A population-based modelling approach was used to predict the occurrence of germination in Solanum sarrachoides (SOLSA) for different treatments. Seeds collected in Toledo (Spain) were exposed to constant temperatures, to temperatures alternating between 10 and 30°C and to gibberellins (GAs; 0, 50, 100, 150 and 1000 ppm) during a 24-h imbibition period. The following parameters were measured: base temperature (Tb), mean thermal time (θT (50)) and the standard deviation of thermal time (σθT). The SOLSA seeds only germinated at constant temperatures when the highest GA concentration was applied. The thermal model suggests that the induction and loss of physiological dormancy following seed dispersal is achieved when temperatures vary and when a mean thermal time of 66 growing degree-days (d°C) and a Tb value of 16°C are achieved when no GA treatment was added. The concentration of GA applied under conditions of alternating temperatures has an additive effect, reducing θT (50) up to threefold, from basal level (66 d°C) to 19.40 d°C, when the 1000 ppm GA treatment was applied. In this last case, the germination was accelerated by reducing Tb to 14°C. A 5–10°C change in temperature and a range of average temperatures of 20–27.5°C promoted the germination of SOLSA seeds to the greatest extent in the absence of GA. However, these conditions are not frequently encountered in the irrigated areas of the studied region; this finding could explain the limited ability of SOLSA to expand its range within this area.

Smoke-water (SW), karrikinolide (KAR1) and vermicompost leachate (VCL) have been reported to possess gibberellic acid-like activity. The effects of these plant growth-promoting substances (PGPSs) on biochemical changes occurring during seed germination of Phaseolus vulgaris were assessed. Seeds were incubated/germinated under dark conditions in water (control) or with different concentrations of SW, KAR1, VCL and gibberellic acid (GA3) for 7 d. The maximum seedling fresh weight (1.863 g) was recorded for the SW (1:750 v/v) treatment. The longest seedling axes (9.9 cm) and the highest number of adventitious roots (16.3) were recorded for VCL-treated seedlings (1:10 and 1:5 v/v, respectively). Analysis of two important hydrolytic enzymes, acid phosphatase and alpha-amylase, showed maximum activity [1856 nkat mg− 1 and 3.225 mg min− 1(g FW)− 1, respectively] in the seeds incubated with 10− 8M KAR1. In all the treated seedlings, proline content was significantly reduced [43.67 μg (g FW)− 1; VCL (1:20 v/v)] in comparison to the control [87 μg (g FW)− 1] but there was an increase in amino acids with some concentrations of PGPSs. The tested PGPSs significantly influenced various biochemical parameters that play a significant role in seed germination and plant growth. This study indicates that PGPSs may act via stress-relieving biochemical pathways during seed germination.

A clear understanding of the mechanisms involved in releasing seed dormancy is important for effective plant conservation and regeneration of desert species. The dormancy types and dormancy release mechanisms of 19 Fabaceae species from four cold deserts in Xinjiang province, China, were studied. An imbibition experiment was performed to determine the presence or absence of physical dormancy. Other treatments included mechanical scarification, sulphuric acid scarification and simulation of summer temperatures typically encountered in the field. High summer temperature treatments included a maximum soil temperature of 80°C, 65°C and 40°C, conducted under dry and wet conditions. The results showed that all the species had orthodox seeds. Caragana korshinskii, Caragana intermedia, Caragana microphylla and Onobrychis taneitica seeds were not dormant. Glycyrrhiza uralensis, Ammodendron bifolium, Vicia costata and Eremosparton songoricum seeds had combinational dormancy (PY+PD) whereas the other 11 species had physical dormancy. The mechanical scarification and sulphuric acid scarification treatments were significant to break dormancy (>80%). Melilotus suaveolens and Oxytropis sp. seeds were temperature-insensitive. Sophora alopecuroides, S. salsula, E. songoricum, A. bifolium and Cassia tora seeds were wet-heat sensitive. Glycyrrhiza glabra seeds were dry-heat sensitive, while those of Halimodendron halodendron and Astragalus lehmannianus were sensitive to both dry and wet heat. Although high temperatures, typical of summer, promoted the breaking of physical dormancy, the proportion of seeds of which dormancy was broken differed among species. These differences were indicative of the conditions in which the species are found naturally. Species may have different adaptations that promote favourable regeneration. Given suitable temperature and moisture conditions similar to those in their natural environment, most species were capable of regeneration.

Seeds of Dodonaea viscosa (Sapindaceae) have a water-impermeable seed coat, i.e. physical dormancy (PY). Although mechanical scarification, dry heat, sulphuric acid and hot water treatment make seeds permeable under laboratory conditions, the mechanisms by which dormancy is alleviated in natural environments have not yet been understood completely. The present investigation aims to understand the pattern of dormancy alleviation in D. viscosa seeds using an artificial burial approach for 2 years. Freshly collected seeds held in hydrated soil at 10/20°C, 15/20°C, 15/30°C, 20/35°C and 25°C for 32 weeks germinated to less than 15%, irrespective of storage temperature. Dry storage of seeds at 15, 20, 25 and 30°C for 1 year did not break dormancy. Hot water treatment at 80 and 90°C for 30 s broke dormancy in 90% of the seeds. On the other hand, burying seeds at a depth of 3–5 cm in the natural environment for 2 years increased germination from 7 to 71%. In particular, seeds exhumed after summer in both years showed a significant increase in germination percentage (P< 0.05). However, seeds buried after summer did not germinate to a higher percentage when exhumed prior to summer. We suggest that a high summer temperature, rising above 60°C in the top soil layer of the tropics, is a likely factor breaking dormancy. Most seeds germinated during burial, which indicates that light is not a cue for germination. We conclude that germination of D. viscosa following summer is an adaptive mechanism to tolerate summer droughts, which are common in the dry tropics.

Structural studies in seeds with physical dormancy (PY) are important to better understand its causes and release when subjected to treatments for dormancy breaking. The aims of this study were to (1) characterize the PY break; (2) examine the role of different seed structures in water uptake; and (3) identify the water gap in Senna multijuga seeds. Imbibition patterns of dormant and non-dormant (subjected to dormancy breaking treatments) seeds and the morphological changes during dormancy breaking and germination were evaluated. To identify the water gap, the micropyle and lens were blocked separately, and the water absorption by seed parts was determined. Structural characteristics of the seed coat were also examined. Immersion in water at 80°C was efficient in breaking seed dormancy and imbibition occurred first at the hilar region, through the lens. Water was not absorbed through the micropyle or the extra-hilar region. S. multijuga seeds have a testa with a linearly aligned micropyle, hilum and lens. The seed coat consisted of a cuticle, macrosclereids, one (hilar region) or two (extra-hilar region) layer(s) of osteosclereids and parenchyma cell layers. The lens has typical parenchyma cells underneath it and two fragile regions comprised of shorter macrosclereids. Heat treatment stimulated the lens region, resulting in the opening of fragile regions at the lens, allowing water to enter the seeds. It is concluded that short-term exposure to a hot water treatment is sufficient for the formation of a water gap in S. multijuga seeds, and only the lens acts in the imbibition process.

Temperate wetland species often require light and warm temperatures for seed germination. However, recent studies indicate that species which specialize on permanently saturated wetlands that are maintained by groundwater discharge (fens, seeps and mountain springs), rather than wetlands with surface-water-driven hydrologic regimes, diverge from the typical wetland germination niche by germinating at cool temperatures and lacking photoblastic seeds. We conducted laboratory experiments that manipulated stratification conditions (non-stratified versus cold stratification in light and darkness), thermal regime (15/6, 25/15 and 35/20°C), and light (14 h photoperiod versus continuous darkness) to test whether seeds of the North American calcareous fen specialist Parnassia grandifolia diverged from the typical temperate wetland germination niche. After 30 d, fresh seeds were conditionally dormant and could only germinate to high percentages in light at 25/15°C. During 16 weeks of incubation, non-stratified seeds germinated to low percentages ( < 40%) at all thermal regimes in darkness. In contrast, cold-stratified seeds germinated to high percentages in both light and darkness at all thermal regimes, although germination was incomplete (no cotyledon emergence) at 35/20°C. Further, seeds did not require light during cold stratification to germinate to high percentages when incubated in light or darkness. Thus, seeds diverged from the typical temperate wetland germination syndrome in lacking a light and warm temperature requirement for germination. Our results reinforce previous work from European fens and Mediterranean wetlands. This indicates that multiple germination strategies are found in fen wetlands that are maintained by the continuous or near-continuous discharge of cool groundwater.

Seed size is one of the most important traits in the regenerative phase of a plant's life cycle; however, for cactus species the relationship of seed size and germination characteristics and seedling traits is still unclear. We studied the relationship between seed mass and germination and seedling characteristics in 17 cactus species from central Argentina, belonging to different genera and life forms. We measured seed mass, total seed germination, light requirements for germination and mean time to germination for these 17 cacti species; in addition, we recorded seedling size and shape in 15 species. To test light requirements we performed germination experiments under laboratory conditions at 25/15°C (day/night temperatures) and under light or dark conditions. We also calculated seedling volume by measuring seedling height and width. A shape index was obtained by dividing height by width (a value of 1 indicates ‘globose’ seedlings, whereas, as this value increases, seedlings become ‘columnar’). We found no significant relationship between seed mass and any of the germination characteristics considered. However, species with heavier seeds produced bigger seedlings, which were more cylindrical. Adult growth was not totally determined by seedling ‘growth form’, because some species that had globose seedlings were columnar at the adult stage.

Rouya polygama (Apiaceae) is an endangered Mediterranean species of great phytogeographical and ecological interest, growing on coastal sandy dunes. Intraspecific variability in the responses to constant temperatures (5–25°C) and an alternating temperature regime (25/10°C), salt stress (0–600 mM NaCl) and recovery of seed germination was evaluated among six populations from Sardinia and Corsica. Seeds were non-dormant and germination percentages ranged from 10 to 83%, depending on temperature and population. Differences in germination percentages were mainly due to different seed mortality among seed lots. R. polygama seeds germinated in salt concentrations up to 200 mM NaCl, whereas higher salt concentrations totally inhibited germination. Salt affected seed viability, and the recovery response decreased with increasing salinity and temperature. Inter-population variability and different sensitivity to NaCl in seed germination were detected. Our results are consistent with field germination in a period from autumn to spring, when water is available in the soil and temperatures are not prohibitive for seedling establishment, representing an advantageous ecological adaptation for seedling establishment to the unpredictable Mediterranean rainfall pattern. Further studies on R. polygama are needed to investigate germination requirements at temperatures higher than 25°C and its germination in the field, and to clarify genetic inter-population variability, considering a higher number of populations and possibly extending to North African populations.

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is an important technology to analyse gene expression levels during plant development or in response to different treatments. An important requirement to measure gene expression levels accurately is a properly validated set of reference genes. In this context, we analysed the potential use of 17 candidate reference genes across a diverse set of samples, including several tissues, different stages and environmental conditions, encompassing seed germination and seedling growth in Ricinus communis L. These genes were tested by RT-qPCR and ranked according to the stability of their expression using two different approaches: GeNorm and NormFinder. GeNorm and Normfinder indicated that ACT, POB and PP2AA1 comprise the optimal combination for normalization of gene expression data in inter-tissue (heterogeneous sample panel) studies. We also describe the optimal combination of reference genes for a subset of root, endosperm and cotyledon samples. In general, the most stable genes suggested by GeNorm are very consistent with those indicated by NormFinder, which highlights the strength of the selection of reference genes in our study. We also validated the selected reference genes by normalizing the expression levels of three target genes involved in energy metabolism with the reference genes suggested by GeNorm and NormFinder. The approach used in this study to identify stably expressed genes, and thus potential reference genes, was applied successfully for R. communis and it provides important guidelines for RT-qPCR studies in seeds and seedlings for other species (especially in those cases where extensive microarray data are not available).

Whether or not it is time for germination is a dilemma for annuals since they have only a single opportunity to reproduce successfully. This is critical for species living in stressful and unpredictable environments such as Mediterranean habitats. In order to clarify the environmental cues of germination, four annuals of different families, all of them occurring very frequently on gypsum soils, were selected and their germination observed under 14 climate scenarios, mimicking temperature and photoperiod conditions from autumn to spring, at two levels of water availability (continuous irrigation versus an initial single irrigation event followed by a progressive soil desiccation). In spring scenarios, two seed-storage conditions were compared: dry cold and room conditions. In the absence of water limitation, germination decreased from early autumn to late spring. Water scarcity always reduced germination, especially in early spring. Our results suggest a facultative winter germination behaviour and highlight the crucial role of dry cold storage in reducing spring germination. In conclusion, Mediterranean ephemerals showed a very plastic germination response that allows them to take advantage of favourable environmental conditions from autumn to spring. This environmental cueing is combined with the ability to dilute the risk through a variable rate of seed dormancy that, according to bet-hedging strategies, increases from secure autumn to riskier spring.

Recently, a list has been published of angiosperms capable of germinating in less than 24 h (‘very fast germination’). Here, we add three families and 23 species to that list. The main extra families complement ecological groups already recognized – the Cactaceae into the aridity-adapted group and the Tamaricaceae into the floodplain-adapted group. These amended findings on very fast germination (VFG) are integrated into the recent work on the functional ecology of embryo size. They confirm the important connection between germination speed and embryo to seed ratio. Plotting the plant orders containing VFG species on a phylogenetic tree shows that VFG has evolved multiple times throughout angiosperm history, including at least three times within the Caryophyllales. The fact that species with VFG are mainly restricted to advanced clades shows that VFG is a derived trait that evolved as an adaptation to either arid, saline or floodplain habitats.

The understanding that many aspects of the spatial and temporal patterns of epiphyte communities may be explained by the comportment of early life stages has given rise to a considerable number of germination studies in recent years. Unfortunately, protocols frequently use unproven assumptions and arbitrary experimental conditions. To make future studies as ecologically meaningful as possible we address a number of potential pitfalls with a series of experiments with seeds from a total of 16 species. We show that it is safe to collect capsules for experiments before natural dehiscence – there is afterripening even in the case of very early collections. The application of fluctuating temperatures is not imperative, because there is no consistent difference in the germination response under constant versus fluctuating temperatures. The effects of different osmotic potentials and intermittent drought of varying intensity on germination are qualitatively, but not quantitatively, comparable. Due to the greater ecological realism, we encourage the use of the latter. However, care must be taken to use realistic temperatures – the impact of intermittent drought on germination is modulated by temperature. This highlights the need for data on the in situ temperature regimes during germination as an important prerequisite towards more realistic experiments in the field of germination ecology of vascular epiphytes.

The role of smoke in fire-stimulated germination in the Mediterranean Basin has often been underestimated. A few records on smoke-enhanced germination are present in Mediterranean Lamiaceae species, but there is still a shortage of information to allow generalizations about this family to be made. To test the hypothesis that smoke enhances germination in Mediterranean Lamiaceae species, we performed a germination experiment, including aqueous smoke treatments in various concentrations (1:1, 1:10 and 1:100) on seven eastern Mediterranean Lamiaceae taxa. Six of the studied taxa (Lavandula stoechas, Origanum onites, Phlomis bourgaei, Stachys cretica ssp. smyrnaea, Satureja thymbra, Teucrium lamiifolium ssp. stachyophyllum) showed significant increments in germination percentage in at least one smoke treatment, as compared to the control. Moreover, L. stoechas, S. thymbra and T. lamiifolium ssp. stachyophyllum displayed faster germination in at least one smoke treatment than in the control. Of the species showing significant increments in germination percentages after aqueous smoke application, at least one single concentration of smoke solution did not stimulate germination, except in L. stoechas and S. thymbra which responded positively to all smoke treatments. Therefore, the concentration of aqueous smoke that improved germination was species-specific. Our results contribute to the current limited knowledge on smoke-enhanced germination in Mediterranean Lamiaceae, and support the idea that smoke is an important germination cue for this family.

Birds are important seed dispersers for fleshy fruits through their transportation of ingested seeds. The seeds of many species germinate faster and in greater proportions after passing through a digestive tract, although the effects of this passage vary amongst bird and plant species. Many factors determine the germination success of ingested seeds, such as seed scarification during the digestion process, the fertilizing effect of droppings and the removal of pulp surrounding the seeds. In central Spain, the great bustard (Otis tarda) may act as a disperser of European black nightshade (Solanum nigrum). We analysed the germination success of ingested and non-ingested S. nigrum seeds. The fertilizing effect of bustard droppings and the disinhibition effect of the removal of Solanum pulp on final germination percentage, germination speed and viability were also assessed. Although ingested seeds germinated faster than non-ingested seeds, the former showed a lower germination percentage than the latter: 80–87% versus 99%. Droppings and fruit pulp showed no effect on germination enhancement, except in one aspect: the germination speed of non-ingested seeds decreased when they were sprayed with a fruit extract. We confirm that seeds ingested by great bustards had lower germination success than non-ingested seeds. Although seed ingestion by great bustards reduced seedling emergence, the number of emerged seedlings was still quite large. Thus, great bustards may play a role as a S. nigrum seed dispersal vector.