This paper describes the exploratory use of isothermal micro-calorimetry (IMC) to measure directly the heat flow produced as seeds age. Heat flow was recorded in primed and non-primed (control) seeds of Ranunculus sceleratus L., aged in a micro-calorimeter at 35°C at three different seed water contents [c. 0.12, 0.075 and 0.045 g H2O (g dw)−1]. The rate of heat flow and total heat generated (an indicator of extent of reaction) were generally greater in control seeds, which aged at a faster rate, than in primed seeds. Total heat generated over a given period also increased with increasing water content. The power–time curves did not indicate first- or second-order rate kinetics, consistent with the probability that seed ageing is complex and involves a number of reactions. Even after the capacity to germinate had ceased, there was a residual power signal. As a method, IMC gave consistent results using independent samples at different times. Therefore, short-term experiments at relatively high water contents and/or temperatures may have the potential to predict the relative longevity of seed-lots, at least within a species.

Although the underdeveloped embryo, and thus morphological (MD) or morphophysiological (MPD) seed dormancy, is basal in angiosperms, it also occurs in advanced groups. A synthesis of the literature, combining phylogeny and the kind of seed dormancy in the highly evolutionarily advanced order Dipsacales, shows that MPD (or MD) occurs throughout all clades except the most advanced one, Valerina. Seeds of taxa in the Valerina clade have fully developed embryos and physiological dormancy (PD) or are non-dormant (ND); thus, PD and ND are derived conditions in Dipsacales. Assuming that types of seed dormancy have not changed since the Early Tertiary, the fossil record suggests that MPD (or MD) was present in extant genera of Dipsacales by the Palaeocene, but PD (or ND) not until the Miocene. Molecular dating indicates that the ages of dipsacalean lineages with MPD and PD are older than those indicated by the fossil evidence.

Desiccation-tolerant organisms, or life-cycle stages of particular taxa, occur among animals, higher and lower plants, terrestrial micro-algae, lichens and bacteria. Recent investigations have revealed that a number of mechanisms conferring desiccation tolerance appear to be common to the diversity of life forms able to survive extreme dehydration. In particular, parallel processes involving late embryogenic abundant (LEA) or LEA-like proteins, accumulation of sugars, aspects of active oxygen species (AOS) and non-enzymic and enzymic antioxidants have been the recent focus of attention, some across a diversity of organisms. The present contribution considers advances made from the study of these processes, particularly in enhancing current understanding of the composition of, and protection afforded by, the glassy state in desiccated organisms. Strong evidence that proteins, particularly LEAs, are implicated in glass formation is reviewed, and behaviour of such proteins upon dehydration is discussed in this context. The question of the ability of cells to survive complete water removal is considered in the context that it is unlikely, and that the basis of the deterioration of very dry seeds results from abstraction of water necessary to maintain the integrity of the intracellular glassy state. Finally, the revelation that desiccated seeds deteriorate with time, even under extremely good genebanking conditions, is discussed.

After-ripening, the loss of dormancy under dry conditions, is associated with a decrease in mean base water potential for germination of Bromus tectorum L. seeds. After-ripening rate is a linear function of temperature above a base temperature, so that dormancy loss can be quantified using a thermal after-ripening time (TAR) model. To incorporate storage water potential into TAR, we created a hydrothermal after-ripening time (HTAR) model. Seeds from two B. tectorum populations were stored under controlled temperatures (20 or 30 °C) and water potentials (−400 to −40 MPa). Subsamples were periodically removed from each storage treatment and incubated at 15 or 25 °C to determine germination time courses. Dormancy status (mean base water potential) was calculated from each time course using hydrothermal time equations developed for each seed collection. Seeds stored at −400 MPa did not after-ripen. At water potentials from −400 to −150 MPa, the rate of after-ripening increased approximately linearly with increasing water potential. Between −150 and −80 MPa, there was no further increase in after-ripening rate, while at −40 MPa seeds did not after-ripen and showed loss of vigour. These results suggest that the concept of critical water potential thresholds, previously shown to be associated with metabolic activity and desiccation damage in partially hydrated seeds, is also relevant to the process of after-ripening. The HTAR model generally improved field predictions of dormancy loss when the soil was very dry. Reduced after-ripening rate under such conditions provides an ecologically relevant explanation of how seeds prolong dormancy at high summer soil temperatures.

The processes involved in rice (Oryza sativa L.) panicle ripening vary with time and topological grain position. Methods to describe the functioning and connectivity of the grains on a panicle could aid the analysis of these processes. Hence, we addressed the difficulty of encoding and representing panicle topology. Array-based decomposition and computational methods were developed to encode and analyse panicle topology and grain traits. The technique, applied to the analysis of dry matter accumulation, clearly represented the basipetal succession of asynchronous grain ripening on a panicle. These methods should be useful for the spatial and temporal analysis of a number of panicle processes and attributes, including molecular ones, involved with ripening.

Investigations of physiological functions of seed lipoxygenase during germination and early seedling growth are complicated by the presence of multiple isoforms with different product specificity. The combination of isoelectric focusing, lipoxygenase in-gel activity staining and mass spectrometry showed that only two seed lipoxygenases, LOX-2 and LOX-3, were active at neutral pH in protein extracts from dry and germinating pea seeds (Pisum sativum L.), with LOX-3 more abundant than LOX-2. Each active seed LOX appeared at multiple pIs, probably resulting from post-translational modifications. The abundance of pea seed lipoxygenases was lower in embryonic shoots than in other parts of the axis. After radicle emergence, seed LOXs were degraded, as revealed by Western blotting using antibodies specific to pea seed LOXs. Two new vegetative isoforms, LOX-N3 and LOX-g, appeared in the shoot and gradually replaced the pea seed lipoxygenases as the principal contributors to LOX enzyme activity during early seedling growth. Activities of LOX-N3 and LOX-g were abundant in shoot tips, but not root tips, in pea seedlings. Thus, during germination, there is a shift of LOX activity from radicles to shoots that accompanies the transition from seed LOXs to vegetative LOXs. These results confirmed that identification of pea LOXs based solely on pI may be misleading; however, mass spectrometry enabled more accurate identification of these highly homologous proteins to be made.

Frequently, in studies on seed germination ecology, mistakes are made that lead to problems in determining the kind of dormancy and in extrapolating data to the field situation. In this paper, we discuss 22 of these issues and offer suggestions on how to avoid them.

This study investigated the effects of 3-methyl-2H-furo[2,3-c]pyran-2-one, a germination active butenolide present in plant-derived smoke, gibberellic acid and smoke water on seeds of Australian Asteraceae exposed to different light regimes. Seeds of all species required light, with maximum germination occurring under white light, or light dominated by 640 nm. Compared to untreated seeds, butenolide increased germination of Angianthus tomentosus, Gnephosis tenuissima, Myriocephalus guerinae, Podolepis canescens and Rhodanthe citrina at suboptimal light wavelengths and in the dark to a level equal to, or greater than, smoke water. Germination of Erymophyllum glossanthus and Gnephosis acicularis was not promoted by butenolide or smoke water under any light regime. The action of gibberellic acid was compared to that of butenolide for three species (Angianthus tomentosus, Myriocephalus guerinae and Podolepis canescens), and both compounds were found to stimulate germination. This study provides evidence that butenolide can act in a similar fashion as gibberellic acid in promoting seed germination of light-sensitive seeds. The ecological significance of these findings is discussed.

The long-term utility of DNA banks is predicated on the stability of DNA during storage. The quality and yield of DNA extracted from seeds from four garden species, which varied in age from 1 to 135 years old, was used to examine the early stages of DNA degradation. Seeds that were 70 years old yielded high molecular weight DNA, which permitted amplification of a 650 bp segment of the internal transcribed spacer (ITS) region of the nuclear ribosomal DNA and a 487 bp segment of the plastid-encoded large subunit of rubisco (rbcL). DNA extracted from seeds estimated to be about 135 years old was degraded, but still permitted amplification when routine extraction procedures were used. The rbcL sequences obtained from c. 135-year-old cucurbit seed DNA were consistent with species identifications based on seed morphological features; however, ITS sequences from the same samples were determined to be of fungal origin. A comparison of our results with studies of DNA stability in leaf specimens suggested that DNA degraded within both tissues, but perhaps more slowly within seeds. Evidence that high-quality DNA can be extracted from old, non-viable seeds expands the utility of seed banks in preserving genetic resources.

Raffinose family oligosaccharides (RFO) accumulate in many developing seeds and are degraded during seed germination. However, acidic α-galactosidase (AGAL) activity and subcellular location do not correlate with raffinose depletion; alkaline α-galactosidases (AGA) may be responsible for RFO hydrolysis in germinating seeds. Three cDNA clones for AGA/SEED IMBIBITION PROTEIN were obtained from the Pioneer Hi-Bred maize expressed sequence database. Two of the clones were expressed in Escherichia coli, and the recombinant proteins, when incubated with naturally occurring galactosides or p-nitrophenyl α-d-galactose, exhibited AGA activity with maximum catalysis at pH 7.5 (ZmAGA1) or pH 8.5 (ZmAGA3). No raffinose biosynthetic capacity was observed with either enzyme. Maximal α-galactosidase activity in mature dehydrated, germinating and germinated maize (Zea mays) seeds occurred at pH 7.5. ZmAGA1 was the sole family member detected in seeds and maize Hi-II, embryo-derived, callus cells. Its transcript accumulated when seed germination was interrupted by heat, cold or dehydration stress, but not in response to NaCl. Tissue prints localized transcripts to the scutellum or the embryo axis, depending on the stress applied. In maize Hi-II callus cells, transcripts accumulated when callus was subjected to heat stress (42 °C), during which ZmAGA1 transcript accumulation was further induced by sucrose. Galactosides in a variety of forms, including raffinose, partially repressed the sucrose-induced accumulation of transcript in heat-stressed callus.

Seed respiration has been utilized as an index of seed vigour and has correlated well with seedling development. We examined the respiration process as affected by temperature and osmopriming during imbibition in two cultivars of sorghum [Sorghum bicolor (L.) Moench] differing in capacity to germinate at low temperatures. Respiration of imbibing seeds was measured with a specially built apparatus linked to an infrared gas analyser. Seed respiration at the beginning of the imbibition process strictly depended on temperature (T). T also greatly affected the total CO2 respired to reach the first germination event, which significantly increased as T declined from the optimum. Osmopriming affected seed respiration differently in the two cultivars. In the cold-tolerant cultivar (Brandes), osmopriming reduced the total thermal requirements and, at T close to the minimum for germination, the energy required to achieve the first radicle protrusion. In the cold-sensitive cultivar (H128), osmopriming lowered base T for respiration and reduced the requirements for total CO2 respired, allowing germination even at 10°C. The cold-tolerant cultivar always maintained higher respiration rates than the cold-sensitive one, showing a greater capability of mobilizing energy from reserves. Germination time (i.e. GR50) related well to cumulative seed respiration down to a threshold value of cumulative CO2, after which GR50 was more affected by T than was cumulative respiration. Seed water uptake significantly slowed with decreasing T, and was accelerated by osmopriming. However, water imbibition rate influenced the start of seed respiration, but not the subsequent rate of metabolic activity preceding germination. Seed respiration was a good indicator of the activation of germinative metabolic processes, although under extreme T conditions the seed populations still respired, even if at low levels, but did not germinate rapidly or completely.

We tested the germination of fresh and frost-treated seeds of 26 species of southern Mongolian mountain and desert steppes, covering the major growth forms of woody and herbaceous perennials and short-lived species in the region. In the field, germination depends on rains that are largely restricted to the summer months between June and August. Thus, germination tests were performed at alternating conditions of 10°C in darkness and 20°C in light (12 h/12 h), which correspond to temperatures at the study site in early and late summer. Seeds of both woody and herbaceous perennials germinated well under the chosen conditions and apparently did not require stratification or scarification. In contrast, germination of annual species was mostly below 30%, while seed viability was equally high in all three growth forms. Winter conditions, simulated by freezing dried seeds at −18°C, hardly changed seed germination in the perennial species, but several short-lived species responded with increased germination. Short-lived species are not abundant in the real vegetation, which is governed by perennials. Thus, we conclude that the important species in Mongolian mountain steppes germinate readily without a dormancy-breaking treatment. A review of the available literature revealed that a complete lack of dormancy, or presence of only conditional dormancy, is also widely described for other species of Central Asian deserts and steppes, which is in contrast to data from North American prairies.

Seed remains of common millet (Panicum miliaceum L.) were excavated from sites of ad 4th-century Darhan (Mongolia), and ad 15th-century Budapest (Hungary). Because the 15th-century medieval grains looked so intact, a germination test was carried out under aseptic conditions, which resulted in swelling of the grains but no cell proliferation or germination. Ancient DNA (aDNA) was extracted from the aseptic grains; analysed for amplified fragment length polymorphisms (AFLP), simple sequence repeats (SSR) and mitochondrial DNA (mtDNA); and compared with the modern millet cultivar ‘Topaz’. AFLP analysis revealed that extensive DNA degradation had occurred in the 4th-century ancient millet, resulting in only 2 (1.2%) AFLP fragments (98.8% degradation) amplified by MseCAA–EcoAGT, compared to the 15th-century medieval millet, with 158 (40%) fragments (60% degradation), and modern millet cultivar ‘Topaz’ with 264 fragments (100%). EcoAGT–MseCAA was found to be the most effective selective-primer combination for the analysis of medieval and modern millet. Eight AFLP fragments were sequenced after re-amplification and cloning. Microsatellite (SSR) analysis at the nuclear gln4, sh1, rps28 and rps15 loci revealed one SNP (single nucleotide polymorphism) at the 29th position (A→G) of rps28 locus, compared to modern millet. An mtDNA fragment (MboI), amplified at the 18S–5S ribosomal DNA (rDNA) locus in the medieval millet, showed no molecular changes compared to modern millet. The results underline the significance of aDNA extraction and analysis of excavated seeds for comparative analysis and molecular reconstruction of ancient and extinct plant genotypes.

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.

The effects of alternating temperatures on seed dormancy changes, germination and seedling emergence were investigated in ‘Arctic’ and ‘Lineta’ orchardgrass (Dactylis glomerata L.). Thermal time models were successfully developed for 0, 5, 10 and 15°C temperature amplitudes, using 28 constant and alternating temperature regimes. These models were then modified by linking seed germination in Petri dishes and seedling emergence in soil. A field experiment was conducted with four seeding dates over 2 years to validate the modified thermal time models. Temperature regimes with a 5–15°C amplitude enhanced seed germination percentages of orchardgrass, indicating that the conditional dormancy was released by these temperature regimes. Base temperatures decreased with increasing temperature amplitude. Seeds germinated more rapidly under alternating temperatures than under constant temperatures. The dual effects of temperature for dormancy breaking and germination were accounted for by thermal time models based on alternating temperature regimes, which accurately predicted the timing and percentage of ‘Arctic’ and ‘Lineta’ orchardgrass seedlings emerging in the field (R2≥0.88).

Malva parviflora L. (Malvaceae) is rapidly becoming a serious weed of Australian farming systems. An understanding of the variability of its seed behaviour is required to enable the development of integrated weed management strategies. Mature M. parviflora seeds were collected from four diverse locations in the Mediterranean-type climatic agricultural region of Western Australia. All of the seeds exhibited physical dormancy at collection; manual scarification or a period of fluctuating summer temperatures (50/20°C or natural) were required to release dormancy. When scarified and germinated soon (1 month) after collection, the majority of seeds were able to germinate over a wide range of temperatures (5–37°C) and had no light requirement. Germination was slower for seeds stored for 2 months than seeds stored for 2 years, suggesting the presence of shallow physiological dormancy. Seed populations from regions with similar annual rainfall exhibited similar dormancy release patterns; seeds from areas of low rainfall (337–344 mm) were more responsive to fluctuating temperatures, releasing physical dormancy earlier than those from areas of high rainfall (436–444 mm). After 36 months, maximum seedling emergence from soil in the field was 60%, with buried seeds producing 13–34% greater emergence than seeds on the surface. Scanning electron microscopy of the seed coat revealed structural differences in the chalazal region of permeable and impermeable seeds, suggesting the importance of this region in physical dormancy breakdown of M. parviflora seeds. The influence of rainfall during plant growth in determining dormancy release, and hence, germination and emergence timing, must be considered when developing management strategies for M. parviflora.

Amaranthus tuberculatus is a weed species that has shifted emergence patterns over the past few years, presumably due to changes in seed dormancy in response to selection in agricultural fields. Although it is recognized that the seed dormancy phenotype is greatly affected by the environment, it is also acknowledged that the genotype plays a significant role. However, the importance of the genotype in determining intra-population seed dormancy variability, and the effect on emergence patterns, is not well understood. The objective of the present study was to determine the importance of the genotype on deep dormancy and the stratification-mediated dormancy alleviation in A. tuberculatus. Wild populations differing in seed dormancy were crossed and F2 families were generated. These families were used to determine narrow sense heritability of dormancy and stratification-mediated dormancy alleviation at the individual (hi2) and family (hf2) levels. hi2 ranged from 0.13 to 0.4 and 0.04 to 0.06 for the dormancy and stratification response, respectively. In the case of hf2, the values ranged from 0.76 to 0.91 for deep dormancy and from 0.33 to 0.58 for the stratification response. The genetic correlation between these two traits was below 0.075, indicating that different genes control them. High temperature strengthened the dormancy of deeply dormant seeds, making them less sensitive to stratification. However, high temperature promoted the germination of non-deeply dormant seeds. It is proposed that delayed weed emergence can be generated by selecting genes that control stratification response, and not necessarily only the genes that are directly responsible for deep dormancy.

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.

It has been hypothesized that fluctuations in soil water content may affect the dormancy status of weed seed banks under field conditions. In this paper, we present results showing that fluctuations in soil water content affect the dormancy status of buried seeds of Polygonum aviculare L. stored at dormancy-releasing temperatures. Effects of fluctuations in soil water content on the dormancy status of P. aviculare seeds were evaluated by comparing changes in the range of temperatures and water potentials permissive for germination, and in the sensitivity to fluctuating temperatures, between seeds subjected to a moist soil regime (MS) or to a fluctuating soil water content regime (FS). In comparison to the dormancy release pattern observed for seeds subjected to MS, seeds subjected to FS generally showed an increase in their dormancy level after periods of storage under dry soil conditions, and a decrease in their dormancy level after periods of storage under moist soil conditions. These effects were more pronounced during early stages of the storage period, producing larger changes in the thermal and water potential range for seed germination than in the sensitivity of seeds to fluctuating temperatures. Seeds subjected to FS generally exhibited a lower mean low-limit temperature, lower mean thermal time and hydrotime requirements for germination, and a higher proportion of the seed population with the capacity to germinate in situ, than seeds subjected to MS. The results obtained suggested that fluctuations in soil water content could be an additional factor affecting dormancy and weed emergence patterns under field conditions.