Solute leakage is used as an indicator of membrane damage during desiccation of recalcitrant seeds. We re-examined this phenomenon and its utility by comparing recalcitrant Spartina alterniflora and orthodox Spartina pectinata seeds, as well as drying isolated embryos or embryos isolated after whole-seed drying. During drying, intact seed leakage (electrical conductivity and absorbance at 280 nm) was independent of seed moisture content, dormant/non-dormant state or desiccation tolerance. Embryos from both Spartina species, isolated after drying within the intact seed, exhibited increased leakage, especially below 40% (dry weight basis, DWB) seed moisture, the critical water content for viability loss in Spartina alterniflora. When isolated embryos were dried, the pattern and extent of increased solute leakage were similar in both Spartina species, even though S. pectinata embryos were >95% viable and S. alterniflora embryos were < 20% viable. We conclude that increased solute leakage is an artefact of embryo excision and not an accurate indicator of desiccation damage to recalcitrant Spartina alterniflora seeds.

Lipid peroxidation and membrane damage are often proposed as causes of recalcitrant seed death, and the thiobarbituric acid reactive substances (TBARS) assay is commonly used to measure lipid peroxidation. However, several artefacts can cause an overestimation of TBARS values, and these have not been routinely addressed in experiments with recalcitrant seeds. In the present report, TBARS was assayed as recalcitrant Spartina alterniflora and orthodox S. pectinata seeds were dried rapidly. Using the traditional Heath and Packer (1968) protocol with tissue extraction at 4°C, S. alterniflora had higher overall TBARS values than S. pectinata, and TBARS products increased when recalcitrant S. alterniflora and orthodox S. pectinata seeds were dried. However, when corrections for interfering substances, such as sugars and anthocyanins, were made, the TBARS values between the two species were almost identical. When seeds were freeze-clamped in liquid nitrogen prior to extraction, TBARS did not increase during desiccation for either species. These findings may indicate that lipid peroxidation is not the cause of desiccation-induced death in S. alterniflora. Therefore, freeze-clamping during tissue extraction and corrections for TBARS interfering substances must be applied to avoid overestimation of lipid peroxidation values.

Convolvulaceae is the only family in the asterid clade with species that produce physically dormant seeds, and most studies on germination in this family have focused on scarified seeds. However, no study has been done on the taxonomic/evolutionary pattern of seed dormancy in Convolvulaceae. We determined the moisture content of non-treated seeds and water uptake and germination percentages for non-treated and manually scarified seeds of 46 species in 11 of the 12 tribes in this family. Germination was tested over a range of temperatures in light/dark and in the dark. The effect of drying and storage at low temperatures was tested on seeds of Erycibe henryi and Maripa panamensis, the only species with high initial moisture content. Non-treated fresh seeds of Bonamia menziesii, M. panamensis and E. henryi imbibed water, whereas those of the other 43 species did not. Manually scarified seeds of all these 43 species took up large amounts of water. Therefore, seeds of 43 of the 46 species are physically dormant, and three are non-dormant. Seeds of all 46 species germinated after imbibition, except those of Cuscuta europaea, which are reported to have combinational dormancy. M. panamensis and E. henryi seeds are recalcitrant and those of the other 44 species orthodox. In Convolvulaceae, basal tribes or tribes derived from basal tribes contain species with non-dormant recalcitrant, physically dormant orthodox and combinationally dormant orthodox seeds. Physical dormancy of seeds in this family possibly evolved from a non-dormant recalcitrant seed-producing ancestor closely related to Erycibeae.

The fruits of Quercus robur are shed containing seeds at high moisture contents which remain desiccation sensitive and exhibit recalcitrant storage behaviour. Little is known of the control of seed development in these and other recalcitrant seeds. In the present work the changing concentrations of four plant growth regulators during seed reserve accumulation was studied over five years on seeds from the same tree. The pattern of reserve accumulation and changing ABA content in seeds differed between years. Although ABA content in the cotyledons increased in line with increasing dry weight to different contents at shedding, similar concentrations existed in cotyledons in each year. Thus ABA did not appear to be influencing dry weight accumulation. However, unlike orthodox seeds the decline in ABA concentration prior to shedding was limited and consistent with a continuing role for ABA in preventing precocious germination. An earlier peak in ABA concentration was associated with greater desiccation tolerance at shedding across years. The concentrations of zeatin and zeatin riboside in cotyledons were similar in each year and declined during reserve accumulation in a similar fashion to that reported for orthodox seeds. By contrast, IAA concentration increased in both the cotyledons and axes in the latter stages of seed development, opposite to that reported for orthodox seeds. It is possible that the increasing IAA concentration in cotyledons and axes and the stable concentration of zeatin and zeatin riboside throughout the latter stages of development in the axes of Q. robur are linked to the maintenance of active metabolism for the rapid initiation of germination upon shedding observed in seeds of this species.

The effect of rate of dehydration was assessed for embryonic axes from mature seeds of Camellia sinensis and the desiccation sensitivity of axes of different developmental stages was estimated using electrolyte leakage. Rapidly (flash) dried excised axes suffered desiccation damage at lower water contents (0.4 g H2O (g DW)−1) than axes dried more slowly in the whole seed (0.9 g H2O (g DW)−1). It is possible that flash drying of isolated axes imposes a stasis on deteriorative reactions that does not occur during slower dehydration. Differential scanning calorimetry (DSC) of the axes indicated that the enthalpy of the melting and the amount of non-freezable water were similar, irrespective of the drying rate.

Very immature axes that had completed morphogenesis and histodifferentiation only were more sensitive to desiccation (damage at 0.7 g H2O (g DW)−1) than mature axes or axes that were in the growth and reserve accumulation phase (damage at 0.4 g H2O (g DW)−1). As axes developed from maturity to germination, their threshold desiccation sensitivity increased to a higher level (1.3−1.4 g H2O (g DW)−1). For the very immature axes, enthalpy of the melting of tissue water was much lower, and the level of non-freezable water considerably higher, than for any other developmental stage studied.

There were no marked correlations between desiccation sensitivity and thermal properties of water. Desiccation sensitivity appears to be related more to the degree of metabolic activity evidenced by ultrastructural characteristics than to the physical properties of water.

Mangosteen (Garcinia mangostana L.), rambai (Baccaurea motleyana Muell.- Arg.) and jelentik (Baccaurea polyneura Hook. f.) are tropical fruit species believed to have recalcitrant seeds. The seeds showed no dormancy; they germinated easily. At harvest, the mean moisture contents (fresh weight basis) were 53.54, 51.20 and 44.90% for G. mangostana, B. motleyana and B. polyneura, respectively. G. mangostana seeds lost viability when their moisture content fell to about 24% while B. motleyana seeds lost viability below 35.5% moisture content. However, for B. polyneura, the seeds could be dried to low moisture content with high survival. The viability was still high when the moisture content was reduced to 13.46%. At this moisture content, the percentage germination was 91.76% and it was found that the seeds survived cyopreservation with 8.3% viability. For B. motleyana axes, the loss of viability occurred when their moisture content fell to about 36% (15% viability with predominantly callus formation) while for B. polyneura axes, viability was reduced to 33-67% when the moisture was 27–30%. At various moisture contents, the seeds of the fruit species studied were exposed for 48 h to 7°C and −4°C. G. mangostana seeds did not survive either temperature. Baccaurea seeds survived 7°C but failed to survive −4°C. No axes from B. motleyana seeds at various moisture contents survived cryopreservation. However, some viability (20–30%) was observed in B. polyneura axes cryopreserved at a moisture content of about 27%. At this moisture content no normal growth was obtained; callus formation was observed. It appears that the seeds vary in the degree of desiccation sensitivity. They also vary in size. Seeds of G. mangostana are larger than Baccaurea seeds and thus, more sensitive to desiccation. Seed and embryonic axis structure may also play a role in desiccation sensitivity.

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 presence of dehydrins could not be demonstrated in axes of mature, undried recalcitrant seeds of the tropical wetland species Avicennia marina, Barringtonia racemosa, Bruguiera exaristata, Bruguiera cylindrica, Bruguiera gymnorrhiza, Ceriops tagal, Rhizophora apiculata, Rhizophora mucronata and Rhizophora stylosa, but were present in the temperate species Acer saccharinum, Aesculus hippocastanum, Araucaria angustifolia, Camellia sinensis, Castanea sativa, Poncirus trifoliata and Zizania palustris. They were also present in axes of Castanospermum australe (of tropical origin) seeds which underwent development in a temperate climate, and were produced in response to drying in axes of Barringtonia racemosa but not Avicennia marina. The presence of dehydrins was associated with high abscisic acid contents. These proteins may provide protection against low temperatures in temperate seeds and against water loss to which the seeds may be naturally exposed. The presence of dehydrins was unrelated to the evolutionary status of the families studied.

Seeds were collected at weekly intervals from mid-maturation to the fully ripened stage. As seed development progressed, desiccation tolerance increased. Desiccation tolerance of C. lansium seeds was greatest at 67 days after anthesis (DAA), when they tolerated air drying for 9 days; 74 DAA was considered as physiological maturity, and their full viability was only maintained for up to 3 days of drying; overripened seeds (88 DAA) had the lowest desiccation tolerance. In L. chinensis, the desiccation sensitivity of seeds at 98 DAA (fully mature) was higher than that at 84 and 91 DAA (less mature); among the excised embryonic axes at different developmental stages, the less mature ones were less sensitive to desiccation than the fully mature ones; excised embryonic axes of the same stage were more tolerant of desiccation than whole seeds.

Quercus robur L. fruits are desiccation sensitive and shed from the plant at high moisture content (c. 48%). Water relations measurements were taken at intervals during the germination of prematurely-harvested immature fruits and naturally-shed mature fruits. In fruits from both harvests the pericarp and seed coat delayed germination by providing physical barriers to emergence of the radicle and by restricting the rate of imbibition. Although immature fruits could germinate in the absence of water they germinated more rapidly in the presence of an external water supply. The fully-mature fruit required a supply of external water, but germination was more rapid than in immature fruits. Substantial physiological changes, resulting in the accumulation of solutes, and increased axis size and extensibility occurred before splitting of the pericarp and subsequent germination of immature fruits. Such changes did not occur in fully-mature fruits, but fruits, from both harvests imbibed water and splitting of the pericarp appeared to result from increased embryo size and tissue pressure. Splitting was minimal prior to germination in immature fruits without an external water supply. It is suggested that the resulting reduction in Ψp when the pericarp splits was greater in the axis than in the cotyledons because of its greater capacity for expansion. This would create a Ψw gradient driving water flow to the axis from the cotyledons allowing continued radicle growth for germination.

The present study assessed the desiccation sensitivity of the excised embryonic axes of the following Amaryllidaceae species: Scadoxus puniceus, Amaryllis belladonna, Nerine huttoniae, N. humulus, N. bowdenii, N. filifolia, Haemanthus humulus humulus, H. coccineus, H. deformis, H. bakerae, Brunsvigia gregaria, Brunsvigia orientalis, Boophane disticha, Strumaria discifera, Crinum macowanii and C. bulbispermum. Excised embryonic axes were rapidly dehydrated (flash-dried). Curves of water content (g g− 1) and viability (%) versus drying time (min) were used to interpolate the water content (WC) and drying time (DT) values corresponding to 80% viability (WC/DT, V = 80) for individual species, and used to compare desiccation sensitivity between developmental stages and among years, provenances, species and genera. The seeds of all 16 species were shed with high axis water contents (2.25 ± 1.34 to 6.95 ± 0.89 g g− 1) and were sensitive to desiccation, with WC V = 80 values ranging from 0.13 to 3.15 g g− 1. Germination in amaryllids is unusual in that part of the cotyledon, to which the embryonic axis is attached, grows right out of the seed. It is at this stage that the axes were found to be more desiccation-sensitive in six out of nine species investigated. Seed fresh mass (SFM), axis shedding water content (SWC) and the degree of desiccation sensitivity were species characteristic. SWC, SFM and drying time were not significantly related to desiccation sensitivity. Parent plants were not confined to any particular biome type, occurring in areas characterized by seasonal dry spells. Parent plant habitat and seed characteristics were not useful in predicting the degree of desiccation sensitivity across species belonging to the same family.

Three questions are considered in the context of the possible effects of seed-associated mycoflora, typified by Fusarium moniliforme, during hydrated storage of recalcitrant propagules of the tropical species, Avicennia marina. These pertain to storage lifespan, whether seed susceptibility to fungal attack changes and the possibility of discriminating ultrastructurally between inherent deteriorative changes and those that are fungallyinduced. The data indicate unequivocally that if fungal activity is curtailed, then the hydrated storage lifespan of A. marina seeds can be considerably extended. When inoculated immediately with F. moniliforme, newly harvested seeds were extremely susceptible to the adverse effects of the fungus, while seeds that had been wet-stored for 4 d showed a considerably heightened resilience to the effects of the fungus when inoculated at that stage. The enhanced resilience, although declining, persisted in seeds stored hydrated for up to 10 d prior to inoculation, being lost after 12 d. After 14 d of hydrated storage, seeds became more susceptible to the effects of the fungus than those in the newly harvested condition. The resilience of seeds that had been stored in the short-term was associated with ultrastructural changes indicative of enhanced metabolic activity associated with the onset of germination. However, with the sustained stress imposed by wet-storage conditions, the seeds became increasingly badly affected by the fungus. While it was not possible to discriminate with certainty among deteriorative subcellular events ascribable to inherent deterioration or the effects of the fungus, it is concluded that a comparison of the timing of the onset of degeneration may well be diagnostic of its cause.

Despite the importance of the palm family, Arecaceae, little has been systematically documented about the seed behaviour of the many species. The post-harvest seed behaviour of Phoenix reclinata, the highly utilized wild date palm species distributed along the eastern seaboard of Africa, is investigated in the present study. While both embryo and endosperm water concentration declined as the seeds of Phoenix reclinata matured, they remained relatively high: this is a characteristic of (but not confined to) non-orthodox seeds. The ultrastructure of embryo cells, and the finding that negligible water uptake was required for the initiation of germination, were in keeping with the possible non-orthodox nature of the seeds. A developmental study revealed that between the acquisition of full germinability and complete pre-shedding maturity, germination performance appeared to be constrained, suggesting the presence of an inhibitor. Pre-treatment by soaking, mechanical or acid scarification had no significant promotory effect on either rate or totality of germination of mature P. reclinata seeds, while use of water transiently at 100°C was highly deleterious. However, germination of partially dehydrated seeds was initiated sooner if they had been soaked or scarified. Mature P. reclinata seeds tolerated dehydration to a mean embryo water concentration of 0.40 g g–1 (dry mass basis; dmb), but at 0.14 g g–1, both rate and totality of germination were adversely affected. However, viability of seeds dehydrated to the mean embryo water concentration 0.40 g g–1 declined during storage for 16 weeks. It is concluded that P. reclinata seeds are non-orthodox, and are best categorized as showing intermediate post-harvest behaviour.

Embryonic axes of tea (desiccation sensitive) and pea (desiccation tolerant) were dried at different rates or stored at different water contents to distinguish between damage associated with the immediate effects of water loss and the longer-term effects of a partially hydrated state. No loss of viability was observed if pea axes were dried sufficiently rapidly (from 1.8 to 0.1 g H2O g-1dry mass (g/g) within 5 d). However, viability was lost in tea axes dried below 0.5 g/g (approximately -15 MPa) even if axes were dried within 1 h. Death in tea axes dried to moisture contents less than 0.5 g/g probably resulted from the removal of water necessary for cellular structural integrity (i.e. desiccation damage sensu stricto). When axes of both species were dried at slower rates, viability losses were observed at water potentials between about -3 and -15 MPa. The timing for this type of damage was species dependent, occurring within 2 d for tea and after 5 d for pea, and may be explained by higher oxidative activity in tea compared to pea. Embryos of both species with water potentials below -3 MPa were lethally damaged if oxygen consumption exceeded 1000–5000 μmol O2 g-1dry mass. Recalcitrant seeds are different than orthodox seeds because the former do not survive drying below a critical water content, regardless of the drying rate. Rapid drying is required for accurate assessment of the critical water content. Slow drying leads to metabolic imbalance and artefactual assessment of the critical water content for desiccation damage. Both tea and pea seeds were susceptible to damage from metabolic imbalances, suggesting that the predominant stress from slow drying is ageing.

The histological and biochemical characteristics of cacao (Theobroma cacao) endosperm were determined at different stages of seed development. At various stages, the endosperm was analysed for minerals, organic acids, carbohydrates, free amino acids, abscisic acid (ABA), indole-3-acetic acid (IAA) and cytokinins. Cacao endosperm was coenocytic and became progressively cellularized from the micropylar zone. From stage I to IIz, the endosperm started to degenerate all around the embryo. Among the minerals detected, potassium and sulphate were the most abundant. Ammonium was the major source of mineral nitrogen, and glutamine was the most abundant free amino acid. Total nitrogen concentration was quite low. No sulphur-containing amino acids could be detected. Malate was present at all endosperm stages and was the most abundant organic acid. The sugars in cacao endosperm were mainly reducing sugars: glucose and fructose. ABA was only detected from stage IIz. IAA and three cytokinins [zeatin, zeatin-riboside and isopentenyl adenosine (iPA)] were present at all stages of development. Cacao endosperm resembled the endosperm of other species in (1) the abundance of organic acids and phosphate; and (2) a unique combination of sugars. However, three features seemed to be specific to cacao: (1) nitrogen was supplied mainly in the form of ammonium and amino acids, whereas, in other species, amino acids represent the predominant form; (2) there was a relatively small amount of nitrogen, and (3) a high concentration of sulphate and the absence of sulphur amino acids.

At shedding, the moisture content (MC) of Avicennia marina (Forssk.) Vierh. propagules was 65% (fresh mass basis), and there was no significant difference in the MC of four tissues (hypocotyl, cotyledons, plumule and root primordia). Viability declined as the propagules were dried below 60% MC, so that only 40%of seeds were capable of germination at 54% MC. At 47% MC all the seeds had died. The four tissues dried at the same constant rate of 0.02 g water g dwt-1 h–1 throughout this range of MCs. There was no significant depletion of the free-radical-quenching mechanisms measured. In each tissue an organic free-radical was detected by electron paramagnetic resonance (EPR). In the plumule the amplitude of the signal increased by a further 50% at MCs where viability was lost, but there was no increase in the other tissues. There was a concurrent increase in the amount of tocopherol and the activity of superoxide dismutase in the plumule. Lipid peroxidation, assessed by the amount of thiobarbituric acid-reactive substances, also increased in advance of viability loss, suggesting that propagules were experiencing oxidative stress. However, lipid peroxidation decreased at 54–57% MC, where most seeds lost viability. The results presented are consistent with a propagule reacting to oxidative stress, but overtaken by more catastrophic physical damage.

The osmotin gene was introduced into tea [Camellia sinensis (L.) O. Kuntze] somatic embryos at the globular stage of development via microprojectile bombardment, to determine the effects of this stress-tolerance-related gene on the accumulation of storage reserves and acquisition of desiccation tolerance during embryo maturation. Changes in total soluble sugars, starch and proteins in response to osmotin were estimated in the stably transformed embryos, using standard biochemical and histochemical methods, after the globular embryos had matured to the heart stage of development. These changes also were compared with two types of controls, comprising (1) untransformed embryos and (2) embryos that were bombarded without osmotin DNA. The control embryos showed poor reserve accumulation and negligible germination but, in contrast, the embryos bombarded with the osmotin gene showed a several-fold increase in storage reserves, normal maturation and 57–63% germination. When these transformed embryos were desiccated to 15.5% moisture content, only 40% germinated, although a further increase in the accumulation of storage reserves occurred. In contrast, the control embryos became necrotic and failed to germinate. This study demonstrates the use of somatic embryogenesis as a tool for understanding seed recalcitrance in tea, and suggests that transformation with stress-tolerance genes can overcome the developmental problems associated with recalcitrant seeds.

Because recalcitrant seeds are not desiccation-tolerant they must be stored moist. Their limited storage potential presents significant practical problems, but the cause of viability loss is not known. It has been suggested that a stress-induced metabolic imbalance can develop during storage that results in free-radical generation and consequent damage. To investigate this hypothesis, the presence of a stable free radical, lipid peroxidation and representative enzymatic and nonenzymatic protection mechanisms against oxidative attack were monitored in nondormant recalcitrant seeds during moist storage. A comparison was made between seeds of a short-lived sub-tropical species (Avicennia marina) and two longer-lived temperate species (Quercus robur and Castanea sativa). As a test of the hypothesis, seeds of both temperate species were held under conditions of elevated temperature and oxygen concentration to develop different rates of respiration during storage. The number of normal seedlings produced from seeds of the two temperate species declined during storage, but viability remained high, so effects of ageing were not confounded with an increasing proportion of dead seeds in the population. Under these conditions, lipid peroxidation changed little over the storage period, although there was evidence of accumulation of a stable free radical in Q. robur axes. However, this response was not affected by storage conditions that elevated respiration rates. In the shorter-lived A. marina seeds viability declined soon after the start of storage, but the significant increase in free radicals shown by EPR measurement was only evident when an increasing percentage of the seed population was no longer viable. Changes in the activity of scavenging enzymes and the concentration of antioxidants were time-dependent and not related to respiration rates. Therefore, in the present work, no consistent evidence was found to show that metabolism-induced free-radical activity was a significant contributing factor to pre-mortem deterioration in moist-stored recalcitrant seeds.