Comparing Salt Tolerance at Seedling and Germination Stages in Local Populations of Medicago ciliaris L. to Medicago intertexta L. and Medicago scutellata L.

Salt stress is one of the most serious environmental stressors that affect productivity of salt-sensitive crops. Medicago ciliaris is an annual legume whose adaptation to agroclimatic conditions has not been well described. This study focused on the salinity tolerance of M. ciliaris genotypes compared to M. intertexta and M. scutellata in terms of plant growth, physiology, and biochemistry. Salt tolerance was determined at both germination and early seedling growth. Germination and hydroponic assays were used with exposing seeds to 0, 50, 100, 150, and 200 mM NaCl. Among seven genotypes of M. ciliaris studied, Pop1, 355, and 667, were most salt tolerant. Populations like 355 and 667 showed marked tolerance to salinity at both germination and seedling stages (TI ≤1, SI(FGP) > 0 increased FGP ≥ 20% and SI(DW) < 0 (DW decline ≤ 20%); at 100 mM); while Pop1 was the most salt tolerant one at seedling stages with (TI =1.79, SI(FGP) < 0 decline of FGP ≤ 40% and with increased DW to 79%); at 150 mM NaCl). The genotypes, 306, 773, and M. scutellata, were moderately tolerant to salt stress depending on salt concentration. Our study may be used as an efficient strategy to reveal genetic variation in response to salt stress. This approach allows selection for desirable traits, enabling more efficient applications in breeding methods to achieve stress-tolerant M. ciliaris populations.


Introduction
In recent decades, the cumulative effects of climate change have led to water shortages, soil pollution and increased soil and water salinization. Loss of available arable land and increased Medicago are well adapted to many soil types ranging in texture from sandy loam to clays. The increment of annual Medicago as Medicago ciliaris into farming system in dry land is a possible mean to slow or stop the spread of dryland salinity. The selection of tolerant populations to salt stress and selecting near isogenic lines are a valuable resource for genetic studies which can be introduced in green farming systems.
To select salt-tolerant varieties, it is necessary to establish an effective screening method for identification of useful salt tolerance parameters for analysis at the molecular level [14,16,24]. An understanding of the responses of different crops to adverse conditions at different growth stages is essential for selecting spices or varieties able to sustain crop production under abiotic stress [16,25]. The goal of this investigation was to determine the effects of salinity stress on spontaneous forage legumes at germination and early growth stages through physiological characterization. We compared the seed germination and the early seedling growth of seven populations of Medicago ciliaris to one population of M. intertexta and one population of M. scutellata. The aim was to select salt-tolerant ecotypes of annual Medicago species that can be sown on saline soil or after using salt irrigation in order to restore soil fertility and reclaim marginal lands.

Effect of Salt on Seed Germination
The effects of salt stress on seed germination of seven populations of Medicago ciliaris and those of Medicago intertexta and Medicago scutellata were studied, and the main results are discussed below.

Salt Stress Effect on Final Germination Percentage (FGP)
In controls without salt treatment, the FGP varied from 62.5% in 667 to 100% in Pop2, Pop3, and M. intertexta (M. int), showing a significant variability among the populations. In the presence of NaCl, the FGP decreased significantly depending on the concentration: 56.7% at 50 mM NaCl in 773 and 21% at 200 mM NaCl in 355, M. scut, and Pop1. At 150 mM NaCl, the FGP was > 50% in Pop2, Pop3, and 355 of M. ciliaris and M. scut, indicating a better capacity for germination under saline conditions compared to the other populations ( Table 1). Analysis of the two-factor classification variance (salt treatment, populations) showed highly significant effects of salt treatment, populations, and their interaction on the final germination percentage of all populations (Table 2).   Analysis of variance with two factors (salt treatment, populations) showed a significant effect (p < 0.05) of salt and populations on the germination rate (T50), but their interaction was not significant (p > 0.05) ( Table 3). The T50 varied among populations and increased with increasing salt concentration while the level varied from 1.9 days at 50 mM to 2.3 days at 200 mM of NaCl (Table 3). In absence of salt, the T50 varied from 1.2 days in Pop2 and 773 to 2 days in 667. At 100 mM, the highest value of T50 was observed in Pop3 and 306 populations; hence, at 150 mM and 200 mM of NaCl, the T50 increased for the majority of populations (except Pop1 and 355 at 200 mM). The average T50 was significantly higher at 150 mM (2.6 days) compared to that of the control (1.7 days). Hence, Pop3, 355, and M. scut reached 50% of germination (T50) after >3 days at 150 mM.  Salt changes the osmolarity of the soil which affects the ability of the seeds to imbibe water, thus delaying germination.

Sensitivity Index to Salt Stress for Seed Germination (SI)
For a better screening of populations to salt stress tolerance at germination stage, we calculated the sensitivity index using the germination rate at all salt concentrations. Figure 1 showed a negative SI (FGP) for all populations at all salt concentration except for 355 which had a positive SI (≥ 20%) at all treatments and 667 at only 50 and 100 mM. The percentage of reduction of germination rate did not exceeded 35% for these populations (Figure 1). The values are the average of 10 seeds replicated 5 times.

Effect of NaCl on Growth
Responses of plants to salt stress is a result of the complex interaction among different morphological, physiological, and biochemical processes [26]. Globally, there is a genotypic difference in response of legume species and cultivars to salt stress. Our study showed a wide variation between populations in terms of dry weight, epicotyl, and radicle length in control and under salt stress conditions. Under salt stress, dry weight was lower than controls in all populations. Overall, the two-factor (treatment, population) ANOVA showed a highly significant effect of salt treatment and population on growth in terms of plant biomass, epicotyl, and radicle length (Table 4).

Effect of NaCl on Growth
Responses of plants to salt stress is a result of the complex interaction among different morphological, physiological, and biochemical processes [26]. Globally, there is a genotypic difference in response of legume species and cultivars to salt stress. Our study showed a wide variation between populations in terms of dry weight, epicotyl, and radicle length in control and under salt stress conditions. Under salt stress, dry weight was lower than controls in all populations. Overall, the two-factor (treatment, population) ANOVA showed a highly significant effect of salt treatment and population on growth in terms of plant biomass, epicotyl, and radicle length (Table 4). The ranking of populations according to their tolerance to NaCl showed a reduction of dry weight with increasing salt concentrations ( Figure 2).
The ranking of populations according to their tolerance to NaCl showed a reduction of dry weight with increasing salt concentrations ( Figure 2).
In control without salt, the plant dry weight was variable between populations, it was high in Pop2, 773, and M. scutellata ( Figure 2). In the presence of salt, the plant dry weight was very variable; it increased in some populations (Pop1, 667) and decreased in others (Pop2, Pop3, 306, 773, M. int, and M. scut). Salt stress significantly affected the biomass in M. int and M. scut. With the application of low doses of salt, a decrease in the weights of plants was observed in all populations except in Pop1, 355, 667. The variability in biomass production is essential for the selection of salinity-tolerant populations that can enhance recovery of saline soil fertility.
The classification of the different populations according to their tolerance to NaCl showed a regression of the biomass, length of epicotyl, and length of radicle ( Figure 2). However, the largest reductions were noted at 50 mM NaCl in Pop2 (50%) and in all populations at higher NaCl concentrations. On the other hand, 355 and 667 seemed to be less affected by salt, at least for this trait and displayed the lowest values of reduction. Pop1 did not show a reduction for this parameter, but there was an improvement. The percentage of reduction in this case for all populations varied from 10% at 50 mM to 50% at 100 mM for all populations, and to 50% or more for M. scut and M. int at 150 mM NaCl and higher. The same reduction was also found for all populations of M. ciliaris at 200 mM NaCl ( Figure 2).  In control without salt, the plant dry weight was variable between populations, it was high in Pop2, 773, and M. scutellata ( Figure 2). In the presence of salt, the plant dry weight was very variable; it increased in some populations (Pop1, 667) and decreased in others (Pop2, Pop3, 306, 773, M. int, and M. scut). Salt stress significantly affected the biomass in M. int and M. scut. With the application of low doses of salt, a decrease in the weights of plants was observed in all populations except in Pop1, 355, 667. The variability in biomass production is essential for the selection of salinity-tolerant populations that can enhance recovery of saline soil fertility.
The classification of the different populations according to their tolerance to NaCl showed a regression of the biomass, length of epicotyl, and length of radicle ( Figure 2). However, the largest reductions were noted at 50 mM NaCl in Pop2 (50%) and in all populations at higher NaCl concentrations. On the other hand, 355 and 667 seemed to be less affected by salt, at least for this trait and displayed the lowest values of reduction. Pop1 did not show a reduction for this parameter, but there was an improvement. The percentage of reduction in this case for all populations varied from 10% at 50 mM to 50% at 100 mM for all populations, and to 50% or more for M. scut and M. int at 150 mM NaCl and higher. The same reduction was also found for all populations of M. ciliaris at 200 mM NaCl ( Figure 2).
As roots are in direct contact with the soil solution, the root and shoot length provides an important clue to the response of plants to salt stress. A reduction in epicotyl and radicle length at all salt doses was noted in all populations except in Pop1, 355, and 667, where the reduction did not occur until salt concentrations reached 100 mM NaCl (Figures 3 and 4).
Plants 2020, 9, 526 7 of 23 As roots are in direct contact with the soil solution, the root and shoot length provides an important clue to the response of plants to salt stress. A reduction in epicotyl and radicle length at all salt doses was noted in all populations except in Pop1, 355, and 667, where the reduction did not occur until salt concentrations reached 100 mM NaCl (Figures 3 and 4).
The ranking of the different populations according to their tolerance to salinity showed a regression of the epicotyl length of the plantlet for the different populations studied ( Figure 3). However, Pop1, 667, and 355 were less affected by salt, at least for this feature, and displayed the lowest values for plant growth reduction, between 10% and 30%, at all NaCl doses. In the case of population Pop1, this parameter seemed to be not affected by salt concentration.

The Sensitivity Index to Salt Stress for Growth (SI), Noted SI(DW)
To select the populations for their tolerance to salt stress, we calculated the index of sensitivity (under hydroponic conditions) using the dry weight of the plants after growth at 50, 100, 150, and 200 mM NaCl ( Figure 5). At all NaCl concentrations, Pop1 had a positive sensitivity index SI(DW) (SI ≥ The ranking of the different populations according to their tolerance to salinity showed a regression of the epicotyl length of the plantlet for the different populations studied ( Figure 3). However, Pop1, 667, and 355 were less affected by salt, at least for this feature, and displayed the lowest values for plant growth reduction, between 10% and 30%, at all NaCl doses. In the case of population Pop1, this parameter seemed to be not affected by salt concentration.
Increasing salt concentration in Hoagland's solution decreased significantly the seedling root length (RL) in all populations ( Figure 4). For radical length, a large variation between populations was observed in absence and in presence of salt ( Figure 4). In control conditions, Pop2 had the longest radical while in presence of salt, 773 had the longest radical at 50 mM NaCl. The highest reduction of radical length was observed in Pop1 and 773 (50%) at the highest concentrations (100 and 150 mM) while the reduction is less in Pop3, 306, and 355. The calculations of plant biomass production throughout the experimental period revealed that Medicago ciliaris populations, as well as M. int and M. scut were able to maintain growth at all salinity levels and even increased it for some populations like Pop1 at low NaCl concentrations ( Figure 2). The values of SI (DW) for Pop1 were positive ( Figure 5), indicating an enhanced growth compared to control plants and showing that Pop1 is the most tolerant population of Medicago ciliaris compared to others at the seedling stage, and this is probably due to its origin from saline soil.

The Salt Tolerance Index at Early Growing Stage (TI)
The one-way ANOVA revealed significant differences among the studied populations of M. ciliaris in all growth parameters measured with respect to their salt tolerance index and for different NaCl concentrations (p > 0.05). Pop1 had the highest tolerance index (TI) at all salinity levels, almost two times that of the rest of populations, while 667 and 355 showed intermediate values ( Figure 6).

Chlorophyll a Content
Analysis of two-way ANOVA (treatment, population) showed a significant effect of salt stress and population (p < 0.0001 and p < 0.0001 respectively) as well as their interaction on Chl a content (Table 5). In the absence of salt, the Chl a content varied from 0.3 mg g −1 FW in 355 and M. int to 2.3 mg g −1 FW for 306. The population 306 was the richest population in Chl a. In general, the value decreased at 50 mM NaCl compared to the control, but there was some increase at 100 mM in most populations. The lowest values were observed at 150 and 200 mM NaCl in all population except that of M. scut which had lower Chl a content in all salt treatments, most likely due to its naturally occurring yellow leaf colorant; this is related to the yellow leaf color of population M. scut in nature.

Chlorophyll b and Total Chlorophyll Content
The population 667 was the richest in Chl b. Salt stress (50 mM) decreased Chl b in all populations except Pop2 and 306 and increased it at 200 mM in all populations except in Pop3 (Table 5). Two-way ANOVA analysis of Chl b and total chlorophyll content demonstrated that the salt stress effect was highly significant. The effect of population and interaction with salt concentrations was also Plants 2020, 9, 526 9 of 23 highly significant (p < 0.01). Populations Pop3 of M. ciliaris and M. scut presented the lowest values of Chl a, Chl b, and total chlorophyll (Table 5).

The Salt Tolerance Index at Early Growing Stage (TI)
The one-way ANOVA revealed significant differences among the studied populations of M. ciliaris in all growth parameters measured with respect to their salt tolerance index and for different NaCl concentrations (p > 0.05). Pop1 had the highest tolerance index (TI) at all salinity levels, almost two times that of the rest of populations, while 667 and 355 showed intermediate values ( Figure 6).

Chlorophyll a Content
Analysis of two-way ANOVA (treatment, population) showed a significant effect of salt stress and population (p < 0.0001 and p < 0.0001 respectively) as well as their interaction on Chl a content (Table 5). In the absence of salt, the Chl a content varied from 0.3 mg g -1 FW in 355 and M. int to 2.3 mg g -1 FW for 306. The population 306 was the richest population in Chl a. In general, the value decreased at 50 mM NaCl compared to the control, but there was some increase at 100 mM in most populations. The lowest values were observed at 150 and 200 mM NaCl in all population except that of M. scut which had lower Chl a content in all salt treatments, most likely due to its naturally occurring yellow leaf colorant; this is related to the yellow leaf color of population M. scut in nature.

Proline Content
Proline content was measured in leaf tissue at an early vegetative stage to determine its association with salt tolerance. Two-way ANOVA analysis (salt stress, population) showed a significant effect of salt and population on proline content (p < 0.0001) and their interaction (p < 0.001).

PCA Analysis for Traits
The PCA analysis revealed variation among the traits for each factor, and a loading value above 0.5 was considered significant. The first component (PC1) and second component (PC2) explained 69.6% of the total variance, and data were used to make a score plot (Figure 9). NaCl concentrations less than 150 mM are on the right side of the score plot, and the populations are closely spaced, indicating that their responses to salinity were similar. This is attributable to their similar geographic origin (north Tunisia), climate, and environmental conditions and confirms that the environment of origin influences the effect of salinity on adaptation among different genotypes. The loading plot of all the measured variables included in the PCA is shown in Figure 9. The variables with the highest scores in PC1 at positive side, are plant biomass production (DW), tolerance index (TI), epicotyl length (SL), radicle length (RL), and proline content (Pro). In PC2, the highest score was for chlorophyll content   ANOVA two-way (salt stress, population) analysis showed a significant effect of salinity and population on sugar content (p < 0.0001) and their interaction effect (p < 0.001). Carbohydrate content in salt-stressed plants increased in all populations of M. ciliaris (except in 306 and 355) and M. int and was dependent of salt concentration. While for M. scut, sugar content increased significantly at 200 mM (Figure 8), in population 306, sugar content decreased with increasing salinity, and in 355, there is no significant change in sugar content according to salinity (Figure 8). A positive correlation between sugar content and plant dry weight (DW) (r = 0.37; p = 0.03; n = 32) was observed.  The PCA analysis revealed variation among the traits for each factor, and a loading value above 0.5 was considered significant. The first component (PC1) and second component (PC2) explained 69.6% of the total variance, and data were used to make a score plot (Figure 9). NaCl concentrations less than 150 mM are on the right side of the score plot, and the populations are closely spaced, indicating that their responses to salinity were similar. This is attributable to their similar geographic origin (north Tunisia), climate, and environmental conditions and confirms that the environment of origin influences the effect of salinity on adaptation among different genotypes. The loading plot of all the measured variables included in the PCA is shown in Figure 9. The variables with the highest scores in PC1 at positive side, are plant biomass production (DW), tolerance index (TI), epicotyl length (SL), radicle length (RL), and proline content (Pro). In PC2, the highest score was for chlorophyll content (Chl) and sugar content (

Discussion
In this research, we studied the effect of salt stress on physiological and biochemical parameters in some populations of Medicago ciliaris compared to populations of M. intertexta and M. scutellata. Analysis of the final germination percentage (FGP) and germination rate (T50), showed significant variability among populations with respect to salt concentration. These results are consistent with those of [27] who showed significant variability in salinity tolerance in Phaseolus at the germination

Discussion
In this research, we studied the effect of salt stress on physiological and biochemical parameters in some populations of Medicago ciliaris compared to populations of M. intertexta and M. scutellata. Analysis of the final germination percentage (FGP) and germination rate (T50), showed significant variability among populations with respect to salt concentration. These results are consistent with those of [27] who showed significant variability in salinity tolerance in Phaseolus at the germination stage. A study of the salt tolerance of ten native and six exotic potato genotypes in Bangladesh measured stress tolerance trait indices (STTIs) in four groups-tolerant, moderately tolerant, sensitive, and very sensitive-and the data were useful for improving potato yield [28]. FGP decreased significantly depending on salt concentration: 56.7% at 50 mM NaCl in 773 and 21% at 200 mM NaCl in 355, M. scut and Pop1. Salt stress from 150 mM NaCl affected FGP in most populations except Pop2, 355, and M. int which can maintain an FGP > 50%, thus showing a good capacity for germination in the presence of salt. However, above 100 mM, this rate dropped to less than 67.5% for some varieties while for others, the germination rate exceeded 70%. The presence of NaCl also resulted in a significant decrease in germination in five legume species [29]. On the contrary, seeds exposed to a low concentration of 25 mM NaCl showed an increase in percent germination and germination index [30]. The coefficients of genotypic determination of six natural populations of a Fabaceae plant (Stylosanthes humilis) from three ecogeographic regions were high for five of the populations (except population Tamandaré) both for germination percentage (0.89) and germination rate (0.79), indicating the possibility of selection for salt tolerance in these populations [31]. The germination rate (T50) of our study populations increased with increasing salt concentration and varied among the populations, ranging on average from 1.9 to 2.6 days. It was low at (50-100 mM NaCl) in Pop1, Pop3, and 306 and did not differ significantly between populations at 150 and 200 mM. However, in two sensitive glycophytes, Phaseolus vulgaris and Glycine max, salt had a depressive effect that slowed the T50 from 50 mM NaCl with critical thresholds at 150 and 200 mM NaCl [29]. A study of tomato cultivars showed that the genotypes BARI Tomato-2, Mintoo, and Unnoyon were more tolerant to high salt stress in terms of T50 and root and shoot characteristics than other genotypes [32].
Excessive NaCl concentrations may cause seeds to lose viability during salt exposure, thereby slowing the germination process [33,34].
According to [29], the germination rate can be used as an early criterion for selecting salt-tolerant species and populations of legumes. Among our populations, Pop2, 355, 667, and M. int were the most tolerant at germination, with the highest germination percentage and germination ability. However, the kinetics of germination at a concentration of 100 mM NaCl showed a high variability among the genotypes of different populations (data not shown). This genotypic variability, which relates to the speed of germination, is a function of the response to salt stress. Data obtained after ten days of salt exposure indicated that the final seed germination percentage decreased significantly with increasing salinity (Table 1). Thus, not all seeds had the same capacity to tolerate salinity. The genotypes Pop2, 355, 667, and M. int showed positive or near zero sensitivity indices for germination percentage regardless of treatment compared to other populations. The germination tolerance indices (TI) for the three experimental replicates showed that the 355 and 667 genotypes were the most vigorous, while the SI of the others did not exceed 50% until 100 mM NaCl. Applying two-way ANOVA showed a significant effect of genotype and treatment on SI and a highly significant effect of treatment on the final percentage of germination and seedling germination rate (T50). Some populations, such as Pop1, were salt stimulated with increased germination speed and with positive SI after pre-treatment salinity stress, retarding germination and increasing the percentage of recovery of seed germination [35].
Growth parameters including plant biomass (DW), epicotyl length, and root length were studied under salt stress condition. The DW of plants from M. ciliaris, M. int, and M. scut populations varied with salinity concentration. A decrease in the biomass of the plants with exposure to salt was noted in most populations (except Pop1, 355, and 667) whose percentage of reduction in dry weight increased from 50 mM to 200 mM NaCl. At 200 mM, a depressive effect of salt on germination was noted for all population of Medicago ciliaris, M. scut, and M. int. Moreover, a reduction in the length of the epicotyl and the radicle was noted in all populations for all doses of salt, except for Pop1, and a slight reduction for 667 and 355. The detrimental effect of salt occurs at the level of the whole plant and is caused by limiting leaf area expansion to stomatal conductance [36]. In terms of growth, the effect of salt stress is a reduction in biomass and in nutrient homeostasis [37]. This effect is common in glycophytes [38], where the observed decrease in vegetative growth can be explained by an increase in osmotic pressure caused by NaCl, which reduces water absorption by the roots. Plants thus adapt to salt stress by reducing their growth in order to avoid damage [10]. The effects of salinity on the growth of seedlings grown under controlled conditions depend on several factors. They vary according to the NaCl concentration, the species, the provenance, the vegetative stage, and the part of the plant [8]. The effects of salinity are mainly manifested by a slower vegetative growth [37]. The reduction in shoot and root growth by salt stress can be a consequence of unbalanced nutrient uptake by the seedlings; and the inhibition of shoot and root elongation is due to diminished water and essential mineral uptake by plants [15,30]. In general, salt stress inhibits the growth of shoots more than roots. Mahdavi and Sanavy [39] observed significant differences among grass pea (Lathyrus sativus) cultivars under salt stress in terms of coleoptile and root growth, indicating that genetic variation exists within the cultivars and that salt inhibited coleoptile growth more than root growth.
Salt sensitivity index (SI) is a reliable parameter for estimation of salt stress level and identification of potential salt-tolerant cultivars. Considering the SI measured in this study, the best results were observed in 355 and 667, whereas Pop1 had the highest weight for all salt levels, thus showing good tolerance to salt stress at the young seedling stage compared with other populations. Similarly, the genotypes 355 and 667 had lower yield reduction under stress condition and root growth was maintained under salt stress, which helps populations or genotypes to tolerate salt stress by maintaining shoot growth toward salt dilution, by salt exclusion uptake, limiting Na accumulation in shoots and more vigorous shoot growth [37]. In contrast, [40] showed salt stress affected germination, radicle elongation, chlorophyll content, and Net assimilation, indicating that alfalfa (Medicago sativa, China seeds) was relatively sensitive to salt stress during germination and early seedling growth stage.
Medicago ciliaris had higher root indices, highlighting the great importance of roots in salt tolerance. The percentage of reduction in dry matter, generally considered as an index of the sensitivity of plants to salt stress, showed that the 100 mM NaCl concentration was insufficient to cause a relative reduction under 50% compared with controls [41]. Even at lower concentrations like 50 mM, many species showed marked reductions.
Screening for salinity tolerance in diverse alfalfa germplasm under non-stress conditions and salt stress (0 mM and 150 NaCl) [42] showed that highly significant variation was found among alfalfa genotypes and salt treatments for fresh and dry matters. Three tolerant genotypes from coastal origin, which displayed the best tolerance, used an Na + "inclusion mechanism" to prevent salt stress. The decrease in growth of aerial parts could be explained by disturbances in tissue growth regulator levels, particularly secondary metabolites, but also by a decrease in the photosynthetic capacity caused by the decrease in stomatal conductance of CO 2 under salt stress [13]. In all cases, this reduction in the growth of the various aerial parts is considered an adaptive strategy necessary for the survival of plants exposed to salinity [25]. This allows the plant to store the energy needed to cope with stress and to reduce the irreversible damage that occurs when the threshold of lethal concentration is reached [43]. Bukhari et al. [44] showed that, in general, environmental stress affected germination and growth processes of plants.
Analysis of biochemical parameters showed that chlorophyll content (Chl a, Chl b) varied with population and salt levels. According to [45], a reduction of chlorophyll a and chlorophyll b was reported in stressed chili plants compared to the control. The reduction of chlorophyll a and chlorophyll b may be related to the sensitivity of their biosynthesis to sodium chloride, although Chl b is less affected. Salt stress plays a role in decreasing chlorophyll activity and inhibits the synthesis of 5-amino-levulinic acid [46]. The reduction of CO 2 in the leaves is associated with an inhibition of photosynthesis [47].
Our results showed that increasing salt stress increased accumulation of sugars in the leaves, but the effect depended on the population. Pop1 of M. ciliaris accumulated more soluble sugars in the leaves than M. scut and M. int at 200 mM NaCl. The same result was found for Medicago sativa, in which sugar accumulation increased with increasing salt concentration. The concentration of soluble sugars in the leaf tissues of stressed plants is always greater than in stems and plays a dual role in stressed plants as a tolerance characteristic, since they participate in metabolic events and dehydration [48]. According to [49], high levels of osmolyte accumulation in plants were well correlated with stress tolerance through the escape of radicals and protective enzymes. Soluble sugars are indicators of the degree of stress [50]. Because of their significant increase in severe stress, metabolic sugars such as glucose, galactose, sucrose, and fructose are determining factors in the osmotic adjustment for resistance to various stresses [49].
There was an increase in proline in leaves with increasing salt level in populations, except for M. int, M. scut, 667, and 773, which had the lowest proline concentration under salt stress. The highest content was recorded at the 100 mM dose in Pop1 (saline site geographic provenance, Raoued el Hessian) and to a lesser extent for the content in leaves of Pop2, Pop3, and 355. At 200 mM NaCl, the proline content in all populations was comparable. In both control and stressed plants, this amino acid was preferentially concentrated in leaves at significant levels depending on populations. Our results are consistent with those of [39], who showed that proline levels rose with increased salinity level, which correlated to reduction of hypocotyl, radicle length, and dry and fresh weight of different cultivars of grass pea (Lathyrus sativus). The accumulated proline could play an osmotic role in the regulation of cytoplasmic pH or constitute a reserve of nitrogen used by the plant after the period of stress [51]. It neutralizes the ionic and osmotic effects of salt accumulation in the vacuole. Increased proline synthesis is considered a stress marker and an adaptive action taken by plants. It acts as a protective soluble compound without exerting a toxic effect as is the case with ions. Proline is involved in the detoxification of active forms of oxygen and the stabilization of proteins [52,53]. Its content is correlated with salt tolerance [54]. The ability to maintain a high concentration of proline in tissues as well as adequate levels of chlorophyll and water content has been suggested for use as tolerance markers for an alfalfa population tested in different geographic sites of Morocco [33].
Under the salt stress conditions of our experiments, it was established that tolerance at the germination stage did not in all cases reflect what happened later at the vegetative and early plant establishment stages. The population Pop1 was not the most tolerant to salt during germination but showed a significant tolerance towards NaCl at the seedling stage. In contrast, Pop2 is tolerant at germination stage but is not tolerant at early seedling stage. Pop 355 and 667 were recognized by a very marked tolerance to salinity at the germination stage and retained this performance at the advanced stages. However, germination under salt stress is not sufficient to identify salt-tolerant species [40]. In this context, many authors have shown that the response to salinity varied according to the stage of development of the plant [36]. However, germination and the early stages of growth are the most sensitive phases [40].
The traits examined in this study have shown significant variability for most growth parameters and can be used as selection indices to identify germplasm for salt tolerance. By combining of features across all the studied population, natural diversity allowed them to maintain a good relative biomass and have different mechanisms to resist high salinity. PCA analysis performed on annual Medicago populations disclosed that tolerant populations had long epicotyls and radicles and produced more proline, while sensitive ones were rich in sugars. Almost all populations on the right side of the graph were tolerant to 50 to 150 mM NaCl and sensitive to 200 mM, while the populations on the left side were sensitive to 200 mM. This result is accommodated with the PCA score plot (Figure 9), which indicated that 306, 773, and M. scut were moderately tolerant to sensitive to salt stress, depending on salt stress. Because of their high content of proline and salt tolerance index, Pop1, 355, and 667 can be recommended as the most promising local populations, the seeds of which can germinate under salt stress up to 150 mM NaCl. As the need to adapt to climate change continues, the salt tolerant populations identified in this study should serve as viable forage options and future breeding material for areas with excessive salt in the irrigation water and to reclaim arable crop land that has been lost due to high salt content.

Plant Genotypes and Seed Sources
The study was carried out at an experimental station in National Institute of Agriculture

Germination Assays
The experimental design involved nine populations of M. ciliaris, M. intertexta, and M. scutellata (10 seeds from each population replicated 5 times at each treatment of salt stress). Seeds of homogeneous size were scarified with liquid nitrogen in order to facilitate water imbibition. Then they were transferred to Petri dishes (10 seeds/dish) on imbibed-filter paper type Whatman No. 2 moistened with 10 mL distilled water for the control and 10 mL of the saline solution with 50, 100, 150, and 200 mM NaCl (3,6,9,12 g NaCl/L) ( Figure 10). To avoid evaporation, the edges of the Petri dishes were tightly sealed with Parafilm and placed in a germination chamber in the dark for 72 h at 22 ± 2 • C, then transferred into the growth chamber at 22 ± 2 • C under a 12 h daylight photoperiod [43]. Petri dishes were controlled at one-day intervals for solutions content. Germinated seedlings were counted every two days for ten days. Seeds were considered to be germinated when radicles were at least 2 mm long ( Figure 10). total number of seeds germinated during the ten days over the total number of seeds tested for germination was calculated. The germination rate (GR), the number of days to reach 50% germination (average germination time, T50) was also determined, as it expresses the germination energy in terms of the depletion of the seed reserves.

Hydroponic Culture Assay under Salt Stress
The hydroponic assay for testing the effects of salinity on seedlings was conducted using the nutrient solution of [57]. For this test, 25 seeds (5 plants / n=5 repetitions for each salt treatment) (nine populations) were germinated and monitored regularly until the appearance of the first trifoliate leaves, at which stage the salt stress was applied. The seedlings were first transplanted into test tubes containing the nutrient solution and different concentrations of NaCl (0, 50, 100, 150 and 200 mM) and then transferred into plastic containers containing the Hoagland's saline solution and equipped with an aeration system. Salt stress was continued for three days.

Parameters Measured
Seedling shoot length (SL), seedling root length (RL), and dry weight of the whole plant (DW: mg plant -1 ), the parameters typically used for plant growth assessment under salt stress, were measured at the end of the stress period. The dry weights of each whole seedling's weight were determined after drying samples at 70 °C in an oven until a constant weight was achieved.

Proline Content
Free proline was quantified following the protocol of [59]. Approximately 0.5 g of plant tissue was homogenized in 5 mL of 3 % aqueous sulphosalicylic acid filtered through filter paper. Two milliliters of the supernatant was heated with 2 mL of acid ninhydrin, and 2 mL of glacial acetic acid was added in a water bath at 100 °C for 1 h. The reaction was stopped in an ice bath. For extraction, At the end of the germination period, the germination percentage, rate of germination, and the mean germination time (MGT) under different salt concentration were calculated. The rate of germination was estimated by using a modified Timson index of germination velocity: where GP is the percentage of seed germination at each day, and T is the total germination period [34]. Germination percentages were measured every day including the ending day using radicle expulsion (2 mm long) [56]. The final germination percentage (FGP), the cumulative percent of the total number of seeds germinated during the ten days over the total number of seeds tested for germination was calculated. The germination rate (GR), the number of days to reach 50% germination (average germination time, T50) was also determined, as it expresses the germination energy in terms of the depletion of the seed reserves.

Hydroponic Culture Assay under Salt Stress
The hydroponic assay for testing the effects of salinity on seedlings was conducted using the nutrient solution of [57]. For this test, 25 seeds (5 plants/n = 5 repetitions for each salt treatment) (nine populations) were germinated and monitored regularly until the appearance of the first trifoliate leaves, at which stage the salt stress was applied. The seedlings were first transplanted into test tubes containing the nutrient solution and different concentrations of NaCl (0, 50, 100, 150 and 200 mM) and then transferred into plastic containers containing the Hoagland's saline solution and equipped with an aeration system. Salt stress was continued for three days.

Parameters Measured
Seedling shoot length (SL), seedling root length (RL), and dry weight of the whole plant (DW: mg plant −1 ), the parameters typically used for plant growth assessment under salt stress, were measured at the end of the stress period. The dry weights of each whole seedling's weight were determined after drying samples at 70 • C in an oven until a constant weight was achieved.

Chlorophyll Content
Small lamina discs were cut from fresh apical leaves (100 mg of fresh samples), added to 10 mL of 95% acetone, and then kept in the dark at 4 • C for 48 h. Calculation of chlorophyll a (Chl a) and chlorophyll b (Chl b) contents was done by applying the following equations with the appropriate extinction coefficients [58]:

Proline Content
Free proline was quantified following the protocol of [59]. Approximately 0.5 g of plant tissue was homogenized in 5 mL of 3 % aqueous sulphosalicylic acid filtered through filter paper. Two milliliters of the supernatant was heated with 2 mL of acid ninhydrin, and 2 mL of glacial acetic acid was added in a water bath at 100 • C for 1 h. The reaction was stopped in an ice bath. For extraction, 4 mL of toluene was added, and samples were mixed vigorously for 15 to 20 s. Samples were then set aside to allow separation of the organic and aqueous phases. The absorbance of the organic phase was measured at 520 nm with a UV-VIS spectrophotometer (UV-160, Shimadzu, Tokyo, Japan) using toluene as a blank. Proline concentration was determined from a standard curve, and the value was determined using calibration curved expressed based on fresh weight as µmol proline g −1 FW.

Total Sugars
The amount of reducing sugars was estimated by a modified method of [60]. One milliliter of alcoholic extract 80% neutral ethanol was mixed with 1.0 mL of a freshly prepared mixture of 25 parts of reagent "A" (4 g CuSO 4 , 5H 2 O, 24g anhydrous Na 2 CO 3 ) and 1 part of reagent "B" (16 g Na-K-tartarate and 180 g anhydrous Na 2 SO 4 dissolved in 1 L distilled water) (25:1). The samples were incubated in a boiling water bath for 20 min and cooled under running water. One milliliter of arsenomolybdate reagent (25 g Ammonium molybdate dissolved in 450 mL distilled water, 3 g sodium arsenate dissolved in 25 mL distilled water, 21 mL concentrated HCl) was added, and the color allowed to develop for 15 min. The mixture was then diluted to 10 mL and the absorbance at 500 nm was measured with a spectrophotometer. Reagent blanks were used to adjust absorbance to zero. Reducing sugar content was estimated by using a standard curve prepared with dextrose.

Salt Tolerance Index
This value of salt tolerance index (TI) was calculated as the ratio of the total dry weight of plants subjected to different salt concentrations to the total dry weight of control plants [41,61].
Rather than comparing absolute growth rates, salinity tolerance is expressed as a relative reduction in the mass of material as a percentage of control, which indicates the relative vigor of the plant under salt stress conditions. Sensitivity Index (SI%) reduction = 100 × ((salt treatment -control)/control) SI(FGP): sensitivity index for germination SI(DW): sensitivity index for seedling growth

Statistical Analysis
Physiological properties of plants in individual populations were compared using factorial ANOVA and Tukey's post hoc test (p < 0.05) using the Statistica software ver. 12 (Statsoft Inc. 2012). Canoco 5 [62] was used for PCA (Principal Component Analysis) calculated from centered (not standardized) data. This analysis was appropriate for comparing differences between annual Medicago populations in response to salt stress.

Conclusions
To develop salt-tolerant varieties, it is necessary to establish an effective screening method that allows accurate identification of salt tolerance parameters useful for analysis at the molecular level for genetic breeding. Germination and the early growth stages would be the most sensitive phases that are affected by salinity. Varieties tolerating salinity at the germination usually continue resistance in later stages, but sprouting is not sufficient to identify salt stress-tolerant genotypes. Results showed a highly significant effect of salt stress, populations, and their interaction on the germination parameters. For biochemical parameters, the significant effect of salt on chlorophyll b, total chlorophyll, leaf soluble sugars content, and proline content, as well as a significant effect on chlorophyll a, were observed. The morphological parameters were greatly influenced by salinity exposure. Negative correlations were found between soluble root sugars and proline and between soluble leaf sugars and epicotyl length, while positive correlation was obtained between proline and plant weight. In addition, PCA analysis showed that the tolerant populations had longer epicotyls and radicles and produced more proline, while the sensitive ones were rich in sugars. According to these results, three populations of M. ciliaris (Pop1, 355, and 667) among the seven studied were found to be more salt tolerant and could be recommended as the most promising for germination and growth under moderate salt stress. Indeed, Pop1 is not the most tolerant to salt stress during the germination stage, but it did have significant tolerance towards NaCl at the seedling stage. As different genetic mechanisms are likely involved in salt tolerance at the various developmental stages, Pop1 may still serve as a valuable genetic resource for future breeding material. On the other hand, populations 306, 773, and M. scut were identified as moderately tolerant to sensitive to salt stress depending on salt rates.
Annual Medicago as pasture legumes can be sown in dry lands of arid and semi-arid regions of the world, where often soil salinity and climactic conditions limit the options available for forage production. Alfalfa from Northern Tunisia can be successfully raised in dry and rain-fed farming systems. Our results confirmed that the study of germination and early growth of spontaneous legumes may be used as an efficient strategy to reveal genetic variations in these plants that make them resistant to salt stress. This approach allows selection of desirable genotypes, thus enabling more efficient application of various breeding methods to achieve stress-tolerant annual alfalfa genotypes.
Further studies at later stages, such as the reproductive phase, and assessment of their performance under field conditions are needed to evaluate the effect of salt stress on yield. A deeper understanding of the genetic mechanisms through a transcriptomic approach would facilitate the selection of useful germplasm as sources for a salt stress tolerance breeding program. The Pop1, 355, and 667 populations should be retained for their salt tolerance and should be tested at field conditions and under combined stresses such as salt and drought.