Intact seeds (seed+endocarp) from freshly harvested fruits of Prunus campanulata were dormant, and required 4–6 weeks of warm followed by 8 weeks of cold stratification for maximum germination percentage. Removing both endocarp and seed coat, however, promoted germination in a high percentage of non-stratified seeds. Treatment of intact, non-stratified seeds with gibberellic acid (GA3) was only partially effective in breaking dormancy. However, GA3 promoted germination of non-stratified seeds in which the endocarp (but not the seed coat) had been removed. The order of abscisic acid (ABA) concentration in fresh seeds was endocarp > seed coat > embryo, and its concentration in endocarp plus seed coat was about 6.2-fold higher than that in the embryo. Total ABA contents of seeds subjected to warm and/or cold moist stratification were reduced 6- to 12-fold. A higher concentration of GA4 was detected in embryos of non-dormant than in those of dormant seeds. Fluridone, a carotenoid biosynthesis inhibitor, was efficient in breaking dormancy of Prunus seeds. Paclobutrazol, a GA biosynthesis inhibitor, completely inhibited seed germination, and the inhibitory effect could be partially reversed by GA4, but not by GA3. Thus, dormancy in P. campanulata seeds is imposed by the covering layers. Dormancy break is accompanied by a decrease in ABA content of the covering layers and germination by an increase of embryonic GA4 content.

We investigated seed dormancy among species of Melastomataceae from Neotropical montane vegetation of Brazil. Four out of 50 studied species had dormant seeds: Miconia corallina (Miconieae), Tibouchina cardinalis (Melastomeae), Comolia sertularia (Melastomeae) and Chaetostoma armatum (Microlicieae). For these four species, germinability of seeds collected in different years was always < 10% and the percentages of embryoless seeds and non-viable embryos were both insufficient to explain low or null germinability. This is the first unequivocal report of seed dormancy in tropical Melastomataceae. The production of seeds with permeable seed coats and fully developed, differentiated embryos indicates the occurrence of physiological dormancy. The reconstructed phylogenetic tree of the 50 species suggests that physiological dormancy evolved multiple times during the evolutionary history of Melastomataceae in this vegetation. Physiological dormancy evolved in species and populations associated with xeric microhabitats, where seeds are dispersed in unfavourable conditions for establishment. Therefore, drought-induced mortality may have been a strong selective pressure favouring the evolution of physiological dormancy in Melastomataceae. We argue that dormancy may have been independently selected in other lineages of Cerrado plants colonizing xeric microhabitats and dispersing seeds at the end of the rainy season. The contributions of our data to the understanding of seed dormancy in tropical montane vegetation are discussed.

Germination and carbohydrate metabolism were studied in fresh developing pea (Pisum sativum L., cv Baccara) seeds and after artificial drying at 25°C and various relative humidities (20, 75 and 99% RH) to investigate whether the occurrence of desiccation tolerance was related to sucrose, raffinose and stachyose contents. Seeds became completely tolerant to fast drying at 25°C and 20% RH a few days after the end of reserve accumulation, i.e. when their moisture content dropped to approx. 50% (fresh weight basis). This acquisition of desiccation tolerance was associated with an accumulation of raffinose and stachyose, the latter being more abundant in the embryonic axis than in the cotyledons. The (raffinose+stachyose)/sucrose ratio increased during seed development and reached 1.1 in the axis and 0.2 in the cotyledons just before the onset of desiccation tolerance. When the natural acquisition of desiccation tolerance occurred on the mother plant, artificial drying of isolated seeds induced an increase in oligosaccharide content in the cotyledons. Immature seeds, the moisture content of which was higher than about 60% (fresh weight basis), did not tolerate fast drying (25°C and 20 or 75% RH). Such drying did not result in the synthesis of stachyose and induced an increase in electrolyte leakage, a decrease in the ability of seeds to convert 1-aminocyclopropane 1-carboxylic acid (ACC) to ethylene and an increase in ethane synthesis, thus indicating a deterioration of cell membrane properties and lipid peroxidation. In contrast, immature seeds tolerated drying either in the pods or at 25°C and 99% RH, and such drying induced a decrease in sucrose content, an increase in oligosaccharide content and a (raffinose+stachyose)/sucrose ratio higher than around 1. Soluble sugar contents of dried immature seeds depended on the conditions of dehydration. In cotyledons, the (raffinose+stachyose)/sucrose ratio reached 0.61 when seeds were dried at 25°C and 99% RH, whereas it was as low as 0.15 when drying was performed at 25°C and 20% RH. All the results obtained are consistent with the concept that oligosaccharides may well be involved in the protection of membranes during dehydration.

Changes in germination and desiccation sensitivity were measured throughout the seed expansion phase of development in fruits of Quercus robur L. The onset of a reduction in sensitivity to desiccation during development on the tree coincided with acquisition of the capacity for seed germination on moist sand substrate. Tolerance of desiccation then increased throughout development to shedding, but viability was still lost at a relatively high moisture content, and seeds did not therefore pass through a fully desiccation-tolerant phase. These data suggest that desiccation sensitivity in Q. robur may have resulted from the premature termination of development.

No desiccation was required to initiate germination in prematurely harvested fruits orseeds. Germination rate of seeds on moist sand increased at successive harvests during development, and was also increased by presoaking seeds in water. Variation in germination rate following shedding was not related to seed size or moisture content, but was affected by shedding date. This effect was not observed when the pericarp was removed.

Alfalfa (Medicago sativa L.) yield is severely compromised by soil salinity, especially at the level of seedling establishment. This question was addressed by proteomics to decipher whether specific changes in protein accumulation correlate with germination performance of alfalfa seeds submitted to a salinity stress as obtained by imbibing seeds in the presence of NaCl. This study used alfalfa seeds submitted to an osmopriming invigoration treatment that proved very efficient in counteracting the negative effect of salinity stress on germination performance. Comparative proteomic analyses disclosed 94 proteins commonly characterizing the response of both the untreated control and osmoprimed seeds to the experimental salinity stress. Remarkably, many of them, representing 84 proteins, showed contrasting accumulation patterns when comparing the untreated control and osmoprimed seeds submitted to the same salt stress. Thus numerous changes observed in the proteome of the untreated control seeds imbibed in the presence of salt, and presumably accounting for the loss in seed vigour associated with salinity stress, can be substantially reversed in osmoprimed seeds undergoing this stress. These data therefore provide a biochemical understanding of the increase in seed vigour generally observed with primed seeds.

An expressed napin storage protein gene from Brassica rapa, BcNA1, has been cloned and sequenced. The gene is a member of a family of four to seven napin genes in B. rapa and is highly expressed in developing seeds. An expression cassette containing the DNA flanking the napin coding region of BcNA1 has been engineered and demonstrated to function appropriately, as compared with the gene's endogenous expression, in transgenic rapeseed using the β-glucuronidase reporter gene. The B. rapa BcNA1 gene and a B. napus napin gene, gNa, share extremely high nucleotide homology not only throughout their coding regions, but over a DNA locus comprising 4.3 kb. We suggest the gNa gene was contributed by the original B. rapa progenitor of the amphidiploid B. napus.

To determine whether seeds of the weedy shrub Wigandia urens, from the Valley of Mexico, undergo natural priming when buried in soil, comparative experiments were performed with seeds: (1) harvested directly from the plants; (2) buried in three natural habitat conditions; and (3) laboratory primed with polyethylene glycol. Seeds were sown in a growth chamber and in a shade house. Final germination percentages, emergence, germination and emergence rates, survival and initial growth were determined. Burial and priming enhanced the germination and emergence parameters evaluated in the laboratory and in the shade house. Effects of treatments on survival were not significantly different. Nevertheless, burial improved emergence and mean survival, and induced differences in specific leaf area of seedlings that could have ecological significance. Heat-stable proteins were extracted and electrophoresed. Proteins formed in W. urens seeds during burial had molecular weights (14–21 kDa) similar to those reported for late embryogenesis abundant (LEA) proteins induced by priming in other species. Nevertheless, the presence and abundance of proteins expressed (14–23, 36 and more than 45 kDa) differed among control, primed and buried seeds. During soil burial, molecular and physiological responses were induced that were similar to the effects of priming.

A common understanding of the control of germination through dormancy is essential for effective communication between seed scientists whether they are ecologists, physiologists or molecular biologists. Vleeshouwers et al. (1995) realized that barriers between disciplines limited progress and through insightful conclusions in their paper ‘Redefining seed dormancy: an attempt to integrate physiology and ecology’, they did much to overcome these barriers at that time. However, times move on, understanding develops, and now there is a case for ‘Redefining seed dormancy as an integration of physiology, ecology and molecular biology’. Finch-Savage and Leubner-Metzger (2006) had this in mind when they extended and re-interpreted the definition of dormancy proposed by Vleeshouwers et al. (1995), by considering dormancy as a having a number of layers that must be removed, with the final layer of dormancy being synonymous with the stimulation/induction of germination.

Seed germination is responsive to diverse environmental, hormonal and chemical signals. Germination rates (i.e. speed and distribution in time) reveal information about timing, uniformity and extent of germination in seed populations and are sensitive indicators of seed vigour and stress tolerance. Population-based threshold (PBT) models have been applied to describe germination responses to temperature, water potential, hormones, ageing and oxygen. However, obtaining detailed data on germination rates of seed populations requires repeated observations at frequent times to construct germination time courses, which is labour intensive and often impractical. Recently, instruments have been developed to measure repeatedly the respiration (oxygen consumption) of individual seeds following imbibition, providing complete respiratory time courses for populations of individual seeds in an automated manner. In this study, we demonstrate a new approach that enables the use of single-seed respiratory data, rather than germination data, to characterize the responses of seed populations to diverse conditions. We applied PBT models to single-seed respiratory data and compared the results to similar analyses of germination time courses. We found consistent and quantitatively comparable relationships between seed respiratory and germination patterns in response to temperature, water potential, abscisic acid, gibberellin, respiratory inhibitors, ageing and priming. This close correspondence between seed respiration and germination time courses enables the use of semi-automated respiratory measurements to assess seed vigour and quality parameters. It also raises intriguing questions about the fundamental relationship between the respiratory capacities of seeds and the rates at which they proceed toward completion of germination.

Dormancy-breaking requirements and types of dormancy were determined for seeds of Lonicera fragrantissima Lindl. & Paxt., L. japonica Thunb., L. maackii (Rupr.) Maxim. and L. morrowii A. Gray. Seeds of all four species have underdeveloped spatulate embryos that are about 20–40%fully developed (elongated) when dispersed. Embryos in freshly matured, intact seeds grew better at 25/15°C than at 5°C. Gibberellic acid (GA3) (tested only in the light) was more effective in breaking dormancy in L. maackii and L. morrowii than in L. fragrantissima and L. japonica. Warm- followed by cold stratification was required to break dormancy in seeds of L. fragrantissima, whereas seeds of L. japonica required cold stratification only. Thus, seeds of L. fragrantissima have deep simple morphophysiological dormancy (MPD) and those of L. japonica nondeep simple MPD. About 50%of the seeds of L. maackii required warm- or cold stratification only to come out of dormancy and 50% of those of L. morrowii required warm stratification only, whereas the other 50% did not require stratification to germinate. Thus, about half of the seeds of the two species has nondeep simple MPD, and the other half has morphological dormancy (MD). In these laboratory tests, seeds of L. japonica, L. maackii, and L. morrowii generally germinated to significantly higher percentages in light than in darkness; seeds of L. fragrantissima were not tested in darkness. Peaks of germination for seeds of L. fragrantissima, L. japonica, L. maackii and L. morrowii sown on a soil surface and covered with Quercus leaves under near-natural temperature conditions shortly after seed maturity and dispersal in late June 1997, late November 1997, early November 1996 and late June 1998, respectively, occurred in early March 1998, late February 1998, late March 1997 and early October 1998, respectively. The germination phenologies of seeds of the same species and seed lots buried in soil were similar to those of seeds under leaf litter. High percentages of seeds of all four species germinated both under litter (78–96%) and beneath the soil surface (78–97%). These germination patterns correspond closely with the requirements for embryo growth and dormancy break in the four Lonicera species.

After 110 years of hermetic sealed storage at temperatures between 10 and 15°C and at a moisture content of 3.12%, Hordeum vulgare showed 90% germination and Avena sativa 81%. In addition, some seeds of Agrostemma githago, Lolium temulentum, Sinapis alba, Sinapis arvensis, and Vaccaria hispanica were still capable of germinating. This demonstrates that ultra-dry, long-term seed storage under ambient temperature conditions can successfully be achieved with the intention of cutting down risks and costs in germplasm conservation.

A principal goal of seed germination modelling for wild species is to predict germination timing under fluctuating field conditions. We coupled our previously developed hydrothermal time, thermal and hydrothermal afterripening time, and hydration–dehydration models for dormancy loss and germination with field seed zone temperature and water potential measurements from early summer through autumn to develop predictions of germination timing for Bromus tectorum at a semi-arid site in north-central Utah, USA. Model predictions were tested with a validation dataset based on concomitant seed retrieval experiments in 2 years. Predictions were generally in agreement with observed field germination time courses, even though integration across multiple precipitation events was necessary. Success of the modelling effort hinged on two factors. First, we used a soil capacitance sensor that measured seed zone (5 mm soil depth) water content accurately over a wide range. Second, simulations were built using physiologically based threshold models that can incorporate differences in germination timing for multiple germination fractions and for multiple stages of dormancy loss. Our results suggest that simulation models using hydrothermal time concepts can predict field germination phenology accurately. Seeds in this study integrated their experiences in a widely fluctuating environment in a manner consistent with the assumptions of hydrothermal time. Such threshold-based models also have the advantage of generality, as these concepts can be applied to many different species, environments and weather scenarios.

Mature seeds of Norway maple (Acer platanoides L.) are tolerant of desiccation, at least to moisture contents of about 7% (fresh weight basis), but those of sycamore (Acer pseudoplatanus) are killed by drying below about 45% moisture content. Sycamore seeds are thus recalcitrant; while the classification of those of Norway maple as orthodox is confirmed by the fact that between 19% and 7.5% moisture content their longevity is increased in a predictable way by reduction of seed moisturecontent. However, the period of useful storage of the latter in seed banks may be much less than for many crop species. The rates of water loss to a dry environment of both fruits and seeds of sycamore are much less than those of Norway maple, suggesting a degree of desiccationavoidance in the desiccation-intolerant species. Seed physiological maturity (maximum dry weight) occurred 2–3 weeks earlier in Norway maple than insycamore, but in both species this occurred about 150–160 days after peak flowering. Tetrazolium staining is a good indicator of embryo viability in both species, correlating well with germination test results. In Norway maple both methods of viability testing indicated that whole-seed desiccation tolerance coincided with the attainment of maximum dry weight. Tetrazolium staining indicated the development of desiccation tolerance in the radicles/hypocotyls of both species approximately 2–4 weeks before physiological maturity. Possible correlation between changes in the level of embryo dormancy during development and the acquisition of desiccation tolerance are discussed.

Changes in population hydrotime parameters were determined during stratification in Polygonum aviculare L. seeds in order to model dormancy loss. Seeds buried in pots were stored at three temperatures (1.6, 7 and 12°C) for 110 d and were exhumed at regular intervals during the storage period. Exhumed seeds were incubated at different water potentials at 15°C and germination time courses were analysed to determine hydrotime parameters. The population mean base water potential (Ψb(50)) decreased concomitantly with seed dormancy, while the hydrotime constant (θH) and the standard deviation of base water potential (θΨb) displayed only minor changes. Based on these results, a model for simulating P. aviculare seed dormancy loss in relation to low temperature was developed. The model employs Ψb(50) as an index of mean seed population dormancy status. While Ψb(50) was allowed to vary as seeds were released from dormancy, θH and θΨb were held constant. Changes in Ψb(50) were related to the time and temperature, using a previously developed thermal stratification time index (Stt), which quantifies the accumulation of thermal time units below a threshold temperature required for dormancy loss to occur. Therefore, Ψb(50) varied in relation to the accumulation of Stt according to time and temperature. Model performance showed acceptable prediction of timing and percentage of germination of seeds buried in irrigated plots, but did not accurately predict germination of seeds exhumed from rain-fed plots. Thus, environmental factors other than temperature could also be involved in the regulation of dormancy status of buried seeds under field conditions.

Osmopriming of leek seeds (Allium porrum L.) in PEG 6000 solution (−10 bars) at 15°C for 14 days leads to reductions in both the spread of germination and mean time to germination, especially in low-vigour seed lots. In vivo methyl [3H]-thymidine pulse-labelling studies have demonstrated constant and low levels of DNA synthesis in leek embryo tissue during the osmopriming treatment. DNA synthesis during osmopriming was not inhibited by aphidicolin, an inhibitor of nuclear DNA replication. Replicative and repair-type DNA synthesis was investigated using BND-cellulose chromatography and these studies revealed that about 30% of the DNA synthesis after 1 day of priming was of a repair-type. DNA repair-type synthesis contributed to approximately 20% of the [3H]thymidine incorporated into DNA during the rest of the priming period in embryo tissue from both high-vigour and low-vigour seed lots. After 1 day of germination following priming, enhanced levels of both replicative and repair-type DNA synthesis were demonstrated. The replicative type of DNA synthesis detected in leek embyros during the priming period was not inhibited by aphidicolin and appears to represent a significant level of mitochondrial DNA synthesis. DNA synthesis could be detected in both nuclei and mitochondria of leek embryo tissue during the osmopriming treatment in the absence of any detectable cell division.

The temperate grass species, red fescue (Festuca rubra ssp. litoralis), perennial ryegrass (Lolium perenne) and Kentucky bluegrass (Poa pratensis), are often sown in mixtures for turfgrass. Differences in germination response to environmental conditions often result in different establishment success. The three species were compared in a study of imbibition at reduced water potential (Ψ) and in a study of the effects of Ψ and temperature on germination. In all three species, imbibition rate and extent were reduced with decreasing Ψ, causing a prolonged lag-phase before germination, particularly in Kentucky bluegrass. In perennial ryegrass and Kentucky bluegrass, water content at radicle emergence decreased non-linearly with decreasing Ψ, with a lower asymptote expressing the critical water content for germination. Seeds were germinated in factorial combinations of two temperatures (10 and 25°C) and nine Ψ levels (–2 to 0 MPa). Kentucky bluegrass required a longer thermal time to germinate than the other species and exhibited conditional dormancy at 25°C, which affected the use of the thermal time model. The effects of Ψ were described by a hydrotime model. Kentucky bluegrass had higher base water potential (Ψb) and required a longer hydrotime for germination than the other species. However, Ψb increased with temperature in Kentucky bluegrass and red fescue, but decreased in perennial ryegrass, and the base temperatures also changed with Ψ. The interacting effects of Ψ and temperature affected the ability of the hydrothermal time model to predict germination performance across all temperature and Ψ conditions.

Seeds of the common bean, Phaseolus vulgaris, contain two inhibitors of mammalian and insect α-amylases (αAls) that show specificity towards the amylases of different insect species. Expression in pea (Pisum sativum) and azuki bean (Vigna angularis) of a chimeric gene consisting of the cDNA of bean αAl-1 and a seed-specific promoter makes the seeds of these legumes resistant to three species of Old World bruchids whose amylases are inhibited by αAl-1. This was the first successful genetic engineering of insect resistance in seeds. To understand the basis of the specificity between amylases and inhibitors we cloned a second bean inhibitor (αAl-2) with different specificity, and we cloned the cDNA of the New World bruchid, Zabrotes subfasciatus. The amylase of this bruchid is inhibited by αAl-2, but not by αAl-1. Knowledge of the amino acid sequences and of the three-dimensional structure of the pancreatic α-amylase–αAl-1 complex allows us to predict the peptide domains and amino acids of the proteins that are important for protein–protein recognition and inhibition of enzyme activity.

MicroRNAs (miRNAs) are involved in developmental programmes of plants, including seed germination and post-germination. Here, we provide evidence that two different miRNA pathways, miR156 and miR172, interact during the post-germination stages in Arabidopsis. Mutant seedlings expressing miR156-resistant SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE13 (mSPL13), which has silent mutations in the miR156 complementary sequence, over-accumulated SPL13 mRNA and exhibited a delay in seedling development. Microarray analysis indicated that SCHNARCHZAPFEN (SNZ), an AP2-like gene targeted by miR172, was down-regulated in these mutants. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) and miRNA gel blot analyses showed that the MIR172 genes were up-regulated in mSPL13 mutants. These results suggest that the miRNA regulation cascades (miR156⊣SPL13 → miR172⊣SNZ) play a critical role during the post-germination developmental stages in Arabidopsis.

The development of techniques for somatic embryogenesis in conifers has led to rapid advances in the ability to culture conifer tissue in vitro. Somatic embryogenesis now provides a means to clonally propagate commercially valuable conifers of several species and offers an in vitro experimental system for studying embryogenesis. As a result of these developments, the conifer zygotic and somatic embryo system has recently attracted the attention not only of the cell biologist but also of biochemists and molecular biologists. In this review I have attempted to consolidate the recent information on ultrastructure, biochemical and molecular aspects of conifer zygotic and somatic embryogenesis. In the first section, salient features of zygotic embryogenesis are highlighted followed by biochemical and ultrastructural characterization of development with reference to storage reserve deposition. In the second section, detailed characterization of seed storage proteins, changes in gene expression with emphasis on storage protein genes and Lea genes during zygotic and somatic embryogenesis are described. Effect of culture treatments such as abscisic acid, osmoticum and desiccation on storage protein gene expression in somatic embryos is discussed. Based on these studies, comparisons are presented between angiosperm and gymnosperm embryogeny regarding the unique morphological and developmental patterns of conifer embryogenesis. Finally, a brief discussion of recent progress in biochemical and molecular characterization of conifer seed germination is presented.

The expression patterns of β-1,3-glucanases (βGlu) and chitinases (Chn) were investigated during the seed germination of members of the Cestroideae (three Nicotiana species, Petunia hybrida) and the Solanoideae (Capsicum annuum, Physalis peruviana) subgroups of Solanaceous species. Rupture of the micropylar testa (seed coat) and rupture of the micropylar endosperm, i.e. radicle emergence, were distinct and temporally separate events during the germination of Cestroideae-type seeds. βGlu accumulation in imbibed Cestroideae-type seeds, occurring after testa rupture but prior to endosperm rupture, was inhibited by abscisic acid (ABA) and promoted by gibberellins (GA) and light, in strict association with germination, and appeared to be caused by transcriptional regulation of the class I βGlu genes. The micropylar cap of Solanoideae-type seeds does not allow a distinction between testa and endosperm rupture, but βGlu accumulation occurred prior to radicle emergence of pepper and P. peruviana seeds. ABA inhibited endosperm rupture and βGlu accumulation in the micropylar cap of pepper seeds. In contrast to tomato, βGlu accumulation in pepper seeds was not only confined to the micropylar cap, was due to distinct, tissue-specific βGlu isoforms, and was not accompanied by Chn accumulation. In conclusion, ABA inhibition of germination and βGlu accumulation in the micropylar endosperm appears to be a widespread event during the seed germination of Solanaceous species. In contrast, accumulation of Chn and distinct βGlu isoforms in the embryo, prior to germination, appears to be a species-specific phenomenon within the Solanaceae. In addition, a post-germination co-induction of βGlu and Chn in the root of the emerged seedling was found in endospermic and non-endospermic species and could represent an evolutionarily conserved event during dicot seedling development.