Light and fluctuating temperatures are two important factors triggering seed germination. The aim of this work was to: (1) elucidate the effect of temperature, light regime and storage time on seed germination in nine taxa of Verbascum spp., collected from different habitats in the Mediterranean area; and (2) estimate threshold temperatures for seed germination. For all taxa, germination assays were performed at constant and fluctuating temperatures, both in continuous darkness (D) and in alternating light/dark (L/D; 12 h photoperiod). Final germinated proportions (FGPs), base (T b), optimal (T o) and cut-off (T c) temperatures were derived. At constant temperatures, seed germination was strongly suppressed under the D regime. In L/D, the effect of storage time was very small and the highest FGPs were observed from 15 to 30°C (40–100%), depending on the species. T b ranged from below 7 to above 10°C and it appeared to be constant within each seed lot. T o and T c showed some within-lot variability and were higher for fast-germinating seeds in each lot. Considering fluctuating temperatures, germination appeared to be quicker and more complete than at constant temperatures. The germination of Verbascum spp. is favoured in L/D and fluctuating temperatures, which explains their pioneer character when positioned near the soil surface and under a low vegetation canopy. V. arcturus and V. pinnatifidum were shown to be less favoured by L and fluctuating temperatures, which might explain their ability to germinate in rocky areas or sandy dunes, even when they are not directly exposed to the light.

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.

Fluctuating temperatures terminate seed dormancy in many species, including Cynara cardunculus (L.) var. sylvestris. Termination of physiological dormancy requires low ratios of abscisic acid (ABA)/gibberellins (GA). In a previous paper we have shown that physiological responses to fluctuating temperatures comprise a reduction of abscisic acid (ABA) content and sensitivity. However, a possible stimulation of GA synthesis was also suggested as part of the mechanism. That possible stimulation, as well as the identification of potential regulatory sites for ABA and GA metabolism and signalling involved in the termination of dormancy by fluctuating temperatures, are yet to be determined. In this study, we measured GA content and sensitivity in seeds incubated under constant and fluctuating temperatures. We also assessed the expression of several genes involved in ABA and GA metabolism and signalling. Our results show that fluctuating temperatures reduce ABA/GA ratios through a reduction in ABA accumulation during incubation but without altering GA synthesis as compared to that observed under constant temperatures. On the other hand, fluctuating temperatures did not increase sensitivity to GA. Fluctuating temperatures reduced the expression of CycaNCED and CycaABI5 (ABA synthesis and signalling genes) with a temporal pattern that coincides with the interruption of ABA accumulation that precedes germination of seeds incubated under fluctuating temperatures. However, fluctuating temperatures did not modify the expression of CycaCYP707A2 (ABA inactivation) as compared to that observed under constant temperatures. Consistent with our determinations of GA content and sensitivity, fluctuating temperatures did not modify the expression of GA synthesis (CycaGA3ox) and signalling genes (CycaRGL2 and CycaGAI) in relation to that observed at constant temperatures. These results show that fluctuating temperatures terminate dormancy in Cynara cardunculus seeds through an interruption in ABA accumulation and a reduction in ABA signalling exerted at the level of CycaNCED and CycaABI5 expression.

Physiological dormancy of scarified seeds of Townsville stylo (Stylosanthes humilis H.B.K.) is broken by ethylene. When the biosynthesis of this gas was impaired by 2-aminoethoxyvinylglycine (AVG) plus Co2+, the response to ethylene at very low concentrations was appreciable in non-dormant seeds and nil in the dormant ones. Complete inhibition of germination of non-dormant seeds occurred only when they were treated with AVG plus Co2+ under an ethylene-free atmosphere, a condition in which no trace of the gas in the atmosphere of Erlenmeyer flasks could be detected. Injection of ethylene into that system triggered germination of both dormant and non-dormant seeds, demonstrating a requirement for the gas. Non-dormant seeds were at least 50-fold more sensitive to ethylene than the dormant ones. Perception of ethylene occurred within a very short time (at most 15 min), since exposure of both dormant and non-dormant seeds to the gas, at a steeply declining concentration, sufficed to cause substantial germination.

Temperature and water potential strongly influence seed dormancy status and germination of Bromus tectorum. As seeds of this plant can be killed by the ascomycete fungus Pyrenophora semeniperda, this study was conducted to learn how water potential and temperature influence mortality levels in this pathosystem. Separate experiments were conducted to determine: (1) if P. semeniperda can kill dormant or non-dormant seeds across a range of water potentials (0 to − 2 MPa) at constant temperature (20°C); and (2) how temperature (5–20°C) and duration at reduced water potentials (0–28 days) affect the outcome. When inoculated with the fungus at 20°C, all dormant seeds were killed, but fungal stromata appeared more quickly at higher water potentials. For non-dormant seeds, decreasing water potentials led to reduced germination and greater seed mortality. Results were similar at 10 and 15°C. Incubation at 5°C prevented stromatal development on both non-dormant and dormant seeds regardless of water potential, but when seeds were transferred to 20°C, dormant seeds evidenced high mortality. For non-dormant seeds, exposure to low water potential at 5°C resulted in secondary dormancy and increased seed mortality. Increasing incubation temperature, decreasing water potential and increasing duration at negative water potentials all led to increased mortality for non-dormant seeds. The results are consistent with field observations that pathogen-caused mortality is greatest when dormant seeds imbibe, or when non-dormant seeds experience prolonged or repeated exposure to low water potentials. We propose a conceptual model to explain the annual cycle of interaction in the Bromus tectorum–Pyrenophora semeniperda pathosystem.

An image analysis system is described to study the process of imbibition in white cabbage (Brassica oleracea L.) seeds. The system consists of two components: 1) an environmental system where seeds germinate; 2) a computer imaging system, composed of a Charged Coupled Device (CCD)-imaging sensor, an image frame grabber, a computer and a video monitor. The measurement software can determine area, perimeter, width, length and eccentricity as descriptors of changes in seed size during swelling. The captured images of imbibing seeds allowed estimation of timing of ‘visible germination’ occurrence without any subjective evaluation. Using the area parameter, the imbibition process can be described graphically by a series of curves similar to the triphasic pattern of water uptake, with extension and rate depending on the degree of seed viability and germination medium conditions. Imbibitional osmotic stress, provided by -1.5 MPa mannitol, induced a large delay in germination and, upon stress removal, a more uniform radicle emergence. The versatility and the sensitivity of the method, together with the feasibility to investigate germination rate in individual seeds within a population, suggest that image analysis techniques have high potential in seed biology studies.

The aim of this study was to establish associations of kernel weight and colour with ecogeographic factors and molecular markers, based on ten wild barley [Hordeum spontaneum (C. Koch) Thell.] populations sampled in Israel across a southward transect of increasing aridity. Kernel weight and colour category were scored using barley kernels (naked caryopsis). Small kernel sizes (0.011 g kernel–1) and dark kernels were found in xeric populations. A higher variation of kernel weight was observed in xeric populations. A higher proportion of variation occurred within, rather than among, populations. Water, temperature and soil factors were associated with kernel size variation. Among 18 simple sequence repeats (SSRs) investigated, HVM14, HVM36, HVM43, BMS64 and BMS90 were associated with kernel weight, and HVM68 with kernel colour. The results indicated that high phenotypic variation and genetic diversity are related to ecological stress, and that the association of phenotypic traits with molecular markers, based on natural plant populations, should be interpreted cautiously due to the high chance of spurious associations between traits and molecular markers.

Osmotic priming in a polyethylene glycol solution (300 g/kg water) for 48 h resulted in a partial loss of desiccation tolerance for seeds of Vigna radiata (L.) Wilczek (mung bean). The percentage of germination began to decrease after primed seeds were dried down to water contents less than 0.06 g/g DW. As compared with control seeds, primed mung bean seeds also had poorer storage stability. The decline of storage stability after osmotic priming was correlated with the modifications of seed water sorption properties. Priming significantly increased the amount of water associated with the weak water-binding sites, and reduced the amount of water associated with the strong binding sites and multi-molecular binding sites in seed tissues. The enhancement of molecular mobility in seeds, as a result of such water redistribution, probably accelerates seed deterioration and decreases storage stability.

The role of free radical-induced damage as a cause of loss of vigour in seeds is by no means resolved. In this contribution, the effects of environmental treatments known to reduce viability rapidly were compared with the effects of long-term, low-temperature storage on germination, hypocotyl growth and free radical accumulation and lipid peroxidation in soybean seeds. Accelerated aging was achieved by incubating seeds at 35°C and 1% relative humidity over H2SO4 for up to 69 days in the light and in darkness. In contrast, seeds under long-term storage were maintained at 5°C and 6% moisture content in darkness for up to 6 years. At 35°C there were rapid and significant reductions in rates of seed germination and hypocotyl extension. Loss of viability and declining vigour were associated with increases in lipid peroxidation and free radical build-up but the latter, surprisingly, was largely confined to the testa rather than the cotyledon. Exposure to light greatly enhanced lipid peroxidation and increased organic free radical accumulation in the translucent testas of seeds, but not in the cotyledons. Similar responses to light were recorded in testas detached from seeds. These results show that in soybean the testa is a significant locus of free radical degenerative events induced by high temperature combined with low moisture.

Depleting the soil weed seedbank is an important integrated weed management strategy that has the potential to foster lasting weed control. Long-term dormancy and decay resistance of weed seeds pose a challenge to weed eradication efforts. Select soil fungi have been shown to cause significant decay of weed seeds. The physical and chemical mechanisms by which seeds in the seedbank defend themselves against pathogens have been well researched. However, very few studies have purposefully investigated the biochemical defence response of seeds. Enzyme-based biochemical seed defences have been detected in dormant and non-dormant seeds, and research supports their function in pathogen defence. This review summarizes current knowledge of the seed defence enzymes polyphenol oxidase, peroxidase, chitinase and oxalate oxidase. The fungal enzymes chitinase, protease and xylanase that function in pathogenesis of seeds in the soil seedbank are also reviewed. Progress in the development and standardization of in situ enzyme analyses fosters our understanding of actual enzyme activity present in soils, while high-throughput microplate techniques promote efficiency and enable greater scope. Application of genomic, proteomic and transcriptomic techniques to glean a deeper and more holistic understanding of the enzymatic interactions of weed seeds and soil fungi in the soil seedbank will support the development of improved integrated weed management strategies.

In recent years germination experiments have become more and more complex. Typically, they are replicated in time as independent runs and at each time point they involve hierarchical, often factorial experimental designs, which are now commonly analysed by means of linear mixed models. However, in order to characterize germination in response to time elapsed, specific event-time models are needed and mixed model extensions of these models are not readily available, neither in theory nor in practice. As a practical workaround we propose a two-step approach that combines and weighs together results from event-time models fitted separately to data from each germination test by means of meta-analytic random effects models. We show that this approach provides a more appropriate appreciation of the sources of variation in hierarchically structured germination experiments as both between- and within-experiment variation may be recovered from the data.

Abstract An autoradiographic study was made of leucine and thymidine incorporation into the meristematic root primordia and hypocotyl tips of seeds of the recalcitrant mangrove species, Avicennia marina. The investigations show that although there is a temporary reduction of protein synthesis at shedding, root primordia and surrounding hypocotyl cells of the axis never wholly cease incorporation of [3H]leucine and regain pre-shedding levels of activity within a day. Precursor studies using methyl-[3H]thymidine show that, at shedding, there is a temporary cessation of incorporation into root meristem nuclei that lasts no longer than 48 h and, within a day, pre-shedding levels are regained in the meristem nuclei. Analysis of DNA fragmentation patterns in root tips at the time of shedding, and their ability to repair radiation-induced DNA damage, indicate that DNA repair processes are markedly compromised in these cells if water loss reaches 22%. Protein synthesis and DNA replication are reduced by more than half by a water loss of 18% and 16%, respectively. DNA replication does not fully recover on rehydration after only 8% water loss. DNA fragmentation to nucleosomes indicates a programme of cell death at a water loss of 10%. We suggest that the feature of continuous protein synthesis activity with only a temporary interruption in active cell cycling in A. marina root primordia helps to explain both the rapidity in seedling establishment and the extreme vulnerability to desiccation.

The seed reserves of sugar beet (Beta vulgaris L.) are starch, located exclusively in the perisperm, and lipids, proteins and a small quantity of soluble sugars, located mainly in the cotyledons. It is shown that lipids are the main respiratory substrate used during germination whereas starch and remaining lipids are only hydrolysed after root extrusion, to sustain root and hypocotyl growth. By removing the perisperm during imbibition, it was demonstrated that lipids and proteins alone are sufficient for viable seedling development, though such seedlings grew less than those developing in the presence of perisperm. The utilization of seed reserves was followed during seedling development in the dark in various organs. At 20°C, the reserves were sufficient for 8 d of growth in the dark, the hypocotyl attaining a length of 5 cm. Specific problems relating to field establishment of sugar beet are discussed in relation to these results.

Fritillaria tubiformis subsp. moggridgei (Liliaceae) is a rare, endemic species that inhabits open mountains and alpine grasslands of the Ligurian Alps. At the time of seed dispersal, the underdeveloped embryos were 27% the length of the seed. Here we report the results of laboratory experiments carried out to determine the temperature preferences for embryo growth and radicle emergence. Embryo growth commenced immediately after sowing at 4°C. Once the embryo had grown the length of the seed, the radicle emerged. The time required for embryo growth and radicle emergence was longer when seeds were placed through a seasonal sequence of temperatures, commencing with late summer (10/20°C), compared with seeds immediately placed at a temperature to simulate winter conditions (4°C). Prematurely transferring seeds from winter to spring temperatures (5/10°C) also slowed the progress of germination. Radicle emergence did not occur at 10 or − 5°C and less than 20% germination occurred in seeds placed at constant 0°C. Addition of gibberellic acid (GA3) did not promote embryo growth of seeds placed at 20°C. Overall, the temperature preferences for embryo growth and subsequent radicle emergence are such that, in situ, seed germination may occur during the winter under snow cover or at the end of winter to coincide with snow melt and warming temperatures.

Mature seeds of many legume species are normally characterized by water-impermeable seed coats, a form of physical dormancy. However, observations have suggested that the incidence of mature but permeable (non-dormant) seeds is sometimes substantial. Yet, the ecological processes associated with this non-dormancy have received little attention by plant ecologists. In white clover (Trifolium repens), we therefore studied: (1) the occurrence of initially permeable seeds in wild populations; (2) the relative performance of non-dormant and dormant seeds in plant establishment and reproduction in a field-sown experiment; and (3) the extent to which the trait is affected by humidity and plant genotype in a greenhouse experiment. No less than 35% of all viable seeds from the wild populations proved to be water permeable at maturity. The proportion of permeable seeds within inflorescences ranged from 0 to 100%. In the field-sown experiment, autumn-germinated non-dormant seeds had almost equally good chances of establishing as spring-germinated dormant seeds. Due to a marked head start in growth, the former yielded more flowers (and thus seeds) in the first flowering season. However, the greenhouse experiment proved that variation in the proportion of permeable seed between inflorescences represented a plastic response to humidity conditions during seed ripening, rather than variation among clones (broad-sense heritability ≤ 0.025). Thus the trait is not easily subject to selection.

Changes in the properties of water in excised embryos were measured during the late stages of grain development in two cultivars of Zizania palustris and a population of the endangered species Z. texana. The relationships between water content and water activity were determined from water sorption isotherms, measured at temperatures between 35 and 5°C and then derived for lower temperatures. The freezing and melting behaviour of water in embryos at different water contents was determined using differential scanning calorimetry. The moisture content of embryos at high water activities decreased with maturation, as did the moisture content at which freezing transitions were not observed. While the temperatures of freezing and melting transitions decreased as the moisture content of embryos decreased, there were no discernible differences among embryos at different developmental stages. The properties of water measured in maturing Zizania embryos approached those for orthodox seeds as determined from the strength of water sorption, the enthalpy of the melting transition and the moisture content at which water is unfreezable. From these data we conclude that the properties of water in recalcitrant Zizania embryos change with development to resemble those of embryos of desiccation-tolerant seeds, but that the seeds never achieve the orthodox condition. The effects of interactions between moisture content and temperature on desiccation damage, freezing damage and germination in Zizania are predicted, based on the physical properties of water reported here and the correspondence of these properties with physiological function reported for other species. The resulting ‘phase diagram’ defines possible combinations of moisture content and temperature for storage under equilibrium conditions.

The genus Conostylis (Haemodoraceae) is endemic to fire-prone south-western Australia. To gain an understanding of the effect of some fire-related germination cues, eight Conostylis taxa were tested in response to water, nitrate, smoke water and karrikinolide (KAR1) under light and dark conditions, when seeds were freshly collected and after a year of burial. The germination of all taxa tested was higher in response to smoke water and KAR1 than in water alone, whereas nitrate did not stimulate germination. Germination was higher in all taxa following 1 year of burial than in fresh seeds. Recently, glyceronitrile has been identified as another chemical in smoke water, apart from KAR1, that can stimulate the germination of certain species. The relative response of eight Conostylis taxa to KAR1, glyceronitrile and smoke water was examined in laboratory-stored seeds. Germination of these taxa was promoted by both smoke water and KAR1, except for C. neocymosa, which had high germination regardless of treatment. Four of the other seven taxa germinated to higher levels in at least one of the glyceronitrile concentrations tested (10, 50 or 100 μM) than in water alone. However, in only two of these taxa, C. aculeata subsp. septentrionora and C. juncea, was germination in glyceronitrile as high as that in smoke water. Thus, the response to glyceronitrile is not uniform across Conostylis taxa. Generally, germination was higher with KAR1 than glyceronitrile, suggesting that although some Conostylis taxa have the capacity to respond to glyceronitrile, KAR1 is the more important germination stimulant for this genus.

Many research questions focused upon seed dormancy and germination remain unanswered due to problems created by inappropriate assumptions concerning the design and implementation of experiments. Lack of attention to the genetic background and growing conditions required for seed production and subsequent storage may compromise an investigator's ability to pool data or assimilate data trends due to variability in seed performance from year to year. Seed aging events during the afterripening process can be mistaken for those involved in the transition from the dormant to nondormant state. After imbibition, inaccurate criteria for and scoring of the germination event may lead to confusion of seed vs. seedling physiology and biochemistry. In addition, a sufficient number and proper spacing of data points during timecourse experiments are required to properly stage a sequence of events. Studies comparing dormant vs. nondormant seeds must be supplemented with kinetic experiments examining the effect of a dormancy-breaking treatment upon the transition between the dormant and nondormant states. In comparisons of the relative efficacy of various dormancy-breaking chemical treatments, the physical properties of substances under evaluation (e.g. lipophilicity) should be considered. In addition, since many dormancy-breaking chemicals are toxic to vegetative growth, there is a danger of introducing pharmacological artifacts that confuse interpretation of the results obtained. These toxic properties also necessitate the use of viability tests during construction of dose-response curves to avoid confusing seed death with dormancy. Seed scientists must also avail themselves of the extensive literature on developmental arrest in other organisms, which may generate fresh ideas for experimentation.

Crop seeds accumulate soluble carbohydrates as part of their maturation process. In legume seeds, the major soluble carbohydrates are sucrose and its galactosides raffinose, stachyose and verbascose. In buckwheat (Fagopyrum esculentum Moench) seeds, the major soluble carbohydrates are sucrose and galactosides of d-chiro-inositol, named fagopyritols. This study was conducted to determine changes in soluble carbohydrate accumulation in embryos of buckwheat seeds after feeding solutions containing the free cyclitols D-chiro-inositol, myo-inositol and d-pinitol to stem–leaf–seed explants. Feeding D-chiro-inositol to explants resulted in a fourfold to fivefold increase in the accumulation of free d-chiro-inositol, fagopyritol A1 and fagopyritol B1 in embryos of mature seeds, but resulted in 30% less embryo dry weight compared to the control treatment without cyclitols. Feeding myo-inositol to buckwheat explants increased d-chiro-inositol in leaves and increased accumulation of fagopyritol A1 and fagopyritol B1 fivefold in embryos, fagopyritol A2 and fagopyritol B2 fourfold; fagopyritol A3 and fagopyritol B3 were also detected, with no reduction in accumulated embryo dry weight. Feeding d-pinitol to buckwheat explants resulted in accumulation of free d-pinitol in mature embryos, but not galactopinitols. D-Pinitol, galactopinitol A and galactopinitol B were not detected in embryos from explants fed solutions without d-pinitol. Feeding d-pinitol also resulted in reduced D-chiro-inositol accumulation by buckwheat seeds. The results indicate that myo-inositol may be the precursor to d-chiro-inositol synthesis, and fagopyritols accumulated in response to d-chiro-inositol availability in the embryo. We suggest that increasing myo-inositol in buckwheat maternal tissues may be an effective means to enhance the accumulation of D-chiro-inositol and fagopyritols in seeds, compounds that may be beneficial for the treatment of non-insulin-dependent diabetes mellitus.

In contrast to reports in the literature that seeds of Dodonaea viscosa from China and Pakistan are non-dormant, or nearly so, we found that a high percentage of seeds of this species collected in north-western India have a water-impermeable seed coat at maturity, i.e. physical dormancy. Thus, seeds that were mechanically scarified and boiled (to open a ‘water gap’ in the seed coat) germinated to much higher percentages (84% and 77%, respectively) than did those that were non-scarified (24%). Our results agree with studies of seed dormancy in this species in various other parts of its large geographical range.