Field Treatment Effects on Seed Germination and Early Growth Traits of Berseem Clover under Salinity Stress Conditions

Seed performance refers to a capacity of seeds to germinate under various environmental conditions and relates to a critical period of a plant’s life cycle which is eminently important in ecological and agronomic plant growth [1]. There is a general agreement that the performance and viability of seeds during their early stages of germination may be related to the conditions under which seeds are formed, developed, and matured [2]. As seed germination is the beginning of a plant’s life cycle, proper seedling emergence is critically important for a decent establishment of plant population [3]. Many researchers have reported that adverse Abstract


Introduction
Seed performance refers to a capacity of seeds to germinate under various environmental conditions and relates to a critical period of a plant's life cycle which is eminently important in ecological and agronomic plant growth [1]. There is a general agreement that the performance and viability of seeds during their early stages of germination may be related to the conditions under which seeds are formed, developed, and matured [2]. As seed germination is the beginning of a plant's life cycle, proper seedling emergence is critically important for a decent establishment of plant population [3]. Many researchers have reported that adverse environmental conditions affect crop growth and productivity, postponing the seed germination and reducing the germination rate [4][5][6][7]. Plants experience a variety of abiotic stresses such as high/low temperatures, drought and salinity stress during their lifecycles which have a profound effect on their viability, growth, morphology, and reproduction [8]. In arid and semi-arid regions, seed germination and emergence are considered as critical stages for plant establishment and crop growth, which can elevate the crops' density and yields Shaygan 2017. In these regions, soil salinization is detrimental to plant growth and productivity [9,10].
Plants usually take up salt (Na + and Clions) from water which is detrimental to germination [11][12][13]. Plant growth in saline dry and semi-dry soils can be further damaged by water scarcity due to high osmotic pressure, which prevents adequate water uptake [14]. (Tables 1 & 2) present the summary of recent reports onthe adverse effects of salt/drought stress on seed germination and establishment [15][16][17][18]. For instance, the seed germination, emergence rate and growth of young seedlings of sugar beet (Beta vulgaris L.) [19,20], Phaseolus mungo [21] and Dianthus chinensis L. [22] decreased by NaCl treatments. Quantitatively, the salinity condition decreased the seed germination of nut (atropha curcas L.) by 50% [23], coriander (Coriandrum Sativum L.) by 65.67%, cultivar Kalmiand Pant Haritma by 55.56% [24], oat (Avena sativa L.) by 60.87% [25] etc. Berseem clover (Trifolium alexandrinum L.) is one of the best forage sources for feeding live stock and is considered as anon-bloating and high-quality forage [26]. It is moderately tolerant to salinity compared to wheat and strawberry clover [27]. Despite the advantages, studies on the effects of salt stress have been mostly carried out on agricultural crops [28][29][30] (Tables 1 & 2). Thus, there is limited information on the effect of field treatments (seed production conditions) on subsequent salt stress tolerance at the germination stage and 4/14 seedling establishment of legume forage crops such as berseem clover. Water shortage and saline soil are known as one of the main natural hazards especially in arid and semiarid regions which delay seed germination and seedling establishment [31]. The aims of this three-year study are to determine factors responsible for the failure of germination and early growth characteristics of berseem clover seeds under saline/osmotic conditions. (Tables 1   & 2) present the results of recent studies on the reduction effect of salinity and drought conditions on different plants traits (Tables  1 & 2). Compared to these results, the present study proposesane co-friendly surfactant solution to mix with irrigation water at a very low rate of 1mg per liter (1ppm) to substantially reduce the severe effects of salinity and drought stresses on seed germination and growth of berseem clover.

Seed materials
Berseem clover (Trifolium alexandrinum L.) seeds obtained from our field experiments during the 2013-2015 growing seasons at the Research Farm of the College of Agriculture, University of Tehran, Karaj, Iran (N 35º56', E 50º58') [32] Daneshnia 2016. The climate at this site is considered as arid to semiarid with a longterm (50years) mean air temperature of 13.5°C, soil temperature of 14.5°C, and 262mm of mean annual rainfall. In the field experiments, three levels of irrigation were applied to the main plots, including normal irrigation I 100 (replenishment of 100% of weekly evaporation and plant water requirement), moderate irrigation (I 75 ) and limited irrigation (I 50 ). Two types of water treatment, including tap water and water+surfactant (added at a rate of 1ppm), were applied to the subplots. The experiments were carried out in split-split plots based on a completely randomized block design with three replications. Likewise, the irrigation treatments (I 100 , I 75 , and I 50 ) were scheduled based on the common practice of the area, once a week, when the plants reached the 4 to 6-leaf growth stage. The amount of required irrigation water was calculated using the following equation [26,32].
In is the volume of irrigation water (gallons), 0.623 is a constant, A is the canopy area (sq.ft.), Kc is the crop coefficient, ET0 is the weekly potential evapo transpiration (inches), and IE is the irrigation efficiency.

Salt stress germination test
The effects of six parental treatments (three irrigation levels and two water treatments) under salinity conditions were systematically studied during the germination process. These experiments were performed at the Seed Research Laboratory 6/14 .98 gr/kg NaCl in dematerialized water. 50 seeds from each parental treatment were placed in 9cm sterile Petri dishes which contained two what man No.1 filter papers, and then placed in seed germination at 20oC. 10ml solution from each test was poured onto the plate; the papers were altered once after every 2 days to prevent salt accumulation [33]. The 42 treatments (6 parental treatments and 7 salinity levels) were arranged on a factorial base with a completely randomized design and three replications. To maintain water and salt concentrations near the target levels, required dematerialized or saline water was added to the Petri dishes during the daily monitoring. The number of germinated seeds was recorded every 24 hours for two weeks. A seed was considered as germinated when an emerging radical was longer than 2mm. Then, the germination parameters, such as final germination percentage (G%) and weighted germination index (WGI) [34], were determined with higher weights assigned to the seeds that germinated early and less to those that germinated late. The final WGI values (after 14 days) were calculated using the method proposed by Bu [34] ( ) Here, n 1 , n 2 , ...., n 10 is the number of seeds that germinated on the 1 st , 2 nd and subsequent days until the 10 th day, respectively, N is the total days of germination process, and N´ is the total number of seeds placed in incubation. In Eq.1, the final WGI value is the product of the experimental value of WGI and the percentage of a live seeds in each treatment.

Early growth characterization of berseem clover seedlings under saline condition
Following the salt stress germination test, a series of pot experiments were conducted to evaluate the interaction between field treatment, salinity, and surfactant in the early growth of berseem clover seedlings. These experiments were performed at the Research Greenhouse of the College of Agriculture, University of Tehran. The greenhouse temperature, humidity and photosynthetic photon flux density (PPFD) for day/night were 25/20°C, 65/60%, and 300-600 µmol m −2 s −1 , respectively. The salinity treatments comprised of either intact soil from the field (soil with an electrical conductivity (EC) of 0.075 MPa and a primary osmotic potential of (-0.083MPa) or saline soil with an osmotic potential of -0.4MPa. The osmotic potential of salinity stress (-0.4MPa) was chosen on the basis of the optimum results of osmotic potential treatments from saline stress in the germination tests. In the following, the salinity level of the pots was kept at -0.4MPa throughout the experimental period. After a few preliminary tests, the EC of soil was adjusted to -0.4MPa by adding a solution of 5.3g NaCl+100 ml distilled water to all the pots which contained the saline-treated soil. All the pots were filled with 2.43kg of air-dried soil collected from the field.
The soil of the pots was sieved (with a mesh diameter of 1 cm) and loaded in plastic pots 15cm in diameter and 12cm in height.
The field soil texture was clay loam with the following physicochemical characteristics: pH=7.8, EC=2.31 dSm −1 , total nitrogen (N)=0.09%, available phosphorus (P)=8.87 mgkg −1 , and available potassium (K)=225mgkg −1 . The soil in each pot was fertilized with 1 liter of basal nutrient solution (BNS) which mainly consisted of both ammoniums (NH4) and nitrate (NO3) as the source of nitrogen [35]. To study the effect of surfactant on seedling establishment of parental seeds in saline environments, the pots were sprinkled with different levels of fresh water with/without surfactant additive. The soil was watered once every three days to replenish 100% of the soil moisture to ensure the plants were under no drought stress. The seeds used in this experiment were the same as used in the germination test. The nonionic surfactant marketed with the name Golden Irrig. Aid was provided by Aquatrols Corporations, New Jersey, USA (10% Alkoxylatedpolyols, 7% Glucoethers, Inter ingredient 83% water). The surfactant was applied at a rate of 1mg per liter (1ppm) to the irrigation water for water treatment purposes [36,37].

Statistical Analysis
The ANOVA technique with three replications was applied to the randomized design using the Proc GLM procedure SAS [38]. Mean comparison was implemented using Duncan's test at the 95% level of probability.

Salt stress germination test
The laboratory experimental results showed that the germination is completely suppressed by -1 and -1.2 MP a salinity stress in the presence of the highest concentrations of NaCl. As the salinity increases, the germination percentage followed a decreasing trend across all field treatments. However, berseem clover can tolerate NaCl up to -0.4 MPa saline stresses with a moderate decrease in its germination stage ( Figure 1). As the salinity stress increased from -0.4 to-0.6MPa, G(%) diminished significantly in all seed sources; the least (8.3%) and highest (33.3%) reductions were observed in I 50 (with and without surfactant) and full irrigation (I 100 ) (with and without surfactant)treatments, respectively. It is not able that under a higher salinity stress (-0.8 MPa), germination percentage increased across all parental seeds compared with -0.6MP a salinity stress. Parental seeds, collected from limited irrigation treatments ( I 75 and I 50 ) under various water treatments (with/without surfactant application), were influenced by the salinity level (Figure 1). Results indicated that the seeds, developed under drought stress during grain filling, were more tolerant to osmotic stress especially when the surfactant was added to the system. Under high salinity stress (-0.6MPa), seeds developed by severely limited irrigation (I 50 ) have more tolerance to the osmotic pressure.    This showed a reasonable germination percentage (75%) when the utilized surfactant was added to the system. As the level of salinity stress increased, the application of surfactant led to a better performance in the preservation of the seeds germination under harsh conditions. As shown in Figure 2 (a-d), seeds developed with irrigation water plus surfactant in both full and moderately limited irrigation treatments (I 100 and I 75 ) have better germination rates, compared with their counterpart treatments with no added surfactant under saline stress (-0.4 and -0.8MPa). In all parental treatments, as salinity stress intensity increased from 0 to -1.2MPa, WGI followed a decreasing trend (Figure 3). The highest WGI (87) was gained from I 100 +s under a moderate salinity stress (-0.4MPa). Under higher osmotic stresses, seeds produced by surfactant had a higher WGI across all parental treatments compared to their 8/14 counter parts from non-surfactant treatments. This is the indication of an enhanced germination rate in parental seeds upon the application of surfactant in irrigation water. The WGI in moderate water potentials of -0.2 and -0.4 MPa, induced by NaCl, was higher compared to that under more stressed levels ( Figure 3). Table 3 indicates that the seeds produced under deficit irrigation regimes (75% and 50%) with surfactant application (parental treatments) have better tolerance to salinity stress. This is also seen in Figure 4 where seeds, collected from plants grown with the irrigation treatments of I 75 and I 50 , have a better performance under salinity stress. The highest seedling height (10.2cm) is gained from I 75 +s where the pots are water reducing surfactant. Figure 5 presents the evidence where the application of surfactant significantly increased the berseem clover seedling height when subjected to saline stress treatments, compared to the control. Under saline conditions, surfactant application increased the seedling height by 43.7 and 31.5% in I 100 and I 75 , respectively, compared to control. In fact, after the application of a surfactant, berseem clover height significantly increased across all treatments. Figure 6 shows that the seeds developed under moderate water stress had a better seedling growth and development with surfactant application ( I 75 +s), compared to the control.    Seedlings from seeds developed under limited irrigation treatments (I 50 and I 75 ), had lower shoot/root ratios compared to those developed in I 100 treatment (Figure 7). This phenomenon verifies the higher vegetative growth potential of seeds developed under full irrigation treatment (I 100 ) (Figure 8). It is assumed that when the soil water content is inadequate to facilitate nutrient uptake by roots, plants face difficulties in absorbing the nutrients necessary for their growth. The higher vegetative growth potential of seeds is confirmed in (Figure 8) where the developed seeds have a lower root weight under a full irrigation treatment (I 100 ). As shown in Figure 8, when the pots were watered with a surfactant, the plants' shoot/root ratio significantly reduced by the favorable effects of surfactant on increasing the root growth under different growing conditions. Figure 9 clearly shows that seeds, developed under deficit irrigations ( I 75 and I 50 with surfactant), produced plants with higher shoot/root ratios compared to fully irrigated seeds. Using surfactant during the grain filling in parental plants induced a higher shoot/root ratio in developed seedlings, compared to their counterpart treatments.

Discussion
It was previously reported that the application of surfactant had a modifying effect against the adverse condition of severely limited irrigation (I 50 ) compared to full irrigation (I 100 ); this resulted in maintaining the forage quality and quantity [26,32]. In this study, we conducted systematic experiments to specify the response of parental seeds (from deficit irrigation field treatments) to salinity stress.

Increased Salinity Tolerance of Seeds Under Field Treatments
Drought and salinity create multifaceted stresses that cause a significant reduction in crops yield depending on the plant growth stage, stress duration and severity Muscolo 2014. Germination is the most critical and sensitive stage in the life cycles of plants [38,39]. Seeds, exposed to unfavorable environmental conditions such as salinity and drought, may exhibit a change in the subsequent seedling establishment processes [40,41]. Our results highlighted the effects of field treatments (drought stress and surfactant application) on increasing seed germination under salinity stress. This study demonstrated that the seeds, developed under drought stress during grain filling, are more tolerant to osmotic stress which agrees with the report from Maleki [42]. The inhibitory effect of NaCl is because of the osmotic stress imposed on the seed/plant during the early phases of germination. At the early stages of imbibitions, the seed respiration and metabolic processes are highly correlated with the water uptake [43]. A threshold level of hydration is required in the initial steps of germination and their subsequent radical elongation [44]. By ensuring turgor maintenance, 'osmotic adjustment' may reduce sensitivity to water stress or allow the plant growth to continue even at a slow rate under stress conditions [45].
Salinity can affect the germination of seeds by disruption to the structure of enzymes and other macromolecules, damaging the cell organelles and the plasma membrane, disrupting the respiration, photosynthesis, and protein synthesis [46][47][48]. In the salinity stress of -0.6 to -1.2 MPa, a sharp reduction of WGI was observed across all parental seed resources. These results corresponded with the findings of Cavallaroa [49], who asserted that, due to osmotic pressure, the seed imbibitions becomes slower; thus, seed metabolism activation is postponed, and germination is taken place with a delay [50]. The amount of WGI resulted from the osmotic pressure of -0.2 and -0.4MPa, induced by NaCl, is higher compared to that under more stress levels. This phenomenon can be the result of increases in the expression of aquaporin and Adenosinetri phosphates (ATPase), as well as a rise in protease and lipase activities [51,52].

Improved seedling growth under parental treatments
Plants grown in the field under I 75 + surfactant application had the highest seedling height (10.2cm). As explained by Maleki Farahani 2010, seeds germinate faster under severe water stress and have a shorter mean germination time (MGT), compared with the seeds produced under full irrigation. Probably, the increased physiological activity, due to the greater absorption of moisture by the treated seeds, is responsible for the improvement of germination and seedling. These results support the idea that the surfactant application in irrigating water in dry regions with salinity problems can maintain agricultural sustainability. The surfactant has a constructive modifying effect on the adverse effects of salinity stress compared with control (Table 3). According to a report from Demie [53], the positive effects of surfactant on improving the water movement and storage in soil could explain the results of the present study in the field. As such, surfactants have the positive effect of increasing the uniformity of water content in soil under drought conditions [54].
Researchers have reported that in response to soil salinity, the seedling growth, leaf area, root and shoot biomass are all reduced [55]. However, in the present study, the irrigation with surfactant successfully maintained a higher resistance of the seeds to saline stress conditions throughout the course of their growth and development ( Table 3). As presented in Figure 5, the application of surfactant increased the berseem clover seedling height significantly while subjected to saline stress treatments, compared to the control. When the soil wet ability is less than the water requirement of plants, the use of surfactant in combination with appropriate irrigation and soil cultivation practices improves the soil hydrological behavior; this also improves the irrigation efficiency and water conservation [56,57]. Reported that salinity reduced water potential in the leaves of clovers (Trifolium sp.); shortened the length and lowered the dry mass of the stem, and undesirably affected the length and conductivity of plant roots. However, in this study, when the pots were treated with a surfactant, the plants' shoot/root ratio significantly reduced under all growing conditions. This result can be explained by the favorable effects of surfactant on increasing the root growth in plants (Figure 8).
Poulter [58] stated that the surfactants decrease the surface tension of water and, in this way, the penetration of water into the soil profile becomes easier, thus, the wetting area of soil increases. With the addition of a surfactant, the plant roots can find water in a wider soil profile, which leads to a better vegetative and generative growth and a higher efficiency in water usage. Figures 7 & 9 are a witness for promoting effects of surfactant on better root development in this experiment [59]. These results clearly show the efficiency of surfactant in increasing both the aboveground and underground components of plants in semi-arid regions where water is limited [60][61][62][63][64]. Our data indicate that those plants irrigated by surfactant and produced from seeds developed with surfactant have a higher shoot/root ratio under saline stress condition. The authors are currently carrying out physiological studies to better understand the function of surfactants in plants and its role in seed germination by tracking the absorption of surfactant into plants and seeds ( Figure 10). This investigation is necessary to determine the contribution of surfactants to the metabolism of plants and seeds and other internal changes which lead to a better performance under saline stress conditions [65][66][67][68][69][70][71].

Conclusion
Improving the seed tolerance to saline soil or water shortage can enhance forage production and agricultural sustain ability in arid and semi-arid areas. In this study, the application of surfactant increased the tolerance of berseem clover seeds to salinity stress under full, I 100 , medium, I 75 , and severe, I 50 , irrigation treatments. Parental seeds developed under the severely limited irrigation in the field had a higher germination percentage (75%) compared to normal and moderately irrigated seeds [72,73]. Enhanced WGI of seeds, produced by surfactant across all parental treatments, indicated the desired effect of surfactant application. Plants treated by surfactant in irrigation water had a higher shoot/root ratio under saline stress condition. The results clearly showed the enhancement effect of surfactant application on improving seed performance and plant components in semi-arid regions where limited irrigation water and salinity are the main issues [74][75][76][77][78][79][80][81].