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.

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.

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.

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.

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.

The heat production of seeds during germination comes from metabolism as well as hydration. Previous studies either lack continuous measurements, or are based on samples composed of more than one seed, thus failing to characterize differences among the critical phases of germination. This study examines the potential of isothermal calorimetry to characterize water uptake and metabolism in single seeds. Seeds of malting barley (Hordeum vulgare L.) and winterfat [Krascheninnikovia lanata (Pursh) A.D.J. Meeuse & Smit], two species with contrasting seed size, structure, composition and selection history, were used to determine patterns of heat production rate by isothermal calorimetry during water uptake and germination. Embryos of malting barley contributed less than 4% of total seed weight, and metabolic heat production during Phase I of germination was negligible compared to that due to hydration. Embryos accounted for 74% of seed mass for winterfat, and the majority of heat produced in Phase I was due to metabolic heat release. The total heat production rate in Phase I decreased rapidly in malting barley due to slowing of hydration reactions, but increased gradually in winterfat due to an increasing metabolic rate. The heat production rate at the end of Phase II was about twice as high in malting barley as in winterfat. This indicates a higher metabolic activity for malting barley than for winterfat seeds during germination, which may have also contributed to the rapid increase in the heat production rate of malting barley seedlings during Phase III, compared to the gradual increase in heat production rate of winterfat. The comparison between excised embryos and intact seeds indicates that the covering tissues delay radicle emergence in malting barley, but not in winterfat, due to differences in seed structure between the two species.

The seed bank of Heracleum mantegazzianum, native to the western Caucasus and invasive in Europe, was studied in the Slavkovský les 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 laboratory conditions (10°C, 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 m2) 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.

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?