy and germination in a laboratory test, and seedling growth and morphology traits in a nursery common-garden test was examined. Populations originating from warmer and drier sites (mostly from the northern region), compared to those from the opposite end of climatic gradient, germinated later, with a lower success, and produced seedlings with shorter and tougher roots. They had deeper dormancy and poorer seed germination capacity, and are likely more vulnerable to environmental changes. The climatic conditions at the origin shape the intraspecific variation of seed germination and seedling traits, and may limit regeneration from seed and affect adaptation potential of beech to increasing temperatures and decreasing precipitation.

Development of functionalized poly(lactide) films with chitosan via SI-SARA ATRP as scaffolds for neuronal cell growth.

Polylactic acid (PLA), a polymer derived from renewable resources, is gaining increasing attention in the development of biomedical devices due to its cost-effectiveness, low immunogenicity, and biodegradability. However, its inherent hydrophobicity remains a problem, leading to poor cell adhesion features. On this basis, the aim of this work was to develop a method for functionalizing the surface of PLA films with a biopolymer, chitosan (CH), which was proved to be a material with intrinsic cell adhesive properties, but whose mechanical properties are insufficient to be used alone. The combination of the two polymers, PLA as a bulk scaffold and CH as a coating, could be a promising combination to develop a scaffold for cell growth. The modification of PLA films involved several steps: aminolysis followed by bromination to graft amino and then bromide groups, poly(glycidyl methacrylate) (PGMA) grafting by surface-initiated supplemental activator and reducing agent atom transfer radical polymerization (SI-SARA ATRP) and finally the CH grafting. To prove the effective adhesive properties, conjugated and non-conjugated films were tested in vitro as substrates for neuronal cell growth using differentiated neurons from human induced pluripotent stem cells. The results demonstrated enhanced cell growth in the presence of CH.

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.

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.

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.

Successful cryopreservation of Quercus robur plumules.

Successful cryopreservation of Q. robur germplasm as plumules (i.e. shoot apical meristems of embryos) is described in this paper. After excision from the recalcitrant seeds and preliminary storage in 0.5 M sucrose solution (18 h), the plumules were subjected to cryoprotection (in 0.75 M sucrose, followed by 1.0 M sucrose and 1.5 M glycerol solutions), and next to desiccation (over silica gel or in nitrogen gas) and cooling (in slush at -210°C or in vials filled with liquid nitrogen, LN, -196°C), and were then cryostored for 24 h. High percentage of survival was obtained after cryostorage (21-67%, depending on pretreatment, assessed in vitro by greening plumules that increased in size). Desiccation of plumules over silica gel resulted in significantly higher survival after cryopreservation (58%) in comparison with desiccation in nitrogen gas (29%), with regrowth (shoots with leaves) 5-18%. The extent of plumule desiccation was comparable in both methods, in which drying of plumules for 20 min decreased the water content to 0.5-0.6 g H(2)O g(-1) dry weight before LN exposure. The type of LN exposure did not significantly influence plumule survival and regrowth after cryostorage. Plumules isolated from acorns of four provenances survived cryostorage after cryoprotection followed by desiccation over silica gel and direct cooling in vials with LN (survival 51-76%, regrowth 8-20%). Normal plants developed from the recovered shoots after rooting. The presented protocol for Q. robur plumule cryopreservation may offer a potential approach for establishing germplasm conservation in gene banks for Quercus species.