Germination and Dormancy in Annual Halophyte Juncus ranarius Song & Perr

The effects of cold stratification and gibberellic acid (GA3) on dormancy breaking for seeds of the annual halophyte species Juncus ranarius were tested. Germination percentage and recovery responses of salt stressed seeds were also tested. Freshly collected seeds germinated slowly under all incubation conditions. Thus, the seeds of J. ranarius have physiological dormancy, e.g. they are water permeable, have a fully developed embryo and require cold stratification to come out of dormancy. Furthermore, promotion of germination by GA3 after-ripening in dry storage also indicated that these seeds have non-deep physiological dormancy. In general, the higher the GA3 concentration, the more germination occurred within the studied range. Juncus ranarius demonstrated a germination preference for light. The highest germination percentage and rate of germination were recorded under constant light conditions at 22 °C after 24 weeks of cold stratification. In saline solutions, the highest percentage of germination was obtained at 25 mM L NaCl, and further increase in salinity resulted in a gradual decrease in germination. However, ungerminated seeds were not damaged by salt, showing a high level of recovery. The greater the reduction in salinity, the better the germination rate became. It was concluded that dormancy could be completely broken by cold stratification, indicating spring germination. Juncus ranarius can grow well at lower NaCl concentrations under constant light conditions at 22 °C.


Introduction
Halophytes, plants capable of growing and reproducing under saline conditions, vary in their upper limits of salt tolerance, while an increase in salinity usually delays seed germination (Gul et al., 2013;Khan and Ungar, 1996;Khan and Ungar, 1998;Ungar and Riehl, 1980;Ungar, 1995); other effects include reduction of the percentage of germinating seeds, delay in the initiation of the germination process and seeds' dormancy.Therefore, the seeds of halophytes germinate only when soil salinity levels are reduced, condition which usually occurs in saline environments in spring or during a season with high precipitation.The low soil salinity levels will befit seedling survival.Moreover, salinities beyond the tolerance limits of a given species can cause complete inhibition of the germination process and lead to loss of seed viability (Ungar, 1978(Ungar, , 1982)).
The seeds of halophytes under natural conditions are subjected to saline stress, which is dominated usually by NaCl.The effect of salinity on seed germination can be attributed to hyperosmotic stress resulting from more negative soil water potential and/or a specific ion toxicity (sodium, chloride), depending on a plant species (Huang and Redmann, 1995;Petruzzelli et al., 1992;Poljakoff-Mayber et al., 1994;Ungar and Hogan, 1970;Poljakoff-Mayber et al., 1994;Verslues et al., 2006;Zekri, 1993).Seeds of many halophytic species are reported to germinate best under fresh water conditions or at salinities below 100 mM L -1 NaCl (Ungar, 1982(Ungar, , 1991)), although storage conditions may also influence the germination response to salt (Katembe et al., 1998;Li et al., 2002;Rozema, 1975;Wetson et al., 2008).The highest salinity concentration at which a seed was reported to germinate was 1.7 M NaCl (Khan and Gul, 2006).
Germination can only occur when dormancy is lost and specific environmental conditions are present (Baskin and Baskin, 1998).Five classes of seed dormancy are now recognized: morphological (MD), physiological (PD), morpho-physiological (MPD), physical (PY) and combinational dormancy (PY + PD) (Baskin andBaskin, 1998, 2004).There are different mechanisms that prevent seed germination until after the right environmental cues have occurred.In morphological dormancy, an embryo is underdeveloped and needs to reach a specific size or developmental stage to germinate.The embryo needs a long period of favourable conditions to grow and then germinate, not just dormancy-breaking treatment.When a seed experiences physiological dormancy, germination is prevented by physiological inhibition radical emergence.Morphophysiological dormancy is due to an underdeveloped embryo that is physiologically dormant.Physical dormancy is caused by a water-impermeable palisade cell layer(s) in seed or fruit coats.Prior to germination, the seed or fruit coat of species with PY must become permeable in order to imbibe water.Physiological and physical dormancy together is called combinational dormancy (PY + PD).Seeds with (PY + PD) have a water impermeable seed or fruit coat (as in PY) and a physiologically dormant embryo (Baskin and Baskin, 2004;Bewley, 1997).
The available information on germination of halophytic seeds is far from complete (Khan and Ungar, 1999).From a total of about 2,400 species of halophytes reported by Lieth et al. (1999), germination data are available for a few hundred species (Ungar, 1995).Some studies have evaluated the germination characteristics of all halophytic Juncus species (Greenwood and MacFarlane, 2006;Jones and Richards, 1954).Seeds of Juncus acutus germinated well without salt (95% in the control treatment), while high salt concentrations prevented them from germination (Vicente et al., 2007).Boscaiu et al. (2011) demonstrated that in two related species of Juncus, J. acutus and J. maritimus, germination was optimal under non-saline conditions, while it was reduced by about 50% in the presence of 1.2% (200 mM L -1 ) NaCl, and completely inhibited by NaCl concentrations above 1.7% (300 mM L -1 ).Other authors (Dghim et al., 2012) reported that both Juncus species had high tolerance to NaCl at concentrations, up to 0.6% (100 mM L -1 ).Variations of light and temperature under saline conditions also affected germination of Juncus species.Absence of light almost completely inhibited seed germination of J. acutus (Jones and Richards, 1954).However, in freshwater at 15-25 °C, Martinez-Sanchez et al. (2006) reported < 75% germination under dark conditions.Clark and Hannon (1970) and Zedler et al. (1990) documented J. kraussii germination success at NaCl concentrations of ≤ 10 ppt, but not ≥ 20 ppt.
It is important to know the kind of seed dormancy for successful propagation of plants (Baskin and Baskin 1998), 440 but there is little information about ecological and physiological indices of seeds of J. ranarius Song and Perr.(also known as J. ambiguus Guss.), especially in relation to seed dormancy and germination.Although J. ranarius has been described as a halophyte (Jakubowska-Gabara et al., 2011;Piernik, 2012;Szafer et al., 1988), the species tolerance to salinity has yet to be estimated.According to Ellenberg's salinity indicator values, J. ranarius had the value of 4, which indicates that a species is encountered mainly in saline areas (Ellenberg et al., 1992).Therefore, the objective of the study was to better understand the seed dormancy and germination characteristics of J. ranarius by: (1) determining whether the seeds are water-permeable or water-impermeable via measurements of imbibitions; (2) testing the effects of cold stratification pre-treatment on dormancy break in the seeds; and (3) evaluating the effect of GA3, salinity, light and temperature on seed germination.

Study species
Juncus ranarius is a small, dark green, annual tufted up to 17 cm high.The leaves are linear with full margins, arranged opposite one another.White flowers are in a compound cyme.The plants bloom from June to August.They are wind-pollinated, as most of Juncaceae.The seeds (nuts) are 0.33-0.44× 0.25-0.35mm, ovoid in shape, almost smooth, indistinctly reticulated (Cope andStace, 1978, 1983), they weigh approx.0.017 mg each (our measurements, five replications of 100 seeds each), they ripen between mid-August and early September (our field observations in 2013).The seeds have relatively small embryos, with starchy endosperm, which is soon used up (Crocker and Barton, 1953).
J. ranarius is a typical halophyte, occurring on the coast on mud-and sand-flats above the high-water mark and on the margins of saline and brackish lakes.It is also found on bare mud and waste-ground associated with inland salt-flashes and saltworkings.It is distributed in Europe, parts of North Africa, Asia and North America (Cope and Stace, 1978).

Seed collection and field site description
Mature inflorescences were collected on 1 September 2013 from at least 30 plants of J. ranarius growing at inland saline meadow near Pełczyska village (latitude/longitude 52°00'N; 19°11'E) in central Poland.Immediately after collection, the seeds Fig. 1. Mean (2000-2010) minimum and maximum temperatures (A) and precipitation (B) at the nearest meteorological station Lodz were separated from inflorescences, hand sorted to eliminate broken, small and infected ones.Seeds were sterilized in 1% (v/v) sodium hypochlorite solution for 10 min and washed three times with sterile distilled water and then dry stored in paper bags at room temperature.
According to the observations in the field during the current study, J. ranarius occurs in almost pure patches or co-occurs with other halophyte species such as Glaux maritima and Spergularia salina along a gradient of reduced salinity.Minimum and maximum soil salinities (in the 0-2 cm soil layers) in the studied site were 0.1% (22 mM L -1 ) and 0.6% (100 mM L -1 ) NaCl respectively, varying markedly through the year.
Experiment 1: seeds imbibition This experiment was done to determine whether seeds were non-dormant or dormant (due to a water-impermeable seed coat).This test was conducted at room temperature (~24 °C).Four replicates of 150 seeds each were weighed (to the nearest 0.001 mg), moistened for 5 min in individual Petri dishes of 5 cm in diameter, lined with moist filter papers, removed from the dishes, blotted dry and reweighed (time 0).The seeds were reweighed again after 1, 6, 12, 24, 48, 72 and 96 h of water absorption.Percentage increase in fresh weight of the seeds was calculated as follows: [(W i -W d )/W d ] × 100, where W i and W d = weight of imbibed and dry seeds, respectively (Turner et al., 2006).
Experiment 2: light and temperature effects on seed germination Freshly matured seeds (14 days after harvest) were placed in individual Petri dishes of 5 cm in diameter with four filter paper discs moistened with distilled water until saturated.Twenty-five seeds were placed in each dish.To assess the effect of light and temperature on germination, four Petri dishes were covered with double layers of aluminium foil to ensure no light penetration (dark treatment) and four dishes were sealed with parafilm (light treatment).Light treated dishes were placed on top of dark treated ones and placed in a room with one of 3 constant temperature regimes: 5, 10, or 22 °C under a cycle of 14 h of light (about 30 µmol m -2 s -1 provided by fluorescent lamps) and 10 h of darkness.This temperature regimen was chosen to replicate the mean monthly temperatures recorded close-by at a climatological station during the growing season: 4.4 °C (early April), 10.3 °C (early May), 21.6 °C (July), when most seeds germinate in the natural habitat (Fig. 1A).Moisture was maintained with distilled water.The dishes were inspected daily and germinated seeds were counted during 20 days from the start of the test.Germinated seeds were counted in the light treatments, whereas counts in the dark treatments were made under dim green safe light.Seed germination was accepted when the radical appeared.The rate of germination was estimated using a modified Timson's index of germination velocity G/t, where G is seed germination percentage each day and t the total germination period (Khan and Ungar, 1997).Therefore if all of the seeds germinated in one day, the Timson's index would be 100 (e.g.2000/20), and a higher value indicates a more rapid germination.

Breaking dormancy of seeds
The low germination percentage ( < 25%) of freshly matured seeds of J. ranarius under both light and dark condition in all temperatures indicated that some seeds were dormant.However, further investigations need to be done in order to find out which dormancy-breaking factor is needed to trigger germination.Juncus ranarius seeds are dispersed in autumn, and thus they probably receive cold stratification after dispersion; 5 °C is near-optimal for many seeds with PD, requiring low moisture and low temperature to break dormancy (Baskin and Baskin, 1998;Stokes, 1965).Gibberellic acid (GA3) is an important endogenous plant growth regulator that can break dormancy in seeds with non-deep PD (Baskin and Baskin 1998;Nikolaeva et al., 1985).Hence, the influence of cold stratification and gibberellic acid (GA3) on seed dormancy breaking was tested.
Experiment 3: cold stratification treatment For this experiment, freshly matured seeds (2 weeks after collection) were placed in each of four Petri dishes, wrapped in aluminium foil and stored at 5 °C in a refrigerator, for 24 weeks.Twenty five seeds were placed in each dish.Next, all Petri dishes were incubated in a growth chamber at constant 5, 10, or 22 °C under a cycle of 14 h of light (about 30 µmol m -2 s -1 provided by fluorescent lamps) and 10 h of darkness.Seeds were considered to be germinated when a radical emerged from the seed coat.The number of germinated seeds was counted daily during 20 days.Germinated seeds were counted in the light treatments, whereas counts in the dark treatments were made under dim green safe light.Filter papers were kept moist with distilled water and germinated seeds were removed during every inspection.
Experiment 4: gibberellic acid (GA 3 ) treatments The GA3 treatments comprised three concentrations of GA3: 0.1, 1 and 10 mM L -1 .For each treatment, four replications of 25 seeds were incubated in 5 ml of each solution mentioned above, for 48 hours.The seeds treated with GA3 solutions and those not treated with GA3 (control) were then placed to germinate in a growth chamber at constant 22 °C under a cycle of 14 h of light (about 30 µmol m -2 s -1 provided by fluorescent lamps) and 10 h of darkness.Preliminary experiments showed that constant 22 °C were optimal for germination of fresh seeds, under the 14 h photoperiod.During experiment, GA3 solution was added whenever was necessary to keep moist use.The seeds were checked every day for emergence of a radical, during 20 days, and discarded when the tip of the radicle emerged.

Experiment 5: germination tests under saline conditions
To determinate the effect of salinity on germination and recovery of J. ranarius seeds, they were germinated in distilled water and 10, 25, 75 and 100 mM L -1 NaCl solutions.Four 25-seed replicates of each treatment were placed on filter paper in 5 cm Petri dishes with 5 mL of the test solution under conditions identical to those described for Experiment 4. The water level was adjusted daily with distilled water to avoid changes in salinity due to evaporation (Mauchamp and Mésleard, 2001;Redondo et al., 2004).Salinity concentrations were chosen to cover variations in inland saline meadow near Pełczyska village.The seeds were counted during 20 days as they germinated and discarded when the tip of a radicle emerged.For the recovery period, ungerminated seeds were removed from the Petri dishes, rinsed three times (10 min each) with sterile deionized water, and put into new 5 cm Petri dishes lined with two sheets of filter paper imbibed with 2.5 mL of sterile deionized water.Recovery percentage was calculated by the following formula: [(A -B)/(C -B)] × 100, where A is the number of seeds that germinated in salt solution plus those that recovered to germinate in distilled water (pH 5.7); B is the number of seeds germinated in salt solution and C is the total number of seeds tested (Gul and Weber, 1999).Final germination was recorded as (A/C) × 100 (Wang et al., 2008).The remaining ungerminated seeds were tested for viability by staining with tetrazolium chloride (Moore, 1962).The rate of germination was determined as described for Experiment 2.

Data analysis
The effects of light, salinity and temperature on the germination and rate of germination were examined using analysis of variance (ANOVA).Prior to analysis, percentage of germination and germination velocity were arcsine-transformed to stabilize the variance.The transformed data were subjected to factorial analysis of variance followed by Tukey's test 'a posteriori' multiple rage test and significances are indicated in the form of letters.All statistical tests were performed using a software package STATISTICA (Statsoft Inc., 2011).

Effect of temperature and light on germination of freshly harvested seeds
Germination of the freshly matured seeds was poor in all constant temperature regime.Significant difference was observed (P < 0.05) in germination percentage between the incubation in light and in dark at all temperature regime.Germination percentage reached 26% at the highest temperature in light (22 °C), but at 10 °C and 5 °C it was only 18% and 3% respectively.In dark, few seeds of J. ranarius germinated at 22 °C, while at 10 °C and 5 °C, germination was completely inhibited (Table 1).

Seed dormancy-break and germination
The difference in the rate and percentage of germination between the fresh seeds and cold stratified ones was significant (P < 0.05).The rate of germination grew after cold stratification, with an increase in the germination index under light from 1 to 58 and under dark from 2 to 11, at 22 °C.The germination rate was slower as the temperature was reduced.The results showed that after cold stratification treatment, germination percentage (P < 0.01) and germination velocity (P <0.05) of the seeds under light and dark conditions were significantly higher than of those that had not been stratified.After the period of 24 weeks of cold stratification, the highest germination percentage (85%) was observed at 22 °C in light followed by 10 °C and 5 °C, which caused germination percentages of 37% and 7% respectively (Table 2).Light conditions (P < 0.001), temperature (P < 0.001), cold stratification (P < 0.001) and their interaction (P <0.05) were all of significant importance to both percentage and rate of germination (Table 3).

Effect of GA 3
The treatment with GA3 was of significant importance for germination.The seed germination percentage was increasing with growing GA3 concentration.An addition of 0.1 mg L -1 GA3 increased germination, while 10 mg L -1 GA3 was optimum (> Fig. 2. Imbibition curve for seeds of J. ranarius in distilled water 7 ± 2 Aa 25 ± 3 Bc Note: Different upper-case letters indicate significant differences by Tukey's test in the germination percentages between the non-treated and 24-week-coldstratified seeds at the same temperature and different lower-case letters in each column with the same light conditions indicate significant differences in the germination percentages of seeds among different temperatures Table 2. Index of germination velocity of the non-treated and 24-week-coldstratified seeds of J. ranarius at three thermo-periods under different light conditions.Each value is a mean ± S.D. of four replicates of 25 seeds

Light condition Temperature
Non-treated seeds 2 ± 1 Aa 11 ± 2 Ba Note: Different upper-case letters indicate significant differences by Tukey's test between germination rate of the non-treated and 24-week-cold-stratified seeds at the same temperature and different lower-case letters in each column with the same light conditions indicate significant differences in germination rate of the seeds among different temperatures 90% germination).The same trend was also visible for seed germination velocity: the Timson's index of the germination velocity was significantly higher after GA3 substrate supplementations compared to the control.In general, the higher the GA3 concentration, the more germination occurred within the studied range (F2.21 = 21.331,P < 0.001) (Table 4).

Effect of salinity on germination and recovery of germination
Under salinity conditions, the seed germination percentage of J. ranarius was significantly higher for 10 mM L -1 and 25 mM L -1 NaCl, while significantly lower for 50, 75 and 100 mM L -1 NaCl, then the corresponding ones in H2O.The highest percentage of germination was obtained at 25 mM L -1 NaCl, and further increase in salinity resulted in a gradual decrease of germination.Less than 5% of seeds germinated at 100 mM L 1 NaCl (Table 5).
The seed germination velocity at the low NaCl concentration (10 mM L -1 ) was the same as for the water control, while at 25 mM L -1 it was significantly higher, and with 50, 75 and 100 mM L -1 NaCl it was significantly lower than the corresponding velocity in H2O.It suggests that J. ranarius seed germination time was shortened at the 25 mM L -1 NaCl, but higher levels of NaCl (> 25 mM L --1 ) prolonged it (Table 5).
When the seeds were transferred to distilled water after twenty-days exposure to salinity, recovery of germination was depended on NaCl concentration.There was little recovery in the lowest (10 mM L -1 ) NaCl variant, but in the highest (100 mM L -1 ) it reached up to 81% (Table 6).
A tetrazolium test was performed on the non-germinated seeds and revealed that these seeds were viable, showing absolute dormancy.

Discussions
Prior to this study, the types of dormancy and methods to release it in J. ranarius seeds were not known.In the present study, it was noticed that the seeds produced by J. ranarius are dormant on maturity and require pretreatment to stimulate germination.The embryo of J. ranarius is fully developed, suggesting that the seeds have neither morphological, nor morphophysiological dormancy.Since the seed coat of J. ranarius is water permeable, neither physical dormancy, nor combinatorial dormancy appears likely, and thus, these seeds exhibit non-deep PY (sensu Baskin and Baskin, 2004).The result is consistent with the report of Polyakoff-Mayber et al. (1992), who suggested that seeds of some species of Juncus are dormant at harvest.In contrast to this finding, in other halophyte Juncus species, for example J. balticus, seeds were considered to be nondormant (Necajeva and Levinsh, 2008).
GA3 application and cold stratification substantially increased germination of J. ranarius seeds.Thus, the seeds of 443 Table 3. Three-way ANOVA of the effects of temperature (T), light conditions (L, light vs. darkness), cold stratification (CS, non-treated vs. 24-week-cold-stratified) seeds of J. ranarius and their interactions on seed germination and rate of germination    Rozema (1975) studying several species of Juncus.
Annual Juncus species vary in their level of salt tolerance at the germination stage.For example, J. gerardii and J. acutus are very salt intolerant with germination severely depressed by even moderate salinities (Rozema and Blom, 1977;Shumway and Bertness, 1992).In the present study, germination of J. ranarius seeds was considerably reduced at the salinity level of 50 mM L -1 NaCl and was completely inhibited by 100 mM L -1 NaCl, because it probably exceed individual tolerance limits.Another halophyte from the area, Spergularia salina was reported to germinate best under fresh water conditions or at salinities below 100 mM L -1 NaCl (Bakker et al., 1985).Keiffer and Ungar (1997) pointed out that halophytes germinate during spring, rather than in summer, in order to avoid the increase in salt concentration in soil solution caused by high evaporation.Some plants that have been classified as salt sensitive can germinate under high concentrations of NaCl, while other tolerant species are more sensitive during germination.
Several investigators reported that the germination process was delayed under salt stress (Keiffer and Ungar, 1997;Khan and Ungar, 1997).However, in some halophytic species, the presence of sodium ions even at low concentrations could have positive effect on seed germination (Ungar 1991), increasing its rate over the distilled water control (Sabahat and Khan, 2004).The results obtained in the present study showing that germination in low salinity (10 and 25 mM L -1 ) began earlier than in the non-saline control support this finding.It has been suggested that fast germination ensures rapid seedling establishment, which can minimize competition (Rogers et al., 1995).Distilled has zero osmosis potential and some seeds show lower rate of germination at this potential (Sedghi et al., 2010).
Juncus ranarius seeds, when transferred to fresh water, showed an enhanced rate of germination after pretreatment with various salinity concentrations.This is in agreement with the results of experiments in which seeds were subjected to salinity and then placed in fresh water (Clarke and Hannon, 1970;Keiffer and Ungar, 1997;Khan and Ungar, 1997;Ungar, 1995;Woodell, 1985).This response suggests that the inhibition of J. ranarius seed germination at high salt concentration was mostly due to osmotic effects and reversible, as found by Ungar (1978Ungar ( , 1996)).It is likely that after immersion in salt water, seeds will be soon washed by rain, which will stimulate their germination.
The response to salinity and germination recovery patterns allow classification of J. ranarius seeds as having intermediate salinity tolerance (Woodell, 1985).However, not only salt concentrations (or osmotic potential), but also nature of the ions in salt solutions and their interactions may have an impact on germination (Sosa et al., 2005).Ungar (1978) pointed out that seed germination in saltaffected soils was influenced by the total concentration of dissolved salt (or the osmotic pressure) as well as by the type of salts involved.
Since NaCl is the major component of most saline soils, it was used to challenge the plants.However, such investigations may not allow to infer germination responses of plants under field conditions, because field soil contains different salts, which collectively influence seed germination in a different way than each one of them separately (Ungar, 1978).
Light (photoperiod) is another important regulatory environmental signal in germination of many halophytes (Ungar, 1978).Light also stimulates germination of Haloxylon recuvrum and Triglochin maritima (Khan and Ungar, 1997).In the present work, germination was found to be controlled by light.This suggests that successful J. ranarius germination and establishment require high-light environment where soils are bare and exposed.This germination response may limit this species colonisation to open locations.A light requirement for seed germination is common, especially in species that have small seeds (Taylorson, 1987).Light was reported as necessary for germination of many Juncus species (Burkart et al., 2010;Lazenby, 1955;Martinez-Sanchez et al., 2006;Richards and Clapham, 1941b;Richards, 1943) including J. effusus (Richards and Clapham, 1941a).
Temperature is an important environmental signal regulating germination of many herbaceous plants from temperate regions.Some species require cold stratification to initiate germination, or to increase their germination rate in spring (Baskin and Baskin, 1998).Our data clearly indicate that J. ranarius had increased germination following cold stratification.Improved germination after short cold stratification is typical of species with non-deep physiological dormancy (Baskin and Baskin, 1998).
Best seed germination of most temperate species occurred at 15-30 °C (Khan and Gul, 2006) with an average of 21 °C (Baskin and Baskin, 1998).It was shown in this study that germination and rate of germination increased with rising temperature and the optimal germination was obtained at a constant temperature of 22 °C.In early spring, lower temperatures can cause high mortality.Field observations suggested that germination only occurred when a certain critical temperature was reached.Seedlings, first seen at the beginning of May, were estimated to have germinated approximately on 20 -th April, at which period maximum day temperatures reached 22 °C and night minima was 5 °C (Fig. 1).

Conclusions
The findings of this study indicate that salt stress, light, temperature and cold stratification are critical determinants of the germination of J. ranarius seeds.Most of the seeds produced by J. ranarius are dormant on maturity and require pretreatment to stimulate germination.These results suggested that J. ranarius becomes established in vegetation gaps during spring through germination of seeds originating from a soil seed bank preserved over winter and brought into light by some local disturbance.Maximum seed germination was obtained in 25 mM L -1 NaCl salinity, similar to the minimum soil salinities at the study site.The high salinities (> 50 mM L -1 NaCl) typical of locally disturbed microsites, would not allow the seeds to germinate.However, inter-annual variations in climatic conditions may influence substrate salinity during the period of seed germination and, consequently, cause significant interannual fluctuations in population size.
Note: Each value is a mean ± S.D. of four replicates of 25 seeds.Data sharing the same letter in each column are not significantly different (Tukey's test, P < 0.05) Table5.Seed germination and rate of germination in J. ranarius seeds at value is a mean ± S.D. of four replicates of 25 seeds.Data sharing the same letter in each column are not significantly different (Tukey's test, P < 0.05)

Table 1 .
Germination percentage (mean ± S.D.) of the non-treated (freshly matured) and 24-week-cold-stratified seeds of J. ranarius at three thermo-periods in light and dark

Table 4 .
Germination percentage and index of germination velocity in J. ranarius

Table 6 .
Germination of the seeds of J. ranarius in relation to salinity and germination recovery in distilled water Note: Each value is a mean ± S.D. of four replicates of 25 seeds.Data sharing the same letter in each column are not significantly different (Tukey's test, P < 0.05) this species have non-deep physiological dormancy.Similar results were obtained by