Showing papers in "Seed Science Research in 2005"

Plant hormone interactions during seed dormancy release and germination

Abstract: This review focuses mainly on eudicot seeds, and on the interactions between abscisic acid (ABA), gibberellins (GA), ethylene, brassinosteroids (BR), auxin and cytokinins in regulating the interconnected molecular processes that control dormancy release and germination. Signal transduction pathways, mediated by environmental and hormonal signals, regulate gene expression in seeds. Seed dormancy release and germination of species with coat dormancy is determined by the balance of forces between the growth potential of the embryo and the constraint exerted by the covering layers, e.g. testa and endosperm. Recent progress in the field of seed biology has been greatly aided by molecular approaches utilizing mutant and transgenic seeds of Arabidopsis thaliana and the Solanaceae model systems, tomato and tobacco, which are altered in hormone biology. ABA is a positive regulator of dormancy induction and most likely also maintenance, while it is a negative regulator of germination. GA releases dormancy, promotes germination and counteracts ABA effects. Ethylene and BR promote seed germination and also counteract ABA effects. We present an integrated view of the molecular genetics, physiology and biochemistry used to unravel how hormones control seed dormancy release and germination.

Longevity of seeds stored in a genebank: species characteristics

Abstract: Seeds of different species are believed to have characteristic shelf lives, although data confirming this are scarce, and a mechanistic understanding of why this should be remains elusive. We have quantified storage performance of c. 42,000 seed accessions, representing 276 species, within the USDA National Plant Germplasm System (NPGS) collection, as well as a smaller experiment of 207 cultivars from 42 species. Accessions from the NPGS collection were harvested between 1934 and 1975, and had relatively high initial germination percentages that decreased at a variable rate during storage at both 5 and –18°C. Germination time courses, which represent the average performance of the species, were fitted to Avrami kinetics, to calculate the time at which germination characteristically declined to 50% (P50). These P50 values correlated with other longevity surveys reported in the literature for seeds stored under controlled conditions, but there was no correlation among these studies and seed persistence observed in the classic buried seed experiment by Duvel. Some plant families had characteristically short-lived (e.g. Apiaceae and Brassicaceae) or long-lived (e.g. Malvaceae and Chenopodiaceae) seeds. Also, seeds from species that originated from particular localities had characteristically short (e.g. Europe) or long (e.g. South Asia and Australia) shelf lives. However, there appeared to be no correlation between longevity and dry matter reserves, soluble carbohydrates and parameters relating to soil persistence or resource allocation. Although data from this survey support the hypothesis that some species tend to survive longer than others in a genebank environment, there is little information on the attributes of the seed that affect its storage performance.

Defining transient and persistent seed banks in species with pronounced seasonal dormancy and germination patterns

Abstract: The most often used time-line for distinguishing a transient seed bank from a persistent seed bank is one calendar year. Thus, species whose seeds live in or on the soil for ,1 year have a transient seed bank, whereas those whose seeds live for $1 year have a persistent seed bank. However, dormancy cycling of seeds buried in soil has not been given due consideration in these models. When dormancy cycling is considered, it is shown that seeds of both autumn-germinators and spring-germinators are in the dormant state when they are 1 year old. Thus, unless the seeds live until at least the second germination season (i.e. usually 16–18 months following dispersal), they are, in effect, part of a transient seed bank, having lived through only one germination season. We propose that for seeds of such species to be considered part of a short-term persistent seed bank, they should remain viable and germinable until at least the second germination season, and to be part of a long-term persistent seed bank, until at least the sixth germination season. Our definitions are applicable to seeds with physiological, physical or morphophysiological dormancy, which often require .1 year after maturity to come out of dormancy in nature. We discuss modifications of the seedling emergence method for detection of a soil seed bank, so that they correspond to our definitions of seed-bank strategies.

Seeds and seasons: interpreting germination timing in the field

Abstract: This paper discusses how field and laboratory experiments, using a variety of genetic material, can be combined to investigate the genetic basis of germination under realistic ecological conditions, and it reviews some of our recent work on germination phenology of Arabidopsis thaliana in the field. Our results indicate that the genetic basis of germination depends on the environment. In particular, the conditions during seed maturation interact with post-dispersal environmental factors to determine germination phenology, and these interactions have a genetic basis. Therefore genetic studies of germination need to consider carefully the environment – both during seed maturation and after dispersal – in which the experiments are conducted in order to characterize genetic pathways involved with germination in the field. Laboratory studies that explicitly manipulate ecologically relevant environmental factors can be combined with manipulative field studies. These studies can identify the particular environmental cues to which seeds respond in the field and characterize the genetic basis of germination responses to those cues. In addition, a variety of genetic material – including mutant and transgenic lines, intact natural genotypes, recombinant genotypes, and near isogenic lines – can be used in field studies as tools to characterize genetic pathways involved in germination schedules under natural ecological conditions.

Structural, physiological and molecular aspects of heterogeneity in seeds: a review

Abstract: Higher plants have several strategies to perpetuate themselves under adequate ecophysiological conditions. The production of heterogeneous seeds is one such strategy. That is, to ensure the survival of the next generation, an individual plant might produce seeds that are heterogeneous with respect to the extent of dormancy, dispersion and persistence within the seed bank. Heterogeneity can affect not only certain physiological and molecular properties related to seed germination, but also such characteristics as colour, size and shape, parameters commonly used to differentiate morphs within a heterogeneous seed population. In heterogeneous seeds, the above features determine seed behaviour and alter their mechanism of germination. In this work, emphasis is placed on the existence of seed mutants having major alterations in characteristics of the testa and hormonal response. These mutants constitute a valuable tool for elucidating the mechanism of dormancy, germination and perpetuation of seeds. Finally, ontogeny and heterogeneity are reviewed, providing the first data related to the possible hormonal control of heterogeneity in seeds. These results raise the hypothesis that one of the factors triggering differences in germination among heterogeneous seeds may be an alteration in the signalling and action mechanism of ethylene and abscisic acid (ABA).

Sheep gut passage and survival of Mediterranean shrub seeds

Abstract: Although viable seeds of Mediterranean dry-fruited shrubs are found in herbivore dung, the ecological importance of this observation is still not well understood. We analysed seed retrieval percentages, defecation time and germinability after sheep gut passage for the five most common shrub species of an area in central Spain (Retama sphaerocarpa, Cytisus scoparius, Halimium umbellatum subsp. viscosum, Cistus ladanifer and Lavandula stoechas subsp. pedunculata). Five ewes were fed seeds, and their dung was collected regularly during the following week. Seeds were hand-sorted from dung subsamples and tested for germinability. The defecated seeds were clustered in time, with a majority retrieved in the 24–40 h period, although over 1% of the seeds were retained in the gut for more than 72 h. Data suggested a possible link between seed size and retrieval, with medium-sized seeds less damaged (16–23%) than larger and smaller seeds (10–12%), although only a small number of species were studied. Germination results showed an increased percentage of germination after gut passage for H. umbellatum (x2 test, P<0.05) and a marginally significant difference for C. scoparius (P<0.1). Soft-seeded L. stoechas did not germinate after gut passage. The results indicate a potential role of herbivore endozoochory for the long-distance dispersal of dry-fruited shrubs and their potential colonization of distant sites.

Seed bank of an invasive alien, Heracleum mantegazzianum, and its seasonal dynamics

Abstract: The seed bank of Heracleum mantegazzianum, native to the western Caucasus and invasive in Europe, was studied in the Slavkovskyles Protected Landscape Area, the Czech Republic, during the course of two growing seasons. In each of seven study sites with dominating stands of the species, soil samples were taken in October (after the fruit release), April (before germination) and June-July (before the fruit release). Ten samples were taken from each site using a soil hand bore, 4.6 cm in diameter. Seeds elutriated from the samples were immediately germinated in labora- tory conditions (108C, 12 h light/12 h dark); those that germinated during 1 month were considered as non- dormant. Non-germinated but viable seeds, tested for viability by tetrazolium, were considered as dormant. The number of dead seeds was also recorded. The total number of seeds significantly increased with mean density of flowering plants at a site. Of the total variation in seed-bank size, about four-fifths were attributed to that among sites, and one-fifth to that within sites. The number and proportion of living seeds differed significantly between years in summer, but not in autumn and spring. Total number and the numbers of dormant, non-dormant, living and dead seeds significantly differed among the autumn, spring and summer samples. The percentage of living seeds in the total seed bank decreased from 56% in autumn to 42% in spring to 15% in summer. The percentage of non-dormant seeds among those living was 0.3% in autumn, it increased to 87.5% in the spring sample, and decreased to 3% in summer. Pooled across all sites, the average seed numbers (expressed per m 2 ) were 6719 ^ 4119 (mean ^ SD) in autumn, 4907 ^ 2278 in spring and 1301 ^ 1036 in summer for the total number of seeds, and 3759 ^ 2906, 2044 ^ 1198 and 192 ^ 165, respectively, for living seeds. The majority of seeds (95%) were concentrated in the upper 5 cm soil layer. However, some seeds were present in lower soil layers, which indicates a short-term persistent seed bank. The present data are the first quantitative estimate of the seed bank in H. mantegazzianum, and show that the reproductive potential of the species is enormous, which seems to be a crucial feature of its invasion success.

Physical dormancy in seeds of six genera of Australian Rhamnaceae

Abstract: Physical dormancy (PY) was identified in six genera representative of Australian Rhamnaceae and subsequently was broken, based on identification of key seed dormancy characteristics: (1) isolation and classification of embryo features; (2) imbibition experiments to determine the rate and amount of water uptake in seeds; and (3) determination of optimum temperature regimes for germination. All six species had relatively large spatulate embryos. Imbibition studies showed all species possessed PY (i.e. a water-impervious seed coat) that was broken by a hot-water treatment. Alleviation of PY resulted in high germination (<70%) at 7/18°C, temperatures similar to winter in south-west Western Australia. Germination was suppressed at higher temperatures and in the presence of light. The study adds information to our knowledge of seed dormancy in Australian Rhamnaceae, and highlights the benefits of understanding dormancy states in seeds prior to evaluating dormancy-release mechanisms on wild species used in restoration ecology and horticulture.

Hydrothermal time analysis of seed dormancy in true (botanical) potato seeds

Abstract: As seed dormancy is released within a seed population, both the rate and percentage of germination increase progressively with increasing dose of a dormancy-breaking treatment or condition. Population-based models can account for this behaviour on the basis of shifting response thresholds as dormancy is alleviated. In particular, hydrothermal time analysis of germination sensitivity to water potential (Ψ) and temperature (T) can describe these features of seed behaviour. We used the hydrothermal time model to analyse the effects of dormancy-breaking treatments on germination of dormant true (botanical) potato (Solanum tuberosum L.) seeds (TPS). After-ripening (37°C and 4% seed moisture content) of TPS for 7 or 30 days partially or fully alleviated primary dormancy. The median base water potential required to prevent germination [Ψb(50)] decreased from –0.25 MPa in control seeds to –0.87 MPa and –1.83 MPa after 7 and 30 days of after-ripening, respectively. In contrast, the base temperature for germination (Tb) was relatively unaffected (0–3.3°C). Fluridone (50 μM), an inhibitor of abscisic acid (ABA) biosynthesis, also promoted germination of dormant TPS and lowered Ψb(50), indicating a role for de novo synthesis of ABA during dormancy maintenance. Moist chilling (3 days at 4°C) or gibberellin (100 μM) alleviated secondary dormancy and lowered Ψb(50) values from –0.08 MPa to –0.36 and –0.87 MPa, respectively. The hydrothermal time model allows quantification of dormancy levels and explains why changes in germination speed and percentage are closely correlated during dormancy alleviation.

Recent advances in understanding cotton fibre and seed development.

Abstract: The unique feature of the seed of tetraploid cotton (Gossypium hirsutum and Gossypium barbadense) is that about 30% of the seed coat epidermal cells develop into cellulose-enriched fibres, while the embryos synthesize oils and proteins. Hence, both the maternal and filial tissues of the cotton seed are of significant economic value. After initiation from the ovule epidermis at or just before anthesis, the single-celled fibres elongate to 2.5–6.0 cm long in the tetraploid species before they switch to intensive secondary cell wall cellulose synthesis. Thus, apart from its agronomic importance, the cotton fibre represents a model single-cell system to study the control of cell differentiation and elongation, carbon partitioning to cellulose synthesis and also the interaction between maternal (fibre) and embryonic tissues in seeds. Over the past decade or so, significant effort has been made to understand the cellular and molecular basis of cotton fibre development and oil biosynthesis in the embryo. Metabolic engineering of the oil biosynthetic pathway in cotton seed has successfully produced healthier and stable oils. A number of candidate genes and cellular processes that potentially regulate various aspects of fibre development have been identified. Further elucidation of the in vivo functions of those candidate genes could significantly deepen our understanding of fibre development and offer potential for improvement of fibre quality through genetic engineering or marker-assisted breeding approaches.

Dormancy release in Australian fire ephemeral seeds during burial increases germination response to smoke water or heat

Abstract: Fire ephemerals are short-lived plants that primarily germinate after fire. Fresh and laboratory-stored seeds are difficult to germinate ex situ, even in response to fire-related cues such as heat and smoke. Seeds of eight Australian fire ephemeral species were buried in unburnt and recently burnt sites of natural bushland during autumn. Seeds were exhumed after 6 and 12 months and incubated in water and smoke water, either with or without a heat treatment at 70 degrees C for 1 h. Generally, germination did not increase after 6 months of burial, but after 12 months of burial germination was enhanced in seven of the eight species. Actinotus leucocephalus produced higher germination following 12 months of burial without any further treatment, and smoke water and heat further improved germination. The four Gyrostemonaceae species, Codonocarpus cotinifolius, Gyrostemon racemiger, Gyrostemon ramulosus and Tersonia cyathiflora, only germinated in the presence of smoke water, and their germination was enhanced by burial. Burial improved germination in response to a heat treatment in Grevillea scapigera and Alyogyne huegelii seeds, but did not enhance Alyogyne hakeifolia germination. During concurrent dry laboratory storage of seeds at 15 degrees C, only Actinotus leucocephalus produced increased germination in response to smoke water and heat over time. In summary, soil burial can alter the dormancy status of a number of Australian fire ephemeral seeds, rendering them more responsive to germination cues such as smoke water and heat. The requirement for a period of burial before seeds become responsive to smoke and/or heat would ensure that seeds persist in the soil until a subsequent fire, when there is an increase in nutrients available for growth and reduced competition from other plants.

Urban domestic gardens (VII): a preliminary survey of soil seed banks

Abstract: As part of a larger survey of biodiversity in private gardens in Sheffield, UK, we examined the composition and diversity of the soil seed banks in each of 56 gardens. Six soil cores from each garden revealed 2759 seedlings of 119 taxa, although the real species richness is likely to be much higher than this. By far the most abundant species were weedy natives, while the most common alien was Buddleja davidii. Seeds of perennial herbs were more abundant than hundreds of all other life forms combined. More frequent species were also more abundant, but the relationship was weak. Numbers of species in the seed bank and in the garden flora were positively but very weakly related. Seeds were quite evenly distributed between 0‐5 cm and 5‐10 cm soil layers, and most seeds were of species known to have persistent seed banks. Seeds of some species were largely confined to gardens in which the plant was growing, but others were not.

Salinity tolerance during germination of seashore halophytes and salt-tolerant grass cultivars

Abstract: Direct sowing is the simplest method of plant establishment for restoration and remediation purposes, but relatively few plants can establish under high salinity conditions. In this study, the ability of different seashore plants and grass cultivars to germinate in different dilutions of seawater (0–400 mM NaCl) was tested. Highest germination was found in distilled water or seawater dilutions up to 100 mM NaCl. When seawater concentrations were increased from 100 to 200 mM NaCl, a strong decline in germination percentage and rate was observed in less salt-tolerant species, such as Matricaria maritima and Achillea millefolium. The more salt-tolerant species, Plantago maritima, Juncus gerardii, Artemisia vulgaris, Agrostis spp. and Rumex spp., had a threshold salinity, where germination was significantly decreased in seawater dilutions between 200 and 400 mM NaCl. Even among the salt-tolerant species, only two, Agrostis stolonifera and Artemisia vulgaris, germinated at 400 mM. Variation in salinity response was observed among populations of Artemisia vulgaris and among cultivars of Festuca spp. Increasing salinity to 200 mM NaCl delayed germination in most species. Ungerminated seeds of most salinity-tolerant species were still viable after 21 d at the highest salinity (400 mM), and showed a rapid and high germination when transferred to distilled water. These species would be able to survive high salinity and germinate when the salinity of the sediments decreases through dilution or leaching of salts. The experiment revealed species and cultivars that will be of interest in further testing for restoration and remediation in saline habitats.

Spatial coherence between seasonal seed banks in a semi-arid gypsum community : density changes but structure does not

Abstract: Seed banks play a crucial role in arid plant communities because they confer stability and long-term persistence. However, seed banks have high temporal and spatial variability, with dramatic changes in density and composition. The aim of this study was to test whether seasonal change affected seed bank community structure and spatial pattern. Moreover, we wanted to know if the effect driven by environmental factors on the seed bank was constant year round. We sampled the seed bank at 188 points along seven parallel transects through a gypsum system in central Spain. Soil samples were taken twice (September and April) in contiguous plots. In each plot we measured environmental parameters, including micro- and macroslope, vegetation band, shrub cover, lichen crust cover and landform. A nearly threefold decrease in seed bank density occurred between September (16,230 seeds m–2) and April (5960 seeds m–2). Seasonal changes in density varied widely among species; however, a seed bank was present for most species at both sampling dates. For several well-studied species (Lepidium subulatum and Helianthemum squamatum), seed losses were within the range of losses by emergence reported in the literature. In both seasons, seed bank composition was controlled mainly by community band and microslope. Sampling season had a significant, but minor effect on seed bank composition. Moreover, a high spatial correlation existed in terms of seed density and richness through the two studied seasons. These results show that the seed bank keeps a constant structure even under substantial variation in density.

Trait stasis versus adaptation in disjunct relict species: evolutionary changes in seed dormancy-breaking and germination requirements in a subclade of Aristolochia subgenus Siphisia (Piperales)

Abstract: There are two ideas regarding changes in the physiological and ecological tolerances and requirements within plant lineages through geological time. One is that these attributes have changed very little, or not at all (trait stasis), and the other is that they have undergone considerable change (adaptation), as plants shifted to new climatic and vegetation zones. We tested these ideas for seed dormancy-breaking and germination requirements of four species in a subclade of Aristolochia subgenus Siphisia: the three temperate species, A. macrophylla and A. tomentosa (the basal species in the subclade) of eastern USA and A. manshuriensis of East Asia, and the Mediterranean-climate species A. californica endemic to California, USA. A long period at cold-stratifying temperatures was required for growth of the underdeveloped embryo and seed germination in A. californica, whereas embryos grew and seeds germinated in the other three species at warm temperatures, either before or after they were cold stratified. Thus, seeds of A. californica have either intermediate or deep complex morphophysiological dormancy (MPD), whereas those of the three temperate species have either morphological dormancy or non-deep simple MPD. Further, there were quantitative differences in temperature requirements for dormancy-break and germination between the Appalachian A. macrophylla, which did not differ from its sister species A. manshuriensis, and the lowland A. tomentosa. Thus, within this lineage there has been both trait stasis and divergence (adaptation) in the physiology and ecology of seed dormancy and germination.

Germination of drupelets in multi-seeded drupes of the shrub Leptecophylla tameiameiae (Ericaceae) from Hawaii: a case for deep physiological dormancy broken by high temperatures

Abstract: This study addressed the difficulty of germinating drupelets (hereafter seeds) in the multi-seeded stony dispersal units (drupes) of Leptecophylla tameiameiae (Ericaceae). Embryos in fresh seeds were 77% the length of the endosperm, and seeds inside the intact drupes imbibed water. We monitored germination at 15/6, 20/10 and 25/15°C for 162 weeks, after which each drupe was cut open and ungerminated seeds counted. Drupes contained 1–6 seeds, and the total number of seeds in all treatments and controls was 1977, with 20, 29, 25, 18, 7 and <1% of them occurring in one-, two-, three-, four-, five- and six-seeded drupes, respectively. The percentage of seeds germinating in one-, two-, three-, four-, five- and six-seeded drupes was 74, 66, 65, 72, 56 and 0, respectively. Neither warm nor cold stratification for 6 or 12 weeks significantly increased germination percentages, compared to controls incubated continuously at 25/15°C for 162 weeks, where 72% of the seeds in the drupes germinated. At 25/15°C, 24–49 weeks were required for 20% of the seeds to germinate. Warm followed by cold stratification did not promote germination, and there was no widening of the temperature range for germination. Like seeds of other species known to have deep physiological dormancy (PD), those of L. tameiameiae required extended periods of time (16 to ≥162 weeks) to come out of dormancy and germinate, gibberellic acid (GA3) did not promote germination and excised embryos failed to grow. Thus, we conclude that seeds of L. tameiameiae have deep PD. However, unlike seeds of other species with deep PD, those of L. tameiameiae required an extensive period of warm rather than of cold stratification to come out of dormancy. It is suggested that a subtype a (seeds require a long period of cold stratification to come out of dormancy) and a subtype b (seeds require a long period of exposure to warm stratification to come out of dormancy) of deep PD be recognized in the Nikolaeva formula system for classifying seed dormancy.

Development and potential of genetically engineered oilseeds

Abstract: Oilseed crops are major sources of oils for human nutrition, and an increasing proportion is also being utilized for industrial purposes. Recent advances in our understanding of the basic biochemistry of seed oil biosynthesis, coupled with identification of genes for oilseed modification, have set the stage for the genetic engineering of oilseed crops that produce ‘designer’ plant seed oils tailored for specific applications. In this review we provide an overview of seed oil biosynthesis and highlight the enzymatic steps that have already been targeted for genetic manipulation, with the end goal of producing seed oils containing desired amounts of fatty acid components. Furthermore, we describe the identification of genes from various wild plant species that are capable of producing structurally diverse fatty acids, and how these advances open the door to the production of entirely novel oils in conventional oilseed crops. Transgenic oilseeds producing high amounts of these novel fatty acids represent renewable sources of raw materials that may compete with, and eventually replace, some petrochemicals that are derived from non-renewable crude oil.

Underdeveloped embryos in dwarf seeds and implications for assignment to dormancy class

Abstract: Studies were conducted to determine if small embryos (i.e. low embryo length:seed length ratio) in mature dwarf seeds (0.2–2 mm) are underdeveloped. In this case, they would grow (inside the seed) prior to germination, and seeds would have morphological or morphophysiological dormancy. Prior to radicle emergence, embryo length in seeds of Drosera anglica (Droseraceae), Campanula americana, Lobelia appendiculata, L. spicata (Campanulaceae) and Sabatia angularis (Gentianaceae) increased 0, 103, 182, 83 and 57%, respectively. Since embryo growth did not occur in seeds of D. anglica prior to germination, embryos, although small, are fully developed; seeds have only physiological dormancy. The underdeveloped embryo in seeds of C. americana has little or no physiological dormancy; thus, seeds have morphological dormancy. On the other hand, underdeveloped embryos in seeds of L. appendiculata, L. spicata and S. angularis are physiologically dormant, and seeds have morphophysiological dormancy. Therefore, since small embryos in dwarf seeds may or may not be underdeveloped, assignment of seeds to a dormancy class requires that studies be done to determine if embryos grow inside the seed before germination can occur. Such information is important in understanding the evolutionary relationship of the different kinds of seed dormancy.

Simple purification of small RNAs from seeds and efficient detection of multiple microRNAs expressed in Arabidopsis thaliana and tomato (Lycopersicon esculentum) seeds

Abstract: MicroRNAs (miRNAs) play critical roles in the development of animals and plants. Characterizing the stage- and tissue-specific expression of miRNAs that potentially regulate target transcription factor expression is becoming more important for understanding the regulatory mechanisms of critical events during plant development. A simple method for purifying small RNAs from seeds is described, as well as an efficient non-radioactive labelling system for making miRNA probes. In Arabidopsis thaliana seed extracts, low molecular-weight (LMW) RNAs (e.g. 5S rRNA, tRNA and miRNA) were separated from high molecular-weight (HMW) nucleic acids (e.g. 28S and 18S rRNA, mRNA and genomic DNA) by fractionation using isopropanol. HMW RNAs precipitated in 20% isopropanol, while most LMW RNAs remained in the supernatant. The purified LMW RNAs were used successfully for RNA gel blotting to detect miRNAs expressed in Arabidopsis and tomato (Lycopersicon esculentum) seeds. To increase the detection sensitivity of the microRNA probes, additional digoxigenin-labelled uridine triphosphates (UTPs) were incorporated into the miRNA probes by designing template oligo DNAs with three extra adenines (A) at each end of their sequence. These DNA oligomers were used to make double-stranded DNA templates for miRNA probe synthesis. This probe (termed AAAPLUS) exhibited stronger signals than normal probes. A technique was also developed to quickly screen expressed miRNAs in seeds using a miniblot system, which enabled simultaneous examination with multiple miRNA probes. This method provides a simple alternative to microRNA microarrays to identify the major miRNAs expressed in seeds.

Testing for adventitious presence of transgenic material in conventional seed or grain lots using quantitative laboratory methods: statistical procedures and their implementation

Abstract: When the laboratory methods employed are qualitative, the statistical methodologies used in testing for the adventitious presence (AP) of transgenic material in conventional seed and grain lots are well defined. However, when the response from the method used by the laboratory is quantitative (e.g. percent transgenic DNA), the statistical methodologies developed for qualitative laboratory methods are not fully appropriate. In this paper, we present the details of procedures specific to quantitative laboratory methods. In particular we consider: (1) the assessment of quantitative laboratory method errors using linear modelling; and (2) the process of deciding whether or not a lot meets pre-specified purity standards, including the development of probability calculations needed to develop operating characteristic curves and estimate consumer and producer risks for a given lower quality limit (LQL), acceptable quality limit (AQL) and testing plan. We also describe implementation of this approach in a useful spreadsheet application.

Incubation under fluctuating temperatures reduces mean base water potential for seed germination in several non-cultivated species

Abstract: Seeds of Carduus acanthoides, Cynara cardunculus, Cirsium vulgare, Brassica campestris ,a nd Sisymbrium altissimum were incubated at a range of decreasing osmotic potentials (Co) under fluctuating temperatures or the median temperature of the fluctuation cycle. Fluctuating temperatures promoted total seed germination in water and at reduced osmotic potential. Total germination was reduced as the Co decreased. However, this trend was smallest under fluctuating temperatures, signalling a higher tolerance of seeds to reduced osmotic potential. Effects of osmoticum and temperature were modelled with the hydrotime model. The parameters estimated from the model, the hydrotime constant (uH), the mean base water potential Cb(50) and its standard deviation (sCb) gave good descriptions of germination time courses. For all species, incubation under fluctuating temperatures shifted Cb(50) values downwards without modifying their distribution substantially. This accounted for the greater tolerance of germination to reduced Co under fluctuating temperatures. To confirm that these effects were mediated by temperature fluctuations per se, the behaviour of C. acanthoides and C. cardunculus incubated at the minimum, the mean and the maximum temperature of the fluctuation cycle was also analysed. Constant maximum and minimum temperatures of the cycle did not stimulate germination, nor did they shift Cb(50) towards more negative values. The hydrotime model provides a physiologically based quantitative description for germination promotion due to fluctuating temperature.

Physical dormancy in seeds of Dodonaea viscosa (Sapindaceae) from India

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

Cyclitol galactosides in embryos of buckwheat stem–leaf–seed explants fed D-chiro-inositol, myo-inositol or D-pinitol

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

Endogenous jasmonates and octadecanoids in hypersensitive tomato mutants during germination and seedling development in response to abiotic stress

Abstract: Although jasmonates (JAs) are involved in germination and seedling development, the regulatory mechanism of JAs, and their relation with endogenous level modifications in these processes, is not well understood. We report here the detection of 12-oxo-phytodienoic acid (OPDA), jasmonic acid (JA), 11-hydroxyjasmonate (11-OH-JA), 12-hydroxyjasmonate (12-OH-JA) and methyljasmonate (JAME) in unimbibed seeds and seedlings of tomato Lycopersicon esculentum Mill cv. Moneymaker (wild type) and tss1, tss2, tos1 mutants. The main compounds in wild-type and tss1, tss2, tos1 seeds were the hydroxylate-JAs; 12-OH-JA was the major component in dry seeds of the wild type and in tss2 and tos1. The amounts of these derivatives were higher in seeds than in seedlings. Changes in JAs during wild-type and tss1 imbibition were analysed in seeds and the imbibition water. In wild-type imbibed seeds, 11-OH-JA content was higher than in tss1. 12-OH-JA showed a different tendency with respect to 11-OH-JA, with high levels in the wild type at early imbibition. In tss1, levels of 12-OH-JA rose from 24 to 48 h of imbibition. At 72 h of imbibition, when radicles had emerged, the amounts of both hydroxylates in wild-type and tss1 seeds were minimal. An important release of the hydroxylate forms was observed in the imbibition water. 11-OH-JA decreased in the imbibition water of wild-type seeds at 48 h. On the contrary, a high and sustained liberation of this compound was observed in tss1 after 24 h. 12-OH-JA increased in wild-type as well in tss1 until 24 h. Thereafter, a substantial reduction in the content of this compound was registered. NaCl-treated wild-type seedlings increased their 12-OH-JA, but tss1 seedlings increased their JA in response to salt treatment. In tss2 seedlings, NaCl caused a slight decrease in 11-OH-JA and JAME, whereas tos1 seedlings showed a dramatic OPDA and 12-OH-JA decrease in response to salt treatment. Under salt stress the mutant seedlings showed different patterns of JAs according to their differential hypersensitivity to abiotic stress. The JA-hydroxylate forms found, and the differential accumulation of JAs during germination, imbibition and seedling development, as well as their response to NaCl stress, provide new evidence about the control of many developmental processes by JA.

Annual rhythm of germination of seeds of Mesembryanthemum nodiflorum 32 years after collection

Abstract: Dry plants of Mesembryanthemum nodiflorum L. (Aizoaceae), with mature seeds enclosed in their dry capsules, were collected in the Judean Desert near Jericho, Israel, in the summer of 1972, and have been stored dry under room conditions [15–30°C, 15–25% relative humidity (RH)] since then. More than 31 years after maturation, these unimbibed seeds retained an annual rhythm of germination. A significantly higher percentage of seeds originating from the terminal, central and basal parts of the capsules germinated in February or March compared to other months of the year. This phenomenon was repeated in all three groups of seeds in 2001, 2002 and 2003 for germination in darkness at 15°C, and in terminal seeds germinated at 25°C in darkness for 4 years from 2001 to 2004. The annual rhythm of M. nodiflorum germination may regulate the season and percentage of seeds that germinate at the proper time of the year, even after many years. But the open question still remains: how do dry seeds measure time, and what are the mechanisms involved?

Endo-β-mannanase and β-mannosidase activities in rice grains during and following germination, and the influence of gibberellin and abscisic acid

Abstract: Grains of indica rice (Oryza sativa cv. Peiza 67) exhibit an increase in endo-β-mannanase activity, mostly after the completion of germination. According to tissue blots, the initial increase occurs in association with the embryo, and possibly the scutellum, although the largest sustained increase in activity is in the peripheral regions of the endosperm. The aleurone layer, being the only living region of the endosperm, is presumably the site of synthesis and secretion of the enzyme into the non-living, starch-laden region. β-Mannosidase activity is low throughout germination and subsequent seedling growth, particularly in the endosperm regions. Its activity profile does not mimic that of endo-β-mannanase. In the intact grain, gibberellin (GA) causes a relatively small increase in endo-β-mannanase activity, while abscisic acid (ABA) causes a large decrease; this inhibition is overcome to a considerable extent when GA is supplied along with ABA. β-Mannosidase activity is little affected by either GA or ABA. Embryoless half-grains imbibed in water exhibit only a small increase in endo-β-mannanase activity with time of imbibition, showing the necessity for a stimulus from the embryo for this to occur. Incubating half-grains in the presence of GA results in a large increase in enzyme activity; ABA reduces the amount of activity compared to the water controls. GA is capable of reversing the inhibitory effect of ABA with respect to endo-β-mannanase activity. As in the intact grains, β-mannosidase activity in the half-grains is unaffected by either GA or ABA. It is concluded that the major site for the production of endo-β-mannanase activity is the aleurone layer, and this event is influenced by the presence of the embryo; in the absence of the latter, the increase in enzyme activity is stimulated by GA. β-Mannosidase activity is low throughout germination and post-germination, it is not influenced by GA and ABA, and thus its activity is not regulated in a coordinated manner with that of endo-β-mannanase.

Characterization of water status in primed seeds of tomato (Lycopersicon esculentum Mill.) by sorption properties and NMR relaxation times

Abstract: The enhanced laboratory and field emergence characteristics of osmo- and halo-primed tomato seeds (cv Pusa Ruby) were related to changes in hydration–dehydration kinetics, modified sorption properties and nuclear magnetic resonance (NMR) relaxation behaviour of humidified and imbibed seeds Water sorption isotherms were constructed for primed and unprimed seeds by equilibrating to different water activities (aw) at 25°C Analysis of the isotherms by the D'Arcy–Watt equation revealed that priming reduced the number of strong binding sites and the associated water content, and increased significantly the number of weak binding sites and the associated water content This redistribution of water, which increased the availability of seed water, may be the reason for the higher speed of germination of primed seeds The changes in transverse relaxation time (T2) of seed water and its components, measured in vivo using nuclear magnetic resonance spectroscopy, showed interesting differences between primed and unprimed seeds With an increase in humidification time, the T2 of primed seeds could be resolved into three components with varying mobilities, while the control seeds had only two components until 10 d of humidification During imbibition, the third component appeared after 2 and 6 h in primed and control seeds, respectively This component disappeared after the germination process started in all treatments The third fraction, with very low molecular mobility, which accounted for about 40% of the proton population, was assigned to hydration water of macromolecules Hence, we propose that better performance of primed seeds may be attributed to the modifications of seed water-binding properties and reorganization of seed water during imbibition, so as to increase the macromolecular hydration water required for various metabolic activities related to the germination process

Molecular analysis of the hull-less seed trait in pumpkin: expression profiles of genes related to seed coat development11

Abstract: We undertook a comparative study of molecular changes during development of seed coats in the wild-type and a recessive hull-less mutant of pumpkin (Cucurbita pepo L.), with the goal of identifying key genes involved in secondary cell wall development in the testa. The mature mutant testa has reduced amounts of cellulose and lignin as compared to the wild type. The expression patterns of several genes involved in secondary cell wall biosynthesis during the development of the testa are described. These genes are: CELLULOSE SYNTHASE, PHENYLALANINE AMMONIA-LYASE, 4-COUMARATE-CoA LIGASE, and CINNAMOYL-CoA REDUCTASE. Additionally, the expression patterns of a few genes that were differentially expressed in the two genotypes during testa development (GLUTATHIONE REDUCTASE, ABSCISIC ACID RESPONSE PROTEIN E, a SERINE-THREONINE KINASE, and a β-UREIDOPROPIONASE) are presented. The results show a coordinated expression of several genes involved in cellulose and lignin biosynthesis, as well as marked differences in the level of their expression between the two genotypes during testa development. There is generally a higher expression of genes involved in cellulose and lignin biosynthesis in the wild-type testa as compared to the mutant. The molecular data presented here are consistent with anatomical and biochemical differences between the wild-type and the mutant testae. An understanding of the genes involved in cell wall development in the testa will facilitate the manipulation of seed coat development in Cucurbita and other species for diverse commercial applications.

Effect of temperature on dormancy and germination of Eupatorium cannabinum L. achenes

Abstract: This study investigated the effects of various temperature regimes on dormancy and germination in Eupatorium cannabinum, a common wetland species in Central Europe. Germination of dry-stored achenes was tested at increasing amplitudes of temperature fluctuations, and stratified achenes were germinated at constant temperatures. Dormancy release was examined in the laboratory at constant temperatures of 3, 8, 12, 15 and 18°C. The effect of an increase in temperature during stratification from 5°C to 15, 18 or 20/10°C on dormancy was tested. Moreover, achenes were exposed to yearly seasonal temperature cycles in the soil in an experimental garden. Germination of dry-stored achenes was not promoted by fluctuating temperatures, while stratified achenes germinated (>5%) over a range of constant temperatures between 9 and 36°C. Dormancy was relieved at all stratification temperatures, but temperatures ≤12°C were more effective than higher temperatures, since germination occurred over a wider range of test conditions after these pretreatments. An induction of secondary dormancy occurred only if the temperature was increased during stratification. Annual dormancy cycles were apparent when achenes were germinated at 15°C, while at 25 and 15/5°C germination was always >60%. The germination behaviour of Eupatorium is that of a typical wetland species. Thus, non-dormant achenes germinated at relatively high temperatures, and germination was promoted by fluctuating temperatures. The annual course of dormancy revealed that achenes can germinate throughout the growing season under favourable conditions. Achenes sown in an experimental garden germinated in spring, when daily mean temperatures were around 14°C. A decline in viability of achenes after 19 months' burial indicated that Eupatorium does not form a long-term persistent seed bank.

Dispersal and germination syndromes of tree seeds in a monsoonal forest in northern India

Abstract: This paper describes the dispersal–germination characteristics of seeds of 77 native tree species in a seasonal monsoon forest in Uttaranchal state, northern India. Results indicate that 50% of the species dispersed in the hot, dry summer months, 18% during the rainy season, 23% during the cold season and the remainder in late spring. Germination tests on fresh and laboratory-stored seeds revealed a relationship between morphological features of the fruit and both germination percentage and mean germination time (MGT). Highest mean germination (50%) was for dry-dehiscent fruits with winged wind-dispersed seeds, followed by dry-dehiscent fruits with non-winged seeds (38%) and seeds of dry-indehiscent fruits (37%). Lowest germination (29%) was for seeds from fleshy or pulpy fruits. MGT followed the reverse course. Germination data for seeds stored dry in the laboratory during one seeding cycle indicated six patterns of seed germination: (1) average germination percentage of fresh seeds lower than that of stored seeds, indicating an after-ripening requirement; (2) initial high germination percentage followed by low values, indicating a steep to moderate decline in viability following harvest; (3) no germination after 1 or 2 months of seed storage, due to complete loss of viability, indicating short seed longevity; (4) fresh seed germination in some species equalled the average germination value of stored seeds, indicating constant germination for one whole seeding cycle; (5) germination of both fresh and stored seeds remained consistently low throughout the season, indicating a requirement for some kind of pre-treatment or having poor quality of seeds; (6) initial high germination followed by decline and again increase, showing a seasonal rhythm of germination.