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Seeds of annual halophytes such as Suaeda maritima experience fluctuating salinity, hydration, hypoxia and temperature during dormancy. Germination then occurs in one flush of 2–3 weeks after about 5 months of winter dormancy during which time the seeds can remain in saline, often waterlogged soil. The aim of this study was to investigate the effect of simulated natural conditions during dormancy on germination and to compare this with germination following the usual conditions of storing seeds dry. The effects of hydration, salinity, hypoxia and temperature regimes imposed during dormancy on germination were investigated. Also looked at were the effects of seed size on germination and the interaction between salinity during dormancy and salinity at the time of germination.
Various pre-treatments were imposed on samples of seeds that had been stored dry or wet for different periods of time during the 5 months of natural dormancy. Subsequent germination tests were carried out in conditions that simulated those found in the spring when germination occurs naturally. Various salinities were imposed at germination for a test of interaction between storage salinity and salinity at germination.
A temperature of about 15 °C was needed for germination and large seeds germinated earlier and better than small seeds. Cold seawater pre-treatment was necessary for good germination; the longer the saline pre-treatment during the natural dormancy period the better the germination. There appeared to be no effect of any specific ion of the seawater pre-treatment on germination and severe hypoxia did not prevent good germination. A short period of freezing stimulated early germination in dry-stored seed. Storage in cold saline or equivalent osmotic medium appeared to inhibit germination during the natural dormancy period and predispose the seed to germinate when the temperature rose and the salinity fell. Seeds that were stored in cold wet conditions germinated better in saline conditions than those stored dry.
The conditions under which seeds of S. maritima are stored affect their subsequent germination. Under natural conditions seeds remain dormant in highly saline, anoxic mud and then germinate when the temperature rises above about 15 °C and the salinity is reduced.
Halophytes that inhabit salt marshes are subjected to large fluctuations in salinity due to the twice-daily tidal cycles. This is particularly extreme in the more exposed areas of the upper-marsh where soil water becomes concentrated by evaporation and diluted by rainfall. The surface soil may become up to 100 times more saline than the subsoil, resulting in exposure of seeds to much more extreme conditions than are experienced by the mature plant (Ungar, 1978). Seeds have to survive not only long periods of high salinity but also severely hypoxic conditions in the estuarine mud – challenging conditions for successful germination.
Chapman (1974) reported that halophytes display delayed and reduced germination when subjected to solutions containing >170 mm NaCl. Later reviews of germination (Ungar, 1978, 1987) showed that tolerance to salt at the germination stage follows a continuum from the least-tolerant glycophytes to the most-tolerant salt-marsh halophytes. From a total of about 2400 halophytes species reported by Lieth et al. (1999), germination data are, however, only available for a few hundred species (Baskin and Baskin, 1995). Of these, Ungar (1995) found that seeds of halophytes usually show optimal germination in fresh water, as do glycophytes, but that halophytes do differ in their ability to germinate at higher salinity. Baskin and Baskin (1998) also reported that the salinity of the soil solution is a major factor affecting germination, which is generally better in water than in NaCl solutions. In general, the majority of halophyte species shows some inhibition of germination by salt water and highest percentage germination in fresh water (Khan and Rizvi, 1994; Noe and Zedler, 2000; Davy et al., 2001; Baskin and Baskin, 1998).
The ability of seeds of halophytes such as Haloxylon recurvum (Khan and Ungar, 1996), Arthrocnemum indicum (Khan and Gul, 1998) and Allenrolfea occidentalis (Gul and Weber, 1999) to remain viable in high salinities and germinate when salinity stress is reduced, provides them with opportunities for successful establishment in unpredictable saline environments (Khan and Ungar, 1997). However, the sea grass Zostera marina has been shown to germinate equally well in full seawater and in half-strength seawater, but only in seeds that had spent the winter in the saline sediment (Harrison, 1991). This suggests that the conditions the seeds experience during their dormancy may affect their subsequent germination behaviour. However, although much work has been carried out on the effects of different treatments imposed at the time of the germination test on the seeds of many halophytes, there has been very little investigation into the effect that conditions during the natural dormancy period may have on germination.
Suaeda maritima is an annual halophyte which is found in both coastal and inland wetland habitats in the northern hemisphere and eastern Asia (Clapham et al., 1962), where it exhibits a wide range of salt tolerance (Waisel, 1972). In British salt marshes, S. maritima grows from April to October over the whole tidal range, from the extreme high spring tidemark to low neap tidemark. Suaeda maritima seeds germinate in the UK in late March to April, indicating that they have a dormancy period of about 5 months from around late October to late March. Early work on S. maritima by Yeo (1974) showed that seeds require both cold and salt treatments for germination to take place under saline conditions. An investigation into salt stress limitation of seedling recruitment in a salt-marsh plant community (Tessier et al., 2000) showed S. maritima to be relatively tolerant to salinity at the germination stage in the laboratory but with reduced numbers of seeds germinating at high salinities.
The present work set out to test the hypothesis that the conditions to which Suaeda maritima seeds are exposed during dormancy will alter the subsequent time, rate and final percentage germination of the seeds. The first aim was to investigate the effects of hydration, salinity, hypoxia and temperature regimes of storage/pre-treatment during part or all of the natural dormancy period on subsequent germination under simulated field conditions. Then the importance of seed size, whether the seed had been stored dry or wet and the salinity imposed at the time of germination were studied. Also investigated was the interaction between temperature and hydration during pre-treatment and the salinity imposed at germination.
Plants of Suaeda maritima (L.) Dum. with mature seeds were collected from a salt marsh at Cuckmere Haven, East Sussex (UK, TQ515978) in mid-October 2004 and 2006. After harvest, plants were dried at about 15 °C for 14 d to allow separation of seeds from the rest of the plant by hand. The external appearance of all the seeds was similar, the only visual difference being size: individual plants produced both large and small seeds. Samples of the seeds were stored either in their post-harvest ‘dry’ state in plastic boxes and maintained in their dried state or ‘wet’ in plastic bottles of saline or fresh water. The samples of both dry- and wet-stored seed were subjected to different pre-treatment regimes for part or all of the natural dormancy period prior to testing germination.
Pre-treatments of different temperature, hydration, salinity or hypoxia were imposed as shown in Table 1. Solutions of polyethylene glycol (PEG 1500; BREOX) for pre-treatment PT6 were made up to the same osmotic potential (determined psychrometrically) as seawater (PEG 305 g L−1) and half-strength seawater (PEG 220 g L−1) in distilled water. In pre-treatment PT7 samples were submerged in artificial seawater (ASW) (Harvey, 1966) or in half-strength artificial seawater (1/2ASW) and kept for the total length of the pre-treatment either in normoxic solution or in sealed containers under severe hypoxia. The oxygen concentration was reduced to around 1 mg L−1 in the solution by bubbling nitrogen through the solution before the seeds were put into the solution. The oxygen concentration was regularly checked to ensure that the solution remained at this level of hypoxia.
In order to set the optimum temperature for a standard germination test, dry-stored seeds were tested on a thermogradient bar (constructed at the University of Sussex), consisting of an aluminium bar (70 × 15 × 3 cm) encased in an insulated wooden box and lid (85 × 30 × 17 cm). The bar was connected at one end to a chiller-thermo-circulator (Churchill) which maintained the coldest temperature at that end of the bar. An electronic thermometer (Type 1604 Cr/Al; Comark Electronics Ltd) recorded temperatures on the underside of the bar at 16-cm intervals to give five controlled temperature positions, the range of which could be altered. The temperatures were set at 10, 12·5, 15, 17·5 and 20 °C – the range of temperatures which the seeds might experience at the expected time of germination in the field. From the results, a standard germination test regime of 15/5 °C day/night temperature and 14-h photoperiod was used for all germination tests unless otherwise noted. Mean field temperatures in Sussex at the time of germination were 15/5 °C day/night (Table 2).
Germination tests were carried out in 9-cm plastic Petri dishes on three layers of Whatman qualitative filter paper type 1 (diameter = 90 mm). Stout and Arnon nutrient solution (S&A) in 1/2ASW, 8 mL, was added to each dish unless otherwise stated. Losses due to evaporation were replaced daily with distilled water (DW). Either four or six replicates of 25 seeds were used for each treatment. Nutrient solution and ASW were prepared according to Stout and Arnon (1939) and Harvey (1966), respectively. In all the experiments, germination was considered to have been successful when plumule emergence occurred. Percentage germination was recorded daily until no further germination occurred. Statistical analysis was carried out using ‘Minitab’ v. 14b. A summary of all germination tests is shown in Table 3.
An initial test was carried out to see whether seed size affected germination. Samples representing the extremes of size were selected from seeds that had been stored dry at 4 °C for 16 weeks. Seed diameter was measured using a micrometer and mean dry weight recorded (Table 4). Germination was tested in 1/2ASW. Seeds of both large and small size started to germinate after about 6 d and reached maximum values of 4 % for small seeds and 19 % for large seeds after 19 d. Consequently, all subsequent tests were carried out using large seed.
To set the temperature (within the natural range for springtime in Sussex) for germination tests, experiments were carried out with seed that had been stored dry at 4 °C for 20 weeks. These experiments showed that for this range of temperature the best germination (38 %) occurred at 20 °C. The germination rate and percentage germination were slower as the temperature of germination was reduced: at 18 °C and 15 °C percentage germination was 17 % and 11 %, respectively. Below 15 °C, seeds germinated very poorly, if at all; at 10 °C seeds did not germinate and at 12·5 °C only 1 % germinated (Fig. 1). These findings correlated with records of soil/mud temperature in the field collected before and during the natural germination period. Germination did not start in the field or in the laboratory until the temperature reached 15 °C. This temperature was therefore chosen for all subsequent germination tests.
Seeds used for experimental work on germination are almost invariably stored dry in an attempt to reduce decay and prolong viability. When seeds are stored dry, the storage temperature need not be reduced from ambient temperature to avoid decay. Dry storage is not, however, the natural state in which seeds of Suaeda maritima remain during the winter months of dormancy in the field. Consequently, the effect of storing seeds dry or wet and at cold or warm temperature for the total natural dormancy period was investigated (Fig. 2). Of seeds that had been stored dry at 4 °C (PT1a; Table 1) for 20 weeks 47 % germinated, but only 3 % germination occurred in those stored dry at 17 °C (PT1b; Table 1) and there was no germination at all in those stored dry at –18 °C for the full natural dormancy period of 20 weeks (PT1c; Table 1). Seeds from the same batch stored in cold DW for 20 weeks (PT1d; Table 1) also failed to germinate but the samples stored in cold ASW (PT1e; Table 1) germinated to 97 % after 5 d. The results therefore indicated that cold seawater pre-treatment is needed for good germination but that temperatures well below zero can kill the seed.
To investigate the effect that a short period of freezing might have on germination of seeds, samples of dry/4 °C and dry/17 °C stored seeds were given 2 weeks at –18 °C (PT3; Table 1). Seeds initially stored at 17 °C and then transferred to –18 °C reached a maximum germination of 7 %. Germination rate increased over one and a half times, to 18 % in seed previously stored dry at 4 °C. At the time of the experiment the seeds were only 2 months into the normal 5 months of dormancy and so the low germination rate was not unexpected. However, it does appear that a short period of very low temperature can stimulate germination and partially break dormancy in dry seeds.
Suaeda maritima grows on the salt marsh over the whole tidal range and therefore seeds are shed into substrates that differ in extent of waterlogging and salinity. Seeds from plants growing in the lower marsh will remain in waterlogged, saline mud for almost all of their dormancy period while, at the other extreme, seeds from plants growing in the upper marsh will remain in soil which is only covered by saline water for short periods of time during each tidal cycle. Here they are likely to experience fluctuating salinity due to rainfall. The present investigation focused on the effect that periods of wet pre-treatment of different salinity might have on the germination of previously dry seeds. We investigated whether a short period of cold seawater pre-treatment midway through the natural dormancy period would increase the percentage germination of dry dormant seeds (PT4; Table 1). Seeds did not germinate after 4 and 8 weeks dry storage and after 12 weeks dry storage there was <10 % germination, irrespective of whether the seeds had been stored at 4 °C or 17 °C (Fig. 3). Samples of these dry-stored seed were given a short period of 2 weeks pre-treatment in seawater half way through dormancy. Germination reached 46 % after 13 d in seeds initially stored at 17 °C and 56 % after 13 d in those stored at 4 °C, in contrast to the 10 % germination for samples stored dry (Fig. 3). The short period of seawater pre-treatment also resulted in germination of these seeds starting on day 2, whereas germination of dry stored seeds did not start until day 6 or 7. The ability of seeds to germinate is influenced greatly by the length of the wet saline pre-treatment during dormancy. When seawater pre-treatment was increased from 2 to 6 weeks, germination increased from around 50 % (Fig. 3) to 65–70 % (Fig. 4) and after 12 weeks or more in seawater it was over 90 % (PT7; Table 1 and Fig. 2).
Germination occurring following a reduction in salinity, could be affected by the ions in seawater or by the change in water potential. Consequently the effects of ASW were compared with those of a solution of a relatively inert osmoticum, PEG, made up to the same osmotic potential as seawater (PT6; Table 1). Germination tests showed a very slightly lower rate and final percentage germination in seeds pre-treated for 6 weeks in PEG than in seawater. However, analysis by logistic regression showed no statistically significant difference in germination of seeds pre-treated in seawater, half-strength seawater or the equivalent PEG concentrations (Fig. 4). The findings indicate that for dry seeds of S. maritima, early in dormancy, there is no apparent specific ion effect (for the ions in seawater) on germination.
Suaeda maritima seeds shed in situ from plants of the lower marsh lie buried in severely hypoxic or anoxic mud. Redox potentials recorded at low tide positions in the estuarine mud at the site of seed collection as soon as the tide had receded from each position gave Eh values of 15·2 ± 5·3 mV at 1 cm depth; at 3 cm, 6 cm and 9 cm depths the values were –121·2 ± 12·3 mV, –200·7 ± 11·7 mV and –266·6 ± 12·6 mV, respectively. Germination of seeds was tested after both normoxic and severe hypoxic pre-treatment in ASW or in 1/2ASW at 4 °C for the first 12 weeks of dormancy (PT7; Table 1). Very high germination rates resulted in all samples after 4 d. Seeds in normoxic ASW or in 1/2ASW germinated to 97 % and 96 %, respectively and there was 92 % germination in samples from hypoxic ASW and from 1/2ASW. These seeds appear to be tolerant of extreme anoxia during dormancy.
After finding that large seeds germinated to twice the final percentage of small seeds, the effect of different salinities imposed at germination on both large and small seeds, which had been stored dry or given a short period of hydration in seawater during dormancy, was investigated to find out if there is a correlation between seed size and salt tolerance at germination. Germination was carried out in DW, 1/2ASW and ASW. After 2 weeks of seawater pre-treatment (PT2, PT5; Table 1), both large and small seeds started to germinate by day 2 (Fig. 5B, D) and germinated both faster and to a much higher final percentage than those stored dry (Fig. 5A, C). Increased salinity of the test solution caused some reduction in the rate of germination and final percentage germination in both large and small seeds stored wet but analysis by logistic regression showed no significant effect of salinity on either large or small seeds. It therefore appears that, irrespective of the salinity imposed at germination, the storage hydration (dry or wet) and the size of seed both greatly affect the percentage germination.
A final experiment was conducted to gain better understanding of the effect of the salinity on germination per se. Germination tests were carried out in different concentrations of seawater and NaCl on dry-stored seeds and seeds that previously had 14 d in ASW (PT2, PT5; Table 1). Dry seeds germinated to between 10 % and 23 % (Fig. 6A, C) and seeds pre-treated with ASW to between 33 % and 54 % (Fig. 6B, D) depending on the salinity of the test. The highest percentage germination was always in distilled water and for both dry-stored and seawater-pre-treated seeds, the effect of decreasing the seawater concentration at germination was to increase the germination rate slightly. Germination rates in NaCl were similar to those in the equivalent concentration of ASW. There was a just significant lowering of the percentage germination by the highest concentration of seawater and of NaCl salinities. Seeds appeared to germinate equally well in the osmotic and ionic mix of seawater or in the equivalent molarity of NaCl alone. A short period of pre-treatment in cold seawater greatly increased percentage germination in the range of salinities to which these seeds might realistically be subjected in the field.
This work set out to provide understanding of the factors affecting germination in a well-adapted annual halophyte which has been shown not to have a persistent seed bank. Tessier et al. (2000) found that when salinity is low, germination in the field exhausted the seed bank and suggested that the absence of a persistent seed bank could be explained by the ability to germinate in the favourable conditions of decreased salinity in the upper soil layers after increased rainfall in the spring. The results of our preliminary experiment support these findings and suggest that good germination can occur only after the seeds have had a period of cold seawater submersion followed by a reduction in salinity (Fig. 2): the better germination in reduced salinity is in accordance with the findings for other halophytes. Work by Clarke and Hannon (1970) has shown that the effect of salinity on germination correlates with the soil salinity of the natural habitat.
The rate of germination and final percentage germination of Suaeda maritima were equally good after prolonged submersion in the salinity of seawater, half-strength seawater and in PEG of the same concentrations as seawater and half-strength seawater. These results verify preliminary findings that seawater submersion appears to be necessary for subsequent good germination to occur. The results also indicate that the specific ions found in seawater do not appear to be necessary for good germination to occur after a period of dormancy. The present work on the effects of storage conditions on germination in S. maritima adds to previous work in which different salinity or osmotic equivalent have been imposed only at the time of the germination test. Katembe et al. (1998) looked at the effect of treating seeds of two species of the halophyte Atriplex with different iso-osmotic solutions of NaCl and PEG at the time of the germination test and suggested that NaCl has a combination of osmotic and specific ion effects.
The influence of oxygen availability and salinity has been investigated on germination responses of two salt-marsh grasses (Spartina alterniflora and Phragmites australis; Wijte and Gallagher, 1996) but we are not aware of work on the effect of hypoxia during storage of seed prior to testing for germination. We have shown that seeds tolerate almost complete anoxia during the dormancy period. Field observations have shown that seedlings emerge in lines from the mudflat indicating that they survive the 5-month winter dormancy period in crevices in the mud (Fig. 7).
An interesting finding from the initial investigation was that germination of large seeds was 100–150 % better than that of small seeds from the same population. This agrees with work done on Suaeda salsa by Zhao et al. (2004) who described two seed types distinguished mainly by colour – brown and black. Brown seeds were reported to be larger and heavier, with a thinner, rougher testa, smaller cotyledons and lower initial water content. Germination of these larger brown seeds was much faster and reached a higher final percentage over a wider temperature range than for black seeds, indicating a better germination capacity. Dimorphism has been reported in another species, S. moquinii (Khan et al., 2001), where germination capacity was again shown to be better in brown soft seeds than in black hard seeds. In dimorphic seeds of Atriplex triangularis (Khan and Ungar, 1984) and Salicornia europaea (Philipupillai and Ungar, 1984), large seeds were shown to be more tolerant to NaCl than small ones. The present investigations into the effect of seed size on germination showed a significant correlation between germination and seed size and storage hydration. Seeds of large size given seawater pre-treatment germinated both more quickly and reached a higher final percentage germination than small seeds (Fig. 5), with no significant effect of salinity on the germination rates of different-sized seeds. The present studies on Suaeda maritima found that large and small seeds did not differ in colour or apparent seed coat characteristics. However, large seeds had larger cotyledons and it may be that one reason for better germination of the large S. maritima seeds is that the seed is more mature and has more nutrient reserves on which to draw. In all cases, cotyledons were green (Fig. 8). Other authors (Marin and Dengler, 1972; Shepherd et al., 2005) have reported the presence of green cotyledons in species of the Chenopodiaceae, an uncommon occurrence in angiosperms. It has been suggested that this enables photosynthetic activity at a very early stage of seedling establishment to promote rapid growth.
Noe and Zedler (2000) showed that germination of salt-marsh annuals is differentially affected by a variety of abiotic factors, with different species responding to the interaction of different factors but more species responding to soil salinity and with greater magnitude than to any other factor. The present test of the effect of salinity at germination on seed stored dry or stored in seawater showed that a decrease in salinity at germination resulted in an increased percentage germination in all samples, whether they had been stored dry or in seawater (Fig. 6). Germination rates were not affected significantly by salinity up to 600 mm NaCl and were similar to those in the equivalent concentration of ASW. The present findings agree with Yokoishi and Tanimoto (1994) who showed that S. japonica seeds germinated to about 80 % in solutions up to 0·7 m NaCl. It was found that after survival of dormancy under saline conditions, seeds germinate when salinity is reduced and other conditions such as a rise in temperature are met. The greater the reduction in salinity the better the germination rate becomes. The much higher rate of germination in seed which had been given a period of wet storage agrees with findings from a preliminary experiment (Fig. 2) and the comparison of dry-stored and wet-stored seed (Fig. 3).
Under field conditions, particularly on the lower marsh, the seeds of S. maritima are exposed to a saline, generally waterlogged and therefore severely hypoxic environment throughout the 5-month natural dormancy period, with soil temperatures generally well below 15 °C. Germination of S. maritima in the field occurs in one flush of 2–3 weeks after about 5 months of dormancy. In laboratory germination under simulated natural spring conditions, germination of S. maritima seeds stored dry from the time of harvest was very poor for about 4 months of the natural 5-month dormancy period. Good germination of dry stored seeds (over 85 %), did not occur until after 6 months of dormancy. It seems, therefore, that drying the seeds delays germination for about 1 month.
The germination of Suaeda maritima seeds is affected by the conditions under which the seeds are stored. The laboratory temperatures at which dry seeds were stored during dormancy (cold, 4 °C or warm, 17 °C) had little effect on the timing of subsequent germination. However, a short period of 2 weeks of freezing the dry-stored seeds only 8 weeks into the dormancy period resulted in a large increase in percentage germination, as did a short period of submersion in cold seawater of dry-stored seeds 12 weeks into dormancy (Fig. 3). In dry-stored seeds, therefore, it appears that low temperature and seawater reduce dormancy time and stimulate germination. As in the field, a period of low temperature and saline conditions during dormancy appear to be necessary before good germination can occur when the temperature rises and the salinity is reduced. It has been shown that laboratory germination responses under simulated natural conditions of hypoxia, hydration and temperature correlate strongly with the environment to which the seeds are exposed under field conditions.