Two different pre-emergence treatments preventing radicle protrusion by limiting the extent of imbibition were tested on conifer seeds. Osmotic priming in solutions of polyethylene glycol (PEG) which prevented radicle protrusion during prolonged (3 weeks) treatment resulted in variable percentage germination, mean germination time, and coefficient of uniformity of germination among different seedlots when placed on water. All three germination parameters were either unchanged or inferior in seeds subjected to the various PEG concentrations, treatment temperatures and treatment durations when compared with prechilled controls. In contrast, white spruce and jack pine seeds invigorated at 30% moisture content and 15°C in Gore-Tex membrane tubes consistently germinated faster than prechilled control seeds when subsequently placed on water. Tube invigoration affected neither the ability of seeds to complete germination nor the uniformity of germination. The decrease in mean germination time as treatment duration progressed was always linear and plateaued at approximately 4 days after the tube-invigorated seeds were placed on water regardless of species or seedlot. On average, tube invigoration decreased white spruce mean germination time by 0.13±0.02, and black spruce by 0.23±0.03 day per day of invigoration for up to 20 days of treatment. Jack pine mean germination time decreased 0.2±0.04 day per day of invigoration for up to 14 days of treatment.

During soybean seed germination, the expansive growth potential of the embryonic axes is driven by water uptake while cell wall loosening occurs in cells from the elongation zone (EZ). Expansins are regarded as primary promoters of cell wall remodelling in all plant expansion processes, with the expression profiles of the soybean expansins supporting their cell or tissue specificity. Therefore, we used embryonic axes isolated from whole seed and focused on the EZ to study seed germination. Using a suite of degenerate primers, we amplified an abundantly expressed expansin gene at the EZ during soybean embryonic axis germination, which was identified as EXP1 by in silico analyses. Expression studies showed that EXP1 was induced under germination conditions in distilled water and down-regulated by abscisic acid (ABA), which inhibits soybean germination by physiologically restraining expansion. Moreover, we also identified a time window of ABA responsiveness within the first 6 h of incubation in water, after which ABA lost control of both EXP1 expression and embryonic axis germination, thus confirming the early role of EXP1 in the EZ during this process. By contrast, EXP1 levels in the EZ increased even when germination was impaired by osmotically limiting the water availability required to develop the embryonic axes’ growth potential. We propose that these higher EXP1 levels are involved in the fast germination of soybean embryonic axes as soon as water availability is re-established. Taken together, our results show strong EXP1 expression in the EZ and postulate EXP1 as a target candidate for soybean seed germination control.

The micromorphology of a seed is linked to its dispersal and germination, but evolutionary and ecological aspects in Orchidaceae remain unclear. We investigated the seed characters of Paphiopedilum and Cypripedium that might be associated with life form and involved in possible ecological adaptations. A phylogenetic comparative analysis of nine seed micromorphological characters was performed in 24 species from two genera with close phylogenetic relationships but significant differences in their ecological characteristics. Species within Paphiopedilum had larger embryos and a smaller percentage of air space (AS) than those of Cypripedium species. Compared with 16 terrestrial species, two epiphytic Paphiopedilum species had larger embryos and smaller AS. Those larger embryos might ensure more successful seedling establishment while the higher amount of air space in both terrestrial Paphiopedilum and Cypripedium may increase seed buoyancy and enable them to disperse over longer distances. Whereas AS and seed length (SL) are phylogenetically conservative, most other characters examined here had weak signals, indicating clear convergent evolution. Across species, SL was positively correlated with AS, indicating a high degree of seed size–dispersal coordination. These findings may imply a trade-off of seed characters in relation to the possible ecological adaptations required for seedling establishment versus dispersal.

The seed coat of soybean (Glycine max (L.) Merrill) must protect the seed but allow water intake. Overprotection, causing impermeability, is assumed to be due to the presence of an impermeable layer in the seed coat, although validation of this assumption has relied on imbibition testing, which tends to be influenced by microfractures in the seed coat. Recent micromorphological analyses using laser-assisted topography microscopy revealed links to the surface roughness (SR) of the seed coat. To verify genetic links between hardseededness and SR, we analysed quantitative trait loci (QTLs) governing SR formation using 148 recombinant inbred lines (RILs) with a genetic linkage map covering 2663.6 cM of all 20 linkage groups of soybean, with 355 DNA markers and 5 phenotype markers. Five QTLs were detected, including previously identified hardseededness QTLs for ratio of seeds absorbing water, namely RAS1 and RAS2, which accounted for 20% of the phenotypic variance, and one near a locus inhibiting seed coat colour (I). These results indicate that the impermeability of soybean seed is genetically related to the reduction of SR.

We studied the variability of germination, dormancy and viability loss of Hirschfeldia incana seeds in relation to seed size. Seeds were stored at 35°C under humid [75% relative humidity (RH)] or dry (33% RH) conditions. Seed germination and electrolyte leakage were evaluated periodically. Small seeds had lower longevity at humid or dry storage conditions (5 or 407 days, respectively) than large or intermediate seeds (7–9 or 536–727 days, respectively). Moreover, H. incana shows variability in seed dormancy related to seed size within a population, with small seeds having lower dormancy (13%) than intermediate (50%) or large seeds (72%). Dormancy was partially released after a short storage at 35°C and humid conditions. Under dry storage conditions, endogenous dormancy cycles were observed for over a year, and longer times of storage had a dormancy-breaking effect through dry after-ripening. Results suggest a dual strategy producing non-dormant seeds with low longevity that will germinate immediately after dispersal, and seeds with greater longevity that will delay germination. Membrane permeability increased linearly with ageing at both humid and dry storage (R2 = 0.60). Small seeds showed greater conductivity than intermediate or large seeds (0.7, 0.4 or 0.3 mS g–1 dry weight, respectively, at the 80% germination). The conductivity test could be used to evaluate the quality of H. incana seeds and would allow us to identify dormant (non-germinating) seed lots as viable. However, the influence of storage conditions and variability within a seed population on seed longevity should be taken into account when evaluating seed quality.

The seeds of most Australian acacias have pronounced physical dormancy (PY). While fire and hot water (HW) treatments cause the lens to ‘pop’ almost instantaneously, for many Acacia species the increase in germination percentage can be gradual. If PY is broken instantly by HW treatment, why is germination often an extended process? Control and HW treatments were performed on seeds of 48 species of Acacia. Seeds were placed on a moist substrate and imbibition was assessed by frequently weighing individual seeds. In the two soft-seeded species all control seeds were fully imbibed within 6–24 h, while in hard-seeded species very few control seeds imbibed over several weeks. In 10 species over 50% of the HW-treated seeds imbibed within 30 h, but mostly the percentage of imbibed seeds gradually increased over several weeks. Some seeds in a replicate would imbibe early, while others would remain unimbibed for many days or weeks then, remarkably, become fully imbibed in less than 24 h. While HW treatment broke PY almost instantaneously, it appeared that in many Acacia species some other part of the testa slowed water from reaching the embryo. This process of having staggered imbibition may be a way of ensuring not all seeds in a population germinate after small rain events. Thus it appears the lens acts as a ‘fire gauge’ while some other part of the seed coat acts as a ‘rain gauge’.

The histological and biochemical characteristics of cacao (Theobroma cacao) endosperm were determined at different stages of seed development. At various stages, the endosperm was analysed for minerals, organic acids, carbohydrates, free amino acids, abscisic acid (ABA), indole-3-acetic acid (IAA) and cytokinins. Cacao endosperm was coenocytic and became progressively cellularized from the micropylar zone. From stage I to IIz, the endosperm started to degenerate all around the embryo. Among the minerals detected, potassium and sulphate were the most abundant. Ammonium was the major source of mineral nitrogen, and glutamine was the most abundant free amino acid. Total nitrogen concentration was quite low. No sulphur-containing amino acids could be detected. Malate was present at all endosperm stages and was the most abundant organic acid. The sugars in cacao endosperm were mainly reducing sugars: glucose and fructose. ABA was only detected from stage IIz. IAA and three cytokinins [zeatin, zeatin-riboside and isopentenyl adenosine (iPA)] were present at all stages of development. Cacao endosperm resembled the endosperm of other species in (1) the abundance of organic acids and phosphate; and (2) a unique combination of sugars. However, three features seemed to be specific to cacao: (1) nitrogen was supplied mainly in the form of ammonium and amino acids, whereas, in other species, amino acids represent the predominant form; (2) there was a relatively small amount of nitrogen, and (3) a high concentration of sulphate and the absence of sulphur amino acids.

Germination studies at the community level are crucial for understanding and predicting changes in species distribution patterns, particularly in endangered plant communities. We investigated the effects of dry (11–25% relative humidity) and freezing (–18°C) storage conditions, phylogeny and plant life-history traits (life-form, life-span, microhabitat and seed dispersal mode) on seed germination percentage (GP) and time to reach 50% germination (T50) of 53 species native to pine rocklands in South Florida, USA, a globally critically imperiled ecosystem. Most species we studied (68%) withstood dry and freezing storage conditions and thus ex situ seed banking can assist their long-term conservation. Bayesian mixed effect models revealed that there was a significant phylogenetic signal in GP and T50 across species. Life-history covariates did not explain significant additional variation in GP in models controlling for the phylogenetic relationships among species. T50 differed among species with contrasting dispersal modes, with animal-dispersed seeds exhibiting more delayed germination than wind-dispersed or unassisted seeds. Differential germination responses across species with different seed dispersal modes have implications for potential shifts in species composition under disturbance and climate change. Thus, knowledge of species-relatedness and some life-history traits such as seed dispersal mode can significantly assist management decisions regarding seed storage and conservation of subtropical endangered plants.

Inga vera subsp. affinis (Fabaceae) is a tree species native to riparian forests in Southeast Brazil and is key for the restoration of deforested areas. The species produce seeds that are highly recalcitrant. Extreme sensitivity to desiccation as well as vivipary are commonly observed in mature seeds, which also tend towards polyembryony. Past research has shown that typical strategies to store seeds are inapplicable to Inga vera as viability is completely lost when seeds are either dried to around 28% water content (wet basis) or stored at 5°C for a few weeks. Here, we examine the feasibility of storing the seeds under hydrated conditions but at reduced water potential. Freshly collected seeds were kept under conventional storage conditions (plastic bags in cold chamber, 5°C) and in polyethylene glycol (PEG) solutions (−1.6 and −2.4 MPa) at 10°C. Seed germination was assessed after various intervals of time, until all seeds had lost viability. Before storage, seeds attained 100% germination and produced an average of 1.8 normal seedlings per seed (due to polyembryony). Storage in PEG at −1.6 MPa maintained 90% germination (radicle protrusion) and one normal seedling per seed on average for more than 200 d. Osmotic storage likely slowed down metabolism within the seed and hence consumption of food reserves. The storage time achieved has practical applications for in situ restoration, but cannot address ex situ germplasm conservation. Extending shelf life for an additional 6 months allows tree nurseries to optimize the production of seedlings so that they can be planted during the wet season.

The effect of accelerated aging (AA) and seed priming on specific seed enzymes was studied in soybeans. Aging seeds for 48 and 96 h at 41°C and 100% humidity reduced germination from 92% to 68% and 0%, respectively. Malate dehydrogenase (MDH) glutamate dehydrogenase (GDH), and esterase were assayed in extracts from dry and imbibing seeds. Malate dehydrogenase activity was the least affected by AA treatments. Certain GDH isozyme activity was reduced by 17% after 48 h of AA and almost completely lost after 96 h. Imbibing for 24 h increased GDH activity in 48 h-accelerated-aged seeds but not the 96 h-aged seeds. Two esterase isozymes in dry seeds showed a 77% loss of activity after 48 h of AA. This treatment increased the total esterase activity, with the greatest activity observed after 96 h of AA. This was due to the appearance of a diffuse high molecular weight (HMW) region of activity observed on native polyacrylamide gels and specifically induced in the embryonic axes. Imbibing 48-h-accelerated-aged seeds for 48 h caused a reappearance of the two esterase enzymes lost during aging. The HMW esterase staining region also appeared after imbibing unaged seeds for 24 h. Seed priming, caused a response similar to imbibition for each enzyme except the HMW esterase activity. Priming 48-h-accelerated-aged seeds reduced the HMW esterase activity. This was not observed during imbibition of aged seeds and indicates the first identification of a specific enzymatic response unique to a seed priming treatment.

The wheat transcription factor TaABF1 physically interacts with the protein kinase PKABA1 and mediates both abscisic acid (ABA)-induced and ABA-suppressed gene expression. In bombarded aleurone cells of imbibing grains, the effect of TaABF1 in down-regulating the gibberellin (GA)-induced Amy32b promoter was stronger in the presence of exogenous ABA. As these grains contained low levels of endogenous ABA, the effect of TaABF1 may also be mediated by ABA-induced activation even in the absence of exogenous ABA. Levels of TaABF1 protein decreased slightly during imbibition of afterripened grains. However, TaABF1 levels (especially in aleurone layers) were not substantially affected by exogenous ABA or GA, indicating that changes in TaABF1 protein level are not an important part of regulating its role in hormone signalling. We found that TaABF1 was phosphorylated in vivo in aleurone cells, suggesting a role for post-translational modification in regulating TaABF1 activity. Induction of Amy32b by overexpression of the transcription factor GAMyb could not be prevented by TaABF1, indicating that TaABF1 acts upstream of GAMyb transcription in the signalling pathway. Supporting this view, knockdown of TaABF1 by RNA interference resulted in increased expression from the GAMyb promoter. These results are consistent with a model in which TaABF1 is constitutively present in aleurone cells, while its ability to down-regulate GAMyb is regulated in response to ABA.

Cultivated northern wild rice (NWR; Zizania palustris L.) has been bred and studied since the 1950s. One challenge facing researchers is the lack of storage options, due to the seed's unorthodox behaviour. This study evaluated varying storage temperature and moisture conditions for the maintenance of seed viability and dormancy breaking in Minnesota-grown NWR. First, seeds were placed in non-submerged, freezing storage (NSFS) for 12–26 weeks, then in submerged, cold storage (SCS) for 2 weeks. The addition of SCS increased germination (%G) relative to NSFS alone (<0.1% NSFS, 15% NSFS and SCS), indicating that NSFS does not kill seeds but also does not break seed dormancy. Next, the required length of SCS was evaluated by placing seeds in NSFS for 12 weeks and then in SCS for 2–14 weeks. A longer SCS period increased %G from 3 to 79%, at 2 and 14 weeks of SCS, respectively. Lastly, seeds were placed in NSFS, followed by SCS, at varying intervals over a 29-week period. Across lines, germination increased from 20 to 76% between 4 and 7 weeks of SCS, respectively, then plateaued. The results of this study indicate that NSFS could be used to store NWR seeds, but at least 7 weeks in SCS is required to overcome dormancy. Additionally, while NSFS did not break seed dormancy, physiological changes related to stratification processes were occurring in non-submerged, freezing conditions. Results also suggest that the genotypic variation in NWR could be utilized for selection to improve germination and storage viability.

Using seeds to restore natural ecosystems has a greater chance of success if the seeds used are ready to germinate given appropriate environmental conditions. For Australian native Poaceae species, seed quality and dormancy can impose constraints on restoration success. In this study, germination biology of three Australian native Poaceae species, such as Cymbopogon refractus, Capillipedium spicigerum and Bothriochloa bladhii, was investigated. The seeds were exposed to different germination-enhancing chemicals (GECs, namely smoke water (SW), potassium nitrate (KNO3) or a combination (SW + KNO3)) and treated with three different seed enhancement technologies (SETs, namely seed priming, seed coating or seed cookies) then sown into two contrasting soil types (sodosol or black vertisol). Laboratory germination percentages achieved were <50% for all species, limited by dormant seeds. Incorporating GECs together with seed priming or seed coating treatment significantly increased seedling emergence rates and promoted earlier emergence as compared to the untreated control. For C. refractus and C. spicigerum, priming and/or coating with KNO3 + SW had the highest cumulative emergence. For B. bladhii, total seedling emergence was the highest (36% in both soils) for primed seeds with KNO3. Seedling emergence from seed cookies was low in all three species (<15%). Generally, soil type did not influence emergence rates for either GEC or SET. Understanding the environmental requirements needed for seed germination, together with an appropriate pre-treatment before sowing, can speed up seedling emergence and increase total emergence when using native Poaceae species for seed-based restoration.

Physical dormancy of Robinia pseudoacacia seeds makes it a challenge for scientists and forest managers to obtain a homogeneous germination for larger seed samples. Water imbibition of the seeds can be achieved through manual piercing of the seed coat, but this method remains time consuming and heterogeneous. We tested several ecologically friendly methods to break seed dormancy, including manual pin puncture, water soaking, oven dry-heating (two temperatures) and sanding. Sanding was performed using an automatic grinder to control shaking duration (three durations) and get a homogeneous scraping of the coat. All methods, except dry-heating, resulted in successful dormancy breaking; water soaking was the least efficient method, attaining 57% germination. Sanding proved to be as efficient as puncturing (97%) but long duration sanding (10 or 15 min) could damage cotyledons, which would impede further development of the plant. Short-time sanding (5 min) proved to be the best method to reach high total germination and healthy (undamaged cotyledon) seedlings, and was successfully applied to 500 seeds. The reference puncture method and the automatic sanding were also tested on seeds of nine Fabaceae species and proved to be efficient for some species. Automated sanding can thus be used as a standard to break physical dormancy of black locust or other Fabaceae seeds to allow further comparative studies of plant populations or genotypes.

The soluble selenium (Se) compounds selenic acid, selenious acid, selenium dioxide, selenium tetrachloride, selenomethionine, selenourea, sodium selenate and sodium selenite broke the dormancy of scarified seeds of Townsville stylo (Stylosanthes humilis HBK). Given the chemical diversity of all these molecules, it could be deduced that Se may act as a dormancy-breaking agent of Townsville stylo seeds. Se-stimulated seeds produced great amounts of both ethylene and 1-aminocyclopropane-1-carboxylic acid, which correlated with the process of germination. As specific inhibitors of ethylene biosynthesis and action largely depressed this Se-stimulated germination, it was concluded that Se broke dormancy by triggering ethylene production by the seeds.

Ferula ovina is a perennial Apiaceae with great medicinal and economic value. This study was conducted to better understand the dormancy and germination behaviour of its seeds. Experiments included temperature requirements for both embryo growth and germination under field and laboratory conditions, the effects of warm stratification, gibberellic acid (GA3), dry after-ripening, exogenous hydrogen peroxide (H2O2) and diphenyleneiodonium (DPI) on dormancy, localizing the accumulation of superoxide (O2−) and measuring endogenous contents of H2O2 in embryos during cold and warm stratification. Embryos were under-developed and did not germinate within one month at temperatures higher than 10°C. Among all treatments, cold stratification and exogenous H2O2 could break dormancy. However, the application of DPI reduced growth and the germination of the embryo at 3°C. During cold stratification, the embryonic axes elongated more than the cotyledons, which coincided with earlier production of O2− in the axes. Only these embryos could eventually complete growth and germinate. Moreover, such asymmetric growth between the axes and cotyledons was also observed when seeds were treated with exogenous H2O2. On the other hand, both axes and cotyledons displayed the same growth over treatments with warm stratification and DPI. Overall, seeds of F. ovina exhibit the characteristics of deep complex morphophysiological dormancy. Moreover, the embryos solely require cold to complete growth and to germinate, and this process seems to be mediated by reactive oxygen species. Under natural conditions, seeds germinate during winter in cold soil, and shoots emerge in spring.

Artificial aging of wheat grains (Triticum aestivum cv. Mercia) for 3 d resulted in the gradual loss of seed vigour without loss of viability, but the latter decreased rapidly over the next 24 h. Loss of vigour was expressed as a delay in germination i.e. an increasing ‘lag’ period with aging before the onset of radicle emergence, but the subsequent rate of seedling growth was not affected. There was an overall decrease in the incorporation of 35S-methionine into protein in aged embryos, and associated with the aging-induced delay in the completion of germination (i.e. start of radicle emergence) there was a delay in the production of certain polypeptides. In embryos of both non-aged and aged seeds these polypeptides appeared just prior to radicle emergence but several of these were never synthesized in embryos from non-viable seeds. The synthesis of mRNA coding for in vitro translated polypeptides, or groups of polypeptides, was similarly delayed in lowvigour embryos. Furthermore, some polypeptides were identified which were induced by the accelerated aging treatment. It is concluded that a reduced level of protein and mRNA synthesis in the embryos of low-vigour seeds may result in the delay in the accumulation of specific of mRNAs and polypeptides which are needed for the normal completion of germination, i.e. radicle emergence.

The dispersal of plants within botanically rich grassland is an important area of study if such swards are to be maintained. The application of farmyard manure provides a possible mechanism by which seeds contained within hay can be returned/introduced to grasslands. In this study, hay, dung and manure samples taken from farms with botanically rich meadows contained very limited quantities of seeds of desirable species. Digestion by cattle, and storage within a manure heap for 6 months or longer, further reduced seed germination. Samples were characterized by an abundance of the grass, Poa trivialis, with few forbs present. Confirmation of the negative effect of the digestive processes of cattle on seed viability was achieved using an in vitro laboratory experiment. However, this experiment did show that the perennial herbs, Filipendula ulmaria and Sanguisorba officinalis, were able to survive digestion at least as well as P. trivialis. The burial of known quantities of seeds in a manure heap also showed these perennial herbs to be at least as resistant to damage as P. trivialis. The results demonstrate that, given appropriate timing of the hay cut, seeds of species with high conservation value could become incorporated into manure for subsequent dispersal. However, manure dispersal would appear to be of limited value for many species desirable from a conservation viewpoint.

Seed mass is an important plant functional trait linked to germination. For instance, higher-mass seeds often display greater germination compared to lower-mass seeds when exposed to non-stressful conditions. Yet, knowledge pertaining to germination dynamics for different mass-based seed fractions following exposure to abiotic stress is lacking. Here, we assess the germination response of relatively fresh, mass-separated Rudbeckia mollis (Asteraceae) seeds to various simulated seasonal temperatures, supra-optimal temperatures and increasing ageing stress duration. Air density separation yielded three mass-based classes, called light (393 ± 35 μg), intermediate (423 ± 29 μg) and heavy (474 ± 38 μg). Water uptake kinetics indicated that imbibition (0–6 h) and germination lag (6–24 h) were independent of seed mass. Similarly, germination and viability loss of fresh seeds following exposure to seasonal and supra-optimal constant temperatures were independent of mass. However, seed mass influenced germination following increasing ageing stress, with light seeds germinating to a significantly greater extent than intermediate or heavy seeds. For example, final germination per cent in light-class seeds was about 1.7 times greater than intermediate or heavy seeds after 20 d of saturated salt accelerated ageing (SSAA). Seeds stored for 1 year in the laboratory displayed mass-dependent germination patterns similar to seeds following SSAA. Mass-independent germination responses may be a strategy to maintain an annual life history in otherwise difficult environments when R. mollis seeds are relatively fresh. However, differences in germination response between aged and unaged seeds suggest that mass-dependent viability loss may occur in R. mollis.

Masting and regeneration dynamics were investigated in a long-term perspective using Abies cephalonica as a study tree species. Extensive fieldwork was implemented in Parnitha National Park, Greece, following a large-scale wildfire. Annual cone production was monitored for a 5-year period in 130 tagged trees, in 13 plots with 10 individuals each, established both within the unburned part of the forest and in surviving fragments of the burned area. In the most recent masting year, a high percentage (88%) of cone-bearing trees was recorded, along with a sizeable, average cone production (40.8 cones per tree). In the intermediate, non-masting years, the corresponding values ranged from 2% to 55% and 0.08 to 5.9 cones per tree, respectively. The reproduction process is affected by both tree density and regional climatic conditions, in particular temperature during spring of the maturation year and precipitation during spring and summer of the previous year. For the first time according to our knowledge, natural regeneration was recorded for a 4-year period, in 13 permanent transects within the monitoring plots, in relation with a masting event and the additional implications of a preceding wildfire. Highest mean density of seedlings and saplings (11.4 per m2) was observed during the first spring after masting. In the non-masting years, the corresponding value ranged from 2.1 to 2.9 per m2. Seedling survival during their first summer was considerable (30–76%) but stabilized afterwards (1–3 years) at a lower level (10–20%). The particular post-masting seedling flush was followed by an extremely high mortality rate (88.6%) and cannot represent a major recruitment event.