Examining the Physiological Traits of Callus Tissues from Endosperm- Supported Mature Embryos in Common Wheat (Triticum aestivum L.) under In Vitro Salt Stress Conditions

Examining the Physiological Traits of Callus Tissues from Endosperm- Supported Mature Embryos in Common Wheat (Triticum aestivum L.) under In Vitro Salt Stress Conditions | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Examining the Physiological Traits of Callus Tissues from Endosperm- Supported Mature Embryos in Common Wheat (Triticum aestivum L.) under In Vitro Salt Stress Conditions Fatine Mouhssine, Houda Elyacoubi, Hamada Imtara, Rabab Ez-zriouli, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4368371/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Salt tolerance is a much-needed potential in cereal crops. To date, numerous research protocols have focused on establishing selection programs to increase tolerance and productivity of crops in salt-stressed areas. In this study, we aimed to induce callus from endosperm-supported mature embryos of five common wheat varieties (Nassim, Wissam, Wafia, Rajae, and Tigre) subject directly to various increasing NaCl concentrations (0–9 g/L) under in vitro culture process. We chose callus growth, organic solutes accumulation, and ion content as main traits to evaluate the impact of salinity on stressed calli. Our findings indicate that the accentuated salinity pressure leads to a substantial elevation of Cl − , Na + , proline and soluble carbohydrates content, along with a reduction in callus growth, potassium accumulation, and K + /Na + ratio. Yet, the performance under salt stress was significantly dependent on the varietal effect. At the highest concentration, the marked values of these solutes (12.06 µmol/g FM of proline; 43 µmol/g FM of Total soluble sugar; 31.15 mg/ g DM Na + ; 12.82 mg/g DM of Cl − , 12.61 mg/g DM of K + and 0.40 K+/Na+) were respectively recorded by Rajae. Principal component analysis (PCA) first classified Rajae as the most tolerant followed by wafia as tolerant, while Wissam and Tigre were ranked as sensitive. Whereas, the variety Nassim showed a moderate tolerance. Besides the K+/Na + Ratio and salinity tolerance index (STI), the PCA analysis has shown that all the studied physiological criteria seem worthwhile for better discrimination of the varieties tested according to their reaction to salinity. Biological sciences/Biotechnology Biological sciences/Ecology Bread wheat (Triticum aestivum L.) callus growth organic solutes inorganic solutes salt tolerance sensitivity Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Soil salinization is considered one of the major abiotic stresses restricting crop yields in about 20% of the world's cultivated and irrigated lands due to inappropriate irrigation practices and poor water quality 1 . This environmental constraint disastrously affects crop growth, morphological and physiological characters 2,3 . Several plant species have shown variable physiological and metabolic responses to adverse environmental stresses 4–6 . Regarding the harmful effect of salt stress, the excess of salt causes the reduction of water and nutrient absorption. This leads to the accumulation of toxic ions, nutritional imbalance and water deficit 7 . Further, ion toxicity, evoke cellular interference and excess generation of toxic reactive oxygen species (ROS) which may interrupt cellular functions and negatively causes plasma membrane disruption, hindrance to respiration and damage to enzyme structure 8,9 . Generally, as sessile organisms, plants are faced with several abiotic stresses throughout their life cycle. To maintain their normal basal metabolism, survival and optimum growth, adapted organisms are those that have managed to elaborate a series of coping mechanisms in favor of their defense against such stresses 10 . For instance, salinity generates osmotic and ionic stress, which alters the patterns of nutrient availability and transport, consequently causing enormous changes in plant growth 11 . One of the essential adaptive mechanisms ensuring osmotic balance in a saline condition is stimulated synthesis and accumulation in cytoplasm of small organic molecules, known as compatible solutes/osmolytes 12 . These compounds apart from their fundamental function in osmotic adjustment play several roles, such as maintaining cellular integrity, ensuring normal cellular function, altering antioxidant enzyme activity, and inorganic ion concentration 13,14 . Nowadays, in vitro culture strategies are the most effective means to study plant tolerance to stress. Indeed, Tissue culture techniques allow examining multiple plant production as well as their physiological and biochemical aspects. In particular, the use of tissue culture is essential to monitor their response to stress conditions and to select tolerant species 15 . Furthermore, these methods present an ideal strategy to save time and space. In addition, the use of these techniques with inexpensive laboratory facilities offers the possibility of improving the stress resistance in plants 16 . The aim of this study is to unveil the detrimental salt effect on callus growth, organic and inorganic solutes accumulation involved in salinity tolerance in five bread wheat varieties subjected to different NaCl concentrations in in vitro culture conditions. Results Mass of fresh weight (FW) and dry weight (DW) and water content (WC) The illustrated results Fig. 1 for callus growth in terms of fresh and dry weight of the five varieties indicate the negative influence of salinity as the concentration increases. The ANOVA results for FW and DW showed significant differences among the varieties studied and significant effects of different salt concentrations and their interaction. However, the FW criterion depends on the varietal effect while the DW parameter is controlled by the salinity effect (Table 1 ). In the absence of salt stress (0 g.L − 1 NaCl), the mass of fresh and dry weight is considerably important (values between 901.33 and 447.50 mg of FW and 70.47 and 43.03 mg of DW). By increasing the salt concentration, both parameters showed a more remarkable decrease. Statistically, Rajae variety always maintains a better production in mass of fresh and dry weight (Table 2 ). For example, the values noted for this variety at 3, 6 and 9 g.L − 1 NaCl, are: 632.33 /50.87 mg; /33.27; 267.50/ 29.20 mg unlike Wissam variety which presents the lowest values (209.20/20.90 mg; 118.97/ 13.97 mg; 86.90 / 11.80 mg. Concerning the water content of the callus (Fig. 1 ), this criterion depends on the saline concentration used and the variety tested. The results of the statistical analysis of variance for this parameter indicate significant effects applied by the variety and salinity. However, the interaction of the two factors "Salinity*variety" was found to be statistically insignificant (Table 1 ). Indeed, this parameter decreases progressively when the salinity increases, especially in the Wissam variety compared to the other varieties, which show a slight decrease irrespective of the salinity level (Table 2 ). For example, the values recorded for this parameter at the concentration 9 g.L − 1 NaCl are 89.05; 88.89; 88.76; 87.68 and 86.43% respectively, in the varieties Rajae, Wafia, Nassim, Tigre and Wissam. Table 1 Analysis of variance of the Means for the Fresh weight, dry weight, water content and salinity tolerant index based on the salt concentration effect and the variety. Sum of squares Source of variation DF FW (mg) DW (mg) WC (%) STI (DW) Variety (V) 4 907931.25*** 4869.27*** 36.06* 1838.86*** NaCl (g/L) 3 2396006.65*** 13073.02*** 100.55*** 41966.33*** V*NaCl (g/L) 12 109435,25* 359.18* 6.04 ns 968.20* Model 19 3413373.15*** 18301.47*** 142.65** 44773.39*** Error 40 140181.56 524.93 96.39 1553.08 C. Total 59 3553554.71 18826.40 239.04 46326.47 *, **, ***: significant respectively, at the levels of 5%, 1% and 0.1%; and ns: not significant. DF: Degree of freedom. Table 2 Comparison of the effects of NaCl concentration and the variety on Fresh weight, dry weight, water content and salinity tolerant index. The data are presented by the means (± ES). Parameters NaCl (g/L) FW (mg) DW (mg) WC (%) STI (DW) 0 681.61 ± 46.64 a 56.47 ± 2.93 a 91.51 ± 0.30 a 100 ± 0.00 a 3 400.33 ± 45.37 b 34.97 ± 3.40 b 90.91 ± 0.37 ab 60.44 ± 3.76 b 6 224.67 ± 29.78 c 21.97 ± 2.04 c 89.55 ± 0.47 bc 37.98 ± 1.81 c 9 168.51 ± 19.80 c 19.02 ± 1.76 c 88.16 ± 0.45 c 32.96 ± 1.82 c Varieties Nassim 346.63 ± 63.51 c 31.22 ± 4.77 c 90.37 ± 0.49 ab 55.85 ± 8.42 bc Rajae 549.66 ± 75.71 a 45.95 ± 5.13 a 90.99 ± 0.62 a 65.85 ± 7.49 a Tigre 263.98 ± 54.67 d 25.13 ± 4.12 d 89.46 ± 0.54 ab 52.58 ± 8.60 c Wafia 464.32 ± 72.85 b 40.82 ± 5.07 b 90.51 ± 0.60 ab 62.82 ± 7.82 ab Levels not connected by the same letter are significantly different at p = 0.05 Salinity tolerance index in relation to callus weight (fresh and dry) The results of the callus tolerance index for the five varieties studied (Fig. 2 ) illustrate the negative effect of salinity when its concentration increased in the culture medium. Significant differences for the factors "variety and salinity" as well as for the interaction were revealed for the STI. However, this parameter was more marked by the salinity factor (Table 1 ). Indeed, the STI of treated callus compared to control callus favored the variety Rajae among all the varieties tested. The values recorded for this criterion at the concentration 3 g.L − 1 NaCl, show weaker tolerance reactions compared to the control. However, the two varieties Rajae and Wafia seem to be more adjusted but equally to this salt dose by registering a tolerance value of 72.67%. By increasing the salt concentration to 6 and 9 g.L − 1 of NaCl, the response to salinity is more affected in all the tested varieties by presenting values lower than 35% respectively, for the varieties Wissam, Tigre and Nassim. However, the varieties Rajae and Wafia maintain the highest values (47.41/42.09; 42.09/35.82%). Statistically, Rajae is the best performing variety while Tiger and Wissam are the most susceptible (Table 2 ). Proline and total soluble sugar (µmoles/g FW) The graphics in Fig. 3 and Table 3 shows the evolution of total proline and sugar content as a function of salt concentration and variety. The kinetics obtained are relatively fluctuating. Thus, we noted a high accumulation in the stressed callus compared to the controls at all salt concentrations. The accumulation of these two osmoprotectants is significantly affected according to salt level and the varietal effect (Table 5 ). Statistically, at 9g/l NaCl the highest values were recorded respectively, in the two varieties Rajae (12.06 proline µmol/g FW − 43 µmol golucose/g FW ) and Wafia (10.24 proline µmol/g MF − 40.47 µmol golucose/g MF). Therefore, this response confers them the character of adaptation (Table 4 ) comparatively, with the variety Wissam, which always reserves a position of sensitivity by accumulating the quantity of proline and the lowest total sugar (8,60 proline µmol /g FW − 33,33 µmol golucose/g FW) (Table 4 ). Table 3 Analysis of variance of the Means for proline and total soluble sugar content based on the salt concentration effect and the variety Sum of squares Source of variation DF Proline (µmol/g FW) Total soluble sugar (µmol/g FW) Variety (V) 4 17.63*** 223.32*** NaCl (g/L) 3 454.26*** 4669.02*** V*NaCl (g/L) 12 8.11 ns 62.17 ns Model 19 479.99*** 4954.51*** Error 40 16.01 112.01 C. Total 59 496.00 5066.52 *, **, ***: significant respectively, at the levels of 5%, 1% and 0.1%; and ns: not significant. DF: Degree of freedom. Table 4 Comparison of the effects of NaCl concentration and the variety on proline and total soluble sugar content. Parameters NaCl (g/L) Proline (µmol/g FW) Total soluble sugar (µmol/g FW) 0 2.93 ± 0.09 d 18.22 ± 0.54 d 3 4.36 ± 0.12 c 22.17 ± 0.48 c 6 7.07 ± 0.18 b 35.31 ± 0.66 b 9 10.14 ± 0.38 a 39.43 ± 0.97 a Varieties Nassim 6.05 ± 0.85 bc 29.21 ± 2.91 ab Rajae 6.98 ± 1.00 a 30.97 ± 2.89 a Tigre 5.90 ± 0.86 bc 28.34 ± 2.81 b Wafia 6.36 ± 0.82 ab 30.05 ± 2.71 ab Levels not connected by the same letter are significantly different at p = 0.05 Na+, K + and Cl − content The variation of sodium, potassium and chloride content as a function of salinity concentration and variety tested was shown in the following Fig. 4 . By analyzing the variance for the three variables Na + , K + and Cl − we found significant differences for the effect of variety and salinity. However, the interaction between these two factors was found to be non-significant for both Na + and K + variables and significant for the Cl- variable (Table 5 ). Callus treated with different salt concentrations showed an upward progression in the accumulation of both Na + and Cl − ions parallel to a reduction in K + ions as the salt concentration increased compared to control callus in all varieties. According to all concentrations combined, Rajae variety stands out significantly from the other varieties by exhibiting the lowest Na + and Cl − values and the highest K + values (Table 6 ). For instance, at the high concentration 9 g/L NaCl the values 31, 15 Na + mg/g DW; 12, 61 K + mg/g DW and 12, 82 Cl − mg/g DW are recorded for the Rajae variety. While the values 52.74 Na + mg/g MS; 8.39 K + mg/g DW and 15.24 (Cl − mg/g DW) are correspondent to callus from the variety Wissam. Table 5 Analysis of variance of the Means for inorganic solutes (Na+, K + and Cl − ) and K + /Na + ratio based on the salt concentration effect and the variety Sum of squares Source of variation DF Na + (mg/g DW) K + (mg/g DW) Cl − (mg/g DW) Ratio K + /Na + Variety (V) 4 880.20** 635.80*** 81.07*** 8.67*** NaCl (g/L) 3 10689.17*** 2445.56*** 1136.79*** 85.72*** V*NaCl (g/L) 12 590.71 ns 215.55 ns 11.22*** 4.39*** Model 19 12160.08*** 3296.90*** 1229.08*** 98.79*** Error 40 1674.47 778.55 9.93 0.33 C. Total 59 13834.55 4075.45 1239.01 99.12 *, **, ***: significant respectively, at the levels of 5%, 1% and 0.1%; and ns: not significant. DF: Degree of freedom. Table 6 Comparison of the effects of NaCl concentration and the variety on for inorganic solutes (Na+, K + and Cl − ) and K+/Na + ratio. Parameters NaCl (g/L) Na + (mg/g DW) K + (mg/g DW) Cl − (mg/g DW) Ratio K + /Na + 0 8.69 ± 0.41 c 28.14 ± 1.36 a 3.63 ± 0.43 d 3.34 ± 0.22 a 3 12.45 ± 0.56 c 21.11 ± 1.18 b 5.86 ± 0.39 c 1.72 ± 0.09 b 6 31.90 ± 2.37 b 18.46 ± 2.03 b 12.30 ± 0.30 b 0.62 ± 0.08 c 9 40.84 ± 2.98 a 10.30 ± 0.63 c 14.12 ± 0.24 a 0.27 ± 0.02 d Varieties Nassim 23.53 ± 4.21 abc 18.82 ± 2.07 b 8.76 ± 1.43 c 1.33 ± 0.32 c Rajae 18.58 ± 3.10 c 25.14 ± 2.61 a 7.33 ± 1.40 d 2.13 ± 0.49 a Tigre 26.51 ± 5.06 ab 16.82 ± 1.99 b 9.74 ± 1.22 b 1.19 ± 0.31 d wafia 19.97 ± 3.59 bc 20.72 ± 2.67 ab 8.35 ± 1.34 c 1.68 ± 0.41 b Levels not connected by the same letter are significantly different at p = 0.05 K + /Na + ratio The ratio [K + /Na + ], was significantly reduced in all the callogenic clusters as a function of salinity at different concentrations and variety tested (Fig. 5 ). Statistically (Table 5 ), significant differences are considered for both factors (Variety and Salinity) as well as for their interaction. In the absence of salt stress, the values noted for this criterion are important but variable according to each variety (between 4.66 and 2.41). Furthermore, in comparison with the control, the K + /Na + ratios maintained by the five varieties studied were strongly affected by salinity, especially at the concentration 6 and 9 g.l − 1 NaCl. However, the highest ratio was recorded for the variety Rajae. While the lowest is obtained by Tigre and Wissam. As an example, at the high concentration 9 and 6 g.l − 1 NaCl, the values corresponding to the K+/Na + ratio are 0.40 and 0.16 respectively, for Rajae and Wissam varieties. Statistical results show that Rajae and Wafia varieties perform better in terms of ionic selectivity and have a good uptake of potassium ions compared to the sensitive varieties Wissam and Tigre (Table 6 ). Principal component analysis Principal component analysis (PCA) was used to graphically represent the relationship between agro-physiological characteristics and the different soft wheat varieties. It revealed an apparent correlation between the parameters evaluated and the different varieties studied (Fig. 6 ). The two axes describe a total variation of 98.42%. The first axis (PC1) expresses the greatest variation with 95.79% and shows high values for all the parameters evaluated. On the one hand, it gathers the varieties Wissam and Tigre as salt sensitive varieties thus revealing a high accumulation of Cl- and Na + ions in relation to dry matter. On the other hand, it classifies the varieties Rajae and Wafia as salt tolerant varieties with a high rate of fresh and dry weight and a high accumulation of K + cation in relation to dry weight, proline, and total sugar. Furthermore, it ranks the variety Nassin as moderately salt tolerant. The second axis (PC2) represented a variation of 2.63% that exposes total sugars and K + cation with high scores. In addition, it classifies Rajae as the variety that accumulates high content of both total sugars and K + cation. As for the results related to the correlation analysis (Table 7 ), callus growth in fresh and dry weight mass is strongly and positively correlated (P < 0.05) with water content (WC), proline and total sugars and K + ion content. On the contrary, the correlations are negatively significant between these same growth descriptors (DW and FW) and Na + and Cl- ion contents. Besides, the K + ion content of the callus of all the varieties studied is strictly and positively correlated with the water content (WC) and the proline content (P < 0.05). However, the correlation between soluble sugar content and K + ion content was not significant. The proline content was positively and significantly correlated with the soluble sugars and K + ions content. As for soluble sugars, they show a negative significant correlation with mineral solutes (Na + and Cl − ). The correlation is positively significant (P < 0.05) between the contents of Na + and Cl − ions and negatively significant with K + ions. Table 7 The correlation coefficient between the Agro-physiological parameters for the five studied wheat varieties Parameters FW (mg) DW (mg) WC (%) Proline (µmol/gMF) Total sugar (µmoles/g MF) Na+ mg/gMS k+ mg/gMS CL- mg/gMS FM (mg) 1.000 0.999 *** 0.942* 0.961** 0.900* -0.989** 0.974** -0.969** DM (mg) 1.000 0.934* 0.949* 0.890* -0.989** 0.965** -0.960** WC (%) 1.000 0.942* 0.962** -0.968** 0.905* -0.989** Proline (µmol/gMF) 1.000 0.949* -0.951* 0.964** -0.980** Total sugar (µmoles/g MF) 1.000 -0.938* 0.851 -0.963** Na + mg/gMS 1.000 -0.936* 0.977* k + mg/gMS 1.000 -0.951* CL- mg/gMS 1.000 Discussion Authors should discuss the results and how they can be interpreted from the perspective of previous studies and of the working hypotheses. The findings and their implications should be discussed in the broadest context possible. Future research directions may also be highlighted. Soil salinization is a major process and a significant threat to land degradation, which adversely affects soil fertility, thereby, the agricultural production and yield 17 . It causes osmotic, ionic, and oxidative stress, thus resulting in reduction of plant growth and development. Because of the high salt content, a variety of negative consequences arises. One of the most serious complications is ion imbalance. For example, a high concentration of sodium and Chloride ions can cause biochemical processes that might be lethal at the plant level. To comprehend each tolerance mechanism at the plant level, it is necessary first to understand each tolerance mechanism at the cellular level 18 . In this manner, numerous studies have been focusing on investigating in vitro salt tolerance by measuring the accumulated levels of toxic ions mostly sodium toxicity and its adverse effect on plant growth. In the present work, we aimed to study in vitro the salt effect on bread wheat by determining the contents of compatible solutes such as, proline and soluble total sugars and inorganic solutes like sodium, potassium and chloride. The contents were analyzed individually using callus of five varieties of bread wheat induced directly in the presence of different salt levels (0, 3, 6 and 9 g/l NaCl). Mass of fresh matter (FM), dry matter (DM), water content WC (%) and salt tolerance index (STI) One of the first and most typical reactions to stress is decreased plant growth. Our results indicated a negative effect of salinity on callus growth in terms of fresh and dry matter as the concentration increases in the culture medium especially at 9 g/l NaCl. Golkar et al 19 stated in their finding that the 200 mM NaCl salt concentration had the highest inhibitory effect on callus formation for all genotypes studied. According to Arzani and Ashraf 20 this finding can be explained by the osmotic effect of NaCl, which results in a decrease in the water content of the cytosol in turn causing a decrease in callus growth. On the other hand, Hamedi et al 21 indicated the increase in salt concentration could lead to a reduction in the osmotic potential of the medium, a decrease in turgidity of the growing cells and eventually a decrease in callus growth. On the other hand, researchers such as Cicek and Cakirlar 22 have previously reported that the negative effect of salinity on plant growth might be due to the occurrence of defective metabolism in plant cells. Since the high osmotic pressure resulted from high salinity that prevents plant cells to absorb water and some mineral nutrients dissolved in the culture medium. In addition, in two bread wheat cultivars Al Hattab et al 23 , affirmed that the reduction in fresh and dry weight of callus is due to ionic imbalances within the cells. The accumulations of sodium and chloride ions in the cells will reduce the availability of water, which induces osmotic stress that presents negative effects on cell growth 24 . Actually, in vitro culture techniques have formerly been recognized to provide controllable environmental conditions for examining salt-stress effects at the cellular level 25 . Under salt stress Shibli et al 26 point out that the reduction in the osmotic potential of the media culture could compromise callus growth thus, reinforcing the need to maintain turgor in growing callus cells. A process that consumes the majority of the energy and ultimately leads to callus growth reduction. Moreover, the NaCl induced gradient in water potential between the cell and the nutritional medium has been proven to cause cell dehydration and callus fresh weight reduction 27 . Regarding the STI, the recorded values show a better response to salinity of the variety Rajae followed by Wafia, unlike the other three varieties. Similarly, in their study Haque et al 28 showed that the recorded values for salinity tolerance index and callus growth declined when calluses were grown at higher NaCl levels. They associated this response to the reduction of water availability, and loss of turgor pressure (TP) in callus cells. Proline and total soluble sugars content Being exposed to salt stress, plants accumulates several compounds contributing to the osmotic regulation. Proline is one of the most essential and efficient compatible metabolites amid other organic osmolytes. It plays a vital role in defense by minimizing the salt-stress effects and improve plant growth 29 . Salt pressure affected the accumulation of proline for all the five varieties gradually with increasing NaCl concentration in the culture medium. However, the content values varied according to the response of each variety. Our results, advocate those obtained by Khuder and Hussein 30 , they revealed that the increase in salt causes a high accumulation of proline in the treated calluses while, the lowest concentration of proline was recorded in the control calluses. This elevation can be considered as a clever coping strategy against ion accumulation since it plays an important role in fixing osmosis between the vacuole and the cytoplasm of the cell 31 . Proline is also recorded as an osmoprotectant with significant contribution under salt conditions, especially in assisting with water absorption, protecting cellular functions by scavenging reactive oxygen species (ROS) molecules, stabilizing membranes and subcellular structures in cytosol, modulating cell redox homeostasis, supplying energy, and functioning as a signal 32 . Formerly, Girija et al 33 explained, the increase in proline content under stress conditions may be due to the decrease in the activity of enzymes involved in proline degradation such as proline dehydrogenase and proline oxidase. Moreover, we remarked that two varieties (Rajae and Wafia) accumulate more proline than the other varieties, indicating a tolerance response towards elevated salt concentration. As reported by Shahid et al 18 , improving the ability of cells to make ionic adjustments, proline has to accumulate to higher levels in the cytosol. This accumulation is linearly related to plant stress tolerance. Although acting as an osmolyte, proline is also thought to be a potent antioxidant defense molecule involved in the inhibition of programmed cell death 34 . Plants also engage another important osmoprotector as one of the defense mechanisms, namely soluble sugar, which is generally known to accumulate under stressful conditions. Studies conducted by khuder and Hussein 30 on carbohydrate accumulation in callus from soft wheat, showed that this criterion is controlled by varietal effect. These findings are in concordance with ours. The content of soluble sugar is higher especially at the higher NaCl level 9 g/L. Both Rajae and Wafia accumulates more sugar comparatively with the three other varieties tested. According to Slama et al 35 in the presence of salt stress, sugar accumulation attributes to the maintenance of osmotic and water balance in cells. In addition, Bezirğanoğlu 16 also showed that the highest level of sugar was accumulated in the presence of the high concentration 200 mM. He added that this trait appeared to present an effective marker of salt resistance in embryogenic callus derived from triticale genotypes. Besides, many in vivo and in vitro studies of Physiological, biochemical, and molecular characters conducted under salt conditions, show that osmolites play important roles in the plant’s defense system against stress 36 . Higher proline and total soluble sugar content accumulation was also observed in other plants exposed to salt stress 37,38 . According to Muchate et al 39 , this might be associated with higher energy consumption for osmolyte synthesis, which, could lead to growth decline. On the other hand, these solutes are essential to prevent damage at the cellular level and mitigating the negative effects of excessive ion concentrations on enzyme activities [ 24 ]. Plants can accumulate Na + and Cl- ions to maintain the water potential of the tissues necessary for their growth. While this accumulation, which requires relatively less energy expenditure, must remain compatible with a metabolic tolerance of the resulting concentration or with a compartmentalization between the different components of the cell or plant. Conversely, the synthesis of organic solutes is generated when a metabolic change takes place in the cells under the action of salt pressure. The main objective is to adjust the water potential. Therefore, the main adaptation strategy is the accumulation of osmoprotective agents, mainly amino compounds and sugars known to be low molecular mass in the cytoplasm as mentioned by Ketehouli et al 40 Shahid et al [ 24 ]. Moreover, It has been stated by Gill and Tuteja 41 , Hayat et al 42 and Farissi et al 43 that salinity causes secondary stress, known as oxidative stress, when reactive oxygen species (ROS) are formed in cells, which leads to disrupt normal metabolism by peroxidizing proteins and lipids, damaging DNA, and inactivating enzymes. As a survival behavior, the accumulation of compatible osmolytes is strictly indispensable to protects plant cells from the negative effects of salt 44 . Na+, K + and Cl- content For most plants, responding to salt stress require the restraining of excessive sodium accumulation. Ion homeostasis is in fact one of the effective physiological strategies deployed by plants to ensure tolerance against abiotic stress. As stated by Shahid et al [ 24 ], sodium is not considered as necessary element for plant development. In fact, plants do not require Na, nor do they have a specific Na transport mechanism. However, when the plant is exposed to high concentrations of sodium, it excessively enters the plant cells through various mechanisms. In addition, Na + causes inhibition of the uptake of other cations, notably K+. Indeed, the hydrated ionic radii of K + are similar to those of Na+, making it easily replaced by this cation. Therefore, the transport proteins cannot distinguish between them. Because Na + cannot perform the biological function of K+, reduced K uptake can eventually lead to reduced plant growth and productivity under saline conditions. Indeed, the balance between sodium and potassium in a cell is crucial for plants to survive in saline soil 45,46 . In the present work, the results indicate a significant accumulation of Na + and Cl − ions and a decrease in K + ion content that occurs progressively with increasing salt concentration yet, depending on the varietal reaction. These findings corroborate those obtained by Arzani and Ashraf [ 26 ] who revealed that the reduction in K + concentration in callus cells under salt stress could be explained by alterations in the expression and/or function of transporters as well as ion channels especially those related to K + . In another in vitro experiment under salt stress to select tolerant varieties of durum wheat from mature embryos, Koutoua et al 47 reported that Na + accumulation was greater in salt sensitive calli than in tolerant calli. The behavior of potassium, on the contrary, appears to be variable when compared to sodium. Both tolerant and sensitive calli accumulated less K + than those of control, although the K + content of salt tolerant calli remained higher than that of sensitive ones. As for the low accumulation of K + ions by some of our studied varieties (Tigre and Wissam), Golkar et al [ 25 ] stated that, the imposition of NaCl shock treatment causes tissue damage, which leads to excessive leaching and poor retention of K + in callus from genotypes with low salt stress tolerance. Thus, they display a very strong reduction in K + content as salt concentration increased from the control to the higher levels. On the other hand, certain varieties (Rajae and Wafia) responded better to salt stress by maintaining a higher K + ion content. Indeed, the same researchers found that compared to callus-sensitive genotypes, callus-tolerant genotypes accumulated less Na + and maintained higher levels of K + . This explained the better growth of tolerant callus in the presence of NaCl. Accordingly, higher K + concentration is directly related to higher biomass production 48 . Salt-tolerant plant species have the ability to retain a higher concentration of K. Hence, in psyllium, Karimi and Haghighat-Pak 49 revealed that the K + inclusion mechanism is an indicator of salt tolerance at the whole plant level. In addition to sodium and potassium ions, we noticed that the most sensitive varieties (Tigre and Wissam) accumulate a significant content of chloride compared to the tolerant variety Rajae, which present the lowest values compared to all the varieties tested. On the other hand, for the varieties Wafia and Nassim, the accumulation of chloride is not significant, but keep an intermediate position between the other three tested varieties. Shahid et al [ 24 ] stated that, Chloride also contributes to the undesirable effects of salt stress on plant growth. However, salt-tolerant plant genotypes have the ability to inhibit chloride uptake. In bread wheat and sugarcane, some researchers have found that, the content of Cl − ions contained in the sensitive callus is higher comparatively to the tolerant callus 50,51 . On the contrary, in eight durum wheat varieties tested by Koutoua et al [ 53 ], the tolerant callus of some varieties showed a higher accumulation of Cl − ions compared to the sensitive callus. For some plants, Cl − ion is more harmful than Na + . Apart from their toxicity, Cl ions have a regulatory function in the establishment of turgor, enzyme stability, membrane potential, pH, charge balance, volume control, osmoregulation, and stomatal conductance, all of which contribute to water preservation, high water usage, and photosynthetic efficiency 52,53 . Therefore, the toxicity of both ions must be considered [ 31 ]. K + /Na + ratio Plants adopt various strategies to cope with a high Na ion concentration, such as the restriction of Na + entry into the cell, Na + extrusion, and vacuole compartmentalization. The plant’s capacity to tolerate saline conditions depends on a high K+/Na + cytosolic concentration 54 . This selection criterion has been supported by several researchers since it better expresses ionic selectivity and is therefore, used as a criterion for selecting cell lines and classifying varieties according to their response to salinity 55 . In earlier times, many researchers such as Farukh 56 , Wahid 57 , Ndayiragije, and Lutts 58 used the K+/Na + ratio as a criterion for varietal classification. Zhang et al 59 and Lui et al 60 also reported that salt tolerance is typified by the maintenance of Na+/K + homeostasis. As previously reported by Arzani and Ashraf [ 26 ], the capacity to maintain cellular K +/ Na + homeostasis at a high level for plant survival in saline conditions is a crucial aspect for salt tolerance. According to our study, although salinity negatively affected the behavior of all the varieties tested. However, on the same saline concentrations, the varieties considered as tolerant were able to keep high levels of K+/Na + ratio compared to the sensitive varieties. Therefore, our remarks are similar to those retained by Koutoua el al [ 53 ], who indicated that the K+/Na + ratio decreased as the salt concentration increased. This decrease was more remarkable in sensitive callus than in tolerant ones. Golkar et al [ 25 ] presented explanations that due to the increase of NaCl concentration in the medium, Na + ions are rivaling with K + ions during salt stress for the same transporter since they both share the same transport mechanisms, hence the decrease in K + uptake. The detrimental effect of Na + also depends on its site of accumulation. Its accumulation is toxic to the salt-sensitive callus in which it is transported into the cytoplasm. Yet, it is not toxic to tolerant callus as it is accumulated in their cell vacuoles 61 . Consequently, the increase of osmotic pressure in the cells and the maintenance of a high K+/Na + ratio are strongly linked to vacuolar sequestration, which plays an important role in the maintenance of water balance in the callus. From another perspective, Negrao et al 62 indicated that maintaining ion homeostasis could be particularly challenging for plants under saline conditions since the accumulation of toxic ions (Na + ) can disrupt the plant's ability to control the accumulation of other ions. In addition, in most species, accumulation of Na + ions to toxic levels appears to occur before Cl − .However, the K + /Na + ratio does not only depend on the depressive effect of salinity but also, on the genotype effect. According to Kumar et al 63 , a genotype with the quality of K + ion selection over Na + ion is proven tolerant. The intracellular K + /Na + ratio in plants is a direct indicator of their salt tolerance. Plants tend to find a way to balance their cytosolic K + /Na + ratio by reducing Na + accumulation or compensating for K + loss 64 . Material and methods Plant material The experiments were carried out on mature seeds of five varieties (Nassim, Wissam, Wafia, Tigre and Rajae) of soft wheat ( Triticum aestivum L.) which were provided to us by ONSSA (National Office for Food Safety in Rabat-Morocco). The five varieties were selected after being previously tested for their behaviour under salt stress at the germination stage. Thus, they were classified primarily as tolerant (Rajae and Nassim), intermediate (Wafia and Tigre) and sensitive (Wissam). In addition, all experiments conducted on plants comply with the strict standards of Ibn Tofail University, Morocco and and with international guidelines and legislation. Sterilization and culture Mature seeds were washed with tap water supplemented with a few drops of Tween 20 and rinsed three times with distilled water. The seeds were then soaked in sterile distilled water overnight at room temperature. Under a horizontal laminar airflow cabinet, the seeds were disinfected by soaking and vigorous shaking in 45% commercial bleach (sodium hypochlorite) for 20 minutes, followed by rinsing six times with sterile distilled water under continuous shaking and transferred to sterile Petri dishes with filter paper for drying. After sterilizing the seeds, with a sharp scalpel, several lesions were made in the embryo of the seeds. Explants were cultured on MS medium supplemented with 2 mg.L − 1 2,4-dichlorophenoxyacetic acid (2,4-D), 30 g.L − 1 sucrose 65 . The culture medium was subdivided into three increasing treatments of (3, 6, 9 g.L − 1 ) NaCl and no salt in the control (0 g.L − 1 NaCl). The medium was adjusted to pH 5.7, solidified with 8 g.L − 1 agar, and sterilized by autoclaving at 120 ° C for 20 min at 1 bar pressure. The cultures were incubated in the dark in a thermoregulated chamber at 25°C ± 2. The obtained 10-day-old calli were carefully removed from the rest of the seeds with a sterilized scalpel under aseptic conditions and then subcultured again regularly on fresh media of the same composition with 11/10-day intervals until the end of the incubation period (6 weeks). Physiological traits Callus growth, water content and Salinity tolerance index To quantify the negative impact of salinity on callus growth, the mass of callus matter was weighed before drying (fresh weight). The water content is determined by the percentage of water presented in three calluses per concentration according to the following formula: $$\text{W}\text{C} \left(\text{%}\right)=\left(\frac{\text{F}\text{W}-\text{D}\text{W}}{\text{F}\text{W}} \right)\times 100$$ To record the dry weight, the calli were placed in an oven (75°C) for 48 hours. The salinity tolerance index (STI) in relation to dry weight as described by Goudarzi and Pakniyat 66 was calculated according to the following formula: $$\text{S}\text{T}\text{I} \left(\text{%}\right)=\frac{\text{V}\text{a}\text{r}\text{i}\text{a}\text{b}\text{l}\text{e} \text{m}\text{e}\text{a}\text{s}\text{u}\text{r}\text{e}\text{d} \text{u}\text{n}\text{d}\text{e}\text{r} \text{t}\text{r}\text{e}\text{a}\text{t}\text{m}\text{e}\text{n}\text{t}}{\text{V}\text{a}\text{r}\text{i}\text{a}\text{b}\text{l}\text{e} \text{m}\text{e}\text{a}\text{s}\text{u}\text{r}\text{e}\text{d} \text{u}\text{n}\text{d}\text{e}\text{r} \text{n}\text{o}\text{r}\text{m}\text{a}\text{l} \text{c}\text{o}\text{n}\text{d}\text{i}\text{t}\text{i}\text{o}\text{n}\text{s}}\times 100$$ Accordingly, 60 calli corresponding to three callus replicates per each variety and salt level (Callus X Variety X salt concentration) were used for the weighing of fresh (FW) and dry mass (DW) the water content (WC) and the STI %. Proline content The method used to determine proline was by Dreier and Goring 67 . The extraction was carried out on 100 mg of fresh material (Cal), mixed in the presence of 3 mL of 40% methanol and heated to 85°C in a water bath for 60 min. After cooling in melting ice and centrifugation (4000 T, 10 min), 1 mL of the extract was taken to which was added 1 mL of glacial acetic acid, 25 mg of ninhydrin (C 6 H 6 O 4 ) and 1mL of mixture containing (120 mL of distilled water + 300 mL of acetic acid (CH 3 COOH) + 80 mL of orthophosphoric acid (H 3 PO 4 )). Using a vortex, the solution was well mixed, then boiled at 100°C for 30 min, until the solution turns red. After cooling, 5 mL of toluene were added to the solution (which was stirred), then left to stand for 30 min until the separation of two phases (an upper and a lower phase). The absorbance of the toluene fraction taken from the liquid phase (three replicates per salt level per each variety) was determined with a spectrophotometer at 528 nm (100% toluene was used as a blank and L-proline was utilized as the standard.) Total soluble sugar content Soluble sugars were determined by the method of Dubois et al 68 . Samples of 100 mg of plant material (callus) in test tubes were crushed in 3 mL of 80% ethanol. At the time of dosing, the tubes were placed in the oven at 80°C to evaporate the alcohol. In clean glass tubes, 10 mL of distilled water was added to the extract in each tube to obtain a test solution. 2 mL of the solution to be analyzed was taken and 1mL of 5% phenol and 5 mL of 96% concentrated sulfuric acid were added while avoiding pouring acid against the tubes. The total sugar content was determined at 620 nm with a spectrophotometer using glucose as standard. The tubes containing the solution (yellow-orange color) were subsequently vortexed to homogenize it before being left to stand for 10 min and placed in a water bath for 15 to 20 min at a temperature of 30 ◦C. The total sugar content was determined on three replicates per salt level per each variety at 620 nm with a spectrophotometer. Ions accumulation Na+, K + and Cl- (Dry oven mineralization) Callus were rinsed with cold distilled water accordingly to remove any electrolytes adhering to the tissue surface or accumulated in the apoplast 69 . Sodium and potassium were determined from dried callus (0.2 g dry weight) using 60 samples with 3 replicates per variety per salt concentration. After mineralization at 500°C, Na + and K + dosages were determined by flame photometry. The values obtained were converted into Na + and K + content from the calibration range and the content was given in mg/g FW and then in mg/g DW. The determination of chloride ions was also performed on 60 samples (0.2 g DW) after mineralization at 500°C in the presence of calcium oxide with 3 repetitions per variety per salt concentration. Being soluble, chlorides are extracted by hot water. The Cl − is then determined by volumetric silverometry (precipitation as AgCl in the presence of AgNO 3 ). To visualize the end of precipitation, potassium chromate (K 2 CrO 3 , yellow in color) was used as an indicator which do transform into silver chromate (reddish tint) as soon as all Cl − ions precipitated 70 . Data statistical analysis The normality of the data distribution was analyzed by the Shapiro-wilk test, and normality is fulfilled by transformation when necessary. Analysis of variance (ANOVA) was used to evaluate the effect of salt level, treatment duration, varieties, and their interaction on different parameters. Means were compared by Tukey HSD (Honestly Significant Difference) test at the risk of error of 0.05. The link between the different studied parameters and different varieties was revealed by the principal component analysis (PCA) in terms of correlation (correlations are estimated by the ROW-WISE method). The data was analyzed by JMP SAS Pro software (JMP®, Version SAS Institute Inc.). Conclusions The five wheat varieties showed different behavior under salt stress. Accumulation of organic solutes, namely proline and total soluble sugars, less chloride and maintenance of K+/Na + homeostasis along with high dry mass production were mainly the adaptive mechanisms to tolerate the intensity of salt applied directly and abruptly on bread wheat explants. Only two varieties Rajae and Wafia showed a high response against salt pressure compared to the other three varieties. They were therefore, statistically selected as tolerant. This research showed that in vitro culture could efficaciously contribute to the selection of salt-tolerant varieties by ensuring the investigation of physiological traits involved in stress tolerance. Therefore, biochemical and molecular approaches are strongly recommended for more detailed elucidation of tolerance/sensitivity mechanisms at callus and regenerated plant levels of bread wheat. Declarations Author Contribution Author Contributions: Conceptualization, F.M. and A.R.; methodology, F.M., H.E. and R.E.; formal analysis, F.M., R.E., M.N.A., N.M.A., Y.E. and H.E.; resources, H.I., M.N.A. and A.A.S.; data curation, H.I., A.A.S. and Y.E.; writing—original draft preparation, F.M.; writing—review and editing, H.I., M.N.A., A.A.S., N.M.A. and R.A.; supervision, R.A.. All authors have read and agreed to the published version of the manuscript. Acknowledgement The authors extend their appreciation to Princess Nourah bint Abdulrahman University Re-searchers Supporting Project number (PNURSP2024R103), Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia. Data Availability The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. References Daba, A. W. & Qureshi, A. S. Review of Soil Salinity and Sodicity Challenges to Crop Production in the Lowland Irrigated Areas of Ethiopia and Its Management Strategies. Land 10, 1377 (2021). Abid, M. et al. Effect of Salt stress on growth, physiological and biochemical characters of Four kiwifruit genotypes. Scientia Horticulturae 271, 109473 (2020). Yadav, S. et al. Effect of Abiotic Stress on Crops . Sustainable Crop Production (IntechOpen, 2020). doi: 10.5772/intechopen.88434 . Wahab, A. et al. 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Plant Physiol 68, 1454–1459 (1981). Cotlove, E. Determination of Chloride in Biological Materials. in Methods of Biochemical Analysis 277–391 (John Wiley & Sons, Ltd, 1964). doi: 10.1002/9780470110300.ch6 . Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4368371","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":304252102,"identity":"129eaed9-926b-446f-9ca9-78eda740c1a0","order_by":0,"name":"Fatine Mouhssine","email":"","orcid":"","institution":"Ibn Tofail University","correspondingAuthor":false,"prefix":"","firstName":"Fatine","middleName":"","lastName":"Mouhssine","suffix":""},{"id":304252103,"identity":"de70cce1-240a-4143-8134-e3b9da02df8f","order_by":1,"name":"Houda Elyacoubi","email":"","orcid":"","institution":"Ibn Tofail University","correspondingAuthor":false,"prefix":"","firstName":"Houda","middleName":"","lastName":"Elyacoubi","suffix":""},{"id":304252104,"identity":"1cfa90e3-2337-4f28-8342-d34a63c45c1b","order_by":2,"name":"Hamada Imtara","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABDElEQVRIiWNgGAWjYBACgwNAggfBPyAHJh8QoUUCpsUYTCaQoiWxAUTh1XL8dOKHNzUMdfyzDz+Trqi5kz4/7PBDoC12croN2LXYn8ndLDnnGIOExLk0M8kzx57lbrydZgDUkmxsdgCXw3I3SPOwAR12hsFMsoHtcO7G2QkgLQcSt+HScv7t5t88/xgk5M+wf5Ns+Hc43XB2+gf8Wm7kbpPmbWOQMDjDYybZ2HY4QV46h4AtN95us5zbJyG58QxPsWVj32HDDdI5BQcSDPD45Xzu5htvvtnwy51h33iz4dthefnZ6Zs/fKiwk8OlBQrA0cIiAQkQMIlXORwwfwCR8g3EqR4Fo2AUjIKRAwAYmWlteJyBBgAAAABJRU5ErkJggg==","orcid":"","institution":"Arab American University Palestine","correspondingAuthor":true,"prefix":"","firstName":"Hamada","middleName":"","lastName":"Imtara","suffix":""},{"id":304252105,"identity":"c209167a-eda7-4ff9-92fd-4242916068ba","order_by":3,"name":"Rabab Ez-zriouli","email":"","orcid":"","institution":"Ibn Tofail University","correspondingAuthor":false,"prefix":"","firstName":"Rabab","middleName":"","lastName":"Ez-zriouli","suffix":""},{"id":304252106,"identity":"395bd46d-de85-4253-aa5f-6ac79fb4daf9","order_by":4,"name":"Younes Elgoumi","email":"","orcid":"","institution":"University of Sultan Moulay Slimane, Higher School of Technology of Fkih Ben Saleh, USMS","correspondingAuthor":false,"prefix":"","firstName":"Younes","middleName":"","lastName":"Elgoumi","suffix":""},{"id":304252107,"identity":"7ada45f3-4004-438f-8025-44fa8d4abab6","order_by":5,"name":"Mashail N. AlZain","email":"","orcid":"","institution":"Princess Nourah bint Abdulrahman University","correspondingAuthor":false,"prefix":"","firstName":"Mashail","middleName":"N.","lastName":"AlZain","suffix":""},{"id":304252108,"identity":"6cf88d0d-51b3-424a-9b39-3f740f45ff5c","order_by":6,"name":"Abdelaaty A. Shahat","email":"","orcid":"","institution":"King Saud University","correspondingAuthor":false,"prefix":"","firstName":"Abdelaaty","middleName":"A.","lastName":"Shahat","suffix":""},{"id":304252109,"identity":"8fc00764-420b-48c9-818e-cf852d47d2f5","order_by":7,"name":"Nurah M. Alzamel","email":"","orcid":"","institution":"Shaqra University","correspondingAuthor":false,"prefix":"","firstName":"Nurah","middleName":"M.","lastName":"Alzamel","suffix":""},{"id":304252110,"identity":"f7349640-5c57-4d33-9d26-9288ef965f19","order_by":8,"name":"Atmane Rochdi","email":"","orcid":"","institution":"Ibn Tofail University","correspondingAuthor":false,"prefix":"","firstName":"Atmane","middleName":"","lastName":"Rochdi","suffix":""}],"badges":[],"createdAt":"2024-05-04 12:11:54","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4368371/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4368371/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":56954529,"identity":"7646eeb1-bb20-459a-b962-972ffbd4ce85","added_by":"auto","created_at":"2024-05-22 15:35:56","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":329908,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of different salt concentrations on fresh, dry weight mass and water content in the five varieties depending on salt concentration (Nassim, Wissam, Wafia, Rajae and Tigre).\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4368371/v1/f1cb1b34005595f5767c0cba.jpeg"},{"id":56954535,"identity":"3bdc1309-e193-4864-aea8-8f9118ee0e86","added_by":"auto","created_at":"2024-05-22 15:35:59","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":64483,"visible":true,"origin":"","legend":"\u003cp\u003eSalt tolerance index relative to dry weight mass (STI (DW)) in five wheat varieties as a function of salt concentration\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-4368371/v1/8e9d4f2e73d2844d89e12e7c.png"},{"id":56954533,"identity":"37253e95-975b-4921-9acc-0eacf08a30db","added_by":"auto","created_at":"2024-05-22 15:35:59","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":170793,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of different salt concentrations on proline and total soluble sugar content in callus derived from five common wheat varieties\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4368371/v1/f6b31482e5305e17d46dd0dd.png"},{"id":56954530,"identity":"0d1bece8-2887-429e-a0ee-d21c52bd9dfb","added_by":"auto","created_at":"2024-05-22 15:35:57","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":74371,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of different salt concentrations on Na \u003csup\u003e+\u003c/sup\u003e, K\u003csup\u003e+\u003c/sup\u003e and Cl\u003csup\u003e-\u003c/sup\u003e content in callus from five soft wheat varieties.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-4368371/v1/d3eb27580dc998b0fc110e12.png"},{"id":56954534,"identity":"7d7a64f7-f30b-4ada-b51b-e195fe846fa9","added_by":"auto","created_at":"2024-05-22 15:35:59","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":10261,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of different salt concentrations on the K +/ Na + ratio of five wheat varieties\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-4368371/v1/c1653ef773bb99a085465fe2.png"},{"id":56954532,"identity":"d4c952ab-9496-49f8-ac4b-3d513529c68a","added_by":"auto","created_at":"2024-05-22 15:35:58","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":374180,"visible":true,"origin":"","legend":"\u003cp\u003ePrincipal component analysis for the five varieties tested and the measured parameters included in the analysis (PCA).\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4368371/v1/f069e928835f9b3b4388d3a4.jpeg"},{"id":64618841,"identity":"5ca8ff9e-37bf-453b-b9ce-34f4a070227c","added_by":"auto","created_at":"2024-09-16 15:58:10","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2093151,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4368371/v1/44c31b6b-e718-4e71-b6ef-8f6e82336c0b.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Examining the Physiological Traits of Callus Tissues from Endosperm- Supported Mature Embryos in Common Wheat (Triticum aestivum L.) under In Vitro Salt Stress Conditions","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSoil salinization is considered one of the major abiotic stresses restricting crop yields in about 20% of the world's cultivated and irrigated lands due to inappropriate irrigation practices and poor water quality \u003csup\u003e\u003cb\u003e1\u003c/b\u003e\u003c/sup\u003e. This environmental constraint disastrously affects crop growth, morphological and physiological characters \u003csup\u003e\u003cb\u003e2,3\u003c/b\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eSeveral plant species have shown variable physiological and metabolic responses to adverse environmental stresses \u003csup\u003e\u003cb\u003e4\u0026ndash;6\u003c/b\u003e\u003c/sup\u003e. Regarding the harmful effect of salt stress, the excess of salt causes the reduction of water and nutrient absorption. This leads to the accumulation of toxic ions, nutritional imbalance and water deficit \u003csup\u003e\u003cb\u003e7\u003c/b\u003e\u003c/sup\u003e. Further, ion toxicity, evoke cellular interference and excess generation of toxic reactive oxygen species (ROS) which may interrupt cellular functions and negatively causes plasma membrane disruption, hindrance to respiration and damage to enzyme structure \u003csup\u003e\u003cb\u003e8,9\u003c/b\u003e\u003c/sup\u003e. Generally, as sessile organisms, plants are faced with several abiotic stresses throughout their life cycle. To maintain their normal basal metabolism, survival and optimum growth, adapted organisms are those that have managed to elaborate a series of coping mechanisms in favor of their defense against such stresses \u003csup\u003e\u003cb\u003e10\u003c/b\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eFor instance, salinity generates osmotic and ionic stress, which alters the patterns of nutrient availability and transport, consequently causing enormous changes in plant growth \u003csup\u003e\u003cb\u003e11\u003c/b\u003e\u003c/sup\u003e. One of the essential adaptive mechanisms ensuring osmotic balance in a saline condition is stimulated synthesis and accumulation in cytoplasm of small organic molecules, known as compatible solutes/osmolytes \u003csup\u003e\u003cb\u003e12\u003c/b\u003e\u003c/sup\u003e. These compounds apart from their fundamental function in osmotic adjustment play several roles, such as maintaining cellular integrity, ensuring normal cellular function, altering antioxidant enzyme activity, and inorganic ion concentration \u003csup\u003e\u003cb\u003e13,14\u003c/b\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eNowadays, \u003cem\u003ein vitro\u003c/em\u003e culture strategies are the most effective means to study plant tolerance to stress. Indeed, Tissue culture techniques allow examining multiple plant production as well as their physiological and biochemical aspects. In particular, the use of tissue culture is essential to monitor their response to stress conditions and to select tolerant species \u003csup\u003e\u003cb\u003e15\u003c/b\u003e\u003c/sup\u003e. Furthermore, these methods present an ideal strategy to save time and space. In addition, the use of these techniques with inexpensive laboratory facilities offers the possibility of improving the stress resistance in plants \u003csup\u003e\u003cb\u003e16\u003c/b\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe aim of this study is to unveil the detrimental salt effect on callus growth, organic and inorganic solutes accumulation involved in salinity tolerance in five bread wheat varieties subjected to different NaCl concentrations in \u003cem\u003ein vitro\u003c/em\u003e culture conditions.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMass of fresh weight (FW) and dry weight (DW) and water content (WC)\u003c/h2\u003e \u003cp\u003eThe illustrated results Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e for callus growth in terms of fresh and dry weight of the five varieties indicate the negative influence of salinity as the concentration increases. The ANOVA results for FW and DW showed significant differences among the varieties studied and significant effects of different salt concentrations and their interaction. However, the FW criterion depends on the varietal effect while the DW parameter is controlled by the salinity effect (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In the absence of salt stress (0 g.L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e NaCl), the mass of fresh and dry weight is considerably important (values between 901.33 and 447.50 mg of FW and 70.47 and 43.03 mg of DW). By increasing the salt concentration, both parameters showed a more remarkable decrease. Statistically, Rajae variety always maintains a better production in mass of fresh and dry weight (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). For example, the values noted for this variety at 3, 6 and 9 g.L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e NaCl, are: 632.33 /50.87 mg; /33.27; 267.50/ 29.20 mg unlike Wissam variety which presents the lowest values (209.20/20.90 mg; 118.97/ 13.97 mg; 86.90 / 11.80 mg.\u003c/p\u003e \u003cp\u003eConcerning the water content of the callus (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), this criterion depends on the saline concentration used and the variety tested. The results of the statistical analysis of variance for this parameter indicate significant effects applied by the variety and salinity. However, the interaction of the two factors \"Salinity*variety\" was found to be statistically insignificant (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Indeed, this parameter decreases progressively when the salinity increases, especially in the Wissam variety compared to the other varieties, which show a slight decrease irrespective of the salinity level (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). For example, the values recorded for this parameter at the concentration 9 g.L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e NaCl are 89.05; 88.89; 88.76; 87.68 and 86.43% respectively, in the varieties Rajae, Wafia, Nassim, Tigre and Wissam.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAnalysis of variance of the Means for the Fresh weight, dry weight, water content and salinity tolerant index based on the salt concentration effect and the variety.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"6\" nameend=\"c6\" namest=\"c1\"\u003e \u003cp\u003eSum of squares\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSource of variation\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDF\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFW (mg)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDW (mg)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eWC (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSTI (DW)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eVariety (V)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e907931.25***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e4869.27***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e36.06*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1838.86***\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eNaCl (g/L)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2396006.65***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e13073.02***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e100.55***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e41966.33***\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eV*NaCl (g/L)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e109435,25*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e359.18*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6.04\u003csup\u003ens\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e968.20*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eModel\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3413373.15***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e18301.47***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e142.65**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e44773.39***\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eError\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e140181.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e524.93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e96.39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1553.08\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eC. Total\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3553554.71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e18826.40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e239.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e46326.47\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e*, **, ***: significant respectively, at the levels of 5%, 1% and 0.1%; and ns: not significant. DF: Degree of freedom.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComparison of the effects of NaCl concentration and the variety on Fresh weight, dry weight, water content and salinity tolerant index. The data are presented by the means (\u0026plusmn;\u0026thinsp;ES).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e \u003cp\u003eParameters\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNaCl (g/L)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFW (mg)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDW (mg)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eWC (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSTI (DW)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e0\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e681.61\u0026thinsp;\u0026plusmn;\u0026thinsp;46.64\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e56.47\u0026thinsp;\u0026plusmn;\u0026thinsp;2.93\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e91.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e100\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e400.33\u0026thinsp;\u0026plusmn;\u0026thinsp;45.37\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e34.97\u0026thinsp;\u0026plusmn;\u0026thinsp;3.40\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e90.91\u0026thinsp;\u0026plusmn;\u0026thinsp;0.37\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e60.44\u0026thinsp;\u0026plusmn;\u0026thinsp;3.76\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e6\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e224.67\u0026thinsp;\u0026plusmn;\u0026thinsp;29.78\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e21.97\u0026thinsp;\u0026plusmn;\u0026thinsp;2.04\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e89.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.47\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e37.98\u0026thinsp;\u0026plusmn;\u0026thinsp;1.81\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e9\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e168.51\u0026thinsp;\u0026plusmn;\u0026thinsp;19.80\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e19.02\u0026thinsp;\u0026plusmn;\u0026thinsp;1.76\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e88.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.45\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e32.96\u0026thinsp;\u0026plusmn;\u0026thinsp;1.82\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eVarieties\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eNassim\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e346.63\u0026thinsp;\u0026plusmn;\u0026thinsp;63.51\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e31.22\u0026thinsp;\u0026plusmn;\u0026thinsp;4.77\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e90.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.49\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e55.85\u0026thinsp;\u0026plusmn;\u0026thinsp;8.42\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eRajae\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e549.66\u0026thinsp;\u0026plusmn;\u0026thinsp;75.71\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e45.95\u0026thinsp;\u0026plusmn;\u0026thinsp;5.13\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e90.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0.62\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e65.85\u0026thinsp;\u0026plusmn;\u0026thinsp;7.49\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTigre\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e263.98\u0026thinsp;\u0026plusmn;\u0026thinsp;54.67\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e25.13\u0026thinsp;\u0026plusmn;\u0026thinsp;4.12\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e89.46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.54\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e52.58\u0026thinsp;\u0026plusmn;\u0026thinsp;8.60\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eWafia\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e464.32\u0026thinsp;\u0026plusmn;\u0026thinsp;72.85\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e40.82\u0026thinsp;\u0026plusmn;\u0026thinsp;5.07\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e90.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.60\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e62.82\u0026thinsp;\u0026plusmn;\u0026thinsp;7.82\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eLevels not connected by the same letter are significantly different at \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.05\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eSalinity tolerance index in relation to callus weight (fresh and dry)\u003c/h2\u003e \u003cp\u003eThe results of the callus tolerance index for the five varieties studied (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) illustrate the negative effect of salinity when its concentration increased in the culture medium. Significant differences for the factors \"variety and salinity\" as well as for the interaction were revealed for the STI. However, this parameter was more marked by the salinity factor (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Indeed, the STI of treated callus compared to control callus favored the variety Rajae among all the varieties tested. The values recorded for this criterion at the concentration 3 g.L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e NaCl, show weaker tolerance reactions compared to the control. However, the two varieties Rajae and Wafia seem to be more adjusted but equally to this salt dose by registering a tolerance value of 72.67%. By increasing the salt concentration to 6 and 9 g.L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of NaCl, the response to salinity is more affected in all the tested varieties by presenting values lower than 35% respectively, for the varieties Wissam, Tigre and Nassim. However, the varieties Rajae and Wafia maintain the highest values (47.41/42.09; 42.09/35.82%). Statistically, Rajae is the best performing variety while Tiger and Wissam are the most susceptible (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eProline and total soluble sugar (\u0026micro;moles/g FW)\u003c/h2\u003e \u003cp\u003eThe graphics in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e shows the evolution of total proline and sugar content as a function of salt concentration and variety. The kinetics obtained are relatively fluctuating. Thus, we noted a high accumulation in the stressed callus compared to the controls at all salt concentrations. The accumulation of these two osmoprotectants is significantly affected according to salt level and the varietal effect (Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Statistically, at 9g/l NaCl the highest values were recorded respectively, in the two varieties Rajae (12.06 proline \u0026micro;mol/g FW \u0026minus;\u0026thinsp;43 \u0026micro;mol golucose/g FW ) and Wafia (10.24 proline \u0026micro;mol/g MF \u0026minus;\u0026thinsp;40.47 \u0026micro;mol golucose/g MF). Therefore, this response confers them the character of adaptation (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e) comparatively, with the variety Wissam, which always reserves a position of sensitivity by accumulating the quantity of proline and the lowest total sugar (8,60 proline \u0026micro;mol /g FW \u0026minus;\u0026thinsp;33,33 \u0026micro;mol golucose/g FW) (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAnalysis of variance of the Means for proline and total soluble sugar content based on the salt concentration effect and the variety\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c4\" namest=\"c1\"\u003e \u003cp\u003eSum of squares\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSource of variation\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDF\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eProline (\u0026micro;mol/g FW)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTotal soluble sugar (\u0026micro;mol/g FW)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eVariety (V)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e17.63***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e223.32***\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eNaCl (g/L)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e454.26***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4669.02***\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eV*NaCl (g/L)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8.11\u003csup\u003ens\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e62.17\u003csup\u003ens\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eModel\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e479.99***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4954.51***\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eError\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e16.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e112.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eC. Total\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e496.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5066.52\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e*, **, ***: significant respectively, at the levels of 5%, 1% and 0.1%; and ns: not significant. DF: Degree of freedom.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComparison of the effects of NaCl concentration and the variety on proline and total soluble sugar content.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e \u003cp\u003eParameters\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNaCl (g/L)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eProline (\u0026micro;mol/g FW)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTotal soluble sugar (\u0026micro;mol/g FW)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e0\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e18.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.54\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4.36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e22.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.48\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e6\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e35.31\u0026thinsp;\u0026plusmn;\u0026thinsp;0.66\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e9\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.38\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e39.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.97\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eVarieties\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eNassim\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.85\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e29.21\u0026thinsp;\u0026plusmn;\u0026thinsp;2.91\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eRajae\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6.98\u0026thinsp;\u0026plusmn;\u0026thinsp;1.00\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e30.97\u0026thinsp;\u0026plusmn;\u0026thinsp;2.89\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTigre\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5.90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.86\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e28.34\u0026thinsp;\u0026plusmn;\u0026thinsp;2.81\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eWafia\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6.36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.82\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e30.05\u0026thinsp;\u0026plusmn;\u0026thinsp;2.71\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eLevels not connected by the same letter are significantly different at p\u0026thinsp;=\u0026thinsp;0.05\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eNa+, K\u0026thinsp;+\u0026thinsp;and Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e content\u003c/h2\u003e \u003cp\u003eThe variation of sodium, potassium and chloride content as a function of salinity concentration and variety tested was shown in the following Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. By analyzing the variance for the three variables Na\u003csup\u003e+\u003c/sup\u003e, K\u003csup\u003e+\u003c/sup\u003e and Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e we found significant differences for the effect of variety and salinity. However, the interaction between these two factors was found to be non-significant for both Na\u0026thinsp;+\u0026thinsp;and K\u0026thinsp;+\u0026thinsp;variables and significant for the Cl- variable (Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Callus treated with different salt concentrations showed an upward progression in the accumulation of both Na\u0026thinsp;+\u0026thinsp;and Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e ions parallel to a reduction in K\u003csup\u003e+\u003c/sup\u003e ions as the salt concentration increased compared to control callus in all varieties. According to all concentrations combined, Rajae variety stands out significantly from the other varieties by exhibiting the lowest Na\u0026thinsp;+\u0026thinsp;and Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e values and the highest K\u003csup\u003e+\u003c/sup\u003e values (Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). For instance, at the high concentration 9 g/L NaCl the values 31, 15 Na\u003csup\u003e+\u003c/sup\u003e mg/g DW; 12, 61 K\u003csup\u003e+\u003c/sup\u003e mg/g DW and 12, 82 Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e mg/g DW are recorded for the Rajae variety. While the values 52.74 Na\u003csup\u003e+\u003c/sup\u003e mg/g MS; 8.39 K\u003csup\u003e+\u003c/sup\u003e mg/g DW and 15.24 (Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e mg/g DW) are correspondent to callus from the variety Wissam.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAnalysis of variance of the Means for inorganic solutes (Na+, K\u0026thinsp;+\u0026thinsp;and Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e) and K\u003csup\u003e+\u003c/sup\u003e/Na\u003csup\u003e+\u003c/sup\u003e ratio based on the salt concentration effect and the variety\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"7\" nameend=\"c7\" namest=\"c1\"\u003e \u003cp\u003eSum of squares\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSource of variation\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDF\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNa\u003csup\u003e+\u003c/sup\u003e (mg/g DW)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eK\u003csup\u003e+\u003c/sup\u003e (mg/g DW)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCl\u003csup\u003e\u0026minus;\u003c/sup\u003e (mg/g DW)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eRatio K\u003csup\u003e+\u003c/sup\u003e/Na\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"1\" nameend=\"c7\" namest=\"c7\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eVariety (V)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e880.20**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e635.80***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e81.07***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e8.67***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c7\" namest=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eNaCl (g/L)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10689.17***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2445.56***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1136.79***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e85.72***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c7\" namest=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eV*NaCl (g/L)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e590.71\u003csup\u003ens\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e215.55\u003csup\u003ens\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e11.22***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4.39***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c7\" namest=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eModel\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e12160.08***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3296.90***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1229.08***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e98.79***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c7\" namest=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eError\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1674.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e778.55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e9.93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c7\" namest=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eC. Total\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e13834.55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4075.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1239.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e99.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c7\" namest=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"7\" nameend=\"c7\" namest=\"c1\"\u003e \u003cp\u003e*, **, ***: significant respectively, at the levels of 5%, 1% and 0.1%; and ns: not significant. DF: Degree of freedom.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab6\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 6\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComparison of the effects of NaCl concentration and the variety on for inorganic solutes (Na+, K\u0026thinsp;+\u0026thinsp;and Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e) and K+/Na\u0026thinsp;+\u0026thinsp;ratio.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e \u003cp\u003eParameters\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNaCl (g/L)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNa\u003csup\u003e+\u003c/sup\u003e (mg/g DW)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eK\u003csup\u003e+\u003c/sup\u003e (mg/g DW)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCl\u003csup\u003e\u0026minus;\u003c/sup\u003e (mg/g DW)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRatio K\u003csup\u003e+\u003c/sup\u003e/Na\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e0\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8.69\u0026thinsp;\u0026plusmn;\u0026thinsp;0.41\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e28.14\u0026thinsp;\u0026plusmn;\u0026thinsp;1.36\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.63\u0026thinsp;\u0026plusmn;\u0026thinsp;0.43\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e12.45\u0026thinsp;\u0026plusmn;\u0026thinsp;0.56\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e21.11\u0026thinsp;\u0026plusmn;\u0026thinsp;1.18\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.86\u0026thinsp;\u0026plusmn;\u0026thinsp;0.39\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e6\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e31.90\u0026thinsp;\u0026plusmn;\u0026thinsp;2.37\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e18.46\u0026thinsp;\u0026plusmn;\u0026thinsp;2.03\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e12.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.62\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e9\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e40.84\u0026thinsp;\u0026plusmn;\u0026thinsp;2.98\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.63\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e14.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eVarieties\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eNassim\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e23.53\u0026thinsp;\u0026plusmn;\u0026thinsp;4.21\u003csup\u003eabc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e18.82\u0026thinsp;\u0026plusmn;\u0026thinsp;2.07\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8.76\u0026thinsp;\u0026plusmn;\u0026thinsp;1.43\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eRajae\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e18.58\u0026thinsp;\u0026plusmn;\u0026thinsp;3.10\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e25.14\u0026thinsp;\u0026plusmn;\u0026thinsp;2.61\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7.33\u0026thinsp;\u0026plusmn;\u0026thinsp;1.40\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.49\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTigre\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e26.51\u0026thinsp;\u0026plusmn;\u0026thinsp;5.06\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e16.82\u0026thinsp;\u0026plusmn;\u0026thinsp;1.99\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9.74\u0026thinsp;\u0026plusmn;\u0026thinsp;1.22\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.31\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ewafia\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e19.97\u0026thinsp;\u0026plusmn;\u0026thinsp;3.59\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20.72\u0026thinsp;\u0026plusmn;\u0026thinsp;2.67\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8.35\u0026thinsp;\u0026plusmn;\u0026thinsp;1.34\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.68\u0026thinsp;\u0026plusmn;\u0026thinsp;0.41\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eLevels not connected by the same letter are significantly different at p\u0026thinsp;=\u0026thinsp;0.05\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eK\u003csup\u003e+\u003c/sup\u003e/Na\u003csup\u003e+\u003c/sup\u003e ratio\u003c/h2\u003e \u003cp\u003eThe ratio [K\u003csup\u003e+\u003c/sup\u003e/Na\u003csup\u003e+\u003c/sup\u003e], was significantly reduced in all the callogenic clusters as a function of salinity at different concentrations and variety tested (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Statistically (Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e), significant differences are considered for both factors (Variety and Salinity) as well as for their interaction. In the absence of salt stress, the values noted for this criterion are important but variable according to each variety (between 4.66 and 2.41). Furthermore, in comparison with the control, the K\u003csup\u003e+\u003c/sup\u003e/Na\u003csup\u003e+\u003c/sup\u003e ratios maintained by the five varieties studied were strongly affected by salinity, especially at the concentration 6 and 9 g.l\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e NaCl. However, the highest ratio was recorded for the variety Rajae. While the lowest is obtained by Tigre and Wissam. As an example, at the high concentration 9 and 6 g.l\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e NaCl, the values corresponding to the K+/Na\u0026thinsp;+\u0026thinsp;ratio are 0.40 and 0.16 respectively, for Rajae and Wissam varieties. Statistical results show that Rajae and Wafia varieties perform better in terms of ionic selectivity and have a good uptake of potassium ions compared to the sensitive varieties Wissam and Tigre (Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003ePrincipal component analysis\u003c/h2\u003e \u003cp\u003ePrincipal component analysis (PCA) was used to graphically represent the relationship between agro-physiological characteristics and the different soft wheat varieties. It revealed an apparent correlation between the parameters evaluated and the different varieties studied (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe two axes describe a total variation of 98.42%. The first axis (PC1) expresses the greatest variation with 95.79% and shows high values for all the parameters evaluated. On the one hand, it gathers the varieties Wissam and Tigre as salt sensitive varieties thus revealing a high accumulation of Cl- and Na\u0026thinsp;+\u0026thinsp;ions in relation to dry matter. On the other hand, it classifies the varieties Rajae and Wafia as salt tolerant varieties with a high rate of fresh and dry weight and a high accumulation of K\u0026thinsp;+\u0026thinsp;cation in relation to dry weight, proline, and total sugar. Furthermore, it ranks the variety Nassin as moderately salt tolerant. The second axis (PC2) represented a variation of 2.63% that exposes total sugars and K\u0026thinsp;+\u0026thinsp;cation with high scores. In addition, it classifies Rajae as the variety that accumulates high content of both total sugars and K\u0026thinsp;+\u0026thinsp;cation. As for the results related to the correlation analysis (Table\u0026nbsp;\u003cspan refid=\"Tab7\" class=\"InternalRef\"\u003e7\u003c/span\u003e), callus growth in fresh and dry weight mass is strongly and positively correlated (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) with water content (WC), proline and total sugars and K\u0026thinsp;+\u0026thinsp;ion content. On the contrary, the correlations are negatively significant between these same growth descriptors (DW and FW) and Na\u0026thinsp;+\u0026thinsp;and Cl- ion contents. Besides, the K\u0026thinsp;+\u0026thinsp;ion content of the callus of all the varieties studied is strictly and positively correlated with the water content (WC) and the proline content (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). However, the correlation between soluble sugar content and K\u0026thinsp;+\u0026thinsp;ion content was not significant. The proline content was positively and significantly correlated with the soluble sugars and K\u0026thinsp;+\u0026thinsp;ions content. As for soluble sugars, they show a negative significant correlation with mineral solutes (Na\u0026thinsp;+\u0026thinsp;and Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e). The correlation is positively significant (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) between the contents of Na\u0026thinsp;+\u0026thinsp;and Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e ions and negatively significant with K\u0026thinsp;+\u0026thinsp;ions.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab7\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 7\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe correlation coefficient between the Agro-physiological parameters for the five studied wheat varieties\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"9\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eParameters\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFW (mg)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDW (mg)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eWC (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eProline (\u0026micro;mol/gMF)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eTotal sugar (\u0026micro;moles/g MF)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNa+\u003c/p\u003e \u003cp\u003emg/gMS\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003ek+\u003c/p\u003e \u003cp\u003emg/gMS\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eCL- mg/gMS\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eFM (mg)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.999 ***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.942*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.961**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.900*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-0.989**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.974**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e-0.969**\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eDM (mg)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.934*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.949*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.890*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-0.989**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.965**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e-0.960**\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eWC (%)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.942*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.962**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-0.968**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.905*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e-0.989**\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eProline (\u0026micro;mol/gMF)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.949*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-0.951*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.964**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e-0.980**\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTotal sugar (\u0026micro;moles/g MF)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-0.938*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.851\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e-0.963**\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eNa\u0026thinsp;+\u0026thinsp;mg/gMS\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-0.936*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.977*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ek\u0026thinsp;+\u0026thinsp;mg/gMS\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e-0.951*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCL- mg/gMS\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e1.000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eAuthors should discuss the results and how they can be interpreted from the perspective of previous studies and of the working hypotheses. The findings and their implications should be discussed in the broadest context possible. Future research directions may also be highlighted. Soil salinization is a major process and a significant threat to land degradation, which adversely affects soil fertility, thereby, the agricultural production and yield \u003csup\u003e17\u003c/sup\u003e. It causes osmotic, ionic, and oxidative stress, thus resulting in reduction of plant growth and development. Because of the high salt content, a variety of negative consequences arises. One of the most serious complications is ion imbalance. For example, a high concentration of sodium and Chloride ions can cause biochemical processes that might be lethal at the plant level. To comprehend each tolerance mechanism at the plant level, it is necessary first to understand each tolerance mechanism at the cellular level \u003csup\u003e18\u003c/sup\u003e. In this manner, numerous studies have been focusing on investigating \u003cem\u003ein vitro\u003c/em\u003e salt tolerance by measuring the accumulated levels of toxic ions mostly sodium toxicity and its adverse effect on plant growth. In the present work, we aimed to study \u003cem\u003ein vitro\u003c/em\u003e the salt effect on bread wheat by determining the contents of compatible solutes such as, proline and soluble total sugars and inorganic solutes like sodium, potassium and chloride. The contents were analyzed individually using callus of five varieties of bread wheat induced directly in the presence of different salt levels (0, 3, 6 and 9 g/l NaCl).\u003c/p\u003e \u003cp\u003e \u003cb\u003eMass of fresh matter (FM), dry matter (DM), water content WC (%) and salt tolerance index (STI)\u003c/b\u003e \u003c/p\u003e \u003cp\u003eOne of the first and most typical reactions to stress is decreased plant growth. Our results indicated a negative effect of salinity on callus growth in terms of fresh and dry matter as the concentration increases in the culture medium especially at 9 g/l NaCl. Golkar et al \u003csup\u003e19\u003c/sup\u003e stated in their finding that the 200 mM NaCl salt concentration had the highest inhibitory effect on callus formation for all genotypes studied. According to Arzani and Ashraf \u003csup\u003e20\u003c/sup\u003e this finding can be explained by the osmotic effect of NaCl, which results in a decrease in the water content of the cytosol in turn causing a decrease in callus growth. On the other hand, Hamedi et al \u003csup\u003e21\u003c/sup\u003e indicated the increase in salt concentration could lead to a reduction in the osmotic potential of the medium, a decrease in turgidity of the growing cells and eventually a decrease in callus growth. On the other hand, researchers such as Cicek and Cakirlar \u003csup\u003e22\u003c/sup\u003e have previously reported that the negative effect of salinity on plant growth might be due to the occurrence of defective metabolism in plant cells. Since the high osmotic pressure resulted from high salinity that prevents plant cells to absorb water and some mineral nutrients dissolved in the culture medium. In addition, in two bread wheat cultivars Al Hattab et al \u003csup\u003e23\u003c/sup\u003e, affirmed that the reduction in fresh and dry weight of callus is due to ionic imbalances within the cells. The accumulations of sodium and chloride ions in the cells will reduce the availability of water, which induces osmotic stress that presents negative effects on cell growth \u003csup\u003e24\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eActually, \u003cem\u003ein vitro\u003c/em\u003e culture techniques have formerly been recognized to provide controllable environmental conditions for examining salt-stress effects at the cellular level \u003csup\u003e25\u003c/sup\u003e. Under salt stress Shibli et \u003cem\u003eal\u003c/em\u003e \u003csup\u003e26\u003c/sup\u003e point out that the reduction in the osmotic potential of the media culture could compromise callus growth thus, reinforcing the need to maintain turgor in growing callus cells. A process that consumes the majority of the energy and ultimately leads to callus growth reduction. Moreover, the NaCl induced gradient in water potential between the cell and the nutritional medium has been proven to cause cell dehydration and callus fresh weight reduction \u003csup\u003e27\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eRegarding the STI, the recorded values show a better response to salinity of the variety Rajae followed by Wafia, unlike the other three varieties. Similarly, in their study Haque et al \u003csup\u003e28\u003c/sup\u003e showed that the recorded values for salinity tolerance index and callus growth declined when calluses were grown at higher NaCl levels. They associated this response to the reduction of water availability, and loss of turgor pressure (TP) in callus cells.\u003c/p\u003e\n\u003ch3\u003eProline and total soluble sugars content\u003c/h3\u003e\n\u003cp\u003eBeing exposed to salt stress, plants accumulates several compounds contributing to the osmotic regulation. Proline is one of the most essential and efficient compatible metabolites amid other organic osmolytes. It plays a vital role in defense by minimizing the salt-stress effects and improve plant growth \u003csup\u003e29\u003c/sup\u003e. Salt pressure affected the accumulation of proline for all the five varieties gradually with increasing NaCl concentration in the culture medium. However, the content values varied according to the response of each variety. Our results, advocate those obtained by Khuder and Hussein \u003csup\u003e30\u003c/sup\u003e, they revealed that the increase in salt causes a high accumulation of proline in the treated calluses while, the lowest concentration of proline was recorded in the control calluses. This elevation can be considered as a clever coping strategy against ion accumulation since it plays an important role in fixing osmosis between the vacuole and the cytoplasm of the cell \u003csup\u003e31\u003c/sup\u003e. Proline is also recorded as an osmoprotectant with significant contribution under salt conditions, especially in assisting with water absorption, protecting cellular functions by scavenging reactive oxygen species (ROS) molecules, stabilizing membranes and subcellular structures in cytosol, modulating cell redox homeostasis, supplying energy, and functioning as a signal \u003csup\u003e32\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eFormerly, Girija et al \u003csup\u003e33\u003c/sup\u003e explained, the increase in proline content under stress conditions may be due to the decrease in the activity of enzymes involved in proline degradation such as proline dehydrogenase and proline oxidase. Moreover, we remarked that two varieties (Rajae and Wafia) accumulate more proline than the other varieties, indicating a tolerance response towards elevated salt concentration. As reported by Shahid et al \u003csup\u003e18\u003c/sup\u003e, improving the ability of cells to make ionic adjustments, proline has to accumulate to higher levels in the cytosol. This accumulation is linearly related to plant stress tolerance. Although acting as an osmolyte, proline is also thought to be a potent antioxidant defense molecule involved in the inhibition of programmed cell death \u003csup\u003e34\u003c/sup\u003e. Plants also engage another important osmoprotector as one of the defense mechanisms, namely soluble sugar, which is generally known to accumulate under stressful conditions. Studies conducted by khuder and Hussein \u003csup\u003e30\u003c/sup\u003e on carbohydrate accumulation in callus from soft wheat, showed that this criterion is controlled by varietal effect. These findings are in concordance with ours. The content of soluble sugar is higher especially at the higher NaCl level 9 g/L. Both Rajae and Wafia accumulates more sugar comparatively with the three other varieties tested.\u003c/p\u003e \u003cp\u003eAccording to Slama et al \u003csup\u003e35\u003c/sup\u003e in the presence of salt stress, sugar accumulation attributes to the maintenance of osmotic and water balance in cells. In addition, Bezirğanoğlu \u003csup\u003e16\u003c/sup\u003e also showed that the highest level of sugar was accumulated in the presence of the high concentration 200 mM. He added that this trait appeared to present an effective marker of salt resistance in embryogenic callus derived from triticale genotypes. Besides, many in vivo and in vitro studies of Physiological, biochemical, and molecular characters conducted under salt conditions, show that osmolites play important roles in the plant\u0026rsquo;s defense system against stress \u003csup\u003e36\u003c/sup\u003e. Higher proline and total soluble sugar content accumulation was also observed in other plants exposed to salt stress \u003csup\u003e37,38\u003c/sup\u003e. According to Muchate et al \u003csup\u003e39\u003c/sup\u003e, this might be associated with higher energy consumption for osmolyte synthesis, which, could lead to growth decline. On the other hand, these solutes are essential to prevent damage at the cellular level and mitigating the negative effects of excessive ion concentrations on enzyme activities [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePlants can accumulate Na\u0026thinsp;+\u0026thinsp;and Cl- ions to maintain the water potential of the tissues necessary for their growth. While this accumulation, which requires relatively less energy expenditure, must remain compatible with a metabolic tolerance of the resulting concentration or with a compartmentalization between the different components of the cell or plant. Conversely, the synthesis of organic solutes is generated when a metabolic change takes place in the cells under the action of salt pressure. The main objective is to adjust the water potential. Therefore, the main adaptation strategy is the accumulation of osmoprotective agents, mainly amino compounds and sugars known to be low molecular mass in the cytoplasm as mentioned by Ketehouli et al \u003csup\u003e40\u003c/sup\u003e Shahid et al [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Moreover, It has been stated by Gill and Tuteja \u003csup\u003e41\u003c/sup\u003e, Hayat et al \u003csup\u003e42\u003c/sup\u003e and Farissi et al \u003csup\u003e43\u003c/sup\u003e that salinity causes secondary stress, known as oxidative stress, when reactive oxygen species (ROS) are formed in cells, which leads to disrupt normal metabolism by peroxidizing proteins and lipids, damaging DNA, and inactivating enzymes. As a survival behavior, the accumulation of compatible osmolytes is strictly indispensable to protects plant cells from the negative effects of salt \u003csup\u003e44\u003c/sup\u003e.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eNa+, K\u0026thinsp;+\u0026thinsp;and Cl- content\u003c/h2\u003e \u003cp\u003eFor most plants, responding to salt stress require the restraining of excessive sodium accumulation. Ion homeostasis is in fact one of the effective physiological strategies deployed by plants to ensure tolerance against abiotic stress. As stated by Shahid et al [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], sodium is not considered as necessary element for plant development. In fact, plants do not require Na, nor do they have a specific Na transport mechanism. However, when the plant is exposed to high concentrations of sodium, it excessively enters the plant cells through various mechanisms. In addition, Na\u0026thinsp;+\u0026thinsp;causes inhibition of the uptake of other cations, notably K+. Indeed, the hydrated ionic radii of K\u0026thinsp;+\u0026thinsp;are similar to those of Na+, making it easily replaced by this cation. Therefore, the transport proteins cannot distinguish between them. Because Na\u0026thinsp;+\u0026thinsp;cannot perform the biological function of K+, reduced K uptake can eventually lead to reduced plant growth and productivity under saline conditions. Indeed, the balance between sodium and potassium in a cell is crucial for plants to survive in saline soil \u003csup\u003e45,46\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn the present work, the results indicate a significant accumulation of Na\u003csup\u003e+\u003c/sup\u003e and Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e ions and a decrease in K\u003csup\u003e+\u003c/sup\u003e ion content that occurs progressively with increasing salt concentration yet, depending on the varietal reaction. These findings corroborate those obtained by Arzani and Ashraf [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] who revealed that the reduction in K\u003csup\u003e+\u003c/sup\u003e concentration in callus cells under salt stress could be explained by alterations in the expression and/or function of transporters as well as ion channels especially those related to K\u003csup\u003e+\u003c/sup\u003e. In another \u003cem\u003ein vitro\u003c/em\u003e experiment under salt stress to select tolerant varieties of durum wheat from mature embryos, Koutoua et al \u003csup\u003e47\u003c/sup\u003e reported that Na\u0026thinsp;+\u0026thinsp;accumulation was greater in salt sensitive calli than in tolerant calli. The behavior of potassium, on the contrary, appears to be variable when compared to sodium. Both tolerant and sensitive calli accumulated less K\u0026thinsp;+\u0026thinsp;than those of control, although the K\u0026thinsp;+\u0026thinsp;content of salt tolerant calli remained higher than that of sensitive ones.\u003c/p\u003e \u003cp\u003eAs for the low accumulation of K\u003csup\u003e+\u003c/sup\u003e ions by some of our studied varieties (Tigre and Wissam), Golkar et al [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] stated that, the imposition of NaCl shock treatment causes tissue damage, which leads to excessive leaching and poor retention of K\u003csup\u003e+\u003c/sup\u003e in callus from genotypes with low salt stress tolerance. Thus, they display a very strong reduction in K\u003csup\u003e+\u003c/sup\u003e content as salt concentration increased from the control to the higher levels. On the other hand, certain varieties (Rajae and Wafia) responded better to salt stress by maintaining a higher K\u003csup\u003e+\u003c/sup\u003e ion content. Indeed, the same researchers found that compared to callus-sensitive genotypes, callus-tolerant genotypes accumulated less Na\u003csup\u003e+\u003c/sup\u003e and maintained higher levels of K\u003csup\u003e+\u003c/sup\u003e. This explained the better growth of tolerant callus in the presence of NaCl. Accordingly, higher K\u0026thinsp;+\u0026thinsp;concentration is directly related to higher biomass production \u003csup\u003e48\u003c/sup\u003e. Salt-tolerant plant species have the ability to retain a higher concentration of K. Hence, in psyllium, Karimi and Haghighat-Pak \u003csup\u003e49\u003c/sup\u003e revealed that the K\u003csup\u003e+\u003c/sup\u003e inclusion mechanism is an indicator of salt tolerance at the whole plant level. In addition to sodium and potassium ions, we noticed that the most sensitive varieties (Tigre and Wissam) accumulate a significant content of chloride compared to the tolerant variety Rajae, which present the lowest values compared to all the varieties tested. On the other hand, for the varieties Wafia and Nassim, the accumulation of chloride is not significant, but keep an intermediate position between the other three tested varieties. Shahid et al [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] stated that, Chloride also contributes to the undesirable effects of salt stress on plant growth. However, salt-tolerant plant genotypes have the ability to inhibit chloride uptake. In bread wheat and sugarcane, some researchers have found that, the content of Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e ions contained in the sensitive callus is higher comparatively to the tolerant callus \u003csup\u003e50,51\u003c/sup\u003e. On the contrary, in eight durum wheat varieties tested by Koutoua et al [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e], the tolerant callus of some varieties showed a higher accumulation of Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e ions compared to the sensitive callus.\u003c/p\u003e \u003cp\u003eFor some plants, Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e ion is more harmful than Na\u003csup\u003e+\u003c/sup\u003e. Apart from their toxicity, Cl ions have a regulatory function in the establishment of turgor, enzyme stability, membrane potential, pH, charge balance, volume control, osmoregulation, and stomatal conductance, all of which contribute to water preservation, high water usage, and photosynthetic efficiency \u003csup\u003e52,53\u003c/sup\u003e. Therefore, the toxicity of both ions must be considered [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eK\u003csup\u003e+\u003c/sup\u003e/Na\u003csup\u003e+\u003c/sup\u003e ratio\u003c/h2\u003e \u003cp\u003ePlants adopt various strategies to cope with a high Na ion concentration, such as the restriction of Na\u0026thinsp;+\u0026thinsp;entry into the cell, Na\u0026thinsp;+\u0026thinsp;extrusion, and vacuole compartmentalization. The plant\u0026rsquo;s capacity to tolerate saline conditions depends on a high K+/Na\u0026thinsp;+\u0026thinsp;cytosolic concentration \u003csup\u003e54\u003c/sup\u003e. This selection criterion has been supported by several researchers since it better expresses ionic selectivity and is therefore, used as a criterion for selecting cell lines and classifying varieties according to their response to salinity \u003csup\u003e55\u003c/sup\u003e. In earlier times, many researchers such as Farukh \u003csup\u003e56\u003c/sup\u003e, Wahid \u003csup\u003e57\u003c/sup\u003e, Ndayiragije, and Lutts \u003csup\u003e58\u003c/sup\u003e used the K+/Na\u0026thinsp;+\u0026thinsp;ratio as a criterion for varietal classification. Zhang et al \u003csup\u003e59\u003c/sup\u003e and Lui et al \u003csup\u003e60\u003c/sup\u003e also reported that salt tolerance is typified by the maintenance of Na+/K\u0026thinsp;+\u0026thinsp;homeostasis.\u003c/p\u003e \u003cp\u003eAs previously reported by Arzani and Ashraf [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], the capacity to maintain cellular K +/ Na\u0026thinsp;+\u0026thinsp;homeostasis at a high level for plant survival in saline conditions is a crucial aspect for salt tolerance. According to our study, although salinity negatively affected the behavior of all the varieties tested. However, on the same saline concentrations, the varieties considered as tolerant were able to keep high levels of K+/Na\u0026thinsp;+\u0026thinsp;ratio compared to the sensitive varieties. Therefore, our remarks are similar to those retained by Koutoua el al [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e], who indicated that the K+/Na\u0026thinsp;+\u0026thinsp;ratio decreased as the salt concentration increased. This decrease was more remarkable in sensitive callus than in tolerant ones. Golkar et al [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] presented explanations that due to the increase of NaCl concentration in the medium, Na\u0026thinsp;+\u0026thinsp;ions are rivaling with K\u0026thinsp;+\u0026thinsp;ions during salt stress for the same transporter since they both share the same transport mechanisms, hence the decrease in K\u0026thinsp;+\u0026thinsp;uptake. The detrimental effect of Na\u0026thinsp;+\u0026thinsp;also depends on its site of accumulation. Its accumulation is toxic to the salt-sensitive callus in which it is transported into the cytoplasm. Yet, it is not toxic to tolerant callus as it is accumulated in their cell vacuoles \u003csup\u003e61\u003c/sup\u003e. Consequently, the increase of osmotic pressure in the cells and the maintenance of a high K+/Na\u0026thinsp;+\u0026thinsp;ratio are strongly linked to vacuolar sequestration, which plays an important role in the maintenance of water balance in the callus. From another perspective, Negrao et al \u003csup\u003e62\u003c/sup\u003e indicated that maintaining ion homeostasis could be particularly challenging for plants under saline conditions since the accumulation of toxic ions (Na\u003csup\u003e+\u003c/sup\u003e) can disrupt the plant's ability to control the accumulation of other ions. In addition, in most species, accumulation of Na\u003csup\u003e+\u003c/sup\u003e ions to toxic levels appears to occur before Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e.However, the K\u003csup\u003e+\u003c/sup\u003e/Na\u003csup\u003e+\u003c/sup\u003e ratio does not only depend on the depressive effect of salinity but also, on the genotype effect. According to Kumar et al \u003csup\u003e63\u003c/sup\u003e, a genotype with the quality of K\u003csup\u003e+\u003c/sup\u003e ion selection over Na\u003csup\u003e+\u003c/sup\u003e ion is proven tolerant. The intracellular K\u003csup\u003e+\u003c/sup\u003e/Na\u003csup\u003e+\u003c/sup\u003e ratio in plants is a direct indicator of their salt tolerance. Plants tend to find a way to balance their cytosolic K\u003csup\u003e+\u003c/sup\u003e/Na\u003csup\u003e+\u003c/sup\u003e ratio by reducing Na\u0026thinsp;+\u0026thinsp;accumulation or compensating for K\u003csup\u003e+\u003c/sup\u003e loss \u003csup\u003e64\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e"},{"header":"Material and methods","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003ePlant material\u003c/h2\u003e \u003cp\u003eThe experiments were carried out on mature seeds of five varieties (Nassim, Wissam, Wafia, Tigre and Rajae) of soft wheat (\u003cem\u003eTriticum aestivum\u003c/em\u003e L.) which were provided to us by ONSSA (National Office for Food Safety in Rabat-Morocco). The five varieties were selected after being previously tested for their behaviour under salt stress at the germination stage. Thus, they were classified primarily as tolerant (Rajae and Nassim), intermediate (Wafia and Tigre) and sensitive (Wissam). In addition, all experiments conducted on plants comply with the strict standards of Ibn Tofail University, Morocco and and with international guidelines and legislation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eSterilization and culture\u003c/h2\u003e \u003cp\u003eMature seeds were washed with tap water supplemented with a few drops of Tween 20 and rinsed three times with distilled water. The seeds were then soaked in sterile distilled water overnight at room temperature. Under a horizontal laminar airflow cabinet, the seeds were disinfected by soaking and vigorous shaking in 45% commercial bleach (sodium hypochlorite) for 20 minutes, followed by rinsing six times with sterile distilled water under continuous shaking and transferred to sterile Petri dishes with filter paper for drying.\u003c/p\u003e \u003cp\u003eAfter sterilizing the seeds, with a sharp scalpel, several lesions were made in the embryo of the seeds. Explants were cultured on MS medium supplemented with 2 mg.L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e 2,4-dichlorophenoxyacetic acid (2,4-D), 30 g.L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e sucrose \u003csup\u003e65\u003c/sup\u003e. The culture medium was subdivided into three increasing treatments of (3, 6, 9 g.L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) NaCl and no salt in the control (0 g.L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e NaCl). The medium was adjusted to pH 5.7, solidified with 8 g.L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e agar, and sterilized by autoclaving at 120 \u0026deg; C for 20 min at 1 bar pressure. The cultures were incubated in the dark in a thermoregulated chamber at 25\u0026deg;C\u0026thinsp;\u0026plusmn;\u0026thinsp;2. The obtained 10-day-old calli were carefully removed from the rest of the seeds with a sterilized scalpel under aseptic conditions and then subcultured again regularly on fresh media of the same composition with 11/10-day intervals until the end of the incubation period (6 weeks).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003ePhysiological traits\u003c/h2\u003e \u003cdiv id=\"Sec17\" class=\"Section3\"\u003e \u003ch2\u003eCallus growth, water content and Salinity tolerance index\u003c/h2\u003e \u003cp\u003eTo quantify the negative impact of salinity on callus growth, the mass of callus matter was weighed before drying (fresh weight). The water content is determined by the percentage of water presented in three calluses per concentration according to the following formula:\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\text{W}\\text{C} \\left(\\text{%}\\right)=\\left(\\frac{\\text{F}\\text{W}-\\text{D}\\text{W}}{\\text{F}\\text{W}} \\right)\\times 100$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eTo record the dry weight, the calli were placed in an oven (75\u0026deg;C) for 48 hours. The salinity tolerance index (STI) in relation to dry weight as described by Goudarzi and Pakniyat \u003csup\u003e66\u003c/sup\u003e was calculated according to the following formula:\u003cdiv id=\"Equb\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equb\" name=\"EquationSource\"\u003e\n$$\\text{S}\\text{T}\\text{I} \\left(\\text{%}\\right)=\\frac{\\text{V}\\text{a}\\text{r}\\text{i}\\text{a}\\text{b}\\text{l}\\text{e} \\text{m}\\text{e}\\text{a}\\text{s}\\text{u}\\text{r}\\text{e}\\text{d} \\text{u}\\text{n}\\text{d}\\text{e}\\text{r} \\text{t}\\text{r}\\text{e}\\text{a}\\text{t}\\text{m}\\text{e}\\text{n}\\text{t}}{\\text{V}\\text{a}\\text{r}\\text{i}\\text{a}\\text{b}\\text{l}\\text{e} \\text{m}\\text{e}\\text{a}\\text{s}\\text{u}\\text{r}\\text{e}\\text{d} \\text{u}\\text{n}\\text{d}\\text{e}\\text{r} \\text{n}\\text{o}\\text{r}\\text{m}\\text{a}\\text{l} \\text{c}\\text{o}\\text{n}\\text{d}\\text{i}\\text{t}\\text{i}\\text{o}\\text{n}\\text{s}}\\times 100$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eAccordingly, 60 calli corresponding to three callus replicates per each variety and salt level (Callus X Variety X salt concentration) were used for the weighing of fresh (FW) and dry mass (DW) the water content (WC) and the STI %.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eProline content\u003c/h2\u003e \u003cp\u003eThe method used to determine proline was by Dreier and Goring \u003csup\u003e67\u003c/sup\u003e. The extraction was carried out on 100 mg of fresh material (Cal), mixed in the presence of 3 mL of 40% methanol and heated to 85\u0026deg;C in a water bath for 60 min. After cooling in melting ice and centrifugation (4000 T, 10 min), 1 mL of the extract was taken to which was added 1 mL of glacial acetic acid, 25 mg of ninhydrin (C\u003csub\u003e6\u003c/sub\u003eH\u003csub\u003e6\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e) and 1mL of mixture containing (120 mL of distilled water\u0026thinsp;+\u0026thinsp;300 mL of acetic acid (CH\u003csub\u003e3\u003c/sub\u003eCOOH)\u0026thinsp;+\u0026thinsp;80 mL of orthophosphoric acid (H\u003csub\u003e3\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e)). Using a vortex, the solution was well mixed, then boiled at 100\u0026deg;C for 30 min, until the solution turns red. After cooling, 5 mL of toluene were added to the solution (which was stirred), then left to stand for 30 min until the separation of two phases (an upper and a lower phase). The absorbance of the toluene fraction taken from the liquid phase (three replicates per salt level per each variety) was determined with a spectrophotometer at 528 nm (100% toluene was used as a blank and L-proline was utilized as the standard.)\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eTotal soluble sugar content\u003c/h2\u003e \u003cp\u003eSoluble sugars were determined by the method of Dubois et al \u003csup\u003e68\u003c/sup\u003e. Samples of 100 mg of plant material (callus) in test tubes were crushed in 3 mL of 80% ethanol. At the time of dosing, the tubes were placed in the oven at 80\u0026deg;C to evaporate the alcohol. In clean glass tubes, 10 mL of distilled water was added to the extract in each tube to obtain a test solution. 2 mL of the solution to be analyzed was taken and 1mL of 5% phenol and 5 mL of 96% concentrated sulfuric acid were added while avoiding pouring acid against the tubes. The total sugar content was determined at 620 nm with a spectrophotometer using glucose as standard. The tubes containing the solution (yellow-orange color) were subsequently vortexed to homogenize it before being left to stand for 10 min and placed in a water bath for 15 to 20 min at a temperature of 30 ◦C. The total sugar content was determined on three replicates per salt level per each variety at 620 nm with a spectrophotometer.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eIons accumulation Na+, K\u0026thinsp;+\u0026thinsp;and Cl- (Dry oven mineralization)\u003c/h2\u003e \u003cp\u003eCallus were rinsed with cold distilled water accordingly to remove any electrolytes adhering to the tissue surface or accumulated in the apoplast \u003csup\u003e69\u003c/sup\u003e. Sodium and potassium were determined from dried callus (0.2 g dry weight) using 60 samples with 3 replicates per variety per salt concentration. After mineralization at 500\u0026deg;C, Na\u003csup\u003e+\u003c/sup\u003e and K\u003csup\u003e+\u003c/sup\u003e dosages were determined by flame photometry. The values obtained were converted into Na\u003csup\u003e+\u003c/sup\u003e and K\u003csup\u003e+\u003c/sup\u003e content from the calibration range and the content was given in mg/g FW and then in mg/g DW. The determination of chloride ions was also performed on 60 samples (0.2 g DW) after mineralization at 500\u0026deg;C in the presence of calcium oxide with 3 repetitions per variety per salt concentration. Being soluble, chlorides are extracted by hot water. The Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e is then determined by volumetric silverometry (precipitation as AgCl in the presence of AgNO\u003csub\u003e3\u003c/sub\u003e). To visualize the end of precipitation, potassium chromate (K\u003csub\u003e2\u003c/sub\u003eCrO\u003csub\u003e3\u003c/sub\u003e, yellow in color) was used as an indicator which do transform into silver chromate (reddish tint) as soon as all Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e ions precipitated \u003csup\u003e70\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eData statistical analysis\u003c/h2\u003e \u003cp\u003eThe normality of the data distribution was analyzed by the Shapiro-wilk test, and normality is fulfilled by transformation when necessary. Analysis of variance (ANOVA) was used to evaluate the effect of salt level, treatment duration, varieties, and their interaction on different parameters. Means were compared by Tukey HSD (Honestly Significant Difference) test at the risk of error of 0.05. The link between the different studied parameters and different varieties was revealed by the principal component analysis (PCA) in terms of correlation (correlations are estimated by the ROW-WISE method). The data was analyzed by JMP SAS Pro software (JMP\u0026reg;, Version\u0026thinsp;\u0026lt;\u0026thinsp;14\u0026thinsp;\u0026gt;\u0026thinsp;SAS Institute Inc.).\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThe five wheat varieties showed different behavior under salt stress. Accumulation of organic solutes, namely proline and total soluble sugars, less chloride and maintenance of K+/Na\u0026thinsp;+\u0026thinsp;homeostasis along with high dry mass production were mainly the adaptive mechanisms to tolerate the intensity of salt applied directly and abruptly on bread wheat explants. Only two varieties Rajae and Wafia showed a high response against salt pressure compared to the other three varieties. They were therefore, statistically selected as tolerant. This research showed that \u003cem\u003ein vitro\u003c/em\u003e culture could efficaciously contribute to the selection of salt-tolerant varieties by ensuring the investigation of physiological traits involved in stress tolerance. Therefore, biochemical and molecular approaches are strongly recommended for more detailed elucidation of tolerance/sensitivity mechanisms at callus and regenerated plant levels of bread wheat.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAuthor Contributions: Conceptualization, F.M. and A.R.; methodology, F.M., H.E. and R.E.; formal analysis, F.M., R.E., M.N.A., N.M.A., Y.E. and H.E.; resources, H.I., M.N.A. and A.A.S.; data curation, H.I., A.A.S. and Y.E.; writing\u0026mdash;original draft preparation, F.M.; writing\u0026mdash;review and editing, H.I., M.N.A., A.A.S., N.M.A. and R.A.; supervision, R.A.. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors extend their appreciation to Princess Nourah bint Abdulrahman University Re-searchers Supporting Project number (PNURSP2024R103), Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eDaba, A. W. \u0026amp; Qureshi, A. S. 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Determination of Chloride in Biological Materials. in \u003cem\u003eMethods of Biochemical Analysis\u003c/em\u003e 277\u0026ndash;391 (John Wiley \u0026amp; Sons, Ltd, 1964). doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1002/9780470110300.ch6\u003c/span\u003e\u003cspan address=\"10.1002/9780470110300.ch6\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Bread wheat (Triticum aestivum L.), callus growth, organic solutes, inorganic solutes, salt tolerance, sensitivity","lastPublishedDoi":"10.21203/rs.3.rs-4368371/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4368371/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSalt tolerance is a much-needed potential in cereal crops. To date, numerous research protocols have focused on establishing selection programs to increase tolerance and productivity of crops in salt-stressed areas. In this study, we aimed to induce callus from endosperm-supported mature embryos of five common wheat varieties (Nassim, Wissam, Wafia, Rajae, and Tigre) subject directly to various increasing NaCl concentrations (0\u0026ndash;9 g/L) under \u003cem\u003ein vitro\u003c/em\u003e culture process. We chose callus growth, organic solutes accumulation, and ion content as main traits to evaluate the impact of salinity on stressed calli. Our findings indicate that the accentuated salinity pressure leads to a substantial elevation of Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e, Na\u003csup\u003e+\u003c/sup\u003e, proline and soluble carbohydrates content, along with a reduction in callus growth, potassium accumulation, and K\u003csup\u003e+\u003c/sup\u003e/Na\u003csup\u003e+\u003c/sup\u003e ratio. Yet, the performance under salt stress was significantly dependent on the varietal effect. At the highest concentration, the marked values of these solutes (12.06 \u0026micro;mol/g FM of proline; 43 \u0026micro;mol/g FM of Total soluble sugar; 31.15 mg/ g DM Na\u003csup\u003e+\u003c/sup\u003e; 12.82 mg/g DM of Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e, 12.61 mg/g DM of K\u003csup\u003e+\u003c/sup\u003e and 0.40 K+/Na+) were respectively recorded by Rajae. Principal component analysis (PCA) first classified Rajae as the most tolerant followed by wafia as tolerant, while Wissam and Tigre were ranked as sensitive. Whereas, the variety Nassim showed a moderate tolerance. Besides the K+/Na\u0026thinsp;+\u0026thinsp;Ratio and salinity tolerance index (STI), the PCA analysis has shown that all the studied physiological criteria seem worthwhile for better discrimination of the varieties tested according to their reaction to salinity.\u003c/p\u003e","manuscriptTitle":"Examining the Physiological Traits of Callus Tissues from Endosperm- Supported Mature Embryos in Common Wheat (Triticum aestivum L.) under In Vitro Salt Stress Conditions","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-05-22 15:35:51","doi":"10.21203/rs.3.rs-4368371/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f16727b9-a598-4f60-9ad0-bc3347bc79ad","owner":[],"postedDate":"May 22nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":32119775,"name":"Biological sciences/Biotechnology"},{"id":32119776,"name":"Biological sciences/Ecology"}],"tags":[],"updatedAt":"2024-07-05T08:21:38+00:00","versionOfRecord":[],"versionCreatedAt":"2024-05-22 15:35:51","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4368371","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4368371","identity":"rs-4368371","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}