This work provides insights into the deterioration of cacti seeds of Escontria chiotilla (F.A.C. Weber ex K. Schum) and Stenocereus pruinosus (Otto ex Pfeiff.) Buxbaum stored ex situ at 25 °C, under dry and dark conditions or buried in situ conditions in a xerophytic shrubland. Viability, germination speed, electrolyte leakage and indicators of the redox balance including glutathione content, glutathione half-cell reduction potential (EGSSG/2GSH) and malondialdehyde (MDA), oxidized protein content, together with water-soluble antioxidant enzyme activity were assessed. Over a period of two years of storage, viability was maintained when seeds were stored ex situ at 7–9% water content compared to seeds buried in the soil. A second burial experiment showed that seeds of E. chiotilla maintained viability during a year of storage that included a rainy season followed by a dry season. Thereafter, they died rapidly during the second rainy season. In contrast, those of S. pruinosus started to lose viability after 6 months of burial at the end of the rainy season and were mostly dead at the end of the dry season. This difference in persistence between species was related to a difference in the glutathione content and antioxidant enzyme activities. In both storage experiments, the loss of viability of both species was associated to a EGSSG/2GSH shift to a more oxidative state during burial. Yet, contents in MDA and oxidized soluble proteins were not related to redox imbalance and loss of viability, indicating that these compounds are not good markers of oxidative stress in cacti seeds during storage.

Seed longevity is a complex process of key ecological and agronomic importance. DNA damage is a significant factor affecting seed ageing. Likewise, epigenetic changes can control gene expression and, therefore, seed response to ageing. The aim of the present work was to investigate the effect of ageing on nucleic acid stability and to identify reliable molecular markers that might help to monitor epigenetic changes within plant genetic resources during conservation. DNA profiles, evaluated by RAPD (random amplified polymorphic DNA), and methylation patterns, obtained by MSAP (methylation-sensitive amplification polymorphism), were compared in non-aged and aged Mentha aquatica seeds and plants produced by them. Germination decreased to 50% by storing seeds at 35°C and 12% wc for 28 days. RAPD profiles were 99% similar in these aged seeds compared to non-aged seeds. However, seedlings produced from the aged seeds showed a 13% dissimilarity compared to seedlings produced from the non-aged seeds. About 8% difference in the MSAP epigenetic profile was detected in seeds after storage and 16% difference was detected in the seedlings produced from them. This indicates that stress from high temperature and humidity during storage induced changes on the methylation state of seeds, and that changes were also detectable in the regenerated plants. Our results suggest that DNA integrity was compromised in seeds during ageing, and on seedlings produced by aged seeds. Genotype screening techniques such as RAPD and MSAP have the potential as markers of nucleic acid stability during seed ageing.

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

In fire-prone ecosystems, many species regenerate after fire from persistent soil seed banks. Species with physically dormant (PY) seeds have dormancy broken by fire-related heat. The magnitude of post-fire recruitment, to predict response to varying fire severity, is commonly estimated by testing dormancy-breaking temperature thresholds of fresh PY seeds. However, seeds spend years in the soil during the inter-fire period, and determining whether dormancy-breaking thresholds change over time is essential to accurately predict population persistence. Germination of four south-eastern Australian PY species from the Fabaceae family (Acacia linifolia, Aotus ericoides, Bossiaea heterophylla and Viminaria juncea) were studied. Dormancy-breaking temperature thresholds vary inter-specifically and the species represented either high or low dormancy-breaking threshold classes. Freshly collected seeds, and seeds that had been buried in the field or stored in dry laboratory conditions for 6 and 18 months were subjected to a fire-related range of heat treatments (40–100°C). Seed ageing increased germination response to heat treatments, effectively lowering the dormancy-breaking thresholds of three species. The fourth species, A. linifolia, initially had a relatively large non-dormant fraction which was lost as seeds aged, with older seeds then displaying PY broadly similar to the other study species. Patterns of threshold decay were species-specific, with the thresholds and viability of low-threshold species declining more rapidly than high-threshold species. The non-dormant fraction did not increase over time for any of our study species. Instead of increasing their non-dormant fraction, as is common in other vegetation types, these fire-prone PY species displayed a change of dormancy-breaking temperature thresholds. This is an important distinction, as maintaining dormancy during the inter-fire period is essential for population persistence. While changes in sensitivity to dormancy-breaking treatments have previously been reported as seeds age, our study provides the first test of changes to temperature thresholds, which increases the range of germination response from the seed bank under varying fire severity.

Seed quality standards enable seed users to achieve their objectives in the establishment of uniform seedlings to a high and reliable level for a range of agricultural and horticultural crops, growing systems and market outlets. Quality standards of commercial seed lots are determined by their positions on the seed survival curves and the shape of their germination progress curves. Although comparative descriptions of germination curves can be achieved by the calculation of the mean germination time (MGT; delay to radicle emergence), single early counts of radicle emergence provide a convenient means of predicting MGT and differences between seed lots. Evidence is presented for an ageing and metabolic repair hypothesis as the overall physiological basis to explain the principles behind the standard germination and vigour tests (ageing, electrolyte leakage, cold test, germination rate and seedling size). The work of the International Seed Testing Association (ISTA) in developing convenient, inexpensive and internationally repeatable tests is illustrated.

Winter rape has a life cycle which necessitates rapid testing of seed quality for the seed production sector, namely between July after harvest of the previous crop and August when the next crop establishment begins. As this crop is the fourth most important crop in France, and the most important oilseed crop, the French seed testing station GEVES has managed several research programmes to study seed quality in this species. These collaborative programmes have not only associated seed science with seed technology but also informatics and electronic engineering to provide seed testing tools that are already used by the seed sector, such as conductivity, or innovative tools, such as germination time courses monitored by computer imaging. Forthcoming tools have also been looked at in order to control seed storage potential, which is often used nowadays to anticipate the next season. Biochemical markers have been studied, including seed-specific biotinylated proteins or enzymes involved in free radical (reactive oxygen species) scavenging, such as catalase. Both are well correlated with seed ageing and have been tested to provide an ELISA-like assay for seed testing laboratories. Germination monitoring by computer imaging is now used by research and breeding institutes to phenotype varying seed material, including genotype or mutant collections, primed or ageing seed lots, etc. The next step in seed quality testing in oilseed rape is to characterize variation in germination and seed vigour in order to stabilize optimal yield. This aim constitutes a new international project involving high-throughput phenotyping and genotyping of a large collection of genetically diverse rapeseed genotypes (double haploid populations or diverse cultivars).

To understand the beneficial effects of cold conditions during the alleviation of dormancy of walnut (Juglans regia L.), lipid mobilization, gluconeogenesis and changes related to ageing were compared in dormant kernels incubated in the cold and under deteriorating warm conditions. Stratifying kernels at 5°C for 30 d enhanced their germination capacity, whereas warm-incubated (27°C) kernels turned rancid after 20 d and had reduced germination. Kernel imbibition was sufficient to bring about lipid mobilization, irrespective of temperature of incubation. Although imbibed kernels displayed high isocitrate lyase activity, starch and soluble sugar accumulation occurred only under cold conditions. Deteriorative 64 kDa fatty acyl-ester hydrolase activity declined in cold-stratified kernels. Cold treatment also led to reduced lipid peroxidation and hydrogen peroxide in kernels. The activity of NADP+-isocitrate dehydrogenase, an NADPH-generating enzyme, declined in warm-incubated kernels. Thus, warm-incubated kernels undergo ageing associated with oxidative stress, but there are beneficial effects of cold stratification in preventing deteriorative ageing-related processes. Imbibition is sufficient to allow lipid mobilization to occur in dormant walnut kernels, although cold stratification accompanied by gluconeogenesis is essential for kernel germination.

The effect of natural and artificial seed ageing has been compared in terms of physiological and biochemical responses of several maize genotypes. The physiological parameters were: viability, germinability, emergence in sand and dry matter accumulation. The biochemical parameters were: DNA synthesis and DNA polymerase activity. A close and direct relationship was found between seed deterioration and DNA metabolism in all maize materials which responded to ageing according to their genetic constitution; i.e., vigorous genotypes suffered less severe damage or recovery was faster than in the low-vigour genotypes. Coordination of events at appropriate times would seem a critical factor for proper seed germination.

It has been proposed that sucrose and raffinose play a role in the storability of maize seeds. The levels of these sugars in the embryos and the glassy state were compared in maize seeds of contrasting storage stability to determine the relationship between sugar composition, glassy state and the storability of the seed. Sucrose was the predominant sugar but its content was not correlated with good storage. The content of raffinose as a mass fraction of total sugars and the magnitude of the glassy state showed positive correlations with storage stability. It is suggested that in maize seeds dry storage stability is a reflection not of the total soluble sugars, but of the mixture of sucrose with raffinose. Apparently raffinose amplifies the magnitude of the glass signal and this in turn is associated with enhanced storability.

A characteristic of recalcitrant seeds is that, if they are maintained under storage conditions that prevent water loss, they will ultimately lose viability. A current view is that hydrated recalcitrant seeds are metabolically active and undergo germination-associated changes in storage. Some of these changes, such as extensive vacuolation and increase in cell size, imply a requirement for water additional to that present in the seed on shedding. It is therefore suggested that, in storage, recalcitrant seeds are exposed to an initially mild, but increasingly severe, water stress. Deleterious events associated with a water stress of considerable duration are suggested to lead ultimately to the death of the tissue.

The damage that occurs on prolonged storage is unlikely to be associated with an inability to form glasses or prevent membrane lipid phase changes, as absolute water contents are higher than those at which these mechanisms become important. It is considered that the most likely process leading to death of water-stressed (as opposed to dehydrated) tissue is a breakdown of co-ordination of metabolism, leading to uncontrolled free-radical-mediated oxidative damage.

It is generally difficult to maintain tissue in a mild water-stressed condition for extended periods. Stored, hydrated, recalcitrant seeds may provide an ideal model system for studying the metabolic effects of prolonged mild water stress.

Ageing of wheat seeds (Triticum aestivum cv. Galahad, 1986 harvest) during storage is accompanied by an increase in the proportion of abnormal seedlings produced when aged seed lots germinate. The response of aleurone layers from normally and abnormally germinated seeds to heat shock has been investigated. [14C]-amino acid incorporation into aleurone layers from 3-d normally and abnormally germinated seeds diminished after 4 h at 42°C but the aleurone layers were able to recover significantly during a 5-h period at 20°C following heat shock. [35S]-methionine pulse-labelling of aleurone proteins showed that α-amylase isoenzyme synthesis was abolished upon heat shock in aleurone layers for both normally and abnormally germinated seeds and that only aleurone layers from normally germinated seeds were capable of recommencing substantial α-amylase synthesis during the recovery period. Heat shock caused aleurone layers from abnormally germinated seeds to synthesize polypeptides not synthesized by aleurone layers from normally germinated seeds under the same conditions. The synthesis of certain polypeptides by aleurone layers from abnormally germinated seeds was enhanced at 20°C, 42°C and during recovery compared with normally germinated seeds. These results are discussed in terms of the control of gene expression during heat shock and recovery during seed ageing.

A method, electrical impedance spectroscopy (EIS), is introduced to study seed viability non-destructively. Snap bean (Phaseolus vulgaris L.) seeds were studied by EIS to determine the most sensitive EIS parameter(s) and the optimal range of moisture content (MC) for separation of viable and non-viable seeds. Hydrated seeds exhibited two impedance arcs in the complex plane at the frequency range from 60 Hz to 8 MHz, and impedance spectra of viable and non-viable seeds differed. The hydrated seeds were best-modelled by an equivalent electrical circuit with two distributed circuit elements in series with a resistor (Voigt model). Moisture content and seed viability had strong effects on the EIS parameters. The most sensitive EIS parameters for detecting the differences between viable and non-viable seeds were the capacitance log(C2), the resistance R2, the resistance ratio R2/R1 and the apex ratio, which all represent specific features of the impedance spectrum. The highest differentiation in the EIS parameters between the viable and non-viable seeds occurred in partially imbibed seeds between MC of 40 and 45% (fresh weight basis).

Osmotic priming reduces the longevity of Vigna radiata (mung bean) seeds during subsequent dry storage. The present study has investigated the relationship between volumetric changes, modified water sorption properties and the decrease in seed longevity after priming. Volumetric analysis of control embryos revealed two major dehydration-related volumetric contractions in the biopolymer matrix, reflecting structural reorganization in biopolymer matrix during drying. These contractions occurred at hydration from 0.29 to 0.23 g g–1 dw and from 0.18 to 0.11 g g–1 dw, respectively. Volumetric contractions were reduced significantly after priming and also occurred at higher water contents (i.e. 0.48–0.32 g g–1 dw and 0.21–0.11 g g–1 dw). Consequently, at the same water content, primed seed embryos had higher specific biopolymer volume and lower specific density than control embryos. Water sorption study showed that priming did not change the monolayer hydration value, but altered the properties of multilayer water sorption in seed axes. The analysis of water clustering function suggested that priming enhanced the water–water association in seed axes, but not in cotyledon tissues. Solid-state 1H-NMR (nuclear magnetic resonance) investigation confirmed that priming did not cause a significant change of water dynamic properties in cotyledon tissues at water contents below 0.20 g g–1 dw. The relevance of volumetric modifications of the biopolymer matrix and changes in water clustering function to the reduced seed longevity after priming is discussed. It is proposed that priming-induced increases in surface reactivity may enhance deteriorative chemical reactions in re-dried seed tissues.

In the West, lotus (Nelumbo nucifera Gaertn.) is relatively little known. However, for more than 3000 years, lotus plants have been cultivated as a crop in Far-East Asia, where they are used for food, medicine and play a significant role in religious and cultural activities. Holder of the world’s record for long-term seed viability (1300 years) is a lotus fruit (China Antique) from Xipaozi, Liaoning Province, China. Five offspring of this variety, from 200–500-year-old fruits (14C dates) collected at Xipaozi, have recently been germinated, and are the first such seedlings to be raised from directly dated fruits. The fruits at Xipaozi, preserved in a dry ancient lakebed, have been exposed to low-dose γ-radiation for hundreds of years (having an accumulated soil irradiation of 0.1–1.0 Gy). Offspring from these old fruits show abnormalities that resemble those in various modern seedlings irradiated at much higher doses. Although these lotus offspring are phenotypically abnormal, the viability of old seeds was evidently not affected by accumulated doses of up to 3 Gy. Growth characteristics of first- and second-year lotus offspring of these fruits, products of the longest-term radiobiological experiment on record, are summarized here (rapid early growth, phenotypic abnormalities, lack of vigour, poor rhizome development and low photosynthetic activity during second-year growth). Aspects of their chromosomal organization, phenotype and physiology (rapid recovery from stress, heat-stable proteins, protein-repair enzyme) are discussed. Important unsolved problems are suggested to elicit interest among members of the seed science community to the study of old fruits recently collected at Xipaozi, with particular emphasis on aspects of ageing and repair.

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.