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Background and Aims: Plant regeneration by seeds is driven by a set of physiological traits, many of which show functional intraspecific variation along biogeographic gradients. In many species, germination phenology depends on a germination delay imposed by the need for post-dispersal embryo growth (a.k.a. morphological dormancy). Such growth occurs as a function of environmental temperatures and shows base, optimum and ceiling temperatures (i.e. cardinal temperatures or thermal thresholds). However, the biogeographical variation in such thresholds has not been tested., Methods: We used a thermal time approach and field experiments to assess intraspecific variation at the continental scale in the embryo growth thermal thresholds of the geophyte Conopodium majus (Apiaceae) across its distribution from the Iberian Peninsula to Scandinavia., Key Results: Thermal thresholds for embryo growth varied across the latitudinal gradient, with the estimated optimum temperatures between 2.5 and 5.2 ºC, ceiling temperatures between 12 and 20.5 ºC and base temperatures between -6.6 and -2.7 ºC. Germination in the field peaked in the months of January and February. The limiting factor for embryo growth was the ceiling temperature, which was negatively correlated with latitude and the bioclimatic environment of each population. In contrast, the optimal and base temperature were independent of local climate., Conclusions: These results indicate that thermal thresholds for embryo growth are functional ecophysiological traits that drive seed germination phenology and seed responses to soil climatic environment. Therefore, post-dispersal embryo growth can be a key trait impacting climate change effects on phenology and species distributions., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

A cryopreservation protocol has been developed for embryogenic cultures (ECs) of Castanea mollissima, an important economic species of the Castanea genus in China. We achieved 100 % regrowth when ECs were treated with Plant Vitrification Solution 2 (PVS2) for 30, 60 and 90 min on ice. Optimal PVS2 treatment for cryopreservation was determined to be 30 min on ice based on the highest biomass regrowth after thawing. Fluorescein diacetate (FDA) staining could rapidly and reliably determine post-thaw cell viability and its use facilitated the optimization of the cryopreservation protocols. Although the proliferation rate of the re-established ECs remained largely unchanged compared to non-cryopreserved ECs, the capacity of the re-established ECs to differentiate (on two media) into somatic embryos nearly doubled to approximately 2200-2300 globular somatic embryos per 1 g of re-established ECs. Based on cell cluster size analysis, this enhanced growth is primarily attributed to the presence of significantly greater cell clusters with a diameter of 100-200 μm, which have the highest level of differentiation ability. In order to understand the increased embryogenic potential following cryopreservation, we analyzed the expression of key genes related to somatic embryogenesis. Genes CmWUS and CmABP1 were downregulated while CmLEC1, CmAGL15, CmGRF2, and CmFUS3 were upregulated in re-established ECs when compared to non-cryopreserved ECs., Competing Interests: Declaration of competing interest On behalf of all authors, the corresponding author declares that there are no conflicts of interest., (Copyright © 2024 Society for Cryobiology. Published by Elsevier Inc. All rights reserved.)

Seed deterioration during storage is a major problem in agricultural and forestry production and for germplasm conservation. Our previous studies have shown that a mitochondrial outer membrane protein VOLTAGE-DEPENDENT ANION CHANNEL (VDAC) is involved in programmed cell death-like viability loss during the controlled deterioration treatment (CDT) of elm (Ulmus pumila L.) seeds, but its underlying mechanism remains unclear. In this study, we demonstrate that the oxidative modification of GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE (GAPDH) is functioned in the gate regulation of VDAC during the CDT of elm seeds. Through biochemical and cytological methods and observations of transgenic material [Arabidopsis (Arabidopsis thaliana), Nicotiana benthamiana, and yeast (Saccharomyces cerevisiae)], we demonstrate that cysteine S-glutathionylated UpGAPDH1 interacts with UpVDAC3 during seed aging, which leads to a mitochondrial permeability transition and aggravation of cell death, as indicated by the leakage of the mitochondrial proapoptotic factor cytochrome c and the emergence of apoptotic nucleus. Physiological assays and inductively coupled plasma mass spectrometry analysis revealed that GAPDH glutathionylation is mediated by increased glutathione, which might be caused by increases in the concentrations of free metals, especially Zn. Introduction of the Zn-specific chelator TPEN [(N,N,N',N'-Tetrakis (2-pyridylmethyl)ethylenediamine)] significantly delayed seed aging. We conclude that glutathionylated UpGAPDH1 interacts with UpVDAC3 and serves as a proapoptotic protein for VDAC-gating regulation and cell death initiation during seed aging., Competing Interests: Conflict of interest statement. There are no conflicts of interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Background and Aims: Orchid seeds are reputed to be short lived in dry, cold storage conditions, potentially limiting the use of conventional seed banks for long-term ex situ conservation. This work explores whether Cattleya seeds are long lived or not during conventional storage (predried to ~12 % relative humidity, then stored at -18 °C)., Methods: We explored the possible interaction of factors influencing seed lifespan in eight species of the genus Cattleya using physiological (germination and vigour), biochemical (gas chromatography), biophysical (differential scanning calorimetry) and morphometric methods. Seeds were desiccated to ~3 % moisture content and stored at -18 °C for more than a decade, and seed quality was measured via three in vitro germination techniques. Tetrazolium staining was also used to monitor seed viability during storage. The morphometric and germination data were subjected to ANOVA and cluster analysis, and seed lifespan was subjected to probit analysis., Key Results: Seeds of all Cattleya species were found to be desiccation tolerant, with predicted storage lifespans (P50y) of ~30 years for six species and much longer for two species. Cluster analysis showed that the three species with the longest-lived seeds had smaller (9-11 %) airspaces around the embryo. The post-storage germination method impacted the quality assessment; seeds equilibrated at room temperature for 24 h or in 10 % sucrose solution had improved germination, particularly for the seeds with the smallest embryos. Chromatography revealed that the seeds of all eight species were rich in linoleic acid, and differential scanning calorimetry identified a peak that might be auxiliary to selecting long-lived seeds., Conclusions: These findings show that not all orchids produce seeds that are short lived, and our trait analyses might help to strengthen prediction of seed longevity in diverse orchid species., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Triacylglycerol (TAG) accumulation is frequently triggered in vegetative tissues experiencing heat stress, which may increases plant basal plant thermo-tolerance by sequestering the toxic lipid intermediates that contribute to membrane damage or cell death under stress conditions. However, stress-responsive TAG biosynthesis and the underlying regulatory mechanisms are not fully understood. Here, we investigated the lipidomic and transcriptomic landscape under heat stress in the leaves of sacha inchi (Plukenetia volubilis L.), an important oilseed crop in tropical regions. Under heat stress (45 °C), the content of polyunsaturated TAGs (e.g., TAG18:2 and TAG18:3) and total TAGs were significantly higher, while those of unsaturated sterol esters, including ZyE 28:4, SiE 18:2 and SiE 18:3, were dramatically lower. Transcriptome analysis showed that the expression of PvDGAT2-2, encoding a type II diacylglycerol acyltransferase (DGAT) that is critical for TAG biosynthesis, was substantially induced under heat stress. We confirmed the function of PvDGAT2-2 in TAG production by complementing a yeast mutant defective in TAG biosynthesis. Importantly, we also identified the heat-induced transcription factor PvMYB1 as an upstream activator of PvDGAT2-2 transcription. Our findings on the molecular mechanism leading to TAG biosynthesis in leaves exposed to heat stress have implications for improving the biotechnological production of TAGs in vegetative tissues, offering an alternative to seeds., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Masson SAS.)

Seed storage underpins global agriculture and the seed trade and revealing the mechanisms of seed aging is essential for enhancing seed longevity management. Safflower is a multipurpose oil crop, rich in unsaturated fatty acids that are at high risk of peroxidation as a contributory factor to seed aging. However, the molecular mechanisms responsible for safflower seed viability loss are not yet elucidated. We used controlled deterioration (CDT) conditions of 60% relative humidity and 50 °C to reduce germination in freshly harvested safflower seeds and analyzed aged seeds using biochemical and molecular techniques. While seed malondialdehyde (MDA) and fatty acid content increased significantly during CDT, catalase activity and soluble sugar content decreased. KEGG analysis of gene function and qPCR validation indicated that aging severely impaired several key functional and biosynthetic pathways including glycolysis, fatty acid metabolism, antioxidant activity, and DNA replication and repair. Furthermore, exogenous sucrose and diethyl aminoethyl hexanoate (DA-6) treatment partially promoted germination in aged seeds, further demonstrating the vital role of impaired sugar and fatty acid metabolism during the aging and recovery processes. We concluded that energy metabolism and genetic integrity are impaired during aging, which contributes to the loss of seed vigor. Such energy metabolic pathways as glycolysis, fatty acid degradation, and the tricarboxylic acid cycle (TCA) are impaired, especially fatty acids produced by the hydrolysis of triacylglycerols during aging, as they are not efficiently converted to sucrose via the glyoxylate cycle to provide energy supply for safflower seed germination and seedling growth. At the same time, the reduced capacity for nucleotide synthesis capacity and the deterioration of DNA repair ability further aggravate the damage to DNA, reducing seed vitality.

Introduction: Tilia miqueliana is an endemic species whose population is declining. The permeability barrier and mechanical constraint of the pericarp (seed coat) are important causes of its seed dormancy. Although there has been considerable research on this subject, questions remain regarding how the permeability barrier and mechanical constraint of the seed coat are eliminated during dormancy release and how water enters the seed. Therefore, protecting the species by improving its germination/dormancy breaking in the laboratory is urgent., Methods: In this study, the changes in the cellular structure, mechanical properties, and components of the Tilia miqueliana seed coat after an H 2 SO 4 -gibberellic acid (GA 3 ) treatment were analyzed during dormancy release. Various analyses (e.g., magnetic resonance imaging, scanning electron microscopy, and paraffin section detection) revealed the water gap and water channel., Results: The H 2 SO 4 treatment eliminated the blockage at the micropyle and hilum of the seeds. Water entered the seeds through the water gap (micropyle) rather than through the hilum or seed coat, after which it dispersed along the radicle, hypocotyl, and cotyledon to the endosperm. During the cold stratification period, the cellular structure was damaged and an increasing number of holes appeared on the inner and outer surfaces of the seed coat. Vickers hardness tests showed that GA 3 decreased the seed coat hardness. Additionally, the seed coat lignin and total phenol contents continuously decreased during the cold stratification period. Notably, the Liquid chromatography-mass spectrometry (LC-MS) analysis of the seed coat detected polyethylene glycol (osmoregulator), which may have destabilized the water potential balance inside and outside the seed and increased the water content to levels required for germination, ultimately accelerating seed dormancy release., Discussion: This sophisticated and multi-level study reveals how H 2 SO 4 and GA 3 eliminate the permeability barrier and mechanical constraints of the seed coat during dormancy release of Tilia miqueliana seeds. This will be beneficial to artificially assist the natural regeneration and population expansion of Tilia miqueliana ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Wu, Huang, Peng, Shen, Visscher, Pritchard, Gao, Sun, Wang and Deng.)

Introduction: Cryo treatment of dry seeds is known to attenuate the structure of fruit and seed coats, but little is known about the microstructural impacts of such treatment. The seeds of Tilia miqueliana are dispersed within a hard pericarp, the manual removal (hulling) of which is time-consuming and inefficient. Rapid hulling technology is urgently needed for sustainable production and convenience of edible nuts., Methods: We explored the mechanistic basis of liquid nitrogen (N)-treatment weakening of the pericarp of T. miqueliana fruits using a range of microscopical, biophysical and chemical approaches., Results: Liquid N treatment (40 s) resulted in lower pericarp contents of cellulose and hemicellulose, and increased amounts of lignin. Profound changes in cell structure and mechanical properties included the emergence of large holes and gaps between the mesocarp and endocarp cells. Also, the toughness of the pericarp decreased, whilst the hardness and brittleness increased, thereby changing the fracture type from ductile to brittle. Liquid N treatment of dry fruits followed by tapping with a hammer, reduced the number of damaged seeds three-fold and pericarp peeling time four-fold compared with manual hulling, whilst seed viability was not negatively affected., Discussion: Comparable findings for the efficient and economical removal of hard covering structures from dispersal units of five more species from three other families following liquid N treatment indicates the potential application of our findings to large-scale production of seeds and seedlings for breeding, forestry and conservation/restoration purposes. Furthermore, it introduces a novel concept for postharvest treatment and pre-treatment of deep processing in nuts., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Wu, Sun, Peng, Shen, Visscher, Pritchard, Wang and Deng.)

Persistence in the soil is a function of seed physiology, particularly non-germination and inherent lifespan. However, for seeds with mechanical dormancy, non-germination is also a function of the composition and activity of the soil microbiota. We attempted to screen out microorganisms in the soil that can specifically and rapidly decompose the hard fruit pericarps of Tilia miqueliana Maxim., a unique native tree species in China. Using the classical replica plating method, more than 100 different culturable microorganisms that could rapidly erode the pericarp were collected from the surface of pericarps under different culture conditions. At the same time, we successfully extended the concept of metagenomics and applied it to the identification of mixed artificial cultures. The decomposition process of the pericarps in soil was also simulated artificially. The physical and chemical data suggested a potential mechanism of microbial scarification and cracking in pericarp, whilst the embryos inside the eroded fruits retained good viability. Our discoveries could pave the way for the removal of physical and mechanical obstacles that prevent hard coat seeds from germinating. We anticipate that the use of this technology will improve the germination of other hard coat seeds. More research is needed to investigate the impacts on other seeds. The findings of this research can inform the design of experiments on the seed ecology of persistence., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Wu, Sun, Pritchard, Shen, Wu and Peng.)

Intra-specific variation in seed storage behaviour observed in several species has been related to different maternal environments. However, the particular environmental conditions and molecular processes involved in intra-specific variation of desiccation tolerance remain unclear. We chose Citrus sinensis 'bingtangcheng' for the present study due to its known variability in desiccation tolerance amongst seed lots. Six seed lots of mature fruits were harvested across China and systematically compared for drying sensitivity. Annual sunshine hours and average temperature from December to May showed positive correlations with the level of seed survival of dehydration. Transcriptional analysis indicated significant variation in gene expression between relatively desiccation-tolerant (DT) and -sensitive (DS) seed lots after harvest. The major genes involved in late seed maturation, such as heat shock proteins, showed higher expression in the DT seed lot. Following the imposition of drying, 80% of stress-responsive genes in the DS seed lot changed to the stable levels seen in the DT seed lot prior to and post-desiccation. However, the changes in expression of stress-responsive genes in DS seeds did not improve their tolerance to desiccation. Thus, higher desiccation tolerance of Citrus sinensis 'bingtangcheng' seeds is modulated by the maternal environment (e.g., higher annual sunshine hours and seasonal temperature) during seed development and involves stable expression levels of stress-responsive genes.

Many useful plant species with potential for plant-based bioregenerative life support systems produce extremophile seeds with tolerance to multiple stressors, including desiccation, which allows for their transport through space in a dried state. However, other valuable species produce desiccation-sensitive seeds or are propagated clonally, and life sciences research in space has not yet addressed the challenge of alternative transport methods in microgravity for such material. Although liquid nitrogen storage is used on Earth for desiccation-sensitive germplasm, it poses atmospheric leakage problems to crewed spacecraft and therefore liquid nitrogen-free cryogenic freezing could be an alternative. Another promising approach is slow growth tissue culture, with subculture intervals extended to months or years through the precise control of the culture environment. Whilst the design of innovative systems for the transport of species with desiccation-sensitive germplasm will be demanding, the prospect still remains for their successful growth beyond Earth., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest., (Copyright © 2022 The Committee on Space Research (COSPAR). All rights reserved.)

The lifespan or longevity of a seed is the time period over which it can remain viable. Seed longevity is a complex trait and varies greatly between species and even seed lots of the same species. Our scientific understanding of seed longevity has advanced from anecdotal 'Thumb Rules,' to empirically based models, biophysical explanations for why those models sometimes work or fail, and to the profound realisation that seeds are the model of the underexplored realm of biology when water is so limited that the cytoplasm solidifies. The environmental variables of moisture and temperature are essential factors that define survival or death, as well as the timescale to measure lifespan. There is an increasing understanding of how these factors induce cytoplasmic solidification and affect glassy properties. Cytoplasmic solidification slows down, but does not stop, the chemical reactions involved in ageing. Continued degradation of proteins, lipids and nucleic acids damage cell constituents and reduce the seed's metabolic capacity, eventually impairing the ability to germinate. This review captures the evolution of knowledge on seed longevity over the past five decades in relation to seed ageing mechanisms, technology development, including tools to predict seed storage behaviour and non-invasive techniques for seed longevity assessment. It is concluded that seed storage biology is a complex science covering seed physiology, biophysics, biochemistry and multi-omic technologies, and simultaneous knowledge advancement in these areas is necessary to improve seed storage efficacy for crops and wild species biodiversity conservation.

Germplasm conservation strongly depends on the desiccation tolerance (DT) of seeds. Xerophytic seeds have strong desiccation resistance, which makes them excellent models to study DT. Although some experimental strategies have been applied previously, most methods are difficult to apply to xerophytic seeds. In this review, we attempted to synthesize current strategies for the study of seed DT and provide an in-depth look at Caragana korshinskii as an example. First, we analyze congenital advantages of xerophytes in the study of seed DT. Second, we summarize several strategies used to study DT and illustrate a suitable strategy for xerophytic species. Then, based on our previous studies work with C. korshinskii , a feasible technical strategy for DT re-establishment is provided and we provide illustrate some special molecular mechanisms seen in xerophytic seeds. Finally, several steps to unveil the DT mechanism of xerophytic seeds are suggested, and three scientific questions that the field should consider are listed. We hope to optimize and utilize this strategy for more xerophytic species to more systematically decipher the physiological and molecular processes of seed DT and provide more candidate genes for molecular breeding., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Peng, Huang, Qi, Pritchard and Xue.)

During seed aging, there is a critical node (CN) where the population viability drops sharply. Exploring the specific locations of the CN in different species of plants is crucial for understanding the biological storage properties of seeds and refining seed life span management. Safflower, a bulk oil crop that relies on seeds for propagation, has a short seed life. However, at present, its biological characteristics during storage are not clear, especially the changes in metabolic capability and cell structures. Such knowledge is needed to improve the management of safflower seed life span and effective preservation in gene banks. Here, the seed survival curve of oilseed safflower under the controlled deterioration conditions of 60% relative humidity and 50°C was detected. The seed population showed an inverted S shape for the fall in germination. In the first 12 days of aging, germination remained above 86%. Prior to the CN at approximately day 10 (C10), when viability was in the "plateau" interval, seed vigor reduced at the same imbibition time point. Further analysis of the changes in sugar concentration found that the sucrose content decreased slowly with aging and the content of raffinose and two monosaccharides decreased abruptly at C10. Differentially metabolized lipids, namely lysophospholipids [lyso-phosphatidylcholine (LPC) and lyso-phosphatidylethanolamines (LPE)] and PMeOH, increased at day 3 of aging (C3). Fatty acid content increased by C6, and the content of phospholipids [phosphatidylcholines (PC), phosphatidylethanolamines (PE), and phosphatidylinositols (PI) and glycolipids [digalactosyl diacylglycerol, monogalactosyl diacylglycerol, and sulphoquinovosyl diglycerides (SQDG)] decreased significantly from C10. In addition, the activities of raffinose hydrolase alpha-galactosidase and the glyoxylate key enzyme isocitrate lyase decreased with seed aging. Confocal microscopy and transmission electron microscopy revealed shrinkage of the seed plasma membrane at C10 and the later fragmentation. Seedling phenotypic indicators and 2,3,5-triphenyltetrazolium chloride activity assays also verified that there were significant changes in seeds quality at the CN. In summary, the time point C10 is a CN during seed population aging. Before the CN, sugar and lipid metabolism, especially fatty acid metabolism into sugar, can make up for the energy consumed by aging. After this point, the seeds were irreversibly damaged, and their viability was greatly and rapidly reduced as the cell structure became increasingly destroyed., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Zhou, Lu, Chen, Zhou, Wu, Wen, Chen, Pritchard, Peng, Pei and Yan.)

Background and Aims: European ancient woodlands are subject to land use change, and the distribution of herbaceous understorey species may be threatened because of their poor ability to colonize isolated forest patches. The regeneration niche can determine the species assembly of a community, and seed germination traits may be important descriptors of this niche., Methods: We analysed ecological records for 208 herbaceous species regarded as indicators of ancient woodlands in Europe and, where possible, collated data on seed germination traits, reviewed plant regeneration strategies and measured seed internal morphology traits. The relationship between plant regeneration strategies and ecological requirements was explored for 57 species using ordination and classification analysis., Key Results: Three regeneration strategies were identified. Species growing in closed-canopy areas tend to have morphological seed dormancy, often requiring darkness and low temperatures for germination, and their shoots emerge in early spring, thus avoiding the competition for light from canopy species. These species are separated into two groups: autumn and late winter germinators. The third strategy is defined by open-forest plants with a preference for gaps, forest edges and riparian forests. They tend to have physiological seed dormancy and germinate in light and at higher temperatures, so their seedlings emerge in spring or summer., Conclusion: Seed germination traits are fundamental to which species are good or poor colonizers of the temperate forest understorey and could provide a finer explanation than adult plant traits of species distribution patterns. Seed dormancy type, temperature stratification and light requirements for seed germination are important drivers of forest floor colonization patterns and should be taken in account when planning successful ecological recovery of temperate woodland understories., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Despite the importance of dormancy and dormancy cycling for plants' fitness and life cycle phenology, a comprehensive characterization of the global and cellular epigenetic patterns across space and time in different seed dormancy states is lacking. Using Capsella bursa-pastoris (L.) Medik. (shepherd's purse) seeds with primary and secondary dormancy, we investigated the dynamics of global genomic DNA methylation and explored the spatio-temporal distribution of 5-methylcytosine (5-mC) and histone H4 acetylated (H4Ac) epigenetic marks. Seeds were imbibed at 30 °C in a light regime to maintain primary dormancy, or in darkness to induce secondary dormancy. An ELISA-based method was used to quantify DNA methylation, in relation to total genomic cytosines. Immunolocalization of 5-mC and H4Ac within whole seeds (i.e., including testa) was assessed with reference to embryo anatomy. Global DNA methylation levels were highest in prolonged (14 days) imbibed primary dormant seeds, with more 5-mC marked nuclei present only in specific parts of the seed (e.g., SAM and cotyledons). In secondary dormant seeds, global methylation levels and 5-mC signal where higher at 3 and 7 days than 1 or 14 days. With respect to acetylation, seeds had fewer H4Ac marked nuclei (e.g., SAM) in deeper dormant states, for both types of dormancy. However, the RAM still showed signal after 14 days of imbibition under dormancy-inducing conditions, suggesting a central role for the radicle/RAM in the response to perceived ambient changes and the adjustment of the seed dormancy state. Thus, we show that seed dormancy involves extensive cellular remodeling of DNA methylation and H4 acetylation., (© 2021. The Author(s).)

Halting and reversing the current loss of biodiversity and habitats will be facilitated by a comprehensive valuation of all nature's contributions to people (NCPs), on which we rely. In this context, we explore the full natural capital value of seeds to reveal how this extends far beyond their economic value associated with mainstream agriculture and forestry. Seeds represent the main assets for nature-based solutions at species (i.e., unlocking neglected species properties and via seed banking) and ecosystem level (i.e., ecological restoration). Challenges remain to enhance their sustainable use in nature conservation and in supporting a sustainable development model. Such advances will depend on the comprehensive valuation of the natural capital value of seeds, which has so far been grossly underestimated., Competing Interests: Declaration of interests No interests are declared., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Main Conclusion: Modulation of the gaseous environment using oxygen absorbers and/or silica gel shows potential for enhancing seed longevity through trapping toxic volatiles emitted by seeds during artificial ageing. Volatile profiling using non-invasive gas chromatography-mass spectrometry provides insight into the specific processes occurring during seed ageing. Production of alcohols, aldehydes and ketones, derived from processes such as alcoholic fermentation, lipid peroxidation and Maillard reactions, are known to be dependent on storage temperature and relative humidity, but little is known about the potential modulating role of the gaseous environment, which also affects seed lifespan, on volatile production. Seeds of Lolium perenne (Poaceae), Agrostemma githago (Caryophyllaceae) and Pisum sativum (Fabaceae) were aged under normal atmospheric oxygen conditions and in sealed vials containing either oxygen absorbers, oxygen absorbers and silica gel (equilibrated at 60% RH), or silica gel alone. Seeds of A. githago that were aged in the absence of oxygen maintained higher viability and produced fewer volatiles than seeds aged in air. In addition, seeds of A. githago and L. perenne aged in the presence of silica gel were longer lived than those aged without silica, with no effect on seed moisture content or oxygen concentration in the storage containers, but with silica gel acting as a volatile trap. These results indicate that the use of inexpensive oxygen absorbers and silica gel could improve seed longevity in storage for some species and suggests a potential, and previously unidentified, role for silica gel in ultra-dry storage.

Plant species conservation through cryopreservation using plant vitrification solutions (PVS) is based in empiricism and the mechanisms that confer cell integrity are not well understood. Using ESI-MS/MS analysis and quantification, we generated 12 comparative lipidomics datasets for membranes of embryogenic cells (ECs) of Magnolia officinalis during cryogenic treatments. Each step of the complex PVS-based cryoprotocol had a profoundly different impact on membrane lipid composition. Loading treatment (osmoprotection) remodeled the cell membrane by lipid turnover, between increased phosphatidic acid (PA) and phosphatidylglycerol (PG) and decreased phosphatidylcholine (PC) and phosphatidylethanolamine (PE). The PA increase likely serves as an intermediate for adjustments in lipid metabolism to desiccation stress. Following PVS treatment, lipid levels increased, including PC and PE, and this effectively counteracted the potential for massive loss of lipid species when cryopreservation was implemented in the absence of cryoprotection. The present detailed cryobiotechnology findings suggest that the remodeling of membrane lipids and attenuation of lipid degradation are critical for the successful use of PVS. As lipid metabolism and composition varies with species, these new insights provide a framework for technology development for the preservation of other species at increasing risk of extinction.

Seed size shapes plant evolution and ecosystems, and may be driven by plant size and architecture, dispersers, habitat and insularity. How these factors influence the evolution of giant seeds is unclear, as are the rate of evolution and the biogeographical consequences of giant seeds. We generated DNA and seed size data for the palm tribe Borasseae (Arecaceae) and its relatives, which show a wide diversity in seed size and include the double coconut (Lodoicea maldivica), the largest seed in the world. We inferred their phylogeny, dispersal history and rates of change in seed size, and evaluated the possible influence of plant size, inflorescence branching, habitat and insularity on these changes. Large seeds were involved in 10 oceanic dispersals. Following theoretical predictions, we found that: taller plants with fewer-branched inflorescences produced larger seeds; seed size tended to evolve faster on islands (except Madagascar); and seeds of shade-loving Borasseae tended to be larger. Plant size and inflorescence branching may constrain seed size in Borasseae and their relatives. The possible roles of insularity, habitat and dispersers are difficult to disentangle. Evolutionary contingencies better explain the gigantism of the double coconut than unusually high rates of seed size increase., (© 2020 The Authors New Phytologist © 2020 New Phytologist Trust.)

Predicting seed germination in the field is a critical part of anticipating the impact of climate change on the timing of wild species regeneration. We combined thermal time and soil heat sum models of seed germination for three endemic Mediterranean mountain species with endospermic seeds and morphophysiological dormancy: Aquilegia barbaricina , Paeonia corsica , and Ribes sandalioticum . Seeds were buried in the soil within the respective collection sites, both underneath and outside the tree canopy, and their growth was assessed regularly and related to soil temperatures and estimates of the thermal characteristics of the seeds. The thermal thresholds for embryo growth and seed germination of A. barbaricina assessed in previous studies under controlled conditions were used to calculate soil heat sum accumulation of this species in the field. Thermal thresholds of seed germination for P. corsica and R. sandalioticum were not previously known and were estimated for the first time in this field study, based on findings of previous works carried out under controlled conditions. Critical embryo length and maximum germination for A. barbaricina were reached in April, and in December for R. sandalioticum . Seeds of P. corsica stay dormant in the ground until the following summer, and the critical embryo length and highest germination were detected from September to December. Soil heat sum models predicted earlier germination by one month for all three species under two Intergovernmental Panel on Climate Change (IPCC) scenarios, based on the assumption that the estimated thermal thresholds will remain constant through climate changes. This phenological shift may increase the risk of mortality for young seedlings. The models developed provide important means of connecting the micro-environmental niche for in situ seed germination and the macro-environmental parameters under a global warming scenario.

Orchid pollinia have the potential to make a valuable contribution to current techniques of germplasm storage and assisted reproduction, yet information regarding their preservation and their ability to remain viable over time is currently limited. Dactylorhiza fuchsii and Disa uniflora were used as models for investigating potential techniques for storing orchid pollinia. Initially, freshly harvested pollinia of Dact. fuchsii were incubated at 25 °C and 100% RH (relative humidity) for up to 7 days and germinated in vitro. For pollinia from both species, moisture sorption isotherms were constructed and thermal fingerprints generated using differential scanning calorimetry (DSC). Pollinia were stored at three temperatures (5, - 18 and - 196 °C) after equilibration at four different RHs (5, 33, 50 and 75%) and germinated. The isotherms and DSC results varied between species. Compared with D. uniflora, pollinia of Dact. fuchsii consistently equilibrated at higher moisture content (MC) for each RH, had less detectable lipids by DSC and had shorter lifespans, remaining viable after 3-4 months only at - 20 and - 196 °C and at low RH (5 and 33%). Both species' pollinia stored well at - 20 °C and - 196 °C, although there was some evidence of a small loss of viability under cryopreservation. In conclusion, pollen of these two species can be stored successfully for at least 3-4 months, and to maximize the pre-storage quality, it is recommended that fresh pollen is collected from flowers just prior to anthesis.

Drylands are predicted to become more arid and saline due to increasing global temperature and drought. Although species from the Caatinga, a Brazilian tropical dry forest, are tolerant to these conditions, the capacity for germination to withstand extreme soil temperature and water deficit associated with climate change remains to be quantified. We aimed to evaluate how germination will be affected under future climate change scenarios of limited water and increased temperature. Seeds of three species were germinated at different temperatures and osmotic potentials. Thermal time and hydrotime model parameters were established and thresholds for germination calculated. Germination performance in 2055 was predicted, by combining temperature and osmotic/salt stress thresholds, considering soil temperature and moisture following rainfall events. The most pessimistic climate scenario predicts an increase of 3.9 °C in soil temperature and 30% decrease in rainfall. Under this scenario, soil temperature is never lower than the minimum and seldomly higher than maximum temperature thresholds for germination. As long as the soil moisture (0.139 cm 3  cm 3 ) requirements are met, germination can be achieved in 1 day. According to the base water potential and soil characteristics, the minimum weekly rainfall for germination is estimated to be 17.5 mm. Currently, the required minimum rainfall occurs in 14 weeks of the year but will be reduced to 4 weeks by 2055. This may not be sufficient for seedling recruitment of some species in the natural environment. Thus, in future climate scenarios, rainfall rather than temperature will be extremely limiting for seed germination.

Seed morphology underpins many critical biological and ecological processes, such as seed dormancy and germination, dispersal, and persistence. It is also a valuable taxonomic trait that can provide information about plant evolution and adaptations to different ecological niches. This study characterised and compared various seed morphological traits, i.e., seed and pod shape, seed colour and size, embryo size, and air volume for six orchid species; and explored whether taxonomy, biogeographical origin, or growth habit are important determinants of seed morphology. We investigated this on two tropical epiphytic orchid species from Indonesia ( Dendrobium strebloceras and D. lineale ), and four temperate species from New Zealand, terrestrial Gastrodia cunnninghamii , Pterostylis banksii and Thelymitra nervosa , and epiphytic D. cunninghamii . Our results show some similarities among related species in their pod shape and colour, and seed colouration. All the species studied have scobiform or fusiform seeds and prolate-spheroid embryos. Specifically, D. strebloceras , G. cunninghamii , and P. banksii have an elongated seed shape, while T. nervosa has truncated seeds. Interestingly, we observed high variability in the micro-morphological seed characteristics of these orchid species, unrelated to their taxonomy, biogeographical origin, or growth habit, suggesting different ecological adaptations possibly reflecting their modes of dispersal., Competing Interests: The authors declare no conflict of interest.

Thermal fingerprints for seeds of 20 crop wild relatives of Brassicaceae stored for 8 to 44 years at the Plant Germplasm Bank-Universidad Politécnica de Madrid and the Royal Botanic Gardens, Kew's Millennium Seed Bank-were generated using differential scanning calorimetry (DSC) and analyzed in relation to storage stability. Relatively poor storing oily seeds at -20 °C tended to have lipids with crystallization and melting transitions spread over a wide temperature range (c. 40 °C) that spanned the storage temperature, plus a melting end temperature of around 15 °C. We postulated that in dry storage, the variable longevity in Brassicaceae seeds could be associated with the presence of a metastable lipid phase at the temperature at which they are being stored. Consistent with that, when high-quality seed samples of various species were assessed after banking at -5 to -10 °C for c. 40 years, melting end temperatures were observed to be much lower (c. 0 to -30 °C) and multiple lipid phases did not occur at the storage temperature. We conclude that multiple features of the seed lipid thermal fingerprint could be used as biophysical markers to predict potential poor performance of oily seeds during long-term, decadal storage., Competing Interests: The authors declare no conflict of interest.

Much is known about the inter-specific distribution of life span in a wide diversity of vertebrates and in adult plants, but not for seeds, yet the functional trait seed life span underpins global agriculture, plant species conservation and seed persistence in the soil. We sourced data for five storage conditions (soil seed bank; high temperature - high humidity accelerated ageing; temperate, cooler, open storage; cool, dry, refrigerator; and cold, dry, freezer); and analysed the distribution of orthodox seed life span amongst crop and wild species. In all cases, whether for maximum known in situ life span in the soil seed bank (417 species), or for half-lives (P50s) ex situ (732 species), the distribution is right-skewed. The finding that seeds of > 50% of species are likely to have life spans ≤ 20% of the longest recorded under the same conditions has implications for future research on the evolution of seed traits and gene bank collections management., (© 2019 The Authors. New Phytologist © 2019 New Phytologist Trust.)

To elucidate biochemical mechanisms leading to seed deterioration, we studied 23 wheat genotypes after exposure to seed bank storage for 6-16 years compared to controlled deterioration (CD) at 45 °C and 14 (CD14) and 18% (CD18) moisture content (MC) for up to 32 days. Under two seed bank storage conditions, seed viability was maintained in cold storage (CS) at 0 °C and 9% seed MC, but significantly decreased in ambient storage (AS) at 20 °C and 9% MC. Under AS and CS, organic free radicals, most likely semiquinones, accumulated, detected by electron paramagnetic resonance, while the antioxidant glutathione (GSH) was partly lost and partly converted to glutathione disulphide (GSSG), detected by HPLC. Under AS the glutathione half-cell reduction potential (E GSSG/2GSH ) shifted towards more oxidising conditions, from -186 to -141 mV. In seeds exposed to CD14 or CD18, no accumulation of organic free radicals was observed, GSH and seed viability declined within 32 and 7 days, respectively, GSSG hardly changed (CD14) or decreased (CD18) and E GSSG/2GSH shifted to -116 mV. The pH of extracts prepared from seeds subjected to CS, AS and CD14 decreased with viability, and remained high under CD18. Across all treatments, E GSSG/2GSH correlated significantly with seed viability ( r  = 0.8, p <.001). Data are discussed with a view that the cytoplasm is in a glassy state in CS and AS, but during the CD treatments, underwent transition to a liquid state. We suggest that enzymes can be active during CD but not under the seed bank conditions tested. However, upon CD, enzyme-based repair processes were apparently outweighed by deteriorative reactions. We conclude that seed ageing by CD and under seed bank conditions are accompanied by different biochemical reactions.

Plant persistence and migration in face of climate change depends on successful reproduction by seed, a central aspect of plant life that drives population dynamics, community assembly and species distributions. Plant reproduction by seed is a chain of physiological processes, the rates of which are a function of temperature, and can be modelled using thermal time models. Importantly, while seed reproduction responds to its instantaneous thermal environment, there is also evidence of phenotypic plasticity in response to the thermal history experienced by the plant's recent ancestors, by the reproducing plant since seedling establishment, and by its seeds both before and after their release. This phenotypic plasticity enables a thermal memory of plant reproduction, which allows individuals to acclimatise to their surroundings. This review synthesises current knowledge on the thermal memory of plant reproduction by seed, and highlights its importance for modelling approaches based on physiological thermal time. We performed a comprehensive search in the Web of Science and analysed 533 relevant articles, of which 81 provided material for a meta-analysis of thermal memory in reproductive functional traits based on the effect size Zr. The articles encompassed the topics of seed development, seed yield (mass and number), seed dormancy (physiological, morphological and physical), germination, and seedling establishment. The results of the meta-analysis provide evidence for a thermal memory of seed yield, physiological dormancy and germination. Seed mass and physiological dormancy appear to be the central hubs of this memory. We argue for integrating thermal memory into a predictive framework based on physiological time modelling. This will provide a quantitative assessment of plant reproduction, a complex system that integrates past and present thermal inputs to achieve successful reproduction in changing environments. The effects of a warming environment on plant reproduction cannot be reduced to a qualitative interpretation of absolute positives and negatives. Rather, these effects need to be understood in terms of changing rates and thresholds for the physiological process that underlie reproduction by seed., (© 2018 Cambridge Philosophical Society.)

This study explores the temperature dependency of the aging rate in dry cells over a broad temperature range encompassing the fluid to solid transition (Tg) and well below. Spores from diverse species of eight families of ferns were stored at temperatures ranging from +45�C to approximately -176�C (vapor phase above liquid nitrogen), and viability was monitored periodically for up to 4,300 d (∼12 years). Accompanying measurements using differential scanning calorimetry (DSC) provide insights into structural changes that occur, such as Tg between +45 and -20�C (depending on moisture), and triacylglycerol (TAG) crystallization between -5 and -35�C (depending on species). We detected aging even at cryogenic temperatures, which we consider analogous to unscheduled degradation of pharmaceuticals stored well below Tg caused by a shift in the nature of molecular motions that dominate chemical reactivity. We occasionally observed faster aging of spores stored at -18�C (conventional freezer) compared with 5�C (refrigerator), and linked this with mobility and crystallization within TAGs, which probably influences molecular motion of dried cytoplasm in a narrow temperature range. Temperature dependency of longevity was remarkably similar among diverse fern spores, despite widely disparate aging rates; this provides a powerful tool to predict deterioration of germplasm preserved in the solid state. Future work will increase our understanding of molecular organization and composition contributing to differences in longevity., (Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists 2018.)

Globally, annual expenditure on ecological restoration of degraded areas for habitat improvement and biodiversity conservation is approximately $18bn. Seed farming of native plant species is crucial to meet restoration goals, but may be stymied by the disconnection of academic research in seed science and the lack of effective policies that regulate native seed production/supply. To illustrate this problem, we identified 1,122 plant species important for European grasslands of conservation concern and found that only 32% have both fundamental seed germination data available and can be purchased as seed. The " restoration species pool," or set of species available in practice, acts as a significant biodiversity selection filter for species use in restoration projects. For improvement, we propose: (1) substantial expansion of research and development on native seed quality, viability, and production; (2) open-source knowledge transfer between sectors; and (3) creation of supportive policy intended to stimulate demand for biodiverse seed.

High sensitivity of seeds to water loss is a widespread phenomenon in the world's plant species. The molecular basis of this trait is poorly understood but thought to be associated with critical changes in membrane function. We profiled membrane lipids of seeds in eight species with varying levels of desiccation tolerance and found a close association between reducing seed viability and increasing phosphatidic acid (PA). We applied hydration-dehydration cycles to Arabidopsis seeds, which are normally desiccation tolerant, to mimic the onset of desiccation sensitivity with progression towards germination and examined the role of phospholipase D (PLD) in desiccation stress-induced production of PA. We found that PLDα1 became more abundant and migrated from the cytosol to the membrane during desiccation, whereas PLDδ did not change, and that all desiccation-induced PA was derived from PLDα1 hydrolysis. When PLDα1 was suppressed, the germination level after each hydration-dehydration cycle improved significantly. We further demonstrated that PLDα1-mediated PA formation modulates desiccation sensitivity as applying its inhibitor improved seed desiccation tolerance and its suppression in protoplasts enhanced survival under dehydration. The insights provided by comparative lipidomics enable us to propose a new membrane-based model for seed desiccation stress and survival., (© 2017 John Wiley & Sons Ltd.)

Recruitment from seeds is among the most vulnerable stage for plants as global temperatures change. While germination is the means by which the vast majority of the world's flora regenerate naturally, a framework for accurately predicting which species are at greatest risk of germination failure during environmental perturbation is lacking. Taking a physiological approach, we assess how one family, the Cactaceae, may respond to global temperature change based on the thermal buffering capacity of the germination phenotype. We selected 55 cactus species from the Americas, all geo-referenced seed collections, reflecting the broad environmental envelope of the family across 70° of latitude and 3700 m of altitude. We then generated empirical data of the thermal germination response from which we estimated the minimum (T b ), optimum (T o ) and ceiling (T c ) temperature for germination and the thermal time (θ 50 ) for each species based on the linearity of germination rate with temperature. Species with the highest T b and lowest T c germinated fastest, and the interspecific sensitivity of the germination rate to temperature, as assessed through θ 50 , varied tenfold. A left-skewed asymmetry in the germination rate with temperature was relatively common but the unimodal pattern typical of crop species failed for nearly half of the species due to insensitivity to temperature change at T o . For 32 fully characterized species, seed thermal parameters correlated strongly with the mean temperature of the wettest quarter of the seed collection sites. By projecting the mean temperature of the wettest quarter under two climate change scenarios, we predict under the least conservative scenario (+3.7°C) that 25% of cactus species will have reduced germination performance, whilst the remainder will have an efficiency gain, by the end of the 21st century., (© 2017 John Wiley & Sons Ltd.)

Understanding the key aspects of plant regeneration from seeds is crucial in assessing species assembly to their habitats. However, the regenerative traits of seed dormancy and germination are underrepresented in this context. In the alpine zone, the large species and microhabitat diversity provide an ideal context to assess habitat-related regenerative strategies. To this end, seeds of 53 species growing in alpine siliceous and calcareous habitats (6230 and 6170 of EU Directive 92/43, respectively) were exposed to different temperature treatments under controlled laboratory conditions. Germination strategies in each habitat were identified by clustering with k-means. Then, phylogenetic least squares correlations (PGLS) were fitted to assess germination and dormancy differences between species' main habitat (calcareous and siliceous), microhabitat (grasslands, heaths, rocky, and species with no specific microhabitats), and chorology (arctic-alpine and continental). Calcareous and siliceous grasslands significantly differ in their germination behaviour with a slow, mostly overwinter germination and high germination under all conditions, respectively. Species with high overwinter germination occurs mostly in heaths and have an arctic-alpine distribution. Meanwhile, species with low or high germinability in general inhabit in grasslands or have no specific microhabitat (they belong to generalist), respectively. Alpine species use different germination strategies depending on habitat provenance, species' main microhabitat, and chorotype. Such differences may reflect adaptations to local environmental conditions and highlight the functional role of germination and dormancy in community ecology.

In close agreement with visible germination, orthodox seeds lose desiccation tolerance (DT). This trait can be regained under osmotic stress, but the mechanisms are poorly understood. In this study, germinating seeds of Caragana korshinskii Kom. were investigated, focusing on the potential modulating roles of reactive oxygen species (ROS) in the re-establishment of DT. Germinating seeds with 2 mm long radicles can be rendered tolerant to desiccation by incubation in a polyethylene glycol (PEG) solution (-1.7 MPa). Upon PEG incubation, ROS accumulation was detected in the radicles tip by nitroblue tetrazolium chloride staining and further confirmed by confocal microscopy. The PEG-induced re-establishment of DT was repressed when ROS scavengers were added to the PEG solution. Moreover, ROS act downstream of abscisic acid (ABA) to modulate PEG-mediated re-establishment of DT and serve as a new inducer to re-establish DT. Transcriptomic analysis revealed that re-establishment of DT by ROS involves the up-regulation of key genes in the phenylpropanoid-flavonoid pathway, and total flavonoid content and key enzyme activity increased after ROS treatment. Furthermore, DT was repressed by an inhibitor of phenylalanine ammonia lyase. Our data suggest that ROS play a key role in the re-establishment of DT by regulating stress-related genes and the phenylpropanoid-flavonoid pathway., (© The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Reactive oxygen species (ROS)-related mitochondrial dysfunction is considered to play a vital role in seed deterioration. However, the detailed mechanisms remain largely unknown. To address this, a comparison of mitochondrial proteomes was performed, and we identified several proteins that changed in abundance with accompanying ROS eruption and mitochondrial aggregation and diffusion. These are involved in mitochondrial metabolisms, stress resistance, maintenance of structure and intracellular transport during seed aging. Reduction of ROS content by the mitochondrial-specific scavenger MitoTEMPO suppressed these changes, whereas pre-treatment of seeds with methyl viologen (MV) had the opposite effect. Furthermore, voltage-dependent anion channels (VDAC) were found to increase both in abundance and carbonylation level, accompanied by increased cytochrome c (cyt c) release from mitochondria to cytosol, indicating the profound effect of ROS and VDAC on mitochondria-dependent cell death. Carbonylation detection revealed the specific target proteins of oxidative modification in mitochondria during ageing. Notably, membrane proteins accounted for a large proportion of these targets. An in vitro assay demonstrated that the oxidative modification was concomitant with a change of VDAC function and a loss of activity in malate dehydrogenase. Our data suggested that ROS eruption induced alteration and modification of specific mitochondrial proteins that may be involved in the process of mitochondrial deterioration, which eventually led to loss of seed viability., (Copyright © 2017. Published by Elsevier Masson SAS.)

Seed germination is comprehensively regulated by multiple intrinsic and extrinsic factors, and reactive oxygen species (ROS) are relatively new among these factors. However, the role and underlying mechanisms of ROS in germination regulation remain largely unknown. In this study, we initially found that cold stratification could promote germination and respiration of Hedysarum scoparium seeds, especially at low temperature. We then noted that a ROS environment change induced by hydrogen peroxide (H 2 O 2 ) or methylviologen (MV) could similarly promote seed germination. On the other hand, the ROS scavenger N-acetyl-L-cysteine (NAC) suppressed germination of cold-stratified H. scoparium seeds, indicating a stimulatory role of ROS upon seed germination. An increased accumulation of O 2 - was detected in embryonic axes of cold-stratified seeds, and stratification-induced ROS generation as well as progressive accumulation of ROS during germination was further confirmed at the cellular level by confocal microscopy. Moreover, protein carbonylation in cold-stratified seeds was enhanced during germination, which was reversed by NAC treatment. Finally, the relationship between ROS and abscisic acid (ABA) or gibberellin (GA) in germination regulation was investigated. ABA treatment significantly inhibited germination and reduced the H 2 O 2 content in both cold-stratified and non-cold-stratified seeds. Furthermore, we found that cold stratification mediates the down-regulation of the ABA content and increase of GA, suggesting an interaction between ROS and ABA/GA. These results in H. scoparium shed new light on the positive role of ROS and their cross-talk between plant hormones in seed germination., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.)

Carica papaya (papaya) seed germinate readily fresh from the fruit, but desiccation induces a dormant state. Dormancy can be released by exposure of the hydrated seed to a pulse of elevated temperature, typical of that encountered in its tropical habitat. Carica papaya is one of only a few species known to germinate in response to heat shock (HS) and we know little of the mechanisms that control germination in tropical ecosystems. Here we investigate the mechanisms that mediate HS-induced stimulation of germination in pre-dried and re-imbibed papaya seed. Exogenous gibberellic acid (GA 3 ≥250 µM) overcame the requirement for HS to initiate germination. However, HS did not sensitise seeds to GA 3 , indicative that it may act independently of GA biosynthesis. Seed coat removal also overcame desiccation-imposed dormancy, indicative that resistance to radicle emergence is coat-imposed. Morphological and biomechanical studies identified that neither desiccation nor HS alter the physical structure or the mechanical strength of the seed coat. However, cycloheximide prevented both seed coat weakening and germination, implicating a requirement for de novo protein synthesis in both processes. The germination antagonist abscisic acid prevented radicle emergence but had no effect on papaya seed coat weakening. Desiccation therefore appears to reduce embryo growth potential, which is reversed by HS, without physically altering the mechanical properties of the seed coat. The ability to germinate in response to a HS may confer a competitive advantage to C. papaya, an opportunistic pioneer species, through detection of canopy removal in tropical forests., (© The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Approximately one fifth of the world's plants are at risk of extinction. Of these, a significant number exist as populations of few individuals, with limited distribution ranges and under enormous pressure due to habitat destruction. In China, these most-at-risk species are described as 'plant species with extremely small populations' (PSESP). Implementing conservation action for such listed species is urgent. Storing seeds is one of the main means of ex situ conservation for flowering plants. Spore storage could provide a simple and economical method for fern ex situ conservation. Seed and spore germination in nature is a critical step in species regeneration and thus in situ conservation. But what is known about the seed and spore biology (storage and germination) of at-risk species? We have used China's PSESP (the first group listing) as a case study to understand the gaps in knowledge on propagule biology of threatened plant species. We found that whilst germination information is available for 28 species (23% of PSESP), storage characteristics are only known for 8% of PSESP (10 species). Moreover, we estimate that 60% of the listed species may require cryopreservation for long-term storage. We conclude that comparative biology studies are urgently needed on the world's most threatened taxa so that conservation action can progress beyond species listing.

Orchids (Orchidaceae) are one of the most diverse plant groups on the planet with over 25,000 species. For over a century, scientists and horticulturalists have been fascinated by their complex floral morphology, pollinator specificity and multiple ethnobotanical uses, including as food, flavourings, medicines, ornaments, and perfumes. These important traits have stimulated world-wide collection of orchid species, often for the commercial production of hybrids and leading to frequent overexploitation. Increasing human activities and global environmental changes are also accelerating the threat of orchid extinction in their natural habitats. In order to improve gene conservation strategies for these unique species, innovative developments of cryopreservation methodologies are urgently needed based on an appreciation of low temperature (cryo) stress tolerance, the stimulation of recovery growth of plant tissues in vitro and on the 'omics' characterization of the targeted cell system (biotechnology). The successful development and application of such cryobiotechnology now extends to nearly 100 species and commercial hybrids of orchids, underpinning future breeding and species conservation programmes. In this contribution, we provide an overview of the progress in cryobanking of a range of orchid tissues, including seeds, pollen, protocorms, protocorm-like bodies, apices excised from in vitro plants, cell suspensions, rhizomes and orchid fungal symbionts. We also highlight future research needs., (Copyright © 2016. Published by Elsevier Inc.)

In the context of climate change, food security and long-term human space missions, it is important to understand which species produce seeds that can tolerate extreme environmental conditions. Here we consider dry seed survival of extreme conditions encountered in both natural and artificially controlled environments. Considerable overlap exists between the two: for example, ultra-dry and anoxic conditions can be artificially imposed during seed storage and also occur naturally in the vacuum of space environments. Aside from ultra-drying and anoxia, dry seeds of many species may experience extremely high temperatures due to heat from wildfires or when exposed to solar heat in biomes such as deserts. In addition, seeds can be irradiated by UV-A and UV-B at the surface of the Earth and by the shorter wavelengths of UV-C in outer space. We focus on the effects of these extreme environmental conditions on dry seed lifespan and germination. Although it is clear that seeds from particular plant species and families can tolerate exposures to ultra-drying, high temperatures (at least 32 families) or UV radiation with minimal consequences for subsequent germination ability, further research is needed to elucidate many of the mechanisms underlying extreme tolerance of these environmental conditions found on Earth or in space.

Background and Aims: Environmental temperature regulates plant regeneration via seed in several superimposed ways, and this complex regulation will be disrupted by climate change. The role of diurnally alternating temperatures (ΔT) in terminating dormancy will be a major factor in this disruption, as its effects on seed germination are immediate., Methods: The effect of ΔT on seed germination was modelled using two populations of the wetland sedge Carex diandra, one from a montane site and one from a subalpine site. A cardinal-temperature model was fitted to germination results obtained from a thermal gradient plate, and the model was used to simulate changes in germination under two possible future climate scenarios (RCP2·6 and RCP8·5, for representative concentration pathways) as defined by the Intergovernmental Panel on Climate Change., Key Results: Scenario RCP2·6 projected moderate increases in average temperatures and ΔT, whereas RCP8·5 projected greater warming and higher ΔT. Increasing ΔT decreased the base temperature for seed germination and the thermal time required for germination. The effect of higher ΔT together with the higher temperatures increased germination under both climate scenarios., Conclusions: Carex diandra germination is highly responsive to potential changes in ΔT, and thus this study highlights the role of ΔT in seed responses to climate change. Comprehensive cardinal-temperature models, encompassing the different effects of temperature on seed germination, are needed to understand how climate change will affect plant regeneration., (© The Author 2015. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Previous studies have shown that controlled deterioration treatment (CDT) induces programmed cell death in elm (Ulmus pumila L.) seeds, which undergo certain fundamental processes that are comparable to apoptosis in animals. In this study, the essential characteristics of mitochondrial physiology in elm seeds during CDT were identified by cellular ultrastructural analysis, whole-body optical imaging, Western blotting and semi-quantitative RT-PCR. The alteration in mitochondrial morphology was an early event during CDT, as indicated by progressive dynamic mitochondrial changes and rupture of the mitochondrial outer membrane; loss of mitochondrial transmembrane potential (Δψ(m)) ensued, and mitochondrial ATP levels decreased. The mitochondrial permeability transition pore inhibitor cyclosporine A effectively suppressed these changes during ageing. The in situ localization of production of reactive oxygen species (ROS), and evaluation of the expression of voltage-dependent anion-selective channel and cyclophilin D indicated that the levels of mitochondrial permeability transition pore components were positively correlated with ROS production, leading to an imbalance of the cellular redox potential and ultimately to programmed cell death. Pre-incubation with ascorbic acid slowed loss of mitochondrial Δψ(m), and decreased the effect of CDT on seed viability. However, there were no significant changes in multiple antioxidant elements or chaperones in the mitochondria during early stages of ageing. Our results indicate that CDT induces dynamic changes in mitochondrial physiology via increased ROS production, ultimately resulting in an irreversible loss of seed viability., (© 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd.)

Background and Aims: Pollinator-limited seed-set in some terrestrial orchids is compensated for by the presence of long-lived flowers. This study tests the hypothesis that pollen from these insect-pollinated orchids should be desiccation tolerant and relatively long lived using four closely related UK terrestrial species; Anacamptis morio, Dactylorhiza fuchsii, D. maculata and Orchis mascula., Methods: Pollen from the four species was harvested from inflorescences and germinated in vitro, both immediately and also after drying to simulate interflower transit. Their tolerance to desiccation and short-term survival was additionally assessed after 3 d equilibration at a range of relative humidities (RHs), and related to constructed sorption isotherms (RH vs. moisture content, MC). Ageing of D. fuchsii pollen was further tested over 2 months against temperature and RH, and the resultant survival curves were subjected to probit analysis, and the distribution of pollen death in time (σ) was determined. The viability and siring ability, following artificial pollinations, were determined in D. fuchsii pollen following storage for 6 years at -20 °C., Key Results: The pollen from all four species exhibited systematic increases in germinability and desiccation tolerance as anthesis approached, and pollen from open flowers generally retained high germinability. Short-term storage revealed sensitivity to low RH, whilst optimum survival occurred at comparable RHs in all species. Similarly, estimated pollen life spans (σ) at differing temperatures were longest under the dry conditions. Despite a reduction in germination and seeds per capsule, long-term storage of D. fuchsii pollen did not impact on subsequent seed germination in vitro., Conclusions: Substantial pollen desiccation tolerance and life span of the four entomophilous orchids reflects a resilient survival strategy in response to unpredictable pollinator visitation, and presents an alternative approach to germplasm conservation., (© The Author 2014. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Seed deterioration is detrimental to plant germplasm conservation, and predicting seed germination and vigor with reliability and sensitivity means is urgently needed for practical problems. We investigated the link between hydrogen peroxide (H2O2) flux, oxygen influx and seed vigor of Caragana korshinskii by the non-invasive micro-test technique (NMT). Some related physiological and biochemical changes in seeds were also determined to further explain the changes in the molecular fluxes. The results showed that there was a good linear relationship between germination and H2O2 flux, and that O2 influx was more suitable for assessing seed vigor. H2O2 flux changed relatively little initially, mainly affected by antioxidants (APX, CAT and GSH) and H2O2 content; afterward, the efflux increased more and more rapidly due to high membrane permeability. With the damage of mitochondrial respiration and membrane integrity, O2 influx was gradually reduced. We propose that monitoring H2O2 and O2 fluxes by NMT may be a reliable and sensitive method to evaluate seed germination and vigor.

Heterogeneity in morphology, physiology and cellular chemistry of plant tissues can compromise successful cryoprotection and cryopreservation. Cryoprotection is a function of exposure time × temperature × permeability for the chosen protectant and diffusion pathway length, as determined by specimen geometry, to provide sufficient dehydration whilst avoiding excessive chemical toxicity. We have developed an innovative method of vacuum infiltration vitrification (VIV) at 381 mm (15 in) Hg (50 kPa) that ensures the rapid (5 min), uniform permeation of Plant Vitrification Solution 2 (PVS2) cryoprotectant into plant embryos and their successful cryopreservation, as judged by regrowth in vitro. This method was validated on zygotic embryos/embryonic axes of three species (Carica papaya, Passiflora edulis and Laurus nobilis) up to 1.6 mg dry mass and 5.6 mm in length, with varying physiology (desiccation tolerances) and 80 °C variation in lipid thermal profiles, i.e., visco-elasticity properties, as determined by differential scanning calorimetry. Comparisons between the melting features of cryoprotected embryos and embryo regrowth indicated an optimal internal PVS2 concentration of about 60% of full strength. The physiological vigour of surviving embryos was directly related to the proportion of survivors. Compared with conventional vitrification, VIV-cryopreservation offered a ∼ 10-fold reduction in PVS2 exposure times, higher embryo viability and regrowth and greater effectiveness at two pre-treatment temperatures (0 °C and 25 °C). VIV-cryopreservation may form the basis of a generic, high throughput technology for the ex situ conservation of plant genetic resources, aiding food security and protection of species from diverse habitats and at risk of extinction.

Background and Aims: Mediterranean mountain species face exacting ecological conditions of rainy, cold winters and arid, hot summers, which affect seed germination phenology. In this study, a soil heat sum model was used to predict field emergence of Rhamnus persicifolia, an endemic tree species living at the edge of mountain streams of central eastern Sardinia., Methods: Seeds were incubated in the light at a range of temperatures (10-25 and 25/10 °C) after different periods (up to 3 months) of cold stratification at 5 °C. Base temperatures (Tb), and thermal times for 50 % germination (θ50) were calculated. Seeds were also buried in the soil in two natural populations (Rio Correboi and Rio Olai), both underneath and outside the tree canopy, and exhumed at regular intervals. Soil temperatures were recorded using data loggers and soil heat sum (°Cd) was calculated on the basis of the estimated Tb and soil temperatures., Key Results: Cold stratification released physiological dormancy (PD), increasing final germination and widening the range of germination temperatures, indicative of a Type 2 non-deep PD. Tb was reduced from 10·5 °C for non-stratified seeds to 2·7 °C for seeds cold stratified for 3 months. The best thermal time model was obtained by fitting probit germination against log °Cd. θ50 was 2·6 log °Cd for untreated seeds and 2·17-2·19 log °Cd for stratified seeds. When θ50 values were integrated with soil heat sum estimates, field emergence was predicted from March to April and confirmed through field observations., Conclusions: Tb and θ50 values facilitated model development of the thermal niche for in situ germination of R. persicifolia. These experimental approaches may be applied to model the natural regeneration patterns of other species growing on Mediterranean mountain waterways and of physiologically dormant species, with overwintering cold stratification requirement and spring germination.

Desiccation-tolerant plants are able to withstand dehydration and resume normal metabolic functions upon rehydration. These plants can be dehydrated until their cytoplasm enters a 'glassy state' in which molecular mobility is severely reduced. In desiccation-tolerant seeds, longevity can be enhanced by drying and lowering storage temperature. In these conditions, they still deteriorate slowly, but it is not known if deteriorative processes include enzyme activity. The storage stability of photosynthetic organisms is less studied, and no reports are available on the glassy state in photosynthetic tissues. Here, the desiccation-tolerant moss Syntrichia ruralis was dehydrated at either 75% or <5% relative humidity, resulting in slow (SD) or rapid desiccation (RD), respectively, and different residual water content of the desiccated tissues. The molecular mobility within dry mosses was assessed through dynamic mechanical thermal analysis, showing that at room temperature only rapidly desiccated samples entered the glassy state, whereas slowly desiccated samples were in a 'rubbery' state. Violaxanthin cycle activity, accumulation of plastoglobules, and reorganization of thylakoids were observed upon SD, but not upon RD. Violaxanthin cycle activity critically depends on the activity of violaxanthin de-epoxidase (VDE). Hence, it is proposed that enzymatic activity occurred in the rubbery state (after SD), and that in the glassy state (after RD) no VDE activity was possible. Furthermore, evidence is provided that zeaxanthin has some role in recovery apparently independent of its role in non-photochemical quenching of chlorophyll fluorescence.

Background and Aims: The importance of thermal thresholds for predicting seed dormancy release and germination timing under the present climate conditions and simulated climate change scenarios was investigated. In particular, Vitis vinifera subsp. sylvestris was investigated in four Sardinian populations over the full altitudinal range of the species (from approx. 100 to 800 m a.s.l)., Methods: Dried and fresh seeds from each population were incubated in the light at a range of temperatures (10-25 and 25/10 °C), without any pre-treatment and after a warm (3 months at 25 °C) or a cold (3 months at 5 °C) stratification. A thermal time approach was then applied to the germination results for dried seeds and the seed responses were modelled according to the present climate conditions and two simulated scenarios of the Intergovernmental Panel on Climate Change (IPCC): B1 (+1·8 °C) and A2 (+3·4 °C)., Key Results: Cold stratification released physiological dormancy, while very few seeds germinated without treatments or after warm stratification. Fresh, cold-stratified seeds germinated significantly better (>80 %) at temperatures ≥20 °C than at lower temperatures. A base temperature for germination (T(b)) of 9·0-11·3 °C and a thermal time requirement for 50 % of germination (θ(50)) ranging from 33·6 °Cd to 68·6 °Cd were identified for non-dormant cold-stratified seeds, depending on the populations. This complex combination of thermal requirements for dormancy release and germination allowed prediction of field emergence from March to May under the present climatic conditions for the investigated populations., Conclusions: The thermal thresholds for seed germination identified in this study (T(b) and θ(50)) explained the differences in seed germination detected among populations. Under the two simulated IPCC scenarios, an altitude-related risk from climate warming is identified, with lowland populations being more threatened due to a compromised seed dormancy release and a narrowed seed germination window.

Seed deterioration is poorly understood and remains an active area for research. Seeds of elm (Ulmus pumila L.) were aged at 37 °C above water [controlled deterioration treatment (CDT)] for various lengths of time to assess programmed cell death (PCD) and reactive oxygen species (ROS) product in embryonic tissues during a 5 d period. The hallmarks of PCD were identified in the elm seeds during CDT including TUNEL experiments, DNA laddering, cytochrome c (cyt c) leakage and enzymatic activities. These analyses indicated that PCD occurred systematically and progressively in deteriorated elm seeds. Cyt c release and increase in caspase-3-like/DEVDase activity occurred during CDT, which could be suppressed by ascorbic acid (AsA) and caspase-3 inhibitor Ac-DEVD-CHO, respectively. In situ localization of ROS production indicated that the distinct spatial-temporal signature of ROS during CDT coincided with the changes in PCD hallmark features. Multiple antioxidant elements were activated during the first few days of CDT, but were subsequently depleted as PCD progressed. Taken together, our findings identify PCD as a key mechanism that occurs asymmetrically during elm seeds CDT and suggest an important role for PCD in seeds deterioration., (© 2012 Blackwell Publishing Ltd.)

The volatile compounds released by orthodox (desiccation-tolerant) seeds during ageing can be analysed using gas chromatography-mass spectrometry (GC-MS). Comparison of three legume species (Pisum sativum, Lathyrus pratensis, and Cytisus scoparius) during artificial ageing at 60% relative humidity and 50 °C revealed variation in the seed volatile fingerprint between species, although in all species the overall volatile concentration increased with storage period, and changes could be detected prior to the onset of viability loss. The volatile compounds are proposed to derive from three main sources: alcoholic fermentation, lipid peroxidation, and Maillard reactions. Lipid peroxidation was confirmed in P. sativum seeds through analysis of malondialdehyde and 4-hydroxynonenal. Volatile production by ageing orthodox seeds was compared with that of recalcitrant (desiccation-sensitive) seeds of Quercus robur during desiccation. Many of the volatiles were common to both ageing orthodox seeds and desiccating recalcitrant seeds, with alcoholic fermentation forming the major source of volatiles. Finally, comparison was made between two methods of analysis; the first used a Tenax adsorbent to trap volatiles, whilst the second used solid phase microextraction to extract volatiles from the headspace of vials containing powdered seeds. Solid phase microextraction was found to be more sensitive, detecting a far greater number of compounds. Seed volatile analysis provides a non-invasive means of characterizing the processes involved in seed deterioration, and potentially identifying volatile marker compounds for the diagnosis of seed viability loss.