Excised Helianthus annuus L. embryos became dormant during the third week after anthesis. At this stage a short drying treatment (3 d) led to a slight improvement in germination but a 6-week dry storage caused a complete release from dormancy. The short drying treatment, however, elicited the embryos' response to an exogenous concentration of GAs which was unable to promote germination of fresh embryos. It therefore appeared that a short drying treatment changed the sensitivity to GAs but was not capable of directing embryo metabolism completely towards a germinative mode. Moreover, this drying treatment reduced considerably the ABA content in both the axis and the embryo. Nevertheless, no correlation could be established between germinability and the ABA content since the amount of ABA was not modified by the 6-week dry storage. The key step for predisposing the seeds to germinate is the suppression of the capacity for ABA synthesis in the axis, a suppression which takes place during 6-week dry storage.

Seed germination rates are sensitive to both temperature (T) and water potential (ψ). The times to germination of seeds imbibed at suboptimal T and/or reduced ψ are inversely proportional to the amounts by which T exceeds a base temperature (Tb) and ψ exceeds a base water potential (ψb). Germination rates across a range of suboptimal T and ψ can be normalized on the basis of the hydrothermal time accumulated in excess of these thresholds. However, seeds can also progress metabolically toward germination even at T or ψ too low to allow radicle emergence to occur. Seeds preimbibed at low ψ and dried back, or primed, germinate more rapidly upon subsequent reimbibition. We show here that the increase in germination rates of tomato (Lycopersicon esculentum Mill.) seeds resulting from seed priming is linearly related to the hydrothermal time accumulated during the priming treatment. The threshold temperature (Tmin = 7.05°C) and water potential (ψmin = −2.50 MPa) for metabolic advancement were considerably lower than the corresponding thresholds for radicle emergence of the same seed lot (Tb = 11°C; ψb = −0.71 MPa), allowing the accumulation of hydrothermal priming time that is subsequently expressed as more rapid germination when T or ψ increase. The hydrothermal time model can now be applied to quantify and analyse germination rates of seeds across the entire range of suboptimal T and ψ at which metabolic progress toward radicle emergence is possible.

Both temperature (T) and water potential (ψ) have consistent and quantifiable effects on the rate and extent of seed germination (radicle emergence). Germination at suboptimal T can be characterized on the basis of thermal time, or the T in excess of a base (Tb) multiplied by the time to a given percentage germination (tg). Similarly, germination at reduced ψ can be characterized on a hydrotime basis, or the ψ in excess of a base (ψb) multiplied by tg. Within a seed population, the variation in thermal times to germination for a specific percentage (g) is based upon the normal distribution of ψb values among seeds (ψb(g)). Germination responses across a range of suboptimal T and ψ might be accounted for by a general hydrothermal time model incorporating both T and ψ components. We tested this hypothesis for tomato (Lycopersicon esculentum Mill.) seeds of two genotypes differing in germination rates and tolerance of suboptimal T and ψ. For combinations of T (10−25°C) and ψ (0 to −0.9 MPa), a general hydrothermal time model accounted for approximately 75% of the variation in times to germination within the seed populations of both genotypes, and over 96% of the variation in median germination rates. However, ψb(g) distributions were sensitive to both the T and ψ of imbibition, resulting in a poor fit of the model to specific time course data. Analysis of germination timing separately for low and high ψ ranges within a given T resulted in specific models accounting for 88−99% of the variation in individual germination times and >99% of the variation in madian germination rates. Thus, for a given T and ψ range, the hydrotime model closely matched tomato seed germination time courses. Accumulated hydrothermal time accounted well for germination rates at ψ> −0.5 MPa across suboptimal T if ψb(g) was allowed to vary with T. Germination did not show a consistent response to T at ψ < −0.5 MPa, and estimated Tb values varied over different T ranges. Generalization of the hydrothermal time model across the entire range of suboptimal T and ψ was limited by physiological adjustments of the seeds to their current environment. The hydrothermal time model detected and quantified these adjustment processes that would otherwise not be evident from inspection of germination time courses. Temperature and water potential influence the time to germination via physiological mechanisms that reciprocally interact.

Using flow cytometry, we determined relative DNA amounts expressed as C-values, of large numbers of nuclei isolated from embryo root tips in three seed lots of pepper (Capsicum annuum) and tomato (Lycopersicon esculentum). The nuclei in embryos of fully matured dry pepper and tomato seeds predominantly revealed 2C signals, which indicates that the cells of maturing seeds have arrested at G1 phase of the cell cycle. Priming of pepper and tomato seeds in −1.1, −1.3 or −1.5 MPa polyethylene glycol (PEG) for 14 days reduced the mean time to germination. In all seed lots an induction of 4C signals was found after priming, indicating that during priming the cells of the embryonic root tip had replicated their DNA and arrested at the G2 phase of the cell cycle. For both tomato and pepper, the frequency of 4C signals was highest at the lowest PEG concentration (osmotic potential of −1.1 MPa). A significant inverse correlation was found between the frequency of root tip cells expressing 4C DNA signals and the mean time to germination for each individual pepper and tomato seed lot. However, the effect of a specific osmotic treatment differed between seed lots of the same cultivar, with regard to both the decrease in the mean time to germination and to the percentage of 4C cells, suggesting specific differences among seed lots in the response to priming. The present results indicate that flow cytometry can be used to follow the progress in nuclear replication activities during priming. The advancement in DNA synthesis can thus be used to measure the efficiency of a priming treatment with regard to enhancement of seed lot germination rate.

Pea seed development on the mother plant consists of three phases, all limited by water concentration (WC). The first (P1) or embryogenesis sensu stricto takes place at constant WC (stable at 80%). During the phase P2, cotyledon filling or maturation, WC decreases linearly from 80 to 55% (physiological desiccation) but the water content stays constant while the dry weight increases until it stops abruptly (at 55% WC), at this time, the seed has almost reached its final dry weight, its maturity mass or physiological maturity. The third phase, P3, consists of a fast desiccation which leads to a WC of 18–14%, where the seed is mature and ready to harvest. Similar events occur in other grain legumes, in cereals where mass maturity is attained at a lower WC (close to 40%) and in other species including crop or weed species. An elementary model of pea seed dry-matter accumulation, based on the constancy of water content (P1) and the linear decrease of WC from 80 to 55% (P2), allows us to define a coefficient α linked to WC and to calculate dry matter changes versus α. This model, taking account of WC in other species, can be generalized easily. Maturation of the somatic embryo, occurring under conditions very close to those present in vivo around the zygotic embryo, follows a pattern of decrease of WC similar to that of the zygotic embryo. We expect that if cell number is similar in the somatic and the zygotic embryo, synseeds will be ready for trade in the near future since control of all the processes that lead to zygotic-like embryoids is now available.

Inorganic P, total P and phytic acid-P leakage from imbibing whole soybean, bean, and pea seeds, as well as whole maize and barley grains were measured. The amounts of P and phytic acid leaked during a 6-h soaking period varied for the five species. Soybeans leaked the most P and phytic acid of the species examined. Peas, beans and barley leaked little or no phytic acid. Based on reported levels of total P and phytic acid in the dry seeds/grains that were studied, total P leakage ranged from 0.5 to 1.2%, while phytic acid leakage ranged from 0.01 to 0.5%. No relationship was found between initial levels of total P or phytic acid in the dry seeds/grains and the amount of total P or phytic acid leaked by the imbibed seed/grain. Similarly, the amounts of phytic acid leaked by the seeds/grains of this study did not appear to be related to the water solubility of the phytic acid in the dry seeds/grains.

A recent hypothesis states that the accumulation of amino-carbonyl reaction products of reducing sugars with proteins (Maillard products) is related to the loss of seed vigour and viability during ageing. Since Maillard products are biochemical end products, their accumulation should provide an index of seed deterioration. A simple fluorescence assay has been used to estimate the quantity of Maillard products in aqueous seed extracts. We tested whether an accumulation of fluorescent compounds correlates with the loss of seed viability during ageing. Fluorescence of extracts from lettuce (Lactuca sativa) embryonic axes increased after controlled deterioration, but was not correlated with ageing period. Fluorescence of extracts from whole broccoli (Brassica oleracea var. italica) seeds was either unchanged or declined during controlled deterioration. Extracts from naturally aged whole seeds of carrot (Daucus carota), tomato (Lycopersicon esculentum), and cauliflower (B. oleracea var. botrytis) had higher fluorescence than controls, but there was no consistent increase in fluorescence in proportion to the loss of viability. Extracts of naturally aged onion (Allium cepa) seeds exhibited less fluorescence than extracts from seeds with higher viability. We conclude that the fluorescence assay does not have general utility as an index of seed deterioration.

The desiccation tolerance of Zizania palustris seeds has been debatable, but recently it has emerged that survival to low moisture contents is a function of the temperature during dehydration. Survival of dehydration is greatest at 25–30°C and viability declines as the dehydration temperature is reduced. On the other hand, extended hydrated chilling (stratification) is required to break dormancy. The present contribution examines the ultrastructural condition of embryonic axes of Zizania palustris after dehydration at various temperatures, and following reimbibition and stratification. Axis cells sustained least damage when dehydration was carried out at 25°C and ultrastructural deterioration was more severe with lower temperatures during water loss. Damage sustained as a result of unfavourably low dehydration temperatures was visible when seeds were fixed from the dry state and was generally exacerbated during fully imbibed stratification. However, 25°C represented the optimum dehydration temperature; seeds that had been dried at 30°C also showed considerable ultrastructural disturbance when fixed from the dry state. This was largely reversed during fully imbibed stratification, although signs of the damage that had been sustained still persisted. These observations are in keeping with the germination behaviour of Z. palustris seeds dehydrated at temperatures above and below 25°C. The results are discussed in terms of the predominance of one of two mechanisms of membrane deterioration, depending on the dehydration temperature, which are probably not mutually exclusive. It is hypothesized that, at temperatures above 25°C, damage by free-radical-mediated events may predominate, whereas, at temperatures below the optimum, irreversible lipid-phase transitions may be the major factor resulting in membrane damage.

We report preliminary experiments to test the hypothesis that two sugars are involved in the acquisition of desiccation tolerance in immature maize embryos. Sucrose and raffinose were quantified during embryo development, at 16–27 days after pollination (DAP) and during slow dehydration. Embryos were exposed to slow drying either upon excision (18 and 16 DAP) or after an incubation treatment resulting in a decrease in sucrose (18 DAP). The sugars were separated by HPLC with a refractive-index detector; composition was determined by comparing peak areas of interest with those of individual sugar standards. Although further evidence is needed, our results do not appear to support the hypothesis.

Fruit and seed development in Quercus robur L. were studied on a single tree over five consecutive seasons. Patterns of growth in the cotyledons and embryonic axes differed between years and resulted in seeds of very different sizes. Moisture content at shedding also differed between years, and late-shed seeds had lower moisture contents than early-shed seeds. Moisture content at shedding was negatively correlated with desiccation tolerance. Seed development in Q. robur therefore appeared indeterminate and did not end in a period of rapid desiccation.

Sensitivity to desiccation in Q. robur was not due to an inability to accumulate dehydrin proteins, ABA or soluble sugars, substances that have been linked with the acquisition of desiccation tolerance in orthodox seeds. There were great similarities between several aspects of Q. robur seed development and that of orthodox seeds before the latter entered the terminal phase of rapid desiccation. This pattern of seed development contrasted with that reported for the highly desiccation-sensitive seeds of Avicennia marina.

The relationship between tolerance of seeds to extreme desiccation and the presence of ‘dehydrinlike’ proteins was investigated in groups of related taxa from the unrelated plant families Aceraceae and Gramineae. Dehydrin-like proteins were identified by Western blot analysis using an antibody raised against a synthetic oligopeptide representing the 23-amino acid consensus sequence common to all group 2 late-embryogenesis-abundant (LEA) proteins.

Evidence is presented that seeds of Acer pseudoplatanus and A. saccharinum are desiccation intolerant (recalcitrant) whereas seeds of A. platanoides and A. rubrum are desiccation tolerant (orthodox). Despite these differences, dehydrinlike proteins at 60 and 20 kDa were detected in all four species.

Dehydrins at 20 kDa were also detected in seed samples of two aquatic grasses, Porteresia coarctata and Oryza sativa from the tribe Oryzeae, despite seeds of the former rapidly losing viability on drying, whereas O. sativa is one of the best-known examples of desiccation-tolerant seeds. In O. sativa, there was a correlation between contents of dehydrins detected and the proportion of individuals capable of withstanding extreme drying. However, the possibility of a causal link between these parameters is equivocal. Dehydrin-like proteins were also detected in desiccation-sensitive seeds of Zizania palustris, Z. latifolia and Z. texana and desiccation-intolerant seeds of Spartina anglica, all from the Gramineae.

The presence of group 2 LEAs is clearly not diagnostic of desiccation tolerance in seeds. However, a more direct correlation with the expression of other groups of LEAs cannot be discounted.

Changes in soluble sugars in cauliflower seeds were followed during 50 h of imbibition in relation to desiccation tolerance. Sucrose and stachyose contents decreased, and glucose and fructose accumulated. This occurred in radicles first and subsequently in hypocotyls and cotyledons. Loss of desiccation tolerance in the various seed parts coincided with an increase in glucose and fructose and the complete loss of stachyose, but sucrose content, the major sugar, was still high. Drying imbibed seeds over silica gel did not evoke resynthesis of stachyose, but did increase sucrose and decrease glucose and fructose contents. Seeds primed in solutions of 30% polyethylene glycol for 10 days showed a loss of stachyose, while sucrose remained high and glucose and fructose contents were still very low. Redrying of primed seeds did not change the sugar contents. The primed seeds were still tolerant of desiccation. We conclude that stachyose is not a prerequisite for desiccation tolerance, but that sucrose may be. We suggest that glucose and fructose may be involved in desiccation damage.

The final stage of development of orthodox seeds is maturation drying, a process often accompanied by a dramatic decline in storage-protein synthesis. When desiccation is imposed prematurely at certain stages prior to the completion of development, a switch in synthetic events is elicited. events unique to development, such as synthesis of storage protein, are terminated, while syntheses associated with germination and growth are initiated. We investigated whether desiccation plays a key role in effecting a decline in expression of genes for storage proteins by acting directly upon the regulatory regions of these developmental genes. The desiccation responsiveness of the 5′ and 3′ regulatory regions of the gene for the pea storage protein vicilin was tested in transgenic tobacco seed. Chimaeric genes were introduced into tobacco, these genes consisted of the coding region of the reporter gene for β-glucuronidase (GUS) and 5′ and 3′ regions from the vicilin gene, or, as controls, the same regions derived from constitutively expressed genes, presumed to be desiccation-insensitive – those from the cauliflower mosaic virus (35S) and nopaline synthase genes. In transgenic seed expressing the gene constructs containing the vicilin 5′ upstream region, GUS activities declined dramatically after imbibition in mature seed and after rehydration of prematurely dried tobacco seed. In contrast, GUS activities increased after seed rehydration when the constitutive viral (35S) promoter replaced the vicilin 5′ upstream region. The 3′ downstream region of the gene construct did not significantly affect the patterns of changes in GUS activities after seed rehydration. In studies of transient gene expression in castor bean cotyledons, premature desiccation led to termination of GUS gene expression only when the gene construct contained the vicilin 5′ upstream region. This region may respond directly to desiccation; alternatively, changes to trans-acting factors important for expression of genes for storage-proteins may occur as a result of drying.

This article reviews mechanisms by which specialized cells of different life forms have overcome the lethal effects of dehydration and considers how the maintenance of genetic information is central to survival. As a dynamic and hydrated molecule in vivo, DNA can assume different conformational structures depending upon the water activity, the base sequence and the presence of specific binding proteins. The attainment of stable secondary structures that are resistant to degradation in vivo at low water potentials is proposed as a likely accompaniment to desiccation tolerance. In addition, chemical modification of bases in DNA, the extent of methylation and conformational changes could determine the expression of different gene sequences as cells pass from desiccation-tolerant to desiccation-intolerant states. We monitored the integrity of extracted DNA in embryos of seeds and in wind-dispersed pollen during transition from their desiccation tolerance to desiccation intolerance on hydration and germination. We present evidence to show that the DNA of these two stages is different and that it is the DNA from desiccation-tolerant cells only that retains integrity when the cells are subjected to desiccation regimes. We discuss these findings in relation to certain hydration-sensitive DNA structures and to other relevant biological systems.

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.

Embryos of Araucaria angustifolia seeds showed no dormancy; they germinated easily at temperatures ranging from 10° to 30°C, and the thermal optimum was about 25–30°C; they were recalcitrant. At harvest, their mean moisture content (dry weight basis) was about 120% and they completely lost viability when their moisture content fell to about 30%. The cotyledons were more sensitive to dehydration than the radicle. Dehydration induced deterioration of cell membranes as indicated by a high increase in leakage of solutes. It also resulted in damage in the nuclei, which was not repaired upon rehydration. During desiccation, respiratory activity decreased; however, O2 uptake was not an indication of germination ability, since it was significantly affected only when embryo moisture content reached the critical value of 30%. The decrease in the capacity to convert 1-aminocyclopropane 1-carboxylic acid to ethylene, which was observed at 30–60 min of dehydration, was a very early indicator of deterioration in embryos. Desiccation resulted also in a rapid decrease in the ability for protein synthesis as measured by [35S]methionine incorporation into total protein; 50% inhibition was observed after 30–60 min of desiccation for both axis and cotyledons.

In the presence of abscisic acid (ABA), contents of glucose and fructose decreased in carrot embryoids, whereas umbelliferose increased during dehydration. The acquisition of desiccation tolerance was tested by scoring germinated embryoids. It did not keep pace with the changes in soluble carbohydrate contents during development, which suggests that in these somatic embryoids di- and oligosaccharides are not the determining factors for anhydrobiosis. However, on slow dehydration, umbelliferose increased, while sucrose decreased and the monosaccharides declined completely. These changes were positively correlated with increased desiccation tolerance. A similar analysis of slowly dried, ABA-treated alfalfa embryoids showed that stachyose and sucrose increased and the monosaccharides declined with the acquisition of desiccation tolerance. These data support the contention that carbohydrates are involved in anhydrobiosis.

Changes in the properties of water in excised embryos were measured during the late stages of grain development in two cultivars of Zizania palustris and a population of the endangered species Z. texana. The relationships between water content and water activity were determined from water sorption isotherms, measured at temperatures between 35 and 5°C and then derived for lower temperatures. The freezing and melting behaviour of water in embryos at different water contents was determined using differential scanning calorimetry. The moisture content of embryos at high water activities decreased with maturation, as did the moisture content at which freezing transitions were not observed. While the temperatures of freezing and melting transitions decreased as the moisture content of embryos decreased, there were no discernible differences among embryos at different developmental stages. The properties of water measured in maturing Zizania embryos approached those for orthodox seeds as determined from the strength of water sorption, the enthalpy of the melting transition and the moisture content at which water is unfreezable. From these data we conclude that the properties of water in recalcitrant Zizania embryos change with development to resemble those of embryos of desiccation-tolerant seeds, but that the seeds never achieve the orthodox condition. The effects of interactions between moisture content and temperature on desiccation damage, freezing damage and germination in Zizania are predicted, based on the physical properties of water reported here and the correspondence of these properties with physiological function reported for other species. The resulting ‘phase diagram’ defines possible combinations of moisture content and temperature for storage under equilibrium conditions.

This review focuses on the possible evolutionary status and functional significance of recalcitrance and certain associated ovule/seed characters, within the framework of modern systems of angiosperm classification. The presence of recalcitrant seed viability in 45 dicotyledonous families is significantly associated with bitegmic and crassinucellate ovules and with nuclear endosperm development, all considered ancestral (plesiomorphic) character states of the ovule; as well as with greater seed size, woody habit and tropical habitat, also regarded as ancestral character states in the dicotyledons. In many species with recalcitrant seeds, the predominant storage reserve is carbohydrate. Recalcitrance is significantly associated with the exalbuminous type of reserve storage. It is proposed that in large recalcitrant seeds, the transfer of the main storage function from endosperm to embryo was probably an early development. In many species with recalcitrance, the ovules/seeds are characterized by extensive vascularization of the integument(s)/seed coat or by a pachychalaza. Pachychalazy is proposed to be a significant functional adaptation for a more efficient transfer of nutrients to the embryo/seed. Recalcitrance and some of the other character states proposed to be plesiomorphic in dicotyledons are also present in some gymnosperms, including presumed sister groups of the dicotyledons. In relatively advanced dicotyledonous families, mostly with orthodox seeds, recalcitrance probably persisted only in isolated relict members. Determination of character polarity is particularly problematic at lower taxonomic levels, because there is always the possibility that, in some taxa, a character state such as recalcitrance may have arisen secondarily as a reversal. Available evidence supports our view that seed recalcitrance can be regarded as a relatively ancestral character state in dicotyledons.