Understanding seed moisture desorption and adsorption isotherms is important for seed quality maintenance and better predicting seed storage lifespan. Freshly harvested oilseed rape and barley seeds were dried and then rehydrated twice. Seed equilibrium relative humidity (eRH) and moisture content (MC) were determined at different humidity levels so that two cycles of desorption and adsorption could be constructed. In addition, seeds were dried to 30% RH and then rehydrated to 50% RH for five cycles to determine whether they shift to the adsorption isotherm. Monolayer MC was determined using the Gugenheim-Anderson-de Boer model. Storage experiments were conducted for seeds equilibrated at 30, 40, 50, 60 and 70% RH for two cycles of desorption and adsorption at 45°C. Isotherm curves’ shapes were similar for oilseed rape and barley, although spanning a greater MC range in barley. The hysteresis effect was observed for oilseed rape and barley seeds when dried over silica gel at <10% RH. However, this effect was only observed for barley seeds when dried to 30% RH, but not for oilseed rape seeds. Longevity was greater for adsorbing seeds than desorbing seeds at a given eRH, however, there was no significant difference in σ (the standard deviation of the normal distribution of seed deaths over time)–MC log–log relationship. The relationship shifted for seeds on the second cycle. In conclusion, if seed lots are stored at a specific RH, reaching equilibrium by desorption or adsorption can strongly influence their longevity. Also, when seeds of different species are dried to low RH, they will respond differently to a subsequent increase in RH, which could profoundly affect their longevity.

Understanding the relative longevity of different seed lots, perhaps of different species or genotypes, but also following production under different environments or using different cultivation methods, or following different post-harvest treatments, is relevant to anyone concerned with the retention of seed lot viability and vigour during storage. However, different scientists over the years have used different conditions to assess seed lot longevity, as well as different variables as the measure of ‘longevity.’ Here, we give some of the backgrounds to how two standard protocols, with an open and closed system respectively, were derived, and explain why we consider p50, defined as the time during storage when seed lot viability, as measured through a germination test, has declined to 50%, is a suitable longevity trait parameter.

Vitamin E is known to scavenge lipid peroxy radicals and has a purported role in preventing seed deterioration during storage. In our previous studies using 20 rice varieties from different variety groups, the specific ratio of vitamin E homologues rather than total vitamin E content was associated with seed longevity. To validate this result, we extended the experiment to a rice panel composed of 185 Aus (semi-wild rice) varieties. Seed longevity values were determined through storage experiments at 45°C and 10.9% seed moisture content (MC). Eight types of vitamin E homologues (α-, β-, γ- and δ-tocopherol/tocotrienol) were quantified by ultra-performance liquid chromatography. The theoretical initial viability in NED, Ki, was positively correlated with γ- and δ-tocopherols and negatively correlated with α-tocotrienol. The time for viability to fall to 50% during storage at elevated temperature and relative humidity, p50, was positively correlated with δ-tocopherol. The harvest MC was negatively correlated with all seed longevity traits. Taking this factor into account in a genome-wide association (GWA) analysis, we were able to correct false positives. A consistent major peak on chromosome 4 associated with −σ−1 was detected with a mixed linear analysis. Based on rice genome annotation and gene network ontology databases, we suggest that RNA modification, oxidation–reduction, protein–protein interactions and abscisic acid signal transduction play roles in seed longevity extension of Aus rice. Although major GWA regions were not overlapped across traits, three genetic markers, on chromosomes 1, 3 and 4, were associated with both δ-tocopherol and Ki and two markers on chromosome 1 and 8 were associated with both δ-tocopherol and p50.

This research attempts to systematically establish shape descriptor states through elliptic Fourier analysis (EFA) using pili (Canarium ovatum Engl.) kernel as a model. Kernel images of 53 pili accessions from the National Plant Genetic Resources Laboratory (NPGRL), University of the Philippines Los Baños were acquired using VideometerLab 3. Shape features, such as roundness, compactness and elongation, were extracted from the images. Shapes outlines were characterized using elliptic Fourier coefficients calculated from SHAPE version 1.3 software. Principal component analysis and cluster analysis were used to elucidate clusters representing the shape descriptor states. The first principal component accounts for the variation in length to width ratio; whereas, the second and third principal components explain the variation in the location of the widest portion and the truncation of the apex and base of the kernel, respectively. Cluster analysis separated the different accessions into six distinct clusters at 0.04 Euclidian distance. Six descriptor states, narrowly elliptic, elliptic, widely elliptic, ovate, obovate and lance-ovate, were characterized from the shape outlines and visualized through R's shape on r package. The discrimination between clusters was validated through MANOVA and LDA with 95% correct classification. The Fourier coefficients were also able to represent the variation observed from the physical properties of shape. The method may be used in establishing shape descriptors of all plant parts of all crop species.

Seeds of 15 diverse rice accessions, representing aus, indica, temperate japonica and tropical japonica subpopulations, were produced under temperate climate conditions in Korea and used for vitamin E analysis and seed storage experiments at 45°C and 10.9% seed moisture content. High γ-tocotrienol was significantly positively correlated with seed longevity. In addition, a high β-tocopherol proportion relative to δ-tocopherol was significantly negatively correlated with seed longevity. Using high-density single-nucleotide polymorphism marker data, DNA haplotype analysis showed clear allelic variations in the region of two S-adenosylmethionine synthetase genes: LOC_Os04g42095 and LOC_Os11g15410, which regulate the conversion of δ-tocopherol into β-tocopherol. Four indica accessions with rare and subpopulation-specific alleles showed a 2.3-fold lower β-/δ-tocopherol ratio compared with accessions from other subpopulations.

A systematic method for determining colour descriptor states using image analysis is proposed using pili (Canarium ovatum) as a model. Kernel images of 52 pili accessions from the core collection of the Institute of Crop Science and National Plant Genetic Resources Laboratory, University of the Philippines Los Baños were captured using a calibrated VideometerLab 3 setup. Colour descriptor states were derived from the average International Commission on Illumination lightness (L*), green–red (a*) and blue–yellow (b*) colour component values. Cluster analysis and subsequent colour-parameter averaging per cluster were performed to produce representative colour values of descriptor states. The Euclidian distance (Delta E) of 3.5 was used to cut the cluster into readily distinguishable colour differences resulting to three descriptor states – light brown, brown and dark brown. Continuous colour variation of brown colour was observed indicating a possible quantitative nature of the trait. The use of delta E in elucidating the descriptor lists served as a gauge in successfully identifying distinguishable variations between colours. The method described can be applied to the elucidation of colour descriptor states of all parts of the plant of all crop species.

Post-harvest drying prolongs seed survival in air-dry storage; previous research has shown a benefit of drying moist rice seeds at temperatures greater than recommended for genebanks (5–20°C). The aim of this study was to determine whether there is a temperature limit for safely drying rice seeds, and to explore whether the benefit to longevity is caused by high-temperature stress or continued seed development. Seeds of two rice varieties were harvested at different stages of development and dried initially either over silica gel, or intermittently (8 h day–1) or continuously (24 h day–1) over MgCl2 at temperatures between 15 and 60°C for up to 3 days. Seeds dried more rapidly the warmer the temperature. Subsequent seed longevity in hermetic storage (45°C and 10.9% moisture content) was substantially improved by increase in drying temperature up to 45°C in both cultivars, and also with further increase from 45 to 60°C in cv. ‘Macassane’. The benefit of high-temperature drying to subsequent longevity tended to diminish the later the stage of development at seed harvest. Intermittent or continuous drying at high temperatures provided broadly similar improvements to longevity, but with the greatest improvements detected in a few treatment combinations with continuous drying. Heated-air drying of rice seeds harvested before maturity improved their subsequent storage longevity by more than that which occurred during subsequent development in planta, which may have resulted from the triggering of protection mechanisms in response to high-temperature stress.

Vitamin E (tocols) is a key metabolite for efficient scavenging of lipid peroxy radicals that cause membrane breakdown during seed ageing. However, in rice, this hypothesis has been tested for very few lines only and without considering intraspecific variation in genomic structure. Here, we present a correlation study between tocols and seed longevity using a diverse rice panel. Seeds of 20 rice accessions held in the International Rice Genebank at the International Rice Research Institute, representing Aus, Basmati/Sadri, Indica, temperate Japonica and tropical Japonica variety groups, were used for tocols analysis (quantification of α-, β-, γ- and δ-tocopherol/tocotrienol by ultra-performance liquid chromatography) and storage experiments at 45°C and 10.9% seed moisture content. To examine the effects of DNA-haplotype on the phenotype, the 700 K high-density single-nucleotide polymorphism marker dataset was utilized. Both seed longevity (time for viability to fall to 50%; p50) and tocols content varied across variety groups related to the heterogeneity in the genetic architecture. Among eight types of tocol homologues, α-tocopherol and γ-tocotrienol were significantly correlated with p50 (negatively and positively, respectively). While temperate Japonica varieties were most abundant in α-tocopherol, Indica varieties recorded 1.3- to 1.7-fold higher γ-tocotrienol than those of other groups. We conclude that the specific ratio of tocol homologues rather than total tocols content plays an important role in the seed longevity mechanism.

In each of two experiments, freshly harvested seeds of two cultivars of rice were dried and then rehydrated to different moisture levels. In experiment 1, seed equilibrium relative humidity (eRH) and moisture content (MC) were determined at each moisture level so that moisture desorption and adsorption isotherms could be constructed. Seed storage experiments were also carried out for seeds equilibrated at each moisture level, in sealed aluminium foil packets at 45°C. In experiment 2, storage experiments at 45°C were carried out on seeds dried to 12% MC and seeds dried to lower moisture levels and then rehydrated to 12% MC.

The moisture adsorption isotherm was shifted to lower MC at a given eRH compared with the desorption isotherm. This hysteresis effect was seen both when seeds were dried to <6% MC and then allowed to adsorb moisture to different levels, and when seeds were dried to different levels (10, 8, 6 or 4% MC) and then allowed to adsorb moisture up to 12% MC. The log-log relationship between seed longevity, σ [the standard deviation of the normal distribution of seed deaths over time, as defined in the Ellis and Roberts (1980) seed viability equations] and seed storage MC did not vary depending on whether the seeds were desorbing or adsorbing moisture. The relationship between σ and eRH was better described by a log-log model than a log-linear model and did vary depending on whether seeds were desorbing or adsorbing moisture: at a given eRH, the longevity of adsorbing seeds was greater than that of desorbing seeds. The implications for seed storage are discussed.

We present a method for multispectral seed phenotyping as a fast and robust tool for managing genebank accessions. A multispectral vision system was used to take images of the seeds of 20 diverse varieties of rice (approximately 30 seeds for each variety). This was followed by extraction of feature information from the images. Multivariate analysis of the feature data was used to classify seed phenotypes according to accession. The proportion of correctly classified rice seeds was 93%. We conclude that the multispectral image analysis could play a role in comparing incoming seeds against existing accessions, identifying different seed types within a sample of seeds and/or in checking whether regenerated seeds match the original seeds.

Chlorophyll fluorescence (CF) analysis was explored as a potential tool to identify the optimal time to harvest rice seed germplasm for maximum storage longevity. Seeds of 20 diverse genebank accessions were harvested at 24, 31, 38 and 45 d after peak flowering (DAF) and half of each seed lot was sorted by hand, following normal practice at the T.T. Chang Genetic Resources Center. CF analysis was carried out on both non-sorted and sorted seeds, while storage experiments were carried out on sorted seeds. Seed longevity (the time for viability to fall to 50%, p50) was significantly correlated with the skewness, kurtosis, mode and mean of the CF histograms when the data for every accession at all the harvest times were included in the correlation analysis. However, these correlation coefficients were ≤ 0.481. The correlation coefficient between p50 and DAF was similarly low (0.461). For individual accessions, there was wide variation in the correlation coefficients. While for some accessions, there appeared to be a strong relationship between p50 and mean CF that could be used to guide when to harvest seeds in the field, for other accessions, a unique mean CF to inform when to harvest seeds or for use in seed sorting could not be identified; this was also true for DAF over the harvesting schedule used in this study. Given the number and diversity of accessions managed by a genebank, it seems unlikely that CF analysis would be an appropriate tool to help manage the regeneration or processing of seeds intended for storage in the genebank.

Probit-based models relating a proportional response variable to a temporal explanatory variable, assuming that the times to response are normally distributed within the population, have been used in seed biology for describing the rate of loss of viability during seed ageing and the progress of germination over time in response to environmental signals (e.g. water, temperature). These models may be expressed as generalized linear models (GLMs) with a probit (cumulative normal distribution) link function, and, using GLM fitting procedures in current statistical software, parameters of these models are efficiently estimated while taking into account the binomial error distribution of the dependent variable. The fitted parameters can then be used to calculate the ‘traditional’ model parameters, such as the hydro- or hydrothermal time constant, the mean or median response of the seeds (e.g. mean time to death, median base water potential), and the standard deviation of the normal distribution of that response. Furthermore, through consideration of the deviance and residuals, performing model evaluation and modification can lead to improved understanding of the underlying physiological/ecological processes. However, fitting a binomial GLM is not appropriate for the cumulative count data often collected from germination studies, as successive observations are not independent, and time-to-event/survival analysis should be considered instead. This review discusses well-known probit-based models, providing advice on how to collect appropriate data and fit the models to those data, and gives an overview of alternative analysis approaches to improve understanding of the underlying mechanisms of seed dormancy and germination behaviour.

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.

This study examined whether the restricted habitat preference of the spring-flowering woodland geophyte Anemone ranunculoides L., compared with that of A. nemorosa growing in the same woodlands in northern Italy, could be explained by subtle differences in germination preference and emergence phenology. Immediately after harvest, seeds of A. ranunculoides were either sown on agar in the laboratory under simulated seasonal temperatures or placed in nylon mesh sachets and buried in the wild. Embryos, undifferentiated at the time of seed dispersal, grew during summer in the laboratory and in the wild, culminating in radicle emergence in the autumn, when temperatures fell to c. 15°C. Shoot emergence was delayed under natural conditions until soil temperature had dropped further to c. 10°C. Compared with populations of the closely related Anemone nemorosa L. occupying the same woodland habitat, which have been reported to have non-dormant radicles, A. ranunculoides displayed a narrower temperature tolerance for radicle emergence and high levels of germination were possible only after prolonged exposure to summer conditions, indicating physiological dormancy. However, unlike A. nemorosa, shoot emergence in A. ranunculoides was not dependent on winter temperatures, suggesting weaker epicotyl morphophysiological dormancy. Under a regime of diurnal temperature alternation, simulating the microclimate where there is little plant cover, germination failed almost completely; this could explain the absence of A. ranunculoides in open habitats.

Although various aspects of the biology of Anemone nemorosa have been examined, few studies present data on seed germination, and even then information tends to be rather contradictory. A. nemorosa L. is a spring-flowering, woodland geophyte, widely distributed across much of Europe. Germination phenology, including embryo development and radicle and shoot emergence, were investigated in one mountain and three lowland populations from northern Italy. Immediately after harvest, seeds were either sown on agar in the laboratory under simulated seasonal temperatures, or placed in nylon mesh sachets and buried in the wild. Embryos, undifferentiated at the time of dispersal, grew under summer conditions in the laboratory and in the wild. However, seeds did not germinate under continuous summer conditions. Radicle emergence in the field was first recorded at the beginning of autumn, when soil temperatures had dropped to c. 15°C in the case of the three lowland populations, and to c. 10°C at the mountain site. Shoot emergence was delayed under natural conditions until late autumn/early winter, when soil temperatures had dropped to c. 10°C in the lowlands and c. 6°C at the mountain site. In the laboratory, a period of cold stratification was required for shoot emergence, and this requirement was more pronounced in the mountain population. Seeds of the mountain population completed embryo development, radicle emergence and shoot emergence at cooler temperatures compared with the lowland populations. These results suggest that germination in A. nemorosa is highly adapted and finely tuned to local climate. We conclude that seeds of A. nemorosa display deep, simple epicotyl, morphophysiogical dormancy, and this is the first report of such dormancy for the genus Anemone. However, the continuous development and growth of embryos from the time of natural dispersal, and the lack of evidence of developmental arrest under natural conditions, suggests that radicles are non-dormant.

A UK seed conservation collection of Anemone nemorosa L. seeds held at the Millennium Seed Bank (MSB) showed low viability in its first post-storage test. Because achenes of A. nemorosa are naturally dispersed when they are green, we tested the hypothesis that seeds may not be fully desiccation tolerant and storable at the time of natural dispersal, and that a post-harvest treatment could increase the proportion of desiccation-tolerant seeds. Achenes harvested at the point of natural dispersal in late May in 2003 and 2004 were either placed immediately on 1% water agar at 20°C (‘laboratory’ treatment), or placed in nylon sachets and buried in leaf litter among plants growing in the wild (‘field’ treatment). Samples were withdrawn at intervals over a period of 168 d and tested for desiccation tolerance (drying to 0.059 g H2O (g DW)− 1) and longevity (controlled ageing at 60% relative humidity and 45°C). An initial increase, followed by a decline, in the proportion of seeds surviving desiccation and in the longevity of both laboratory- and field-treated samples coincided with the development of embryos from globular to heart- and then torpedo-shaped. Developmental arrest was not required for the acquisition of desiccation tolerance, and continued growth and development of the embryo resulted in a loss of desiccation tolerance, similar to that seen in orthodox seeds upon radicle emergence. Furthermore, while A. nemorosa seeds, like many from the Ranunculaceae family, might be described as having morphological or morphophysiological dormancy, this lack of developmental arrest does not fit with the usual concept of dormancy. The implications of these results for the classification systems of seed-storage behaviour and dormancy, and for the long-term conservation of seeds of A. nemorosa, are discussed.

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.

Most species of marine turtle breed every two or more years and it is the norm for females to lay more than one clutch of eggs within a nesting season. Knowing the interval between breeding seasons and the clutch frequency (number of clutches laid by an individual in a breeding season) of females allows us to assess the status of a nesting population. At Alagadi Beach, Northern Cyprus, over a period of 6 years (1995–2000), we attributed 96% of green Chelonia mydas and 80% of loggerhead Caretta caretta turtle clutches to known individual females. This intensive level of monitoring enabled us to estimate the clutch frequency for both species. Using four different methods we estimated clutch frequency to be 2.9–3.1 clutches per female for green turtles and 1.8–2.2 clutches per female for loggerhead turtles. The median interval between nesting seasons for green turtles was 3 years, and for loggerhead turtles it was 2 years. Utilizing these parameters and available data from other beaches that are monitored regularly, we estimate that there are 2,280–2,787 logger-head and 339–360 green turtles nesting annually at these sites in the Mediterranean. This highlights the Critically Endangered status of this population of green turtles. Furthermore, as conventional beach patrols underestimate clutch frequency, these population estimates are likely to be optimistic.