Effects of storage temperature on viability, germination and antioxidant metabolism in Ginkgo biloba L. seeds.

The behaviour of the Ginkgo biloba L. seeds was studied during storage at 4 and 25 degrees C. When stored at 25 degrees C, all the seeds died in 6 months. Cold temperatures preserved seed tissue viability for 1 year but did not preserve their capability to germinate, since such capability decreased after 6 months. A significant increase in lipid peroxidation occurred in the seed both in the embryo and in the endosperm. During storage a progressive deterioration of the endosperm tissues was evident. The two major water soluble antioxidants, ascorbate (ASC) and glutathione (GSH), showed different behaviour in the two conditions of storage and in the two main structures of the seed, the embryo and the endosperm. The ASC content of embryos and endosperms remained quite unchanged in the first 9 months at 4 degrees C, then increased. At 25 degrees C a significant decrease in the ASC content in the embryos was evident, whereas it remained more stable in the endosperm. The GSH pool decreased at both storage temperatures in the embryos. As far as the ASC-GSH redox enzymes are concerned, their activities decreased with storage, but changes appeared to be time-dependent more than temperature-dependent, with the exception of the endosperm ascorbate free radical (AFR) reductase (EC 1.6.5.4), the activity of which rapidly decreased at 25 degrees C. Therefore overall the antioxidant enzymes were scarcely regulated and unable to counteract oxidative stress occurring during the long-term storage.     

The effect of the temperature of drying on viability and some factors affecting storability of <Emphasis Type="Italic">Fagus sylvatica</Emphasis> seeds

Beech (Fagus sylvatica L.) seeds, which are tolerant to desiccation, freshly harvested after shedding, were dried at 15 and 30 °C and at the similar rate, to 9% of water content. A slight decrease of germinability was observed in seeds dried at 30 °C. Moreover, there was a notably higher solute leakage and a higher level of lipid hydroperoxides. Seeds dried at 30 °C contained less PC and PE and a lower level of unsaturated fatty acids (18:2 and 18:3), sterols and α-tocopherol. These results as well as changes in ascorbate and glutathione contents provide conclusive evidence for the presence of oxidative stress in beech seeds desiccated at 30 °C, which damaged membranes due to increased lipid peroxidation and changed membrane structure leading to their enhanced sensibility to free radical attack during storage.     

Comparative response of maize and rice genotypes to heat stress: status of oxidative stress and antioxidants

In the present study, two genotypes each of maize and rice were compared for their response to varying degrees of temperature stress (35/30, 40/35, 45/40°C) with controls growing at 30/25°C. At elevated temperatures of 40/35 and 45/40°C, the rice genotypes were inhibited to a significantly higher extent, especially for their shoot growth compared to maize genotypes. The stress injury measured as damage to membranes, loss of chlorophyll and reduction in leaf water status was significantly higher in rice plants, especially at 45/40°C. The components of oxidative stress particularly the level of malondialdehyde was significantly greater in rice plants while the differences for hydrogen peroxide concentrations were small at 40/35 and 45/40°C. The expression of enzymatic antioxidants like catalase, ascorbate peroxidase and glutathione reductase was found to be higher in maize plants compared to rice plants while no variations existed for superoxide dismutase at 45/40°C. In addition, the non-enzymatic antioxidants like ascorbic acid, glutathione and proline were maintained at significantly greater levels at 45/40°C in maize than in rice genotypes. These findings suggested that maize genotypes were able to retain their growth under high-temperature conditions partly due to their superior ability to cope up with oxidative damage by heat stress compared to rice genotypes. Since, maize and rice belong to C₄ and C₃ plant groups, respectively, these observations may also reflect the relative sensitivity of these plant groups to heat stress.     

Lemon flavonoid,eriocitrin, suppresses exercise-induced oxidative damage in rat liver

To examine the preventive effect of the lemon flavonoid, eriocitrin (eriodictyol 7-O-rutinoside), on oxidative stress during acute exercise in vivo, levels of N( epsilon )- (hexanoyl)lysine, HEL; o,o-dityrosine, DT; and nitrotyrosine, NT, as oxidative stress markers, were determined by ELISA in livers of trained rats in addition to thiobarbituric acid-reactive substance (TBARS). Eriocitrin administration prior to exercise significantly suppressed the increases in TBARS caused by lipid peroxidation during acute exercise. The contents of HEL, DT, and NT in rat liver increased dramatically by exercise without eriocitrin administration. However, these increases were significantly suppressed by eriocitrin administration before exercise. Moreover, in this study, to clarify whether eriocitrin influences glutathione metabolite system that is considered to be important for a defense against the damage by oxidative stress, the levels of glutathione in rat liver were determined during exercise. The level of reduced glutathione after exercise was maintained by administration of eriocitrin. The increase in the concentration of oxidized glutathione caused by exercise was significantly suppressed by eriocitrin. This result suggested that eriocitrin might play an important role in the control of the change in glutathione redox status in rat liver during exercise. These findings showed that eriocitrin was effective in the prevention of oxidative damages caused by acute exercise-induced oxidative stress.     

Expression of a bacterial α-amylase gene in transgenic rice seeds

An alpha-amylase gene from Bacillus stearothermophilus under the control of the promoter of a major rice-seed storage protein was introduced into rice. The transgenic line with the highest alpha-amylase activity reached about 15,000 U/g of seeds (one unit is defined as the amount of enzyme that produces 1 mumol of reducing sugar in 1 min at 70 degrees C). The enzyme produced in the seeds had an optimum pH of 5.0-5.5 and optimum temperature of 60-70 degrees C. Without extraction or purification, the power of transgenic rice seeds was able to liquify 100 times its weight of corn powder in 2 h. Thus, the transgenic rice could be used for industrial starch liquefaction.     

Modification of the radiosensitivity of barley seed by post-treatment with caffein. IV. Effect of the moisture content of seed and storage temperature after irradiation.

The oxygen-dependent damage which develops in barley seeds with approximately 7-8 per cent moisture content disappears after post-irradiation storage in vacuo for 48 hours at 40 degrees C and for 24 hours at 50 degrees C. When the diration of storage at 40 degrees C is extended to 384 hours, oxygen-independent damage becomes potentiated. There is oxygen-dependent damage in seeds of approximately 13.3 per cent moisture content and after the seeds have been stored in vacuo at 50 degrees C, the oxygen-dependent damage begins to increase by 168 hours, and it is very significantly potentiated by 192 hours. Under these circumstances, caffeine acts as a radioprotector only as long as the precursors of oxic damage are present in the seeds. Once these sites are lost, caffeine acts only as a radiosensitizer. The oxygen-independent damage which increases with storage at high temperature is further potentiated by caffeine.     

Survival and growth of Enterobacter sakazakii in infant rice cereal reconstituted with water, milk, liquid infant formula, or apple juice

AIMS: To determine survival and growth characteristics of Enterobacter sakazakii in infant rice cereal as affected by type of liquid used for reconstitution and storage temperature after reconstitution. METHODS AND RESULTS: A commercially manufactured dry infant rice cereal was reconstituted with water, apple juice, milk, or liquid infant formula, inoculated with a 10-strain mixture of E. sakazakii at populations of 0.27, 0.93, and 9.3 CFU ml(-1), and incubated at 4, 12, 21 or 30 degrees C for up to 72 h. Growth did not occur in cereal reconstituted with apple juice, regardless of storage temperature, or in cereal reconstituted with water, milk, or formula and stored at 4 degrees C. The lag time for growth in cereal reconstituted with water, milk, or formula was decreased as the incubation temperature (12, 21 and 30 degrees C) was increased. Upon reaching maximum populations of 7-8 log10 CFU ml(-1), in some instances populations decreased to nondetectable levels during subsequent storage which was concurrent with decreases in pH. CONCLUSIONS: Enterobacter sakazakii initially at very low populations can rapidly grow in infant rice cereal reconstituted with water, milk, or infant formula. SIGNIFICANCE AND IMPACT OF THE STUDY: Reconstituted infant rice cereal can support luxuriant growth of E. sakazakii. Reconstituted cereal that is not immediately consumed should be discarded or stored at a temperature at which E. sakazakii and other food-borne pathogens cannot grow.     

Storage-dependent degradation of 57-kDa protein in royal jelly: a possible marker for freshness

In order to find a marker for freshness of royal jelly (RJ), the composition change of RJ during storage was investigated. The contents of 10-hydroxy-2-decenoic acid, a bioactive component of RJ, and several vitamins did not change during storage at 40 degrees C for 7 days. However, a specific protein, designated royal jelly protein-1 (RJP-1), was gradually degraded during storage under various conditions (from 4 degrees C to 50 degrees C for up to 7 days). The specific degradation of RJP-1 was proportional to storage temperature and storage period. RJP-1 was purified to homogeneity and characterized as a monomeric glycoprotein with a molecular mass of 57 kDa. These results suggest that 57-kDa protein in RJ can be used as a marker for freshness of RJ, reflecting the conditions under which RJ has been stored.     

Enzymatic Conversion of Volatile Metabolites in Dry Seeds during Storage

Dry seeds can transform volatile metabolites via enzymes pre-existingin them. The interconversion between acetaldehyde and ethanoloccurred when they were applied gaseously to seeds of rice,lettuce, pea, cocklebur during storage at different relativehumidities (RH) at either 23°C or –3.5°C. Interconversionsbetween the compounds decreased with decreasing RH from 75%to 12% regardless of seed species and storage temperature, butit was still detectable even at 12% RH at –3.5°C.However, the conversion from acetaldehyde to ethanol did notoccur when seeds were killed by heating prior to storage. 2-Methoxyethanol,a competitive inhibitor of alcohol dehydrogenase (ADH) (EC 1.1.1.1 [EC] ),suppressed the transformation from ethanol to acetaldehyde inlettuce seeds. Therefore, ADH pre-existing in dry seeds maybe involved in the interconversion between ethanol and acetaldehydein dry seeds. Propanal, also a substrate of ADH, could be transformedto propanol during storage. However, methanol, which is nota substrate of this enzyme, was hardly converted to formaldehyde.Ethylacetate, applied to seeds during storage, was hydrolyzedby both lettuce and rice seeds, and the amount of hydrolysisincreased with increasing RH. Similarly, other carboxylic esters,such as methylacetate, ethylformate and ethylpropionate, couldbe hydrolyzed by rice, pea, and lettuce seeds. Little hydrolysisof ethylacetate occurred in the seeds killed by heating beforestorage, suggesting that ethylacetate may be hydrolyzed by carboxylic-esterhydrolase (EC 3.1.1.1 [EC] ). On the other hand, the production ofethylacetate through esterification was demonstrated by additionof gaseous acetate and ethanol in lettuce seeds, which occurredmore greatly at 12% RH than at 75% RH. These findings were discussedin relation to the mechanism of seed aging. (Received September 3, 1994; Accepted November 14, 1994)     

Dormancy release during hydrated storage in Lolium rigidum seeds is dependent on temperature, light quality, and hydration status

The influence of temperature, light environment, and seed hydration on the rate of dormancy release in Lolium rigidum (annual ryegrass) seeds during hydrated storage (stratification) was investigated. In a series of experiments, seeds were subjected to a range of temperatures (nine between 5 degrees C and 37 degrees C), light (white, red, far-red, and dark), and hydration (4-70 g H(2)O 100 g(-1) FW) during stratification for up to 80 d. Samples were germinated periodically at 25/15 degrees C or constant 15, 20, or 25 degrees C with a 12 h photoperiod to determine dormancy status. Dark-stratification was an alternative, but not equivalent dormancy release mechanism to dry after-ripening in annual ryegrass seeds. Dormancy release during dark-stratification caused a gradual increase in sensitivity to light, but germination in darkness remained negligible. Germination, but not dormancy release, was greater under fluctuating diurnal temperatures than the respective mean temperatures delivered constantly. Dormancy release rate was a positive linear function of dark-stratification temperature above a base temperature for dormancy release of 6.9 degrees C. Dormancy release at temperatures up to 30 degrees C could be described in terms of thermal dark-stratification time, but the rate of dormancy release was slower at < or =15 degrees C (244 degrees Cd/probit increase in germination) than > or =20 degrees C (208 degrees Cd/probit). Stratification in red or white, but not far-red light, inhibited dormancy release, as did insufficient (<40 g H(2)O 100 g(-1) FW) seed hydration. The influence of dark-stratification on dormancy status in annual ryegrass seeds is discussed in terms of a hypothetical increase in available membrane-bound phytochrome receptors.     

The influence of aerated hydration seed treatment on seed longevity as assessed by the viability equations

Aerated hydration (AH) treatments of cauliflower seeds for 12 h (12AH) and 28 h (28AH) at 20 degrees C resulted in improved or reduced storage potential of low or high vigour seeds, respectively. Seeds were stored at their initial seed moisture content (mean 5.5% mc) or at 12% mc at 10 degrees C for 12 months and at 20 degrees C for 4 months. The improved longevity of low vigour seeds was associated with increased K(i) (initial seed viability) and a reduced rate of deterioration (1/sigma) whereas the K(i) of high vigour seeds fell after 28AH and the rate of deterioration increased such that the time to lose one probit of viability decreased from 28.7 to 5.3 months at 10 degrees C and from 10.4 to 1.2 months at 20 degrees C. The improved K(i) of low vigour seeds could be explained by the reduction in the extent of deterioration after AH, as indicated by the increase in germination after cotrolled deterioration (CD), and the possible activation of metabolic repair during treatment. In contrast the reduced germination after CD of AH-treated high vigour seeds was indicative of deterioration as a result of treatment. Both high and low vigour seeds contained constitutive levels of ss-tubulin which increased during AH treatment, the increase being greater in high vigour seeds. High vigour seeds also showed an increase in the proportion of nuclear DNA present as 4C DNA, from 3% (untreated seeds) to 26% (28AH), indicative of germination advancement from the G(1) to G(2) phase of the cell cycle during treatment. This higher proportion of 4C DNA is correlated with the increased sensitivity of seeds to drying and/or storage after AH, leading to their reduced K(i) and storage potential. In contrast, there was little change in %4C in low vigour seeds. Priming in polyethylene glycol (PEG, -1.0 MPa) for 5 d or 13 d also improved the longevity of low vigour seeds stored at their initial and 12% mc at 10 degrees C for 8 months, as reflected in their laboratory and CD germination. In this case, however, the improved longevity of the low vigour seeds following 13 d priming was associated with an increase in 4C DNA from 4% (dry control) to 56% after treatment. The germination of both untreated and primed high vigour seeds remained high throughout the storage period. Increases in the rate of germination (decreased mean germination time) observed after all AH and PEG treatments were not consistently associated with an increase in the proportion of nuclei containing 4C DNA.     

Heat stress negatively affects physiology and morphology during germination of Ormosia coarctata (Fabaceae,Papilionoideae)

Research on the morphophysiological behavior of forest seeds during germination with respect to climate change is scarce. To date, there have been no studies on the biochemical or morphological aspects of Ormosia spp. In this study, we subjected Ormosia coarctata seeds to various temperature conditions to investigate temperature-dependent impacts on morphology, reactive oxygen species (ROS) generation, antioxidant systems, and storage systems. Analyses were performed on seeds exposed to 25, 35, and 40 °C for 48, 96, and 144 h. The morphology was evaluated by radiation using a Faxitron MX-20 device. ROS production (superoxide anion and hydrogen peroxide), malonaldehyde (MDA), carbonylated proteins, antioxidant enzyme activity (superoxide dismutase [SOD], ascorbate peroxidase [APX], catalase [CAT], and peroxidase [POX]), β-carotene, lycopene, glucose, and reserve enzyme activity (α- and β-amylase, lipase, and protease) were analyzed by spectrophotometry. Heat stress (40 °C) decreased germination by 76.2% and 78.1% (compared to 25 and 35 °C, respectively), caused damage to the external morphology of the seed, increased the content of ROS, MDA, and carbonylated proteins, and reduced APX, CAT, and POX activity. Furthermore, heat stress decreased glucose content and α-amylase activity. These results suggest that an increase of 5 °C in temperature negatively affects germination, promotes oxidative stress, and induces deterioration in O. coarctata seeds.

     

Age-dependent effect of oxidative stress on cardiac sarcoplasmic reticulum vesicles

The oxidative stress hypothesis of aging suggests that accumulation of oxidative damage is a key factor of the alterations in physiological function during aging. We studied age-related sensitivity to oxidative modifications of proteins and lipids of cardiac sarcoplasmic reticulum (SR) isolated from 6-, 15- and 26-month-old rats. Oxidative stress was generated in vitro by exposing SR vesicles to 0.1 mmol/l FeSO4/EDTA + 1 mmol/l H2O2 at 37 degrees C for 60 min. In all groups, oxidative stress was associated with decreased membrane surface hydrophobicity, as detected by 1-anilino-8-naphthalenesulfonate as a probe. Structural changes in SR membranes were accompanied by degradation of tryptophan and significant accumulation of protein dityrosines, protein conjugates with lipid peroxidation products, conjugated dienes and thiobarbituric acid reactive substances. The sensitivity to oxidative damage was most pronounced in SR of 26-month-old rat. Our results indicate that aging and oxidative stress are associated with accumulation of oxidatively damaged proteins and lipids and these changes could contribute to cardiovascular injury.     

C-Phycocyanin Confers Protection against Oxalate-Mediated Oxidative Stress and Mitochondrial Dysfunctions in MDCK Cells

Oxalate toxicity is mediated through generation of reactive oxygen species (ROS) via a process that is partly dependent on mitochondrial dysfunction. Here, we investigated whether C-phycocyanin (CP) could protect against oxidative stress-mediated intracellular damage triggered by oxalate in MDCK cells. DCFDA, a fluorescence-based probe and hexanoyl-lysine adduct (HEL), an oxidative stress marker were used to investigate the effect of CP on oxalate-induced ROS production and membrane lipid peroxidation (LPO). The role of CP against oxalate-induced oxidative stress was studied by the evaluation of mitochondrial membrane potential by JC1 fluorescein staining, quantification of ATP synthesis and stress-induced MAP kinases (JNK/SAPK and ERK1/2). Our results revealed that oxalate-induced cells show markedly increased ROS levels and HEL protein expression that were significantly decreased following pre-treatment with CP. Further, JC1 staining showed that CP pre-treatment conferred significant protection from mitochondrial membrane permeability and increased ATP production in CP-treated cells than oxalate-alone-treated cells. In addition, CP treated cells significantly decreased the expression of phosphorylated JNK/SAPK and ERK1/2 as compared to oxalate-alone-treated cells. We concluded that CP could be used as a potential free radical-scavenging therapeutic strategy against oxidative stress-associated diseases including urolithiasis.     

Effect of temperature on stability of marker constituents in Echinacea purpurea root formulations.

Stability of an alkamide and a phenolic phytochemical marker in a hydro-alcoholic extract of Echinacea purpurea root and a dried powder prepared by evaporation of the extract was assessed in storage for 7 months at three temperature regimes: -20, 25 and 40 degrees Celsius. In the extract, the major alkamide, dodeca-2E, 4E, 8Z, 10E/Z-tetraenoic acid isobutyl amide, was not significantly affected by storage at any of the temperatures, but cichoric acid content declined as significantly (P = 0.05) at both 25 degrees C and 40 degrees C as compared to low-temperature storage. In the powder, the major alkamide showed a significantly reduced level at 25 degrees C and 40 degrees C while cichoric acid did not decline significantly. These results suggest that more attention should be given to the effect of formulation and temperature on storage of Echinacea products.     

Viability loss of neem (Azadirachta indica) seeds associated with membrane phase behaviour.

Storage of neem (Azadirachta indica) seeds is difficult because of their sensitivity to chilling stress at moisture contents (MC) > or =10% or imbibitional stress below 10% MC. The hypothesis was tested that an elevated gel-to-liquid crystalline phase transition temperature (Tm) of membranes is responsible for this storage behaviour. To this end a spin probe technique, Fourier transform infrared microspectroscopy, and electron microscopy were used. The in situ Tm of hydrated membranes was between 10 degrees C and 15 degrees C, coinciding with the critical minimum temperature for germination. During storage, viability of fresh embryos was lost within two weeks at 5 degrees C, but remained high at 25 degrees C. The loss of viability coincided with an increased leakage of K+ from the embryos upon imbibition and with an increased proportion of cells with injured plasma membranes. Freeze-fracture replicas of plasma membranes from chilled, hydrated axes showed lateral phase separation and signs of the inverted hexagonal phase. Dehydrated embryos were sensitive to soaking in water, particularly at low temperatures, but fresh embryos were not. After soaking dry embryos at 5 degrees C (4 h) plus 1 d of further incubation at 25 degrees C, the axis cells were structurally disorganized and did not become turgid. In contrast, cells had a healthy appearance and were turgid after soaking at 35 degrees C. Imbibitional stress was associated with the loss of plasma membrane integrity in a limited number of cells, which expanded during further incubation of the embryos at 25 degrees C. It is suggested that the injuries brought about by storage or imbibition at sub-optimal temperatures in tropical seeds whose membranes have a high intrinsic Tm (10-15 degrees C), are caused by gel phase formation.     

Long-term stability of oxidative stress biomarkers in human serum

The storage time and storage temperature might affect stability of oxidative stress biomarkers, therefore, they have to be analyzed after long-term storage of serum samples. The stability of three biomarkers reflecting oxidative stress: reactive oxygen metabolites (ROM) for hydroperoxides, total thiol levels (TTL) for the redox status and biological antioxidant potency (BAP) for the antioxidant status, was investigated at several time points during 60 months of storage at ?20 and ?80?°C. Biomarkers ROM and BAP showed a very good stability during storage for 60 months at both temperatures. In addition, the correlation of the data after 60 months of storage compared with the starting data was very good with correlation coefficients >0.9. The TTL assay showed good results in serum samples stored at ?80?°C, but not in samples stored at ?20?°C. Serum samples for analysis of the set of oxidative stress biomarkers ROM, BAP and TTL can be stored up to 60 months at ?80?°C. ROM and BAP can also be stored at ?20?°C during this period. The present results are very important for the biomarker-related epidemiological studies that make use of biobanks with samples stored for many years and for new project planning, including sample storage conditions.     

Expression of an Acidothermus cellulolyticus endoglucanase in transgenic rice seeds

The thermostable endo-1,4-β-glucanase (E1) from Acidothermus cellulolyticus, is a useful enzyme for commercial hydrolysis of cellulose into glucose. A codon-optimized synthetic gene encoding this enzyme was transformed into rice (Oryza sativa L. ssp. japonica) under the control of the rice seed storage protein Gt1 promoter. The transgenic line C19 was identified as the one with the highest endoglucanase activity among the total of 36 independent transgenic lines obtained. The cellulase activity in the C19 seeds was estimated at about 830U/g of dried seeds using CMC as substrate. The enzymes produced in the seeds had an optimum pH of 5.0 and optimum temperature of 80°C, which is similar to the enzymes produced by the native bacterium host. This study demonstrates that the transgenic rice seeds could be used as a bioreactor for production of enzymes for cellulosic biomass conversion.     

Promoting glutathione synthesis after spinal cord trauma decreases secondary damage and promotes retention of function.

The study aimed to 1) quantify oxidative stress in spinal cord after crush injury at T6, 2) determine whether the administration of the procysteine compound L-2-oxothiazolidine-4-carboxylate (OTC) would up-regulate glutathione (GSH) synthesis and decrease oxidative stress, and 3) determine whether decreased oxidative stress results in better tissue and function retention. We demonstrate that spinal cord compression (5 s with a 50 g aneurysm clip) at T6 in rats results in oxidative stress that is extensive (significant increases in oxidative stress seen at C3 and L4) and rapid in onset. Indices of oxidative stress used were GSH content, protein carbonyl content, and inactivation of glutathione reductase. Administration of OTC resulted in a marked decrease in oxidative stress associated with a sparing of white matter at T6 (16+/-1.9% retained in OTC-treated animals vs. less than 1% in saline-treated). Behavioral indices in control, saline-treated, and OTC-treated animals after 6 wk were respectively: angle board scores (59 degrees, 32 degrees, and 42 degrees ), modified Tarlov score (7, 2.4, and 4.1), and Basso-Beattie-Bresnahan score (21, 5.3, and 12.9). We conclude that administration of OTC after spinal cord trauma greatly decreases oxidative stress and allows tissue preservation, thereby enabling otherwise paraplegic animals to locomote.     

The changing window of conditions that promotes germination of two fire ephemerals, Actinotus leucocephalus (Apiaceae) and Tersonia cyathiflora (Gyrostemonaceae)

BACKGROUND AND AIMS: Following a period of burial, more Actinotus leucocephalus (Apiaceae) and Tersonia cyathiflora (Gyrostemonaceae) seeds germinate in smoke water. The main aim of this study was to determine whether these fire-ephemeral seeds exhibit annual dormancy cycling during burial. This study also aimed to determine the effect of dormancy alleviation on the range of light and temperature conditions at which seeds germinate, and the possible factors driving changes in seed dormancy during burial. METHODS: Seeds were collected in summer, buried in soil in mesh bags in autumn and exhumed every 6 months for 24 months. Germination of exhumed and laboratory-stored (15 degrees C) seeds was assessed at 20 degrees C in water or smoke water. Germination response to light or dark conditions, incubation temperature (10, 15, 20, 25 and 30 degrees C), nitrate and gibberellic acid were also examined following burial or laboratory storage for 24 months. In the laboratory seeds were also stored at various temperatures (5, 15, 37 and 20/50 degrees C) for 1, 2 and 3 months followed by germination testing in water or smoke water. KEY RESULTS: The two species exhibited dormancy cycling during soil burial, producing low levels of germination in response to smoke water when exhumed in spring and high levels of germination in autumn. In autumn, seeds germinated in both light and dark and at a broader range of temperatures than did laboratory-stored seeds, and some Actinotus leucocephalus seeds also germinated in water alone. Dormancy release of Actinotus leucocephalus was slow during dry storage at 15 degrees C and more rapid at higher temperatures (37 and 20/50 degrees C); weekly wet/dry cycles further accelerated the rate of dormancy release. Cold stratification (5 degrees C) induced secondary dormancy. By contrast, no Tersonia cyathiflora seeds germinated following any of the laboratory storage treatments. CONCLUSIONS: Temperature and moisture influence dormancy cycling in Actinotus leucocephalus seeds. These factors alone did not simulate dormancy cycling of Tersonia cyathiflora seeds under the conditions tested.